U.S. patent application number 12/952091 was filed with the patent office on 2011-06-09 for b lymphocyte stimulator assays.
This patent application is currently assigned to HUMAN GENOME SCIENCES, INC.. Invention is credited to Reinhard Ebner, Jian Ni, Guo-Liang Yu.
Application Number | 20110135639 12/952091 |
Document ID | / |
Family ID | 46303884 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110135639 |
Kind Code |
A1 |
Yu; Guo-Liang ; et
al. |
June 9, 2011 |
B LYMPHOCYTE STIMULATOR ASSAYS
Abstract
The present invention relates to nucleic acid molecules encoding
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides, including
soluble forms of the extracellular domain. Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides are also provided as are vectors,
host cells and recombinant methods for producing the same. The
invention further relates to antibodies or portions thereof that
specifically bind Neutrokine-alpha and/or Neutrokine-alphaSV and
diagnostic and therapeutic methods using these antibodies. Also
provided are diagnostic methods for detecting immune system-related
disorders and therapeutic methods for treating immune
system-related disorders using the compositions of the
invention.
Inventors: |
Yu; Guo-Liang; (Berkeley,
CA) ; Ebner; Reinhard; (Gaithersburg, MD) ;
Ni; Jian; (Germantown, MD) |
Assignee: |
HUMAN GENOME SCIENCES, INC.
Rockville
MD
|
Family ID: |
46303884 |
Appl. No.: |
12/952091 |
Filed: |
November 22, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12210134 |
Sep 12, 2008 |
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12952091 |
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11054539 |
Feb 10, 2005 |
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12210134 |
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09589285 |
Jun 8, 2000 |
6881401 |
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11054539 |
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09589288 |
Jun 8, 2000 |
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12210134 |
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10270487 |
Oct 16, 2002 |
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11054539 |
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09929493 |
Aug 15, 2001 |
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10270487 |
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09588947 |
Jun 8, 2000 |
6562579 |
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09929493 |
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09589285 |
Jun 8, 2000 |
6881401 |
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09588947 |
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09589286 |
Jun 8, 2000 |
6635482 |
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09589285 |
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09589287 |
Jun 8, 2000 |
6403770 |
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09589286 |
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09586288 |
Jun 2, 2000 |
6302742 |
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09589287 |
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12170333 |
Jul 9, 2008 |
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12210134 |
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11382837 |
May 11, 2006 |
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12170333 |
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09589288 |
Jun 8, 2000 |
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11382837 |
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09589288 |
Jun 8, 2000 |
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12210134 |
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09507968 |
Feb 22, 2000 |
6812327 |
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09589288 |
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09507968 |
Feb 22, 2000 |
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09929493 |
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Feb 23, 1999 |
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PCT/US96/17957 |
Oct 25, 1996 |
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Feb 11, 2004 |
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Current U.S.
Class: |
424/133.1 ;
424/130.1; 424/173.1; 424/184.1; 436/501; 436/86 |
Current CPC
Class: |
G01N 2800/24 20130101;
C07K 16/24 20130101; C07K 2317/622 20130101; G01N 2333/52 20130101;
G01N 33/564 20130101; A61K 2039/505 20130101; C07K 2317/21
20130101; A61K 38/00 20130101; C07K 2317/34 20130101; C07K 2317/76
20130101; A61P 37/06 20180101; A61K 49/0004 20130101; G01N 33/56972
20130101; C07K 14/52 20130101; A61K 31/519 20130101; C07K 14/70575
20130101; A61P 35/00 20180101; A61P 37/00 20180101; A61P 35/02
20180101; C07H 21/04 20130101; C07K 16/2875 20130101; C07K 14/715
20130101; A61K 47/6849 20170801 |
Class at
Publication: |
424/133.1 ;
436/86; 424/130.1; 424/173.1; 424/184.1; 436/501 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/68 20060101 G01N033/68; A61K 39/00 20060101
A61K039/00; G01N 33/53 20060101 G01N033/53; A61P 37/00 20060101
A61P037/00; A61P 37/06 20060101 A61P037/06; A61P 35/00 20060101
A61P035/00 |
Claims
1. A method for monitoring B cell levels in a subject comprising
the steps of determining the serum BAFF levels in a test sample of
the subject, determining the serum BAFF levels in a control sample,
and calculating the B cell levels in the subject relative to the
control, which calculation comprises the step of comparing the
serum BAFF level in the test sample to the serum BAFF level in the
control sample.
2. The method according to claim 1, wherein the control sample is
from the subject before treatment with a therapeutic agent and the
test sample is from the subject after treatment with the
therapeutic agent.
3. The method according to claim 1, wherein the test sample is from
the subject who is suffering from a disease and the control sample
is from a subject that is not suffering from the disease.
4. A method for treating a subject suffering from a disease
comprising the steps of (1) administering a therapeutically
effective amount of a therapeutic agent to the subject, (2)
determining the serum BAFF levels in a test sample of the subject,
(3) comparing the B cell level in the test sample relative to a
control sample and (4) administering a therapeutically effective
amount of the same or different therapeutic agent at a time point
dependent on the serum BAFF level in the subject.
5. A method for treating a subject suffering from a disease
comprising the steps of (1) administering a therapeutically
effective amount of a therapeutic agent to the subject, (2)
determining the serum BAFF levels in a test sample of the subject,
and (3) administering a therapeutically effective amount of the
same or different therapeutic agent at a time point dependent on
the serum BAFF level in the test sample.
6. A method of maintenance therapy for a subject previously treated
with a B cell depletion agent for a disease comprising the step of
determining the serum BAFF levels in the subject and treating the
subject with a B cell depletion agent at a time point after maximum
B cell depletion and while serum BAFF levels are decreasing.
7. The method according to claim 4 or 5, wherein the time point is
at the time of or after the maximum B cell depletion phase.
8. The method according to claim 4 or 5, wherein the time point is
during the B cell recovery phase.
9. The method according to claim 8, wherein the B cell recovery
phase is characterized by decreasing serum BAFF levels.
10. The method according to claim 6, wherein the maximum B cell
depletion phase is characterized by maximum levels of BAFF in the
sera of a subject.
11. The method according to claim 7, wherein the maximum B cell
depletion phase is characterized by maximum levels of BAFF in the
sera of a subject.
12. The method according to claim 4 or 5, wherein the therapeutic
agent is a B cell depletion agent.
13. The method according to claim 4 or 5, wherein the therapeutic
agent is not a BAFF antagonist that binds to BAFF.
14. The method according to claim 4 or 5, wherein the therapeutic
agent does bind BAFF and block BAFF from binding to BCMA, TACI or
BR3.
15. The method according to claim 4 or 5, wherein the therapeutic
agent is a B cell promoting agent.
16. The method according to claim 12, wherein the therapeutic agent
is a B cell depletion agent that binds to a B cell surface antigen
selected from the group consisting of CD10, CD19, CD20, CD21, CD22,
CD23, CD24, CD37, CD40, CD52, D53, CD72, CD73, CD74, CDw75, CDw76,
CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85,
CD86, CD180 (RP105), FcRH2 (IRTA4), CD79A, C79B, CR2, CCR6, CD72,
P2.times.5, HLA-DOB, CXCR5 (BLR1), FCER2, BR3 (aka BAFF-R), TACI,
BTLA, NAG14 (aka LRRC4), SLGC16270 (ala LOC283663), FcRH1 (IRTA5),
FcRH5 (IRTA2), ATWD578 (aka MGC15619), FcRH3 (IRTA3), FcRH4
(IRTA1), FcRH6 (aka LOC343413) and BCMA (aka TNFRSF17), HLA-DO,
HLA-Dr10 and MHC ClassII.
17. The method according to claim 16, wherein the B cell depletion
agent is an antibody.
18. The method according to any one of claims 4-6, wherein the
disease is an immunological disorder.
19. The method according to claim 4 or 5, wherein the disease is an
immunodeficiency disease.
20. The method according to any one of claims 4-6, wherein the
disease is an autoimmune disease elected from the group consisting
of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic
lupus erythematosus (SLE), Wegener's disease, inflammatory bowel
disease, idiopathic thrombocytopenic purpura (ITP), thrombotic
thrombocytopenic purpura (TTP), autoimmune thrombocytopenia,
multiple sclerosis, psoriasis, IgA nephropathy, IgM
polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,
Reynaud's syndrome, Sjorgen's syndrome, glomerulonephritis,
Neuromyelitis Optica (NMO) and IgG neuropathy.
21. The method according to any one of claims 4-6, wherein the
disease is a cancer selected from the group consisting of B cell
lymphoma, B cell leukemia and multiple myeloma.
22. The method according to any one of claims 4-6, wherein the B
cells express CD19.
23. The method according to any one of claims 4-6, wherein the B
cells express CD20.
24. The method according to any one of claims 4-6, wherein the
subject is a mammal.
25. The method according to claim 23, wherein the subject is a
human.
26. The method according to claim 12, wherein the B cell depletion
agent is selected from the group consisting of an anti-CD20
antibody, an anti-BR3 antibody, an anti-CD22 antibody and an
anti-CD52 antibody.
27. The method according to claim 26, wherein the anti-CD20
antibody is rituximab or 2H7.
28. The method according to claim 15, wherein the B cell promoting
agent is a cytokine.
29. The method according to any one of claims 4-5, wherein the
different therapeutic agent is selected from the group consisting
of a T cell depleting agent, an immunosuppressive agent, a DMARD
and a vaccine.
30. The method according to any one of claims 4-6, wherein the time
point is before or during tissue B cell recovery that is prior to
peripheral blood B cell recovery.
31. A kit comprising a BAFF binding reagent and a package insert
comprising instructions for determining serum BAFF levels using the
BAFF binding reagent and for relating serum BAFF levels to B cell
levels in the patient after treatment with a B cell depleting or
promoting agent.
32. A method for monitoring B Lymphocyte Stimulator expression in
an individual comprising the steps of assaying the level of B
Lymphocyte Stimulator expression in a biological sample of the
individual using one or more antibodies or fragments thereof that
immunospecifically bind B Lymphocyte Stimulator; and comparing the
level of B Lymphocyte Stimulator assayed in the biological sample
with a standard level of B Lymphocyte Stimulator.
33. The according to claim 32, wherein the level of B Lymphocyte
Stimulator is assayed before a treatment regimen and during or
after the treatment regimen.
34. The according to claim 32, wherein the individual has a disease
and wherein the standard B Lymphocyte Stimulator level is from an
individual without the disease.
35. A method for treating an individual suffering from a disease
comprising the steps of (1) administering a therapeutically
effective amount of a therapeutic agent to the individual; (2)
assaying for the level of B Lymphocyte Stimulator in a biological
sample of the subject; (3) comparing the level of B Lymphocyte
Stimulator with a standard B Lymphocyte Stimulator level; and (4)
administering a therapeutically effective amount of the same or a
different therapeutic agent to the individual.
36. A method for treating an individual suffering from a disease
comprising the steps of (1) administering a therapeutically
effective amount of a therapeutic agent of a treatment regimen to
the subject; (2) assaying for the level of B Lymphocyte Stimulator
in a biological sample of the individual; and (3) administering a
therapeutically effective amount of the same or a different
therapeutic agent to the individual.
37. A method for treating aberrant B Lymphocyte Stimulator levels
in an individual comprising (1) assaying for the level of B
Lymphocyte Stimulator in a biological sample of the individual; (2)
treating the individual with a therapeutically effective amount of
an agent that inhibits B cell proliferation, differentiation,
survival, or activation; (3) assaying for the level of B Lymphocyte
Stimulator in a biological sample of the individual; and (4) if B
Lymphocyte Stimulator levels as determined in (3) are lower than
those in (1), administering a therapeutic agent that inhibits B
cell proliferation, differentiation or survival.
38. The method according to claim 35 or 36, wherein the therapeutic
agent is administered after assayed levels of B Lymphocyte
Stimulator indicate that maximum effectiveness of a therapeutic
agent that inhibits B cell proliferation, differentiation,
survival, or activation has been reached.
39. The method according to claim 35 or 36, wherein the therapeutic
agent inhibits B cell proliferation, differentiation, survival, or
activation.
40. The method according to claim 35 or 36, wherein the therapeutic
agent is not a B Lymphocyte Stimulator antagonist that binds to B
Lymphocyte Stimulator.
41. The method according to claim 35 or 36, wherein the therapeutic
agent binds B Lymphocyte Stimulator and substantially blocks B
Lymphocyte Stimulator from binding to BCMA, BAFF-R, or TACI.
42. The method according to claim 35 or 36, wherein the therapeutic
agent promotes B cell differentiation, proliferation or
activation.
43. The method according to claim 39, wherein the therapeutic agent
binds to an antigen selected from the group consisting of CD19,
CD20, CD22, CD40, CCR6, BAFF-R, TACI, and BCMA, and HLA-Dr10.
44. The method according to claim 43, wherein the therapeutic agent
is an antibody.
45. The method according to any one of claims 35-37, wherein the
disease is an immune disorder.
46. The method according to claim 35 or 36, wherein the disease is
an immunodeficiency disease.
47. The method according to any one of claims 35 or 36, wherein the
disease is an autoimmune disease selected from the group consisting
of rheumatoid arthritis, systemic lupus erythematosus (SLE),
granulomatosis, inflammatory bowel disease, idiopathic
thrombocytopenic purpura (ITP), autoimmune thrombocytopenia,
multiple sclerosis, psoriasis, IgA nephropathy, IgM
polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,
Reynaud's syndrome, Sjorgen's syndrome, and glomerulonephritis.
48. The method according to any one of claims 35-37, wherein the
disease is a cancer selected from the group consisting of B cell
lymphoma, B cell leukemia and multiple myeloma.
49. The method according to any one of claims 35-37, wherein the B
cells express CD19.
50. The method according to any one of claims 35-37, wherein the
biological sample comprises one or more CD20 positive B cells.
51. The method according to any one of claims 35-37, wherein the
subject is a mammal.
52. The method according to claim 51, wherein the subject is a
human.
53. The method according to claim 39, wherein the therapeutic agent
is selected from the group consisting of an anti-CD20 antibody, an
anti-BAFF-R antibody, and an anti-CD22 antibody.
54. The method according to claim 53, wherein the anti-CD20
antibody is rituximab.
55. The method according to claim 42, wherein the therapeutic agent
is a cytokine.
56. The method according to any one of claims 35-37, wherein the
different therapeutic agent is selected from the group consisting
of a T cell suppressive agent, an immunosuppressive agent, a
vaccine, methotrexate, and a steroid.
57. A kit comprising a B Lymphocyte Stimulator binding reagent and
a package insert comprising instructions for determining serum B
Lymphocyte Stimulator levels using the B Lymphocyte Stimulator
binding reagent and for determining whether aberrant levels of B
Lymphocyte Stimulator are present.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of copending U.S.
patent application Ser. No. 12/210,134, filed Sep. 12, 2008, which
is a continuation of copending U.S. patent application Ser. No.
11/054,539, filed Feb. 10, 2005, which claims the benefit of U.S.
Provisional Patent Application No. 60/543,261, filed Feb. 11, 2004;
U.S. Provisional Patent Application No. 60/580,387, filed Jun. 18,
2004; and U.S. Provisional Patent Application No. 60/617,191, filed
Oct. 12, 2004. U.S. patent application Ser. No. 11/054,539 is a
continuation in-part of U.S. patent application Ser. No.
10/270,487, filed Oct. 16, 2002, now abandoned, which claims the
benefit of U.S. Provisional Patent Application No. 60/368,548,
filed Apr. 1, 2002; U.S. Provisional Patent Application No.
60/336,726, filed Dec. 7, 2001; U.S. Provisional Patent Application
No. 60/331,478, filed Nov. 16, 2001; U.S. Provisional Patent
Application No. 60/330,835, filed Oct. 31, 2001; U.S. Provisional
Patent Application No. 60/329,747, filed Oct. 18, 2001; and U.S.
Provisional Patent Application No. 60/329,508, filed Oct. 17, 2001.
U.S. patent application Ser. No. 10/270,487, filed Oct. 16, 2002 is
a continuation-in-part of U.S. patent application Ser. No.
09/929,493, filed Aug. 15, 2001, now abandoned, which claims
benefit of U.S. Provisional Patent Application No. 60/225,628,
filed Aug. 15, 2000; U.S. Provisional Patent Application No.
60/227,008, filed Aug. 23, 2000; U.S. Provisional Patent
Application No. 60/234,338, filed Sep. 22, 2000; U.S. Provisional
Patent Application No. 60/240,806, filed Oct. 17, 2000; U.S.
Provisional Patent Application No. 60/250,020, filed Nov. 30, 2000;
U.S. Provisional Patent Application No. 60/276,248, filed Mar. 16,
2001; U.S. Provisional Patent Application No. 60/293,499, filed May
25, 2001; U.S. Provisional Patent Application No. 60/296,122, filed
Jun. 7, 2001; and U.S. Provisional Patent Application No.
60/304,809, filed Jul. 13, 2001. U.S. patent application Ser. No.
09/929,493 is a continuation-in-part of U.S. patent application
Ser. No. 09/588,947, filed Jun. 8, 2000, issued as U.S. Pat. No.
6,562,579 on May 13, 2003; U.S. patent application Ser. No.
09/589,285, filed Jun. 8, 2000, issued as U.S. Pat. No. 6,881,401
on Apr. 19, 2005; U.S. patent application Ser. No. 09/589,286,
filed Jun. 8, 2000, issued as U.S. Pat. No. 6,635,482 on Oct. 21,
2003; U.S. patent application Ser. No. 09/589,287, filed Jun. 8,
2000, issued as U.S. Pat. No. 6,403,770 on Jun. 11, 2002; and
copending U.S. patent application Ser. No. 09/589,288, filed Jun.
8, 2000. U.S. patent application Ser. No. 12/210,134 also is a
continuation-in-part of copending U.S. patent application Ser. No.
12/170,333, filed Jul. 9, 2008, which is a continuation of U.S.
patent application Ser. No. 11/382,837, filed May 11, 2006, now
abandoned, which is a continuation of copending U.S. patent
application Ser. No. 09/589,288. U.S. patent application Ser. No.
12/210,134 also is a continuation-in-part of copending U.S. patent
application Ser. No. 09/589,288. Each of U.S. patent application
Ser. Nos. 09/589,288, 09/589,285, 09/589,286, 09/588,947, and
09/589,287 is a continuation of U.S. patent application Ser. No.
09/507,968, filed Feb. 22, 2000, issued as U.S. Pat. No. 6,812,327
on Nov. 2, 2004, which claims the benefit of U.S. Provisional
Patent Application No. 60/122,388, filed Mar. 2, 1999; U.S.
Provisional Patent Application No. 60/124,097, filed Mar. 12, 1999;
U.S. Provisional Patent Application No. 60/126,599, filed Mar. 26,
1999; U.S. Provisional Patent Application No. 60/127,598, filed
Apr. 2, 1999; U.S. Provisional Patent Application No. 60/130,412,
filed Apr. 16, 1999; U.S. Provisional Patent Application No.
60/130,696, filed Apr. 23, 1999; U.S. Provisional Patent
Application No. 60/131,278, filed Apr. 27, 1999; U.S. Provisional
Patent Application No. 60/131,673, filed Apr. 29, 1999; U.S.
Provisional Patent Application No. 60/136,784, filed May 28, 1999;
U.S. Provisional Patent Application No. 60/142,659, filed Jul. 6,
1999; U.S. Provisional Patent Application No. 60/145,824, filed
Jul. 27, 1999; U.S. Provisional Patent Application No. 60/167,239,
filed Nov. 24, 1999; U.S. Provisional Patent Application No.
60/168,624, filed Dec. 3, 1999; U.S. Provisional Patent Application
No. 60/171,108, filed Dec. 16, 1999; U.S. Provisional Patent
Application No. 60/171,626, filed Dec. 23, 1999; and U.S.
Provisional Patent Application No. 60/176,015, filed Jan. 14, 2000.
U.S. patent application Ser. No. 09/929,493 is also a
continuation-in-part of U.S. patent application Ser. No.
09/507,968, which is a continuation-in-part of U.S. patent
application Ser. No. 09/255,794, filed Feb. 23, 1999, issued as
U.S. Pat. No. 6,716,576 on Apr. 6, 2004, which is a
continuation-in-part of U.S. patent application Ser. No.
09/005,874, filed Jan. 12, 1998, issued as U.S. Pat. No. 6,689,579
on Feb. 10, 2004, which claims the benefit of U.S. Provisional
Patent Application No. 60/036,100, filed Jan. 14, 1997. U.S. patent
application Ser. No. 09/005,874 is a continuation-in-part of
International Patent Application No. PCT/US96/17957, filed Oct. 25,
1996. Each of the applications identified above is herein
incorporated by reference in their entireties.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 81,196 Byte
ASCII (Text) file named "SequenceListing.TXT," created on Nov. 22,
2010.
FIELD OF THE INVENTION
[0003] The present invention relates to a novel cytokine which has
been designated Neutrokine-alpha ("Neutrokine-alpha"). In addition,
an apparent splicing variant of Neutrokine-alpha has been
identified and designated Neutrokine-alphaSV. In specific
embodiments, the present invention provides nucleic acid molecules
encoding Neutrokine-alpha and Neutrokine-alphaSV polypeptides. In
additional embodiments, Neutrokine-alpha and Neutrokine-alphaSV
polypeptides are also provided, as are vectors, host cells and
recombinant methods for producing the same. Neutrokine-alpha is
also referred to in the art as TALL-1, THANK, BAFF, zTNF4,
TNFSF13B. Neutrokine-alpha is also referred to as the BLyS.TM.
protein, from Human Genome Sciences, Inc.
RELATED ART
[0004] Human tumor necrosis factors (TNF-alpha) and (TNF-beta, or
lymphotoxin) are related members of a broad class of polypeptide
mediators, which includes the interferons, interleukins and growth
factors, collectively called cytokines (Beutler, B. and Cerami, A.,
Annu. Rev. Immunol. 7:625-655 (1989)). Sequence analysis of
cytokine receptors has defined several subfamilies of membrane
proteins (1) the Ig superfamily, (2) the hematopoietin (cytokine
receptor superfamily) and (3) the tumor necrosis factor (TNF)/nerve
growth factor (NGF) receptor superfamily (for review of TNF
superfamily see, Gruss and Dower, Blood 85(12):3378-3404 (1995) and
Aggarwal and Natarajan, Eur. Cytokine Netw., 7(2):93-124 (1996)).
The TNF/NGF receptor superfamily contains at least 10 different
proteins. Gruss and Dower, supra. Ligands for these receptors have
been identified and belong to at least two cytokine superfamilies.
Gruss and Dower, supra.
[0005] Tumor necrosis factor (a mixture of TNF-alpha and TNF-beta)
was originally discovered as a result of its anti-tumor activity,
however, now it is recognized as a pleiotropic cytokine capable of
numerous biological activities including apoptosis of some
transformed cell lines, mediation of cell activation and
proliferation and also as playing important roles in immune
regulation and inflammation.
[0006] To date, known members of the TNF-ligand superfamily include
TNF-alpha, TNF-beta (lymphotoxin-alpha), LT-beta, OX40L, Fas
ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF
ligand superfamily are acidic, TNF-like molecules with
approximately 20% sequence homology in the extracellular domains
(range, 12%-36%) and exist mainly as membrane-bound forms with the
biologically active form being a trimeric/multimeric complex.
Soluble forms of the TNF ligand superfamily have only been
identified so far for TNF, LT-beta, and Fas ligand (for a general
review, see Gruss, H. and Dower, S. K., Blood, 85(12):3378-3404
(1995)), which is hereby incorporated by reference in its entirety.
These proteins are involved in regulation of cell proliferation,
activation, and differentiation, including control of cell survival
or death by apoptosis or cytotoxicity (Armitage, R. J., Curr. Opin.
Immunol. 6:407 (1994) and Smith, C. A., Cell 75:959 (1994)).
[0007] Tumor necrosis factor-alpha (TNF-alpha; also termed
cachectin; hereinafter "TNF") is secreted primarily by monocytes
and macrophages in response to endotoxin or other stimuli as a
soluble homotrimer of 17 kD protein subunits (Smith, R. A. et al.,
J. Biol. Chem. 262:6951-6954 (1987)). A membrane-bound 26 kD
precursor form of TNF has also been described (Kriegler, M. et al.,
Cell 53:45-53 (1988)).
[0008] Accumulating evidence indicates that TNF is a regulatory
cytokine with pleiotropic biological activities. These activities
include: inhibition of lipoprotein lipase synthesis ("cachectin"
activity) (Beutler, B. et al., Nature 316:552 (1985)), activation
of polymorphonuclear leukocytes (Klebanoff, S. J. et al., J.
Immunol. 136:4220 (1986); Perussia, B., et al., J. Immunol. 138:765
(1987)), inhibition of cell growth or stimulation of cell growth
(Vilcek, J. et al., J. Exp. Med. 163:632 (1986); Sugarman, B. J. et
al., Science 230:943 (1985); Lachman, L. B. et al., J. Immunol.
138:2913 (1987)), cytotoxic action on certain transformed cell
types (Lachman, L. B. et al., supra; Darzynkiewicz, Z. et al.,
Canc. Res. 44:83 (1984)), antiviral activity (Kohase, M. et al.,
Cell 45:659 (1986); Wong, G. H. W. et al., Nature 323:819 (1986)),
stimulation of bone resorption (Bertolini, D. R. et al., Nature
319:516 (1986); Saklatvala, J., Nature 322:547 (1986)), stimulation
of collagenase and prostaglandin E2 production (Dayer, J.-M. et
al., J. Exp. Med. 162:2163 (1985)); and immunoregulatory actions,
including activation of T cells (Yokota, S. et al., J. Immunol.
140:531 (1988)), B cells (Kehrl, J. H. et al., J. Exp. Med. 166:786
(1987)), monocytes (Philip, R. et al., Nature 323:86 (1986)),
thymocytes (Ranges, G. E. et al., J. Exp. Med. 167:1472 (1988)),
and stimulation of the cell-surface expression of major
histocompatibility complex (MHC) class I and class II molecules
(Collins, T. et al., Proc. Natl. Acad. Sci. USA 83:446 (1986);
Pujol-Borrel, R. et al., Nature 326:304 (1987)).
[0009] TNF is noted for its pro-inflammatory actions which result
in tissue injury, such as induction of procoagulant activity on
vascular endothelial cells (Pober, J. S. et al., J. Immunol.
136:1680 (1986)), increased adherence of neutrophils and
lymphocytes (Pober, J. S. et al., J. Immunol. 138:3319 (1987)), and
stimulation of the release of platelet activating factor from
macrophages, neutrophils and vascular endothelial cells (Camussi,
G. et al., J. Exp. Med. 166:1390 (1987)).
[0010] Recent evidence implicates TNF in the pathogenesis of many
infections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immune
disorders, neoplastic pathology, e.g., in cachexia accompanying
some malignancies (Oliff, A. et al., Cell 50:555 (1987)), and in
autoimmune pathologies and graft-versus host pathology (Piguet,
P.-F. et al., J. Exp. Med. 166:1280 (1987)). The association of TNF
with cancer and infectious pathologies is often related to the
host's catabolic state. A major problem in cancer patients is
weight loss, usually associated with anorexia. The extensive
wasting which results is known as "cachexia" (Kern, K. A. et al. J.
Parent. Enter. Nutr. 12:286-298 (1988)). Cachexia includes
progressive weight loss, anorexia, and persistent erosion of body
mass in response to a malignant growth. The cachectic state is thus
associated with significant morbidity and is responsible for the
majority of cancer mortality. A number of studies have suggested
that TNF is an important mediator of the cachexia in cancer,
infectious pathology, and in other catabolic states.
[0011] TNF is thought to play a central role in the
pathophysiological consequences of Gram-negative sepsis and
endotoxic shock (Michie, H. R. et al., Br. J. Surg. 76:670-671
(1989); Debets, J. M. H. et al., Second Vienna Shock Forum, p.
463-466 (1989); Simpson, S. Q. et al., Crit. Care Clin. 5:27-47
(1989)), including fever, malaise, anorexia, and cachexia.
Endotoxin is a potent monocyte/macrophage activator which
stimulates production and secretion of TNF (Kornbluth, S. K. et
al., J. Immunol. 137:2585-2591 (1986)) and other cytokines. Because
TNF could mimic many biological effects of endotoxin, it was
concluded to be a central mediator responsible for the clinical
manifestations of endotoxin-related illness. TNF and other
monocyte-derived cytokines mediate the metabolic and neurohormonal
responses to endotoxin (Michie, H. R. et al., N. Eng. J. Med.
318:1481-1486 (1988)). Endotoxin administration to human volunteers
produces acute illness with flu-like symptoms including fever,
tachycardia, increased metabolic rate and stress hormone release
(Revhaug, A. et al., Arch. Surg. 123:162-170 (1988)). Elevated
levels of circulating TNF have also been found in patients
suffering from Gram-negative sepsis (Waage, A. et al., Lancet
1:355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum
p. 715-718 (1989); Debets, J. M. H. et al., Crit. Care Med.
17:489-497 (1989); Calandra, T. et al., J. Infec. Dis. 161:982-987
(1990)).
[0012] Passive immunotherapy directed at neutralizing TNF may have
a beneficial effect in Gram-negative sepsis and endotoxemia, based
on the increased TNF production and elevated TNF levels in these
pathology states, as discussed above. Antibodies to a "modulator"
material which was characterized as cachectin (later found to be
identical to TNF) were disclosed by Cerami et al. (EPO Patent
Publication 0,212,489, Mar. 4, 1987). Such antibodies were said to
be useful in diagnostic immunoassays and in therapy of shock in
bacterial infections. Rubin et al. (EPO Patent Publication
0,218,868, Apr. 22, 1987) disclosed monoclonal antibodies to human
TNF, the hybridomas secreting such antibodies, methods of producing
such antibodies, and the use of such antibodies in immunoassay of
TNF. Yone et al. (EPO Patent Publication 0,288,088, Oct. 26, 1988)
disclosed anti-TNF antibodies, including mAbs, and their utility in
immunoassay diagnosis of pathologies, in particular Kawasaki's
pathology and bacterial infection. The body fluids of patients with
Kawasaki's pathology (infantile acute febrile mucocutaneous lymph
node syndrome; Kawasaki, T., Allergy 16:178 (1967); Kawasaki, T.,
Shonica (Pediatrics) 26:935 (1985)) were said to contain elevated
TNF levels which were related to progress of the pathology (Yone et
al., supra).
[0013] Other investigators have described mAbs specific for
recombinant human TNF which had neutralizing activity in vitro
(Liang, C-M. et al. Biochem. Biophys. Res. Comm. 137:847-854
(1986); Meager, A. et al., Hybridoma 6:305-311 (1987); Fendly et
al., Hybridoma 6:359-369 (1987); Bringman, T S et al., Hybridoma
6:489-507 (1987); Hirai, M. et al., J. Immunol. Meth. 96:57-62
(1987); Moller, A. et al. (Cytokine 2:162-169 (1990)). Some of
these mAbs were used to map epitopes of human TNF and develop
enzyme immunoassays (Fendly et al., supra; Hirai et al., supra;
Moller et al., supra) and to assist in the purification of
recombinant TNF (Bringman et al., supra). However, these studies do
not provide a basis for producing TNF neutralizing antibodies that
can be used for in vivo diagnostic or therapeutic uses in humans,
due to immunogenicity, lack of specificity and/or pharmaceutical
suitability.
[0014] Neutralizing antisera or mAbs to TNF have been shown in
mammals other than man to abrogate adverse physiological changes
and prevent death after lethal challenge in experimental
endotoxemia and bacteremia. This effect has been demonstrated,
e.g., in rodent lethality assays and in primate pathology model
systems (Mathison, J. C. et al., J. Clin. Invest. 81:1925-1937
(1988); Beutler, B. et al., Science 229:869-871 (1985); Tracey, K.
J. et al., Nature 330:662-664 (1987); Shimamoto, Y. et al.,
Immunol. Lett. 17:311-318 (1988); Silva, A. T. et al., J. Infect.
Dis. 162:421-427 (1990); Opal, S. M. et al., J. Infect. Dis.
161:1148-1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30:279-292
(1990)).
[0015] To date, experience with anti-TNF mAb therapy in humans has
been limited but shows beneficial therapeutic results, e.g., in
arthritis and sepsis. See, e.g., Elliott, M. J. et al., Baillieres
Clin. Rheumatol. 9:633-52 (1995); Feldmann M, et al., Ann. N.Y.
Acad. Sci. USA 766:272-8 (1995); van der Poll, T. et al., Shock
3:1-12 (1995); Wherry et al., Crit. Care. Med. 21:S436-40 (1993);
Tracey K. J., et al., Crit. Care Med. 21:S415-22 (1993).
[0016] Mammalian development is dependent on both the proliferation
and differentiation of cells as well as programmed cell death which
occurs through apoptosis (Walker, et al., Methods Achiev. Exp.
Pathol. 13:18 (1988). Apoptosis plays a critical role in the
destruction of immune thymocytes that recognize self antigens.
Failure of this normal elimination process may play a role in
autoimmune diseases (Gammon et al., Immunology Today 12:193
(1991)).
[0017] Itoh et al. (Cell 66:233 (1991)) described a cell surface
antigen, Fas/CD95 that mediates apoptosis and is involved in clonal
deletion of T-cells. Fas is expressed in activated T-cells,
B-cells, neutrophils and in thymus, liver, heart and lung and ovary
in adult mice (Watanabe-Fukunaga et al., J. Immunol. 148:1274
(1992)) in addition to activated T-cells, B-cells, neutrophils. In
experiments where a monoclonal Ab is cross-linked to Fas, apoptosis
is induced (Yonehara et al., J. Exp. Med. 169:1747 (1989); Trauth
et al., Science 245:301 (1989)). In addition, there is an example
where binding of a monoclonal Ab to Fas is stimulatory to T-cells
under certain conditions (Alderson et al., J. Exp. Med. 178:2231
(1993)).
[0018] Fas antigen is a cell surface protein of relative MW of 45
Kd. Both human and murine genes for Fas have been cloned by
Watanabe-Fukunaga et al., (J. Immunol. 148:1274 (1992)) and Itoh et
al. (Cell 66:233 (1991)). The proteins encoded by these genes are
both transmembrane proteins with structural homology to the Nerve
Growth Factor/Tumor Necrosis Factor receptor superfamily, which
includes two TNF receptors, the low affinity Nerve Growth Factor
receptor and CD40, CD27, CD30, and OX40.
[0019] Recently the Fas ligand has been described (Suda et al.,
Cell 75:1169 (1993)). The amino acid sequence indicates that Fas
ligand is a type II transmembrane protein belonging to the TNF
family. Thus, the Fas ligand polypeptide comprises three main
domains: a short intracellular domain at the amino terminal end and
a longer extracellular domain at the carboxy terminal end,
connected by a hydrophobic transmembrane domain. Fas ligand is
expressed in splenocytes and thymocytes, consistent with T-cell
mediated cytotoxicity. The purified Fas ligand has a MW of 40
kD.
[0020] Recently, it has been demonstrated that Fas/Fas ligand
interactions are required for apoptosis following the activation of
T-cells (Ju et al., Nature 373:444 (1995); Brunner et al., Nature
373:441 (1995)). Activation of T-cells induces both proteins on the
cell surface. Subsequent interaction between the ligand and
receptor results in apoptosis of the cells. This supports the
possible regulatory role for apoptosis induced by Fas/Fas ligand
interaction during normal immune responses.
[0021] Accordingly, there is a need to provide cytokines similar to
TNF that are involved in pathological conditions. Such novel
cytokines may be used to make novel antibodies or other antagonists
that bind these TNF-like cytokines for diagnosis and therapy of
disorders related to TNF-like cytokines.
SUMMARY OF THE INVENTION
[0022] In accordance with one embodiment of the present invention,
there is provided a novel extracellular domain of a
Neutrokine-alpha polypeptide, and a novel extracellular domain of a
Neutrokine-alphaSV polypeptide, as well as biologically active and
diagnostically or therapeutically useful fragments, analogs and
derivatives thereof.
[0023] In accordance with another embodiment of the present
invention, there are provided isolated nucleic acid molecules
encoding human Neutrokine-alpha or Neutrokine-alphaSV, including
mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and
biologically active and diagnostically or therapeutically useful
fragments and derivatives thereof.
[0024] The present invention provides isolated nucleic acid
molecules comprising, or alternatively, consisting of, a
polynucleotide encoding a cytokine and an apparent splice variant
thereof that are structurally similar to TNF and related cytokines
and have similar biological effects and activities. This cytokine
is named Neutrokine-alpha and the invention includes
Neutrokine-alpha polypeptides having at least a portion of the
amino acid sequence in FIGS. 1A and 1B (SEQ ID NO:2) or amino acid
sequence encoded by the cDNA clone (HNEDU15) deposited on Oct. 22,
1996 assigned ATCC number 97768. The nucleotide sequence determined
by sequencing the deposited Neutrokine-alpha clone, which is shown
in FIGS. 1A and 1B (SEQ ID NO:1), contains an open reading frame
encoding a complete polypeptide of 285 amino acid residues
including an N-terminal methionine, a predicted intracellular
domain of about 46 amino acid residues, a predicted transmembrane
domain of about 26 amino acids, a predicted extracellular domain of
about 213 amino acids, and a deduced molecular weight for the
complete protein of about 31 kDa. As for other type II
transmembrane proteins, soluble forms of Neutrokine-alpha include
all or a portion of the extracellular domain cleaved from the
transmembrane domain and a polypeptide comprising the complete
Neutrokine-alpha polypeptide lacking the transmembrane domain,
i.e., the extracellular domain linked to the intracellular domain.
The apparent splice variant of Neutrokine-alpha is named
Neutrokine-alphaSV and the invention includes Neutrokine-alphaSV
polypeptides comprising, or alternatively, consisting of, at least
a portion of the amino acid sequence in FIGS. 5A and 5B (SEQ ID
NO:19) or amino acid sequence encoded by the cDNA clone HDPMC52
deposited on Dec. 10, 1998 and assigned ATCC number 203518. The
nucleotide sequence determined by sequencing the deposited
Neutrokine-alphaSV clone, which is shown in FIGS. 5A and 5B (SEQ ID
NO:18), contains an open reading frame encoding a complete
polypeptide of 266 amino acid residues including an N-terminal
methionine, a predicted intracellular domain of about 46 amino acid
residues, a predicted transmembrane domain of about 26 amino acids,
a predicted extracellular domain of about 194 amino acids, and a
deduced molecular weight for the complete protein of about 29 kDa.
As for other type II transmembrane proteins, soluble forms of
Neutrokine-alphaSV include all or a portion of the extracellular
domain cleaved from the transmembrane domain and a polypeptide
comprising the complete Neutrokine-alphaSV polypeptide lacking the
transmembrane domain, i.e., the extracellular domain linked to the
intracellular domain.
[0025] Thus, one embodiment of the invention provides an isolated
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide having a nucleotide sequence selected from the group
consisting of: (a) a nucleotide sequence encoding a full-length
Neutrokine-alpha polypeptide having the complete amino acid
sequence in FIGS. 1A and 1B (SEQ ID NO:2) or as encoded by the cDNA
clone contained in the deposit having ATCC accession number 97768;
(b) a nucleotide sequence encoding the predicted extracellular
domain of the Neutrokine-alpha polypeptide having the amino acid
sequence at positions 73 to 285 in FIGS. 1A and 1B (SEQ ID NO:2) or
as encoded by the clone contained in the deposit having ATCC
accession number 97768; (c) a nucleotide sequence encoding a
fragment of the polypeptide of (b) (e.g., amino acids 134-285)
having Neutrokine-alpha functional activity (e.g., biological
activity); (d) a nucleotide sequence encoding a polypeptide
comprising the Neutrokine-alpha intracellular domain (predicted to
constitute amino acid residues from about 1 to about 46 in FIGS. 1A
and 1B (SEQ ID NO:2)) or as encoded by the clone contained in the
deposit having ATCC accession number 97768; (e) a nucleotide
sequence encoding a polypeptide comprising the Neutrokine-alpha
transmembrane domain (predicted to constitute amino acid residues
from about 47 to about 72 in FIGS. 1A and 1B (SEQ ID NO:2) or as
encoded by the cDNA clone contained in the deposit having ATCC
accession number 97768; (f) a nucleotide sequence encoding a
soluble Neutrokine-alpha polypeptide having the extracellular and
intracellular domains but lacking the transmembrane domain; and (g)
a nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e) or (f) above.
[0026] Further embodiments of the invention include isolated
nucleic acid molecules that comprise, or alternatively consist of,
a polynucleotide having a nucleotide sequence at least 80%, 85% or
90% identical, and more preferably at least 95%, 96%, 97%, 98% or
99% identical, to any of the nucleotide sequences in (a), (b), (c),
(d), (e), (f) or (g) above, or a polynucleotide which hybridizes
under stringent hybridization conditions to a polynucleotide in
(a), (b), (c), (d), (e), (f) or (g) above. This polynucleotide
which hybridizes does not hybridize under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
consisting of only A residues or of only T residues.
[0027] Another embodiment of the invention provides an isolated
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide having a nucleotide sequence selected from the group
consisting of: (a) a nucleotide sequence encoding a full-length
Neutrokine-alphaSV polypeptide having the complete amino acid
sequence in FIGS. 5A and 5B (SEQ ID NO:19) or as encoded by the
cDNA clone contained in the ATCC Deposit deposited on Dec. 10, 1998
as ATCC Number 203518; (b) a nucleotide sequence encoding the
predicted extracellular domain of the Neutrokine-alphaSV
polypeptide having the amino acid sequence at positions 73 to 266
in FIGS. 1A and 1B (SEQ ID NO:2) or as encoded by the cDNA clone
contained in ATCC 203518 deposited on Dec. 10, 1998; (c) a
nucleotide sequence encoding a polypeptide comprising the
Neutrokine-alphaSV intracellular domain (predicted to constitute
amino acid residues from about 1 to about 46 in FIGS. 5A and 5B
(SEQ ID NO:19)) or as encoded by the cDNA clone contained in ATCC
No. 203518 deposited on Dec. 10, 1998; (d) a nucleotide sequence
encoding a polypeptide comprising the Neutrokine-alphaSV
transmembrane domain (predicted to constitute amino acid residues
from about 47 to about 72 in FIGS. 5A and 5B (SEQ ID NO:19) or as
encoded by the cDNA clone contained in ATCC No. 203518 deposited on
Dec. 10, 1998; (e) a nucleotide sequence encoding a soluble
Neutrokine-alphaSV polypeptide having the extracellular and
intracellular domains but lacking the transmembrane domain; and (f)
a nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), or (e) above.
[0028] Further embodiments of the invention include isolated
nucleic acid molecules that comprise, or alternatively consist of,
a polynucleotide having a nucleotide sequence at least 80%, 85% or
90% identical, and more preferably at least 95%, 96%, 97%, 98% or
99% identical, to any of the nucleotide sequences in (a), (b), (c),
(d), (e) or (f) above, or a polynucleotide which hybridizes under
stringent hybridization conditions to a polynucleotide in (a), (b),
(c), (d), (e) or (f) above. This polynucleotide which hybridizes
does not hybridize under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence consisting of only A
residues or of only T residues.
[0029] In one embodiment, the invention includes isolated nucleic
acid molecules that comprise, or alternatively consist of, a
polynucleotide having a nucleotide sequence encoding the apparent
splice variant of Neutrokine-alpha comprising, or alternatively
consisting of, at least a portion of the amino acid sequence from
Gly-142 to Leu-266 as shown in FIGS. 5A and 5B (SEQ ID NO:19) or
amino acid sequence encoded by the cDNA clone HDPMC52 deposited on
Dec. 10, 1998 and assigned ATCC Deposit No. 203518.
[0030] In another preferred embodiment, the invention include
isolated nucleic acid molecules that comprise, or alternatively
consist of, a polynucleotide having a nucleotide sequence encoding
the apparent splice variant of Neutrokine-alpha comprising, or
alternatively consisting of, at least a portion of the amino acid
sequence from Ala-134 to Leu-266 as shown in FIGS. 5A and 5B (SEQ
ID NO:19) or amino acid sequence encoded by the cDNA clone HDPMC52
deposited on Dec. 10, 1998 and assigned ATCC Deposit No.
203518.
[0031] In additional embodiments, the nucleic acid molecules of the
invention comprise, or alternatively consist of, a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a Neutrokine-alpha or Neutrokine-alphaSV polypeptide having an
amino acid sequence in (a), (b), (c), (d), (e), (f) or (g) above. A
further nucleic acid embodiment of the invention relates to an
isolated nucleic acid molecule comprising, or alternatively
consisting of, a polynucleotide which encodes the amino acid
sequence of a Neutrokine-alpha or Neutrokine-alphaSV polypeptide
having an amino acid sequence which contains at least one amino
acid addition, substitution, and/or deletion but not more than 50
amino acid additions, substitutions and/or deletions, even more
preferably, not more than 40 amino acid additions, substitutions,
and/or deletions, still more preferably, not more than 30 amino
acid additions, substitutions, and/or deletions, and still even
more preferably, not more than 20 amino acid additions,
substitutions, and/or deletions. Of course, in order of
ever-increasing preference, it is highly preferable for a
polynucleotide which encodes the amino acid sequence of a
Neutrokine-alpha or Neutrokine-alphaSV polypeptide to have an amino
acid sequence which contains not more than 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1 or 1-100, 1-50, 1-25, 1-20, 1-15, 1-10, or 1-5 amino acid
additions, substitutions and/or deletions. Conservative
substitutions are preferable.
[0032] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of Neutrokine-alpha polypeptides by
recombinant techniques.
[0033] In accordance with a further embodiment of the present
invention, there is provided a process for producing such
polypeptides by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a
Neutrokine-alpha or Neutrokine-alphaSV nucleic acid sequence of the
invention, under conditions promoting expression of said
polypeptide and subsequent recovery of said polypeptide.
[0034] The invention further provides an isolated Neutrokine-alpha
polypeptide comprising, or alternatively consisting of, an amino
acid sequence selected from the group consisting of: (a) the amino
acid sequence of the full-length Neutrokine-alpha polypeptide
having the complete amino acid sequence shown in FIGS. 1A and 1B
(i.e., positions 1-285 of SEQ ID NO:2) or as encoded by the cDNA
plasmid contained in the deposit having ATCC accession number
97768; (b) the amino acid sequence of the full-length
Neutrokine-alpha polypeptide having the complete amino acid
sequence shown in SEQ ID NO:2 excepting the N-terminal methionine
(i.e., positions 2 to 285 of SEQ ID NO:2); (c) a fragment of the
polypeptide of (b) having Neutrokine-alpha functional activity
(e.g., biological activity); (d) the amino acid sequence of the
predicted extracellular domain of the Neutrokine-alpha polypeptide
having the amino acid sequence at positions 73 to 285 in FIGS. 1A
and 1B (SEQ ID NO:2) or as encoded by the cDNA plasmid contained in
the deposit having ATCC accession number 97768; (e) an amino acid
sequence encoding the mature soluble form of Neutrokine-alpha
polypeptide having the amino acid sequence at positions 134-285 in
FIGS. 1A and 1B (SEQ ID NO:2); (f) the amino acid sequence of the
Neutrokine-alpha intracellular domain (predicted to constitute
amino acid residues from about 1 to about 46 in FIGS. 1A and 1B
(SEQ ID NO:2)) or as encoded by the cDNA plasmid contained in the
deposit having ATCC accession number 97768; (g) the amino acid
sequence of the Neutrokine-alpha transmembrane domain (predicted to
constitute amino acid residues from about 47 to about 72 in FIGS.
1A and 1B (SEQ ID NO:2)) or as encoded by the cDNA plasmid
contained in the deposit having ATCC accession number 97768; (h)
the amino acid sequence of the soluble Neutrokine-alpha polypeptide
having the extracellular and intracellular domains but lacking the
transmembrane domain, wherein each of these domains is defined
above; and (i) fragments of the polypeptide of (a), (b), (c), (d),
(e), (f), (g) or (h). The polypeptides of the present invention
also include polypeptides having an amino acid sequence at least
80% identical, more preferably at least 85% or 90% identical, and
still more preferably 95%, 96%, 97%, 98% or 99% identical to those
described in (a), (b), (c), (d), (e) (f), (g), (h) or (i) above, as
well as polypeptides having an amino acid sequence with at least
80%, 85%, or 90% similarity, and more preferably at least 95%
similarity, to those above. Additional embodiments of the invention
relates to polypeptides which comprise, or alternatively consist
of, the amino acid sequence of an epitope-bearing portion of a
Neutrokine-alpha polypeptide having an amino acid sequence
described in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above.
Polypeptides having the amino acid sequence of an epitope-bearing
portion of a Neutrokine-alpha polypeptide of the invention include
portions of such polypeptides with at least 4, at least 5, at least
6, at least 7, at least 8, and preferably at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at
least 20, at least 25, at least 30, at least 40, at least 50, and
more preferably at least about 30 amino acids to about 50 amino
acids, although epitope-bearing polypeptides of any length up to
and including the entire amino acid sequence of a polypeptide of
the invention described above also are included in the
invention.
[0035] Highly preferred embodiments of the invention are directed
to nucleic acid molecules comprising, or alternatively consisting
of a polynucleotide having a nucleotide sequence at least 80%, 85%,
90% identical and more preferably at least 95%, 96%, 97%, 98%, 99%
or 100% identical to a polynucleotide sequence encoding the
Neutrokine-alpha polypeptide having the amino acid sequence at
positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2). Preferred
embodiments of the invention are directed to nucleic acid molecules
comprising, or alternatively consisting of a polynucleotide having
a nucleotide sequence at least 90% identical to a polynucleotide
sequence encoding the Neutrokine-alpha polypeptide having the amino
acid sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID
NO:2). More preferred embodiments of the invention are directed to
nucleic acid molecules comprising, or alternatively consisting of a
polynucleotide having a nucleotide sequence at least 95% identical
to a polynucleotide sequence encoding the Neutrokine-alpha
polypeptide having the amino acid sequence at positions 134-285 in
FIGS. 1A and 1B (SEQ ID NO:2). More preferred embodiments of the
invention are directed to nucleic acid molecules comprising, or
alternatively consisting of a polynucleotide having a nucleotide
sequence at least 96% identical to a polynucleotide sequence
encoding the Neutrokine-alpha polypeptide having the amino acid
sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2).
Additionally, more preferred embodiments of the invention are
directed to nucleic acid molecules comprising, or alternatively
consisting of a polynucleotide having a nucleotide sequence at
least 97% to a polynucleotide sequence encoding the
Neutrokine-alpha polypeptide having the amino acid sequence at
positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2). Additionally,
more preferred embodiments of the invention are directed to nucleic
acid molecules comprising, or alternatively consisting of a
polynucleotide having a nucleotide sequence at least 98% to a
polynucleotide sequence encoding the Neutrokine-alpha polypeptide
having the amino acid sequence at positions 134-285 in FIGS. 1A and
1B (SEQ ID NO:2). Additionally, more preferred embodiments of the
invention are directed to nucleic acid molecules comprising, or
alternatively consisting of a polynucleotide having a nucleotide
sequence at least 99% identical to a polynucleotide sequence
encoding the Neutrokine-alpha polypeptide having the amino acid
sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2).
[0036] The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these polynucleotides and nucleic
acid molecules are also encompassed by the invention.
[0037] The invention further provides an isolated
Neutrokine-alphaSV polypeptide comprising, or alternatively
consisting of, an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of the full-length
Neutrokine-alphaSV polypeptide having the complete amino acid
sequence shown in FIGS. 5A and 5B (i.e., positions 1-266 of SEQ ID
NO:19) or as encoded by the cDNA clone contained in ATCC No. 203518
deposited on Dec. 10, 1998; (b) the amino acid sequence of the
full-length Neutrokine-alphaSV polypeptide having the complete
amino acid sequence shown in SEQ ID NO:19 excepting the N-terminal
methionine (i.e., positions 2 to 266 of SEQ ID NO:19); (c) the
amino acid sequence of the predicted extracellular domain of the
Neutrokine-alphaSV polypeptide having the amino acid sequence at
positions 73 to 266 in FIGS. 5A and 5B (SEQ ID NO:19) or as encoded
by the cDNA clone contained in ATCC No. 203518 deposited on Dec.
10, 1998; (d) the amino acid sequence of the Neutrokine-alphaSV
intracellular domain (predicted to constitute amino acid residues
from about 1 to about 46 in FIGS. 5A and 5B (SEQ ID NO:19)) or as
encoded by the cDNA clone contained in ATCC No. 203518 deposited on
Dec. 10, 1998; (e) the amino acid sequence of the
Neutrokine-alphaSV transmembrane domain (predicted to constitute
amino acid residues from about 47 to about 72 in FIGS. 5A and 5B
(SEQ ID NO:19)) or as encoded by the cDNA clone contained in ATCC
No. 203518 deposited on Dec. 10, 1998; (f) the amino acid sequence
of the soluble Neutrokine-alphaSV polypeptide having the
extracellular and intracellular domains but lacking the
transmembrane domain, wherein each of these domains is defined
above; and (g) fragments of the polypeptide of (a), (b), (c), (d),
(e), or (f). The polypeptides of the present invention also include
polypeptides having an amino acid sequence at least 80% identical,
more preferably at least 85% or 90% identical, and still more
preferably 95%, 96%, 97%, 98% or 99% identical to those described
in (a), (b), (c), (d), (e) (f), or (g) above, as well as
polypeptides having an amino acid sequence with at least 80%, 85%,
or 90% similarity, and more preferably at least 95% similarity, to
those above. Additional embodiments of the invention relates to
polypeptides which comprise, or alternatively consist of, the amino
acid sequence of an epitope-bearing portion of a Neutrokine-alphaSV
polypeptide having an amino acid sequence described in (a), (b),
(c), (d), (e), (f), or (g) above. Peptides or polypeptides having
the amino acid sequence of an epitope-bearing portion of a
Neutrokine-alphaSV polypeptide of the invention include portions of
such polypeptides with at least 4, at least 5, at least 6, at least
7, at least 8, and preferably at least 9, at least 10, at least 11,
at least 12, at least 13, at least 14, at least 15, at least 20, at
least 25, at least 30, at least 40, at least 50, and more
preferably at least about 30 amino acids to about 50 amino acids,
although epitope-bearing polypeptides of any length up to and
including the entire amino acid sequence of a polypeptide of the
invention described above also are included in the invention.
[0038] Certain non-exclusive embodiments of the invention relate to
a polypeptide which has the amino acid sequence of an
epitope-bearing portion of a Neutrokine-alpha or Neutrokine-alphaSV
polypeptide having an amino acid sequence described in (a), (b),
(c), (d), (e), (f), (g), (h) or (i) above. In other embodiments,
the invention provides an isolated antibody that binds specifically
(i.e., uniquely) to a Neutrokine-alpha or Neutrokine-alphaSV
polypeptide having an amino acid sequence described in (a), (b),
(c), (d), (e), (f), (g), (h) or (i) above.
[0039] The invention further provides methods for isolating
antibodies that bind specifically (i.e., uniquely) to a
Neutrokine-alpha or Neutrokine-alphaSV polypeptide having an amino
acid sequence as described herein. Such antibodies are useful
diagnostically or therapeutically as described below.
[0040] The invention also provides for pharmaceutical compositions
comprising soluble Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides, particularly human Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides, and/or anti-Neutrokine-alpha
antibodies and/or anti-Neutrokine-alphaSV antibodies which may be
employed, for instance, to treat, prevent, prognose and/or diagnose
tumor and tumor metastasis, infections by bacteria, viruses and
other parasites, immunodeficiencies, inflammatory diseases,
lymphadenopathy, autoimmune diseases, graft versus host disease,
stimulate peripheral tolerance, destroy some transformed cell
lines, mediate cell activation, survival and proliferation, mediate
immune regulation and inflammatory responses, and to enhance or
inhibit immune responses.
[0041] In certain embodiments, soluble Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention, or agonists
thereof, are administered, to treat, prevent, prognose and/or
diagnose an immunodeficiency (e.g., severe combined
immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine
deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia
(XLA), Bruton's disease, congenital agammaglobulinemia, X-linked
infantile agammaglobulinemia, acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.) or
conditions associated with an immunodeficiency.
[0042] In a specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
prognose and/or diagnose common variable immunodeficiency.
[0043] In a specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
prognose and/or diagnose X-linked agammaglobulinemia.
[0044] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
prognose and/or diagnose severe combined immunodeficiency
(SCID).
[0045] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
prognose and/or diagnose Wiskott-Aldrich syndrome.
[0046] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
prognose and/or diagnose X-linked Ig deficiency with hyper IgM.
[0047] In another embodiment, Neutrokine-alpha antagonists and/or
Neutrokine-alphaSV antagonists (e.g., an anti-Neutrokine-alpha
antibody), are administered to treat, prevent, prognose and/or
diagnose an autoimmune disease (e.g., rheumatoid arthritis,
systemic lupus erythematosus, idiopathic thrombocytopenia purpura,
autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,
dermatitis, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, glomerulonephritis (e.g,
IgA nephropathy), an immune-based rheumatologic disease (e.g., SLE,
rheumatoid arthritis, CREST syndrome (a variant of scleroderma
characterized by calcinosis, Raynaud's phenomenon, esophageal
motility disorders, sclerodactyl), and telangiectasia.),
Seronegative spondyloarthropathy (SpA),
Polymyositis/dermatomyositis, Microscopic polyangiitis, Hepatitis
C-associated arthritis, Takayasu's arteritis, and undifferentiated
connective tissue disorder), Multiple Sclerosis, Neuritis, Uveitis
Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein
purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune
Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent
diabetes mellitis, and autoimmune inflammatory eye, autoimmune
thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis,
Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as,
for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c)
insulin resistance, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, scleroderma with anti-collagen
antibodies, mixed connective tissue disease,
polymyositis/dermatomyositis, pernicious anemia, idiopathic
Addison's disease, infertility, glomerulonephritis such as primary
glomerulonephritis and IgA nephropathy, bullous pemphigoid,
Sjogren's syndrome, diabetes millitus, and adrenergic drug
resistance (including adrenergic drug resistance with asthma or
cystic fibrosis), chronic active hepatitis, primary biliary
cirrhosis, other endocrine gland failure, vitiligo, vasculitis,
post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma,
inflammatory myopathies, and other inflammatory, granulamatous,
degenerative, and atrophic disorders) or conditions associated with
an autoimmune disease. In a specific preferred embodiment,
rheumatoid arthritis is treated, prevented, prognosed and/or
diagnosed using anti-Neutrokine-alpha antibodies and/or
anti-Neutrokine-alphaSV antibodies and/or other antagonist of the
invention. In another specific preferred embodiment, systemic lupus
erythemosus is treated, prevented, prognosed, and/or diagnosed
using anti-Neutrokine-alpha antibodies and/or
anti-Neutrokine-alphaSV and/or other antagonist of the invention.
In another specific preferred embodiment, idiopathic
thrombocytopenia purpura is treated, prevented, prognosed, and/or
diagnosed using anti-Neutrokine-alpha antibodies and/or
anti-Neutrokine-alphaSV and/or other antagonist of the invention.
In another specific preferred embodiment IgA nephropathy is
treated, prevented, prognosed and/or diagnosed using
anti-Neutrokine-alpha antibodies and/or anti-Neutrokine-alphaSV
and/or other antagonist of the invention. In a preferred
embodiment, the autoimmune diseases and disorders and/or conditions
associated with the diseases and disorders recited above are
treated, prevented, prognosed and/or diagnosed using
anti-Neutrokine-alpha antibodies and/or anti-Neutrokine-alphaSV
antibodies.
[0048] The invention further provides compositions comprising a
Neutrokine-alpha or Neutrokine-alphaSV polynucleotide, a
Neutrokine-alpha or Neutrokine-alphaSV polypeptide, and/or an
anti-Neutrokine-alpha antibody or anti-Neutrokine-alphaSV antibody,
for administration to cells in vitro, to cells ex vivo, and to
cells in vivo, or to a multicellular organism. In preferred
embodiments, the compositions of the invention comprise a
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotide for
expression of a Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide in a host organism for treatment of disease. In a most
preferred embodiment, the compositions of the invention comprise a
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotide for
expression of a Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide in a host organism for treatment of an immunodeficiency
and/or conditions associated with an immunodeficiency. Particularly
preferred in this regard is expression in a human patient for
treatment of a dysfunction associated with aberrant endogenous
activity of a Neutrokine-alpha, Neutrokine-alphaSV, Neutrokine
alpha receptor, and/or Neutrokine-alphaSV receptor gene (e.g.,
expression to enhance the normal B-cell function by expanding
B-cell numbers or increasing B cell lifespan).
[0049] The present invention further encompasses methods and
compositions for preventing, treating and/or ameliorating diseases
or disorders associated with aberrant or inappropriate
Neutrokine-alpha, Neutrokine-alphaSV, Neutrokine-alpha receptor,
and/or Neutrokine-alphaSV receptor expression or function in an
animal, preferably a mammal, and most preferably a human,
comprising, or alternatively consisting of, administering to an
animal in which such treatment, prevention or amelioration is
desired one or more Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides (including molecules which comprise, or alternatively
consist of, Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
fragments or variants thereof) in an amount effective to treat
prevent or ameliorate the disease or disorder.
[0050] The present invention further encompasses methods and
compositions for killing cells of hematopoietic origin, comprising,
or alternatively consisting of, contacting Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide with cells of hematopoietic origin.
In preferred embodiments, the cells of hematopoietic origin are B
cells.
[0051] The present invention further encompasses methods and
compositions for killing cells of hematopoietic origin, comprising,
or alternatively consisting of, administering to an animal in which
such killing is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide (e.g., a radiolabeled
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide) in an
amount effective to kill cells of hematopoietic origin. In
preferred embodiments, the cells of hematopoietic origin are B
cells.
[0052] The present invention further encompasses methods and
compositions for stimulating immunoglobulin production, comprising,
or alternatively consisting of, contacting an effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV with cells of
hematopoietic origin, wherein the effective amount of the
Neutrokine-alpha and/or Neutrokine-alphaSV binding polypeptide
stimulates Neutrokine-alpha and/or Neutrokine-alphaSV-mediated
immunoglobulin production.
[0053] The present invention further encompasses methods and
compositions for stimulating immunoglobulin production comprising,
or alternatively consisting of, administering to an animal in which
such stimulation is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide in an amount effective to stimulate
immunoglobulin production.
[0054] The present invention further encompasses methods and
compositions for stimulating proliferation of cells of
hematopoietic origin, comprising, or alternatively consisting of,
contacting an effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide with cells of hematopoietic origin,
wherein the effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide stimulates Neutrokine-alpha and/or
Neutrokine-alphaSV-mediated cell proliferation. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0055] The present invention further encompasses methods and
compositions for stimulating proliferation of cells of
hematopoietic origin, comprising, or alternatively consisting of,
administering to an animal in which such stimulation is desired, a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide in an amount
effective to stimulate Neutrokine-alpha and/or
Neutrokine-alphaSV-mediated cell proliferation. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0056] The present invention further encompasses methods and
compositions for increasing activation of cells of hematopoietic
origin, comprising, or alternatively consisting of, contacting an
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide with cells of hematopoietic origin, wherein the
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide increases Neutrokine-alpha and/or
Neutrokine-alphaSV-mediated activation of cells of hematopoietic
origin. In preferred embodiments, the cells of hematopoietic origin
are B cells.
[0057] The present invention further encompasses methods and
compositions for increasing activation of cells of hematopoietic
origin, comprising, or alternatively consisting of, administering
to an animal in which such increase is desired, a Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide in an amount effective to
increase Neutrokine-alpha and/or Neutrokine-alphaSV-mediated
activation of cells of hematopoietic origin. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0058] The present invention further encompasses methods and
compositions for increasing lifespan of cells of hematopoietic
origin, comprising, or alternatively consisting of, contacting an
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide with cells of hematopoietic origin, wherein the
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
binding polypeptide increases Neutrokine-alpha and/or
Neutrokine-alphaSV-regulated lifespan of cells of hematopoietic
origin. In preferred embodiments, the cells of hematopoietic origin
are B cells.
[0059] The present invention further encompasses methods and
compositions for increasing lifespan of cells of hematopoietic
origin, comprising, or alternatively consisting of, administering
to an animal in which such increase is desired, a Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide in an amount effective to
increase Neutrokine-alpha and/or Neutrokine-alphaSV-regulated
lifespan of cells of hematopoietic origin. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0060] The present invention further encompasses methods and
compositions for inhibiting or reducing immunoglobulin production,
comprising, or alternatively consisting of, contacting an effective
amount of Neutrokine-alpha and/or Neutrokine-alphaSV with cells of
hematopoietic origin, wherein the effective amount of the
Neutrokine-alpha and/or Neutrokine-alphaSV binding polypeptide
inhibits or reduces Neutrokine-alpha and/or
Neutrokine-alphaSV-mediated immunoglobulin production. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0061] The present invention further encompasses methods and
compositions for inhibiting or reducing immunoglobulin production
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide in an amount
effective to inhibit ir reduce immunoglobulin production.
[0062] The present invention further encompasses methods and
compositions for inhibiting or reducing proliferation of cells of
hematopoietic origin, comprising, or alternatively consisting of,
contacting an effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide with cells of hematopoietic origin,
wherein the effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide inhibits ir reduces Neutrokine-alpha
and/or Neutrokine-alphaSV-mediated cell proliferation. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0063] The present invention further encompasses methods and
compositions for inhibiting or reducing proliferation of cells of
hematopoietic origin, comprising, or alternatively consisting of,
administering to an animal in which such inhibition or reduction is
desired, a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
in an amount effective to inhibit or reduce Neutrokine-alpha and/or
Neutrokine-alphaSV-mediated cell proliferation. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0064] The present invention further encompasses methods and
compositions for decreasing activation of cells of hematopoietic
origin, comprising, or alternatively consisting of, contacting an
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide with cells of hematopoietic origin, wherein the
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide decreases Neutrokine-alpha and/or
Neutrokine-alphaSV-mediated activation of cells of hematopoietic
origin. In preferred embodiments the cells of hematopoietic origin
are B cells.
[0065] The present invention further encompasses methods and
compositions for decreasing activation of cells of hematopoietic
origin, comprising, or alternatively consisting of, administering
to an animal in which such increase is desired, a Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide in an amount effective to
decrease Neutrokine-alpha and/or Neutrokine-alphaSV-mediated
activation of cells of hematopoietic origin. In preferred
embodiments the cells of hematopoietic origin are B cells.
[0066] The present invention further encompasses methods and
compositions for decreasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide with cells
of hematopoietic origin, wherein the effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV binding polypeptide
decreases Neutrokine-alpha and/or Neutrokine-alphaSV-regulated
lifespan of cells of hematopoietic origin. In preferred embodiments
the cells of hematopoietic origin are B cells.
[0067] The present invention further encompasses methods and
compositions for decreasing lifespan of cells of hematopoietic
origin, comprising, or alternatively consisting of, administering
to an animal in which such reduction is desired, a Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide in an amount effective to
decrease Neutrokine-alpha and/or Neutrokine-alphaSV-regulated
lifespan of cells of hematopoietic origin. In preferred embodiments
the cells of hematopoietic origin are B cells.
[0068] The present invention also provides a screening method for
identifying compounds capable of enhancing or inhibiting a cellular
response induced by Neutrokine-alpha and/or Neutrokine-alphaSV
which involves contacting cells which express Neutrokine-alpha
and/or Neutrokine-alphaSV with the candidate compound, assaying a
cellular response, and comparing the cellular response to a
standard cellular response, the standard being assayed when contact
is made in absence of the candidate compound; whereby, an increased
cellular response over the standard indicates that the compound is
an agonist and a decreased cellular response over the standard
indicates that the compound is an antagonist.
[0069] In another embodiment, a method for identifying
Neutrokine-alpha and/or Neutrokine-alphaSV receptors is provided,
as well as a screening assay for agonists and antagonists using
such receptors. This assay involves determining the effect a
candidate compound has on Neutrokine-alpha and/or
Neutrokine-alphaSV binding to the Neutrokine-alpha and/or
Neutrokine-alphaSV receptor. In particular, the method involves
contacting a Neutrokine-alpha and/or Neutrokine-alphaSV receptor
with a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide of
the invention and a candidate compound and determining whether
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide binding to
the Neutrokine-alpha and/or Neutrokine-alphaSV receptor is
increased or decreased due to the presence of the candidate
compound. The antagonists may be employed to prevent septic shock,
inflammation, cerebral malaria, activation of the HIV virus,
graft-host rejection, bone resorption, rheumatoid arthritis,
cachexia (wasting or malnutrition), immune system function,
lymphoma, and autoimmune disorders (e.g., rheumatoid arthritis and
systemic lupus erythematosus).
[0070] The present inventors have discovered that Neutrokine-alpha
is expressed not only in cells of monocytic lineage, but also in
kidney, lung, peripheral leukocyte, bone marrow, T cell lymphoma, B
cell lymphoma, activated T cells, stomach cancer, smooth muscle,
macrophages, and cord blood tissue. The present inventors have
further discovered that Neutrokine-alphaSV appears to be expressed
highly only in primary dendritic cells. For a number of disorders
of these tissues and cells, such as tumor and tumor metastasis,
infection of bacteria, viruses and other parasites,
immunodeficiencies (e.g., chronic variable immunodeficiency),
septic shock, inflammation, cerebral malaria, activation of the HIV
virus, graft-host rejection, bone resorption, rheumatoid arthritis,
autoimmune diseases (e.g., rheumatoid arthritis and systemic lupus
erythematosus) and cachexia (wasting or malnutrition). It is
believed that significantly higher or lower levels of
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression can be
detected in certain tissues (e.g., bone marrow) or bodily fluids
(e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken
from an individual having such a disorder, relative to a "standard"
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression level,
i.e., the Neutrokine-alpha and/or Neutrokine-alphaSV expression
level in tissue or bodily fluids from an individual not having the
disorder. Thus, the invention provides a diagnostic method useful
during diagnosis of a disorder, which involves: (a) assaying
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression level in
cells or body fluid of an individual; (b) comparing the
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression level
with a standard Neutrokine-alpha and/or Neutrokine-alphaSV gene
expression level, whereby an increase or decrease in the assayed
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression level
compared to the standard expression level is indicative of a
disorder.
[0071] An additional embodiment of the invention is related to a
method for treating an individual in need of an increased or
constitutive level of Neutrokine-alpha and/or Neutrokine-alphaSV
activity in the body comprising administering to such an individual
a composition comprising a therapeutically effective amount of an
isolated Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide of
the invention or an agonist thereof.
[0072] A still further embodiment of the invention is related to a
method for treating an individual in need of a decreased level of
Neutrokine-alpha and/or Neutrokine-alphaSV activity in the body
comprising, administering to such an individual a composition
comprising a therapeutically effective amount of an
Neutrokine-alpha and/or Neutrokine-alphaSV antagonist. Preferred
antagonists for use in the present invention are
Neutrokine-alpha-specific and/or Neutrokine-alphaSV-specific
antibodies.
BRIEF DESCRIPTION OF THE FIGURES
[0073] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0074] FIGS. 1A and 1B show the nucleotide (SEQ ID NO:1) and
deduced amino acid (SEQ ID NO:2) sequences of Neutrokine-alpha.
Amino acids 1 to 46 represent the predicted intracellular domain,
amino acids 47 to 72 the predicted transmembrane domain (the
double-underlined sequence), and amino acids 73 to 285, the
predicted extracellular domain (the remaining sequence). Potential
asparagine-linked glycosylation sites are marked in FIGS. 1A and 1B
with a bolded asparagine symbol (N) in the Neutrokine-alpha amino
acid sequence and a bolded pound sign (#) above the first
nucleotide encoding that asparagine residue in the Neutrokine-alpha
nucleotide sequence. Potential N-linked glycosylation sequences are
found at the following locations in the Neutrokine-alpha amino acid
sequence: N-124 through Q-127 (N-124, S-125, S-126, Q-127) and
N-242 through C-245 (N-242, N-243, S-244, C-245).
[0075] Regions of high identity between Neutrokine-alpha,
Neutrokine-alphaSV, TNF-alpha, TNF-beta, LT-beta, and the closely
related Fas Ligand (an alignment of these sequences is presented in
FIGS. 2A, 2B, 2C, and 2D) are underlined in FIGS. 1A and 1B. These
regions are not limiting and are labeled as Conserved Domain
(CD)-I, CD-II, CD-III, CD-IV, CD-V, CD-VI, CD-VII, CD-VIII, CD-IX,
CD-X, and CD-XI in FIGS. 1A and 1B.
[0076] FIGS. 2A, 2B, 2C, and 2D show the regions of identity
between the amino acid sequences of Neutrokine-alpha (SEQ ID NO:2)
and Neutrokine-alphaSV (SEQ ID NO:19), and TNF-alpha ("TNFalpha" in
FIGS. 2A, 2B, 2C, and 2D; GenBank No. Z15026; SEQ ID NO:3),
TNF-beta ("TNFbeta" in FIGS. 2A, 2B, 2C, and 2D; GenBank No.
Z15026; SEQ ID NO:4), Lymphotoxin-beta ("LTbeta" in FIGS. 2A, 2B,
2C, and 2D; GenBank No. L11016; SEQ ID NO:5), and FAS ligand
("FASL" in FIGS. 2A, 2B, 2C, and 2D; GenBank No. U11821; SEQ ID
NO:6), determined by the "MegAlign" routine which is part of the
computer program called "DNA*STAR." Residues that match the
consensus are shaded.
[0077] FIG. 3 shows an analysis of the Neutrokine-alpha amino acid
sequence. Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown, as predicted for the amino
acid sequence of SEQ ID NO:2 using the default parameters of the
recited computer programs. In the "Antigenic Index--Jameson-Wolf"
graph, the indicate location of the highly antigenic regions of
Neutrokine-alpha i.e., regions from which epitope-bearing peptides
of the invention may be obtained. Antigenic polypeptides include
from about Phe-115 to about Leu-147, from about Ile-150 to about
Tyr-163, from about Ser-171 to about Phe-194, from about Glu-223 to
about Tyr-246, and from about Ser-271 to about Phe-278, of the
amino acid sequence of SEQ ID NO:2.
[0078] The data presented in FIG. 3 are also represented in tabular
form in Table I. The columns are labeled with the headings "Res",
"Position", and Roman Numerals I-XIV. The column headings refer to
the following features of the amino acid sequence presented in FIG.
3, and Table I: "Res": amino acid residue of SEQ ID NO:2 and FIGS.
1A and 1B; "Position": position of the corresponding residue within
SEQ ID NO:2 and FIGS. 1A and 1B; I: Alpha, Regions-Garnier-Robson;
II: Alpha, Regions-Chou-Fasman; III: Beta, Regions-Garnier-Robson;
IV: Beta, Regions-Chou-Fasman; V: Turn, Regions-Garnier-Robson; VI:
Turn, Regions-Chou-Fasman; VII: Coil, Regions-Garnier-Robson; VIII:
Hydrophilicity Plot-Kyte-Doolittle; IX: Hydrophobicity
Plot-Hopp-Woods; X: Alpha, Amphipathic Regions-Eisenberg; XI: Beta,
Amphipathic Regions-Eisenberg; XII: Flexible
Regions-Karplus-Schulz; XIII: Antigenic Index-Jameson-Wolf; and
XIV: Surface Probability Plot-Emini.
[0079] FIGS. 4A, 4B, and 4C show the alignment of the
Neutrokine-alpha nucleotide sequence determined from the human cDNA
deposited in ATCC No. 97768 with related human cDNA clones of the
invention which have been designated HSOAD55 (SEQ ID NO:7), HNEDU15
(SEQ ID NO:1), HSLAH84 (SEQ ID NO:8) and HLTBM08 (SEQ ID NO:9).
[0080] FIGS. 5A and 5B shows the nucleotide (SEQ ID NO:18) and
deduced amino acid (SEQ ID NO:19) sequences of the
Neutrokine-alphaSV protein. Amino acids 1 to 46 represent the
predicted intracellular domain, amino acids 47 to 72 the predicted
transmembrane domain (the double-underlined sequence), and amino
acids 73 to 266, the predicted extracellular domain (the remaining
sequence). Potential asparagine-linked glycosylation sites are
marked in FIGS. 5A and 5B with a bolded asparagine symbol (N) in
the Neutrokine-alphaSV amino acid sequence and a bolded pound sign
(#) above the first nucleotide encoding that asparagine residue in
the Neutrokine-alphaSV nucleotide sequence. Potential N-linked
glycosylation sequences are found at the following locations in the
Neutrokine-alphaSV amino acid sequence: N-124 through Q-127 (N-124,
S-125, S-126, Q-127) and N-223 through C-226 (N-223, N-224, S-225,
C-226). Antigenic polypeptides include from about Pro-32 to about
Leu-47, from about Glu-116 to about Ser-143, from about Phe-153 to
about Tyr-173, from about Pro-218 to about Tyr-227, from about
Ala-232 to about Gln-241; from about Ile-244 to about Ala-249; and
from about Ser-252 to about Val-257 of the amino acid sequence of
SEQ ID NO:19.
[0081] Regions of high identity between Neutrokine-alpha,
Neutrokine-alphaSV, TNF-alpha, TNF-beta, LT-beta, and the closely
related Fas Ligand (an alignment of these sequences is presented in
FIG. 2) are underlined in FIGS. 1A and 1B. These conserved regions
(of Neutrokine-alpha and Neutrokine-alphaSV) are labeled as
Conserved Domain (CD)-I, CD-II, CD-III, CD-V, CD-VI, CD-VII,
CD-VIII, CD-IX, CD-X, and CD-XI in FIGS. 5A and 5B.
Neutrokine-alphaSV does not contain the sequence of CD-IV described
in the legend of FIGS. 1A and 1B.
[0082] An additional alignment of the Neutrokine-alpha polypeptide
sequence (SEQ ID NO:2) with APRIL, TNF alpha, and LT alpha is
presented in FIGS. 7A-1 and 7A-2. In FIGS. 7A-1 and 7A-2, beta
sheet regions are indicated as described below in the legend to
FIGS. 7A-1 and 7A-2.
[0083] FIG. 6 shows an analysis of the Neutrokine-alphaSV amino
acid sequence. Alpha, beta, turn and coil regions; hydrophilicity
and hydrophobicity; amphipathic regions; flexible regions;
antigenic index and surface probability are shown, as predicted for
the amino acid sequence of SEQ ID NO:19 using the default
parameters of the recited computer programs. The location of the
highly antigenic regions of the Neutrokine-alpha protein, i.e.,
regions from which epitope-bearing peptides of the invention may be
obtained is indicated in the "Antigenic Index--Jameson-Wolf" graph.
Antigenic polypeptides include, but are not limited to, a
polypeptide comprising amino acid residues from about Pro-32 to
about Leu-47, from about Glu-116 to about Ser-143, from about
Phe-153 to about Tyr-173, from about Pro-218 to about Tyr-227, from
about Ser-252 to about Thr-258, from about Ala-232 to about
Gln-241; from about Ile-244 to about Ala-249; and from about
Ser-252 to about Val-257, of the amino acid sequence of SEQ ID NO:
19.
[0084] The data shown in FIG. 6 can be easily represented in
tabular format similar to the data shown in Table I. Such a tabular
representation of the exact data disclosed in FIG. 6 can be
generated using the MegAlign component of the DNA*STAR computer
sequence analysis package set on default parameters. This is the
identical program that was used to generate FIGS. 3 and 6 of the
present application.
[0085] FIGS. 7A-1 and 7A-2. The amino-acid sequence of
Neutrokine-alpha (SEQ ID NO:2) and alignment of its predicted
ligand-binding domain with those of APRIL, TNF-alpha, and LT-alpha
(specifically, amino acid residues 115-250 of the human APRIL
polypeptide (SEQ ID NO:20; GenBank Accession No. AF046888
(nucleotide) and AAC6132 (protein)), amino acid residues 88-233 of
TNF alpha (SEQ ID NO:3; GenBank Accession No. Z15026), and LT alpha
((also designated TNF-beta) amino acid residues 62-205 of SEQ ID
NO:4; GenBank Accession No. Z15026)). The predicted
membrane-spanning region of Neutrokine-alpha is indicated and the
site of cleavage of Neutrokine-alpha is depicted with an arrow.
Sequences overlaid with lines (A thru H) represent predicted
beta-pleated sheet regions.
[0086] FIG. 7B. Expression of Neutrokine-alpha mRNA. Northern
hybridization analysis was performed using the Neutrokine-alpha orf
as a probe on blots of poly (A)+ RNA (Clonetech) from a spectrum of
human tissue types and a selection of cancer cell lines. A 2.6 kb
Neutrokine-alpha mRNA was detected at high levels in placenta,
heart, lung, fetal liver, thymus, and pancreas. The 2.6 kb
Neutrokine-alpha mRNA was also detected in HL-60 and K562 cell
lines.
[0087] FIGS. 8A, 8B and 8C. Neutrokine-alpha expression increases
following activation of human monocytes by IFN-gamma. FIGS. 8A and
8B. Flow cytometric analysis of Neutrokine-alpha protein expression
on in vitro cultured monocytes. Purified monocytes were cultured
for 3 days in presence or absence of IFN-gamma (100 U/ml). Cells
were then stained with a Neutrokine-alpha-specific mAb (2E5) (solid
lines) or an isotype-matched control (IgG1) (dashed lines).
Comparable results were obtained with monocytes purified from three
different donors in three independent experiments. FIG. 8C.
Neutrokine-alpha-specific TaqMan primers were prepared and used to
assess the relative Neutrokine-alpha mRNA expression levels in
unstimulated and IFN-gamma (100 U/mL) treated monocytes. Nucleotide
sequences of the TaqMan primers are as follows: (a) Probe: 5'-CCA
CCA GCT CCA GGA GAA GGC AAC TC-3' (SEQ ID NO:24); (b) 5'
amplification primer: 5'-ACC GCG GGA CTG AAA ATC T-3' (SEQ ID
NO:25); and (c) 3' amplification primer: 5'-CAC GCT TAT TTC TGC TGT
TCT GA-3' (SEQ ID NO:26).
[0088] FIGS. 9A and 9B. Neutrokine-alpha is a potent B lymphocyte
stimulator. FIG. 9A. The biological activity of Neutrokine-alpha
was assessed in a standard B-lymphocyte co-stimulation assay
utilizing Staphylococcus aureus cowan 1 SAC as the priming agent.
SAC alone yielded background counts of 1427+/-316. Values are
reported as mean+/-standard deviation of triplicate wells. Similar
results were obtained using recombinant Neutrokine-alpha purified
from stable CHO transfectants and transiently transfected HEK 293T
cells. FIG. 9B. Proliferation of tonsillar B cells with
Neutrokine-alpha and co-stimulation with anti-IgM. The bioassay was
performed as described for SAC with the exception that individual
wells were pre-coated with goat anti-human IgM antibody at 10
micrograms/mL in PBS.
[0089] FIGS. 10A, 10B, 10C, 10D, 10E, 10F and 10G. Neutrokine-alpha
receptor expression among normal human peripheral blood mononuclear
cells and tumor cell lines. FIGS. 10A, 10B, 10C, 10D and 10E. Human
peripheral blood nucleated cells were obtained from normal
volunteers and isolated by density gradient centrifugation. Cells
were stained with biotinylated Neutrokine-alpha followed by
PE-conjugated streptavidin and FITC or PerCP coupled mAbs specific
for CD3, CD20, CD14, CD56, and CD66b. Cells were analyzed on a
Becton Dickinson FACScan using the CellQuest software. Data
represent one of four independent experiments. FIGS. 10F and 10G.
Neutrokine-alpha binding to histiocytic cell line U-937 and the
myeloma line IM-9.
[0090] FIGS. 11A, 11B, 11C, 11D, 11E, and 11F. In vivo effects of
Neutrokine-alpha administration in BALB/cAnNCR mice. FIG. 11A.
Formalin-fixed spleens were paraffin embedded and 5 micrometer
sections stained with hematoxylin and eosin (upper panels). The
lower panels are sections taken from the same animals stained with
anti-CD45R(B220) mAb and developed with horseradish-peroxidase
coupled rabbit anti-rat Ig (mouse adsorbed) and the substrate
diaminobenzidine tetrahydrochloride (DAB). Slides were
counter-stained with Mayer's hematoxylin. CD45R(B220) expressing
cells appear brown. FIGS. 11B and 11C. Flow cytometric analyses of
normal (left panel) and Neutrokine-alpha-treated (right panel)
stained with PE-CD45R(B220) and FITC-ThB (Ly6D). FIGS. 11D, 11E,
and 11F. Serum IgM, IgG, and IgA levels in normal and
Neutrokine-alpha treated mice.
[0091] FIG. 12. Effect of .sup.131I-labeled Neutrokine-alpha (lot
TX1) on the survival of BCL1 tumor-bearing BALB/c mice. Survival
curve expressed in terms of survival probability vs. time. Day 0 is
the first day of tumor cell injection. Differences among the
treatment groups were analyzed using the Log Rank Test for
equality. Treatment with .sup.131I-labeled Neutrokine-alpha (LR131
in figure) at doses of either 11.9 or 15.3 mCi/kg (red and blue
dotted lines, respectively) significantly prolonged survival
(p=0.0162 and p=0.0052, respectively) compared with vehicle-treated
controls (black solid line). In the group of mice that did not have
BCL1 tumors but did receive 15.3 mCi/kg of .sup.131I-labeled
Neutrokine-alpha, 10% of the mice died (yellow dashed line).
[0092] FIG. 13. Effect of .sup.131I-labeled Neutrokine-alpha (lot
TX2) on the survival of BCL1 tumor-bearing BALB/c mice. Survival
curve expressed in terms of survival probability vs. time. Day 0 is
the first day of tumor cell injection. Differences among the
treatment groups were analyzed using the Log Rank Test for
equality. Treatment with .sup.131I-labeled Neutrokine-alpha (LR131
in figure) at a dose of 17.5 mCi/kg (dashed line) significantly
prolonged survival (p=0.0348) compared with the vehicle-treated
controls (black solid line). In the group of mice that did not have
BCL1 tumors but did receive .sup.131I-labeled Neutrokine-alpha,
12.5% of the mice died (dotted line).
[0093] FIG. 14. Effect of .sup.131I-labeled Neutrokine-alpha (lot
TX3) on the survival of BCL1 tumor-bearing BALB/c mice. Survival
curve expressed in terms of survival probability vs. time. Day 0 is
the day the tumor cells were injected. Differences among the
treatment groups were analyzed using the Log Rank Test for
equality. Treatment with .sup.131I-labeled Neutrokine-alpha (LR131
in figure) at a dose of 37.7 mCi/kg (dashed line) significantly
prolonged survival (p=0.0212) compared to the vehicle-treated
controls (solid line). In the group of mice that did not have BCL1
tumors but did receive 131I-labeled Neutrokine-alpha, 12.5% of the
mice died (dotted line).
[0094] FIG. 15 shows a plasmid map of the pML124 vector. The
sequence of this vector is shown in SEQ ID NO:52.
[0095] FIG. 16 shows a plasmid map of the pML124 vector containing
the MBPss-Neutrokine-alpha fusion. The sequence of this vector is
shown in SEQ ID NO:53.
DETAILED DESCRIPTION
[0096] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a Neutrokine-alpha
polypeptides having the amino acid sequences shown in FIGS. 1A and
1B (SEQ ID NO:2), which was determined by sequencing a cDNA clone.
The nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) was
obtained by sequencing the HNEDU15 clone, which was deposited on
Oct. 22, 1996 at the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209, and assigned ATCC
Accession No. 97768. The deposited clone is contained in the
pBluescript SK(-) plasmid (Stratagene, La Jolla, Calif.). The ATCC
deposits were made pursuant to the terms of the Budapest Treaty on
the international recognition of the deposit of microorganisms for
the purposes of patent procedure.
[0097] The present invention also provides isolated nucleic acid
molecules comprising a polynucleotide encoding Neutrokine-alphaSV
polypeptides having the amino acid sequences shown in FIGS. 5A and
5B (SEQ ID NO:19), which was determined by sequencing a cDNA clone.
The nucleotide sequence shown in FIGS. 5A and 5B (SEQ ID NO:18) was
obtained by sequencing the HDPMC52 clone, which was deposited on
Dec. 10, 1998 at the American Type Culture Collection, and assigned
ATCC Accession No. 203518. The deposited clone is contained in the
pBluescript SK(-) plasmid (Stratagene, La Jolla, Calif.). The ATCC
deposits were made pursuant to the terms of the Budapest Treaty on
the international recognition of the deposit of microorganisms for
the purposes of patent procedure.
[0098] The Neutrokine-alpha and Neutrokine-alpha polypeptides of
the present invention share sequence homology with the translation
products of the human mRNAs for TNF-alpha, TNF-beta, LTbeta, Fas
ligand, APRIL, and LTalpha. (See, FIGS. 2A, 2B, 2C, 2D, 7A-1 and
7A-2). As noted above, TNF-alpha is thought to be an important
cytokine that plays a role in cytotoxicity, necrosis, apoptosis,
costimulation, proliferation, lymph node formation, immunoglobulin
class switch, differentiation, antiviral activity, and regulation
of adhesion molecules and other cytokines and growth factors.
Nucleic Acid Molecules
[0099] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc., Foster City, Calif.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
as above. Therefore, as is known in the art for any DNA sequence
determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90%
identical, more typically at least about 95% to at least about
99.9% identical to the actual nucleotide sequence of the sequenced
DNA molecule. The actual sequence can be more precisely determined
by other approaches including manual DNA sequencing methods well
known in the art. As is also known in the art, a single insertion
or deletion in a determined nucleotide sequence compared to the
actual sequence will cause a frame shift in translation of the
nucleotide sequence such that the predicted amino acid sequence
encoded by a determined nucleotide sequence will be completely
different from the amino acid sequence actually encoded by the
sequenced DNA molecule, beginning at the point of such an insertion
or deletion.
[0100] By "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U), where each thymidine deoxyribonucleotide (T) in the
specified deoxyribonucleotide sequence is replaced by the
ribonucleotide uridine (U).
[0101] Using the information provided herein, such as the
nucleotide sequence in FIGS. 1A and 1B, a nucleic acid molecule of
the present invention encoding a Neutrokine-alpha polypeptide may
be obtained using standard cloning and screening procedures, such
as those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule described
in FIGS. 1A and 1B (SEQ ID NO:1) was discovered in a cDNA library
derived from neutrophils. Expressed sequence tags corresponding to
a portion of the Neutrokine-alpha cDNA were also found in kidney,
lung, peripheral leukocyte, bone marrow, T cell lymphoma, B cell
lymphoma, activated T cells, stomach cancer, smooth muscle,
macrophages, and cord blood tissue. In addition, using the
nucleotide information provided in FIGS. 5A and 5B, a nucleic acid
molecule of the present invention encoding a Neutrokine-alphaSV
polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule
described in FIGS. 5A and 5B (SEQ ID NO:18) was discovered in a
cDNA library derived from primary dendritic cells.
[0102] The Neutrokine-alpha plasmid HNEDU15 deposited as ATCC
Accession No. 97768 contains an open reading frame encoding a
protein of about 285 amino acid residues, a predicted intracellular
domain of about 46 amino acids (amino acid residues from about 1 to
about 46 in FIGS. 1A and 1B (SEQ ID NO:2)), a predicted
transmembrane domain of about 26 amino acids (underlined amino acid
residues from about 47 to about 72 in FIGS. 1A and 1B (SEQ ID
NO:2)), a predicted extracellular domain of about 213 amino acids
(amino acid residues from about 73 to about 285 in FIGS. 1A and 1B
(SEQ ID NO:2)); and a deduced molecular weight of about 31 kDa. The
Neutrokine-alpha polypeptide shown in FIGS. 1A and 1B (SEQ ID NO:2)
is about 20% similar and about 10% identical to human TNF-alpha,
which can be accessed on GenBank as Accession No. 339764.
[0103] The Neutrokine-alphaSV plasmid HDPMC52, deposited as ATCC
Accession No. 203518, contains a predicted open reading frame
encoding a protein of about 266 amino acid residues, a predicted
intracellular domain of about 46 amino acids (amino acid residues
from about 1 to about 46 in FIGS. 5A and 5B (SEQ ID NO:19)), a
predicted transmembrane domain of about 26 amino acids (underlined
amino acid residues from about 47 to about 72 in FIGS. 5A and 5B
(SEQ ID NO:19)), a predicted extracellular domain of about 194
amino acids (amino acid residues from about 73 to about 266 in
FIGS. 5A and 5B (SEQ ID NO:19)); and a deduced molecular weight of
about 29 kDa. The Neutrokine-alphaSV polypeptide shown in FIGS. 5A
and 5B (SEQ ID NO:19) is about 33.9% similar and about 22.0%
identical to human TNF-alpha which can be accessed on GenBank as
Accession No. 339764. As one of ordinary skill would appreciate,
due to the possibilities of sequencing errors discussed above, the
actual complete Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides encoded by the deposited cDNAs, which comprise about
285 and 266 amino acids, respectively, may be somewhat shorter. In
particular, the determined Neutrokine-alpha and Neutrokine-alphaSV
coding sequences contain a common second methionine codon which may
serve as an alternative start codon for translation of the open
reading frame, at nucleotide positions 210-212 in FIGS. 1A and 1B
(SEQ ID NO:1) and at nucleotide positions 64-66 in FIGS. 5A and 5B
(SEQ ID NO:18). More generally, the actual open reading frame may
be anywhere in the range of .+-.20 amino acids, more likely in the
range of .+-.10 amino acids, of that predicted from either the
first or second methionine codon from the N-terminus shown in FIGS.
1A and 1B (SEQ ID NO:1) and in FIGS. 5A and 5B (SEQ ID NO:18). It
will further be appreciated that, the polypeptide domains described
herein have been predicted by computer analysis, and accordingly,
that depending on the analytical criteria used for identifying
various functional domains, the exact "address" of the
extracellular, intracellular and transmembrane domains of the
Neutrokine-alpha and Neutrokine-alphaSV polypeptides may differ
slightly. For example, the exact location of the Neutrokine-alpha
and Neutrokine-alphaSV extracellular domains in FIGS. 1A and 1B
(SEQ ID NO:2) and FIGS. 5A and 5B (SEQ ID NO:19) may vary slightly
(e.g., the address may "shift" by about 1 to about 20 residues,
more likely about 1 to about 5 residues) depending on the criteria
used to define the domain. In this case, the ends of the
transmembrane domains and the beginning of the extracellular
domains were predicted on the basis of the identification of the
hydrophobic amino acid sequence in the above indicated positions,
as shown in FIGS. 3 and 6 and in Table I. In any event, as
discussed further below, the invention further provides
polypeptides having various residues deleted from the N-terminus
and/or C-terminus of the complete polypeptides, including
polypeptides lacking one or more amino acids from the N-termini of
the extracellular domains described herein, which constitute
soluble forms of the extracellular domains of the Neutrokine-alpha
and Neutrokine-alphaSV polypeptides.
[0104] As indicated, nucleic acid molecules and polynucleotides of
the present invention may be in the form of RNA, such as mRNA, or
in the form of DNA, including, for instance, cDNA and genomic DNA
obtained by cloning or produced synthetically. The DNA may be
double-stranded or single-stranded. Single-stranded DNA or RNA may
be the coding strand, also known as the sense strand, or it may be
the non-coding strand, also referred to as the anti-sense
strand.
[0105] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule (DNA or RNA), which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. However, a nucleic
acid contained in a clone that is a member of a library (e.g., a
genomic or cDNA library) that has not been isolated from other
members of the library (e.g., in the form of a homogeneous solution
containing the clone and other members of the library) or a
chromosome isolated or removed from a cell or a cell lysate (e.g.,
a "chromosome spread", as in a karyotype), is not "isolated" for
the purposes of this invention. As discussed further herein,
isolated nucleic acid molecules according to the present invention
may be produced naturally, recombinantly, or synthetically.
[0106] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising, or alternatively consisting of,
an open reading frame (ORF) with an initiation codon at positions
147-149 of the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID
NO:1). In addition, isolated nucleic acid molecules of the
invention include DNA molecules which comprise, or alternatively
consist of, a sequence substantially different from those described
above, but which due to the degeneracy of the genetic code, still
encodes the Neutrokine-alpha protein. Of course, the genetic code
is well known in the art. Thus, it would be routine for one skilled
in the art to generate the degenerate variants described above. In
another embodiment, the invention provides isolated nucleic acid
molecules comprising, or alternatively consisting of, a sequence
encoding the Neutrokine-alpha polypeptide having an amino acid
sequence encoded by the cDNA contained in the plasmid having ATCC
accession number 97768. Preferably, this nucleic acid molecule
comprises, or alternatively consists of a sequence encoding the
extracellular domain the mature or soluble polypeptide sequence of
the polypeptide encoded by the cDNA contained in the plasmid having
ATCC accession number 97768.
[0107] Isolated nucleic acid molecules of the present invention
also include DNA molecules comprising an open reading frame (ORF)
with an initiation codon at positions 1-3 of the nucleotide
sequence shown in FIGS. 5A and 5B (SEQ ID NO:18). In addition,
isolated nucleic acid molecules of the invention include DNA
molecules which comprise, or alternatively consist of, a sequence
substantially different from those described above, but which due
to the degeneracy of the genetic code, still encodes the
Neutrokine-alphaSV polypeptide. Of course, the genetic code is well
known in the art. Thus, it would be routine for one skilled in the
art to generate the degenerate variants described above. In another
embodiment, the invention provides isolated nucleic acid molecules
comprising, or alternatively consisting of, a sequence encoding the
Neutrokine-alphaSV polypeptide having an amino acid encoded by the
cDNA contained in the plasmid having ATCC accession number 203518.
Preferably, this nucleic acid molecule comprises, or alternatively
consists of, a sequence encoding the extracellular domain or the
mature soluble polypeptide sequence of the polypeptide encoded by
the cDNA contained in the plasmid having ATCC accession number
203518.
[0108] The invention further provides an isolated nucleic acid
molecule comprising, or alternatively consisting of, the nucleotide
sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) or the nucleotide
sequence of the Neutrokine-alpha cDNA contained in the plasmid
having ATCC accession number 97768, or a nucleic acid molecule
having a sequence complementary to one of the above sequences. In
addition, the invention provides an isolated nucleic acid molecule
comprising, or alternatively, consisting of, the nucleotide
sequence shown in FIGS. 5A and 5B (SEQ ID NO:18) or the nucleotide
sequence of the Neutrokine-alpha SV cDNA contained in the plasmid
having ATCC accession number 203518, or a nucleic acid molecule
having a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, have uses which
include, but are not limited to, as probes for gene mapping by in
situ hybridization with chromosomes, and for detecting expression
of the Neutrokine-alpha and Neutrokine-alphaSV in human tissue, for
instance, by Northern or Western blot analysis.
[0109] In one embodiment, the polynucleotides of the invention
comprise, or alternatively consist of, the sequence shown in SEQ ID
NO:22. The sequence provided as SEQ ID NO:22 was constructed from
several overlapping mouse EST sequences obtained from GenBank
(AI182472, AA422749, AA254047, and AI122485). The EST sequences
were aligned to generate the Neutrokine-alpha-like polynucleotide
sequence provided as SEQ ID NO:22. The amino acid sequence
resulting from the translation of SEQ ID NO:22 is provided as SEQ
ID NO:23. Fragments, variants, and derivatives of the sequences
provided as SEQ ID NO:22 and SEQ ID NO:23 are also encompassed by
the invention.
[0110] In another embodiment, the polynucleotides of the invention
comprise, or alternatively consist of, the sequence shown in SEQ ID
NO:27, and/or a sequence encoding the amino acid sequence disclosed
in SEQ ID NO:28, fragments, variants, and derivatives thereof.
These polynucleotides are also encompassed by the invention. For
example, certain embodiments of the invention are directed to
polynucleotides comprising, or alternatively consisting of, a
sequence encoding a polypeptide sequence that is at least 80%, 85%,
90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids
68-219 of SEQ ID NO:28. The amino acid sequence resulting from the
translation of SEQ ID NO:27 is provided as SEQ ID NO:28.
Polypeptides comprising, or alternatively consisting of, the amino
acid sequence of SEQ ID NO:28, and fragments, variants, and
derivatives of the sequence provided as SEQ ID NO:28 are also
encompassed by the invention. For example, certain embodiments of
the invention are directed to polypeptides comprising, or
alternatively consisting of, a polypeptide sequence that is at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to
amino acids 68-219 of SEQ ID NO:28. A nucleic acid molecule having
the sequence provided as SEQ ID NO:27 was obtained by RT-PCR from
cyanomologous monkey (i.e., Macaca irus) PBMC using two degenerate
primers. Briefly, total RNA was prepared from cyanomologous monkey
PBMC by using Trizol (available from Life Technologies, Inc.,
Rockville, Md.) according to the manufacturer's protocol. Then a
single stranded cDNA was synthesized from the cyanomologous monkey
PBMC preparation using standard methods with an oligo-dT primer.
Neutrokine-alpha-specific primers were designed based on the
conserved region between the mouse and human Neutrokine-alpha
molecules (SEQ ID NOs:22 and 1, respectively). A cyanomologous
monkey Neutrokine-alpha nucleic acid molecule was then generated by
PCR using the cDNA template in combination with the following two
degenerate oligonucleotide primers. 5' primer: 5'-TAC CAG ITG GCI
GCC ITG CAA G-3' (SEQ ID NO:35) and 3' primer: 5'-GTI ACA GCA GTT
TIA IIG CAC C-3' (SEQ ID NO:36). In the sequence of the degenerate
primers (SEQ ID NOs:35 and 36), "I" represents deoxyinosine or
dideoxyinosine.
[0111] In another embodiment, the polynucleotides of the invention
comprise, or alternatively consist of, the sequence shown in SEQ ID
NO:29, and/or a sequence encoding the amino acid sequence disclosed
in SEQ ID NO:30, fragments, variants, and derivatives thereof.
These polynucleotides are also encompassed by the invention. For
example, certain embodiments of the invention are directed to
polynucleotides comprising, or alternatively consisting of, a
sequence encoding a polypeptide sequence that is at least 80%, 85%,
90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids
68-219 of SEQ ID NO:30. The amino acid sequence resulting from the
translation of SEQ ID NO:29 is provided as SEQ ID NO:30.
Polypeptides comprising, or alternatively consisting of, the amino
acid sequence of SEQ ID NO:30, and fragments, variants, and
derivatives of the sequences provided as SEQ ID NO:29 and SEQ ID
NO:30 are also encompassed by the invention. For example, certain
embodiments of the invention are directed to polypeptides
comprising, or alternatively consisting of, a polypeptide sequence
that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%
identical to amino acids 68-219 of SEQ ID NO:30. A nucleic acid
molecule having the sequence provided as SEQ ID NO:29 was obtained
by RT-PCR from rhesus monkey PBMC using two degenerate primers.
Briefly, total RNA was prepared from rhesus monkey PBMC by using
Trizol (available from Life Technologies, Inc., Rockville, Md.)
according to the manufacturer's protocol. Then a single stranded
cDNA was synthesized from the rhesus monkey PBMC preparation using
standard methods with an oligo-dT primer. Neutrokine-alpha-specific
primers were designed based on the conserved region between the
mouse and human Neutrokine-alpha molecules (SEQ ID NOs:22 and 1,
respectively). A rhesus monkey Neutrokine-alpha nucleic acid
molecule was then generated by PCR using the cDNA template in
combination with the following two degenerate oligonucleotide
primers. 5' primer: 5'-TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID
NO:35) and 3' primer: 5'-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID
NO:36). In the sequence of the degenerate primers (SEQ ID NOs:35
and 36), "I" represents deoxyinosine or dideoxyinosine.
[0112] The invention also provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID NO:1
and SEQ ID NO:18 which have been determined from the following
related cDNA clones: HSOAD55 (SEQ ID NO:7), HSLAH84 (SEQ ID NO:8),
and HLTBM08 (SEQ ID NO:9).
[0113] The present invention is further directed to nucleic acid
molecules encoding portions of the nucleotide sequences described
herein, as well as to fragments of the isolated nucleic acid
molecules described herein. In one embodiment, the invention
provides a polynucleotide having a nucleotide sequence representing
the portion of SEQ ID NO:1 which consists of the nucleotides at
positions 1-1001 of SEQ ID NO:1. In another embodiment, the
invention provides a polynucleotide having a nucleotide sequence
representing the portion of SEQ ID NO:18 which consists of
positions 1-798 of SEQ ID NO: 18.
[0114] The present invention is further directed to fragments of
the nucleic acid molecules (i.e. polynucleotides) described herein.
By a fragment of a nucleic acid molecule having, for example, the
nucleotide sequence of the cDNA contained in the plasmid having
ATCC accession number 97768, a nucleotide sequence encoding the
polypeptide sequence encoded by the cDNA contained in the plasmid
having ATCC accession number 97768, the nucleotide sequence of SEQ
ID NO:1, a nucleotide sequence encoding the polypeptide sequence of
SEQ ID NO:2, the nucleotide sequence of the cDNA contained in the
plasmid having ATCC accession number 203518, a nucleotide sequence
encoding the polypeptide sequence encoded by the cDNA contained in
the plasmid having ATCC accession number 203518, the nucleotide
sequence of SEQ ID NO:18, a nucleotide sequence encoding the
polypeptide sequence of SEQ ID NO:19, or the complementary strand
thereto, is intended fragments at least 15 nt, and more preferably
at least 20 nt or at least 25 nt, still more preferably at least 30
nt, and even more preferably, at least 40, 50, 100, 150, 200, 250,
300, 325, 350, 375, 400, 450, or 500 nt in length. These fragments
have numerous uses which include, but are not limited to,
diagnostic probes and primers as discussed herein. Of course,
larger fragments, such as those of 501-1500 nt in length are also
useful according to the present invention as are fragments
corresponding to most, if not all, of the nucleotide sequences of
the cDNA contained in the plasmid having ATCC accession number
97768, the nucleotide sequence of SEQ ID NO:1, the nucleotide
sequences of the cDNA contained in the plasmid having ATCC
accession number 203518, and the nucleotide sequence of SEQ ID
NO:18. Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding polypeptides comprising, or
alternatively, consisting of, epitope-bearing portions of the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide as
identified in FIGS. 1A and 1B (SEQ ID NO:2) and in FIGS. 5A and 5B
(SEQ ID NO:19), respectively, and described in more detail below.
Polypeptides encoded by these polynucleotide fragments are also
encompassed by the invention.
[0115] Also by a fragment of a nucleic acid molecule having, for
example, the nucleotide sequence of SEQ ID NO:21, the nucleotide
sequence of SEQ ID NO:22, the nucleotide sequence of SEQ ID NO:27,
the nucleotide sequence of SEQ ID NO:29, the nucleotide sequence of
SEQ ID NO:37, a nucleotide sequence encoding the polypeptide
sequence of SEQ ID NO:23, a nucleotide sequence encoding the
polypeptide sequence of SEQ ID NO:28, a nucleotide sequence
encoding the polypeptide sequence of SEQ ID NO:30, a nucleotide
sequence encoding the polypeptide sequence of SEQ ID NO:38, a
nucleotide sequence encoding the polypeptide sequence of SEQ ID
NO:39, a nucleotide sequence encoding the polypeptide sequence of
SEQ ID NO:40, a nucleotide sequence encoding the polypeptide
sequence of SEQ ID NO:41, a nucleotide sequence encoding the
polypeptide sequence of SEQ ID NO:42, a nucleotide sequence
encoding the polypeptide sequence of SEQ ID NO:43, a nucleotide
sequence encoding the polypeptide sequence of SEQ ID NO:44, or the
complementary strands thereof, is intended fragments at least 15
nt, and more preferably at least 20 nt or at least 25 nt, still
more preferably at least 30 nt, and even more preferably, at least
40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450, or 500 nt
in length. These fragments have numerous uses which include, but
are not limited to, diagnostic probes and primers as discussed
herein. Of course, larger fragments, such as those of 501-1500 nt
in length are also useful according to the present invention as are
fragments corresponding to most, if not all, of the nucleotide
sequence of SEQ ID NO:21, the nucleotide sequence of SEQ ID NO:22,
the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of
SEQ ID NO:29, the nucleotide sequence of SEQ ID NO:37, a nucleotide
sequence encoding the polypeptide sequence of SEQ ID NO:23, a
nucleotide sequence encoding the polypeptide sequence of SEQ ID
NO:28, a nucleotide sequence encoding the polypeptide sequence of
SEQ ID NO:30 a nucleotide sequence encoding the polypeptide
sequence of SEQ ID NO:38, a nucleotide sequence encoding the
polypeptide sequence of SEQ ID NO:39, a nucleotide sequence
encoding the polypeptide sequence of SEQ ID NO:40, a nucleotide
sequence encoding the polypeptide sequence of SEQ ID NO:41, a
nucleotide sequence encoding the polypeptide sequence of SEQ ID
NO:42, a nucleotide sequence encoding the polypeptide sequence of
SEQ ID NO:43, a nucleotide sequence encoding the polypeptide
sequence of SEQ ID NO:44, or the complementary strands thereof.
Polypeptides encoded by these polynucleotide fragments are also
encompassed by the invention.
[0116] Representative examples of Neutrokine-alpha polynucleotide
fragments of the invention include, for example, fragments that
comprise, or alternatively, consist of, a sequence from about
nucleotide 1 to 50, 51 to 100, 101 to 146, 147 to 200, 201 to 250,
251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to
550, 551 to 600, 600 to 650, 651 to 700, 701 to 750, 751 to 800,
800 to 850, 851 to 900, 901 to 950, 951 to 1000, 1001 to 1050,
and/or 1051 to 1082, of SEQ ID NO:1, or the complementary strand
thereto, or the cDNA contained in the plasmid having ATCC accession
number 97768. In this context "about" includes the particularly
recited ranges, and ranges that are larger or smaller by several
(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both
termini.
[0117] Representative examples of Neutrokine-alphaSV polynucleotide
fragments of the invention include, for example, fragments that
comprise, or alternatively, consist of, a sequence from about
nucleotide 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250,
251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to
550, 551 to 600, 600 to 650, 651 to 700, 701 to 750, 751 to 800,
800 to 850, and/or 851 to 900 of SEQ ID NO:18, or the complementary
strand thereto, or the cDNA contained in the plasmid having ATCC
accession number 203518. In this context "about" includes the
particularly recited ranges, and ranges that are larger or smaller
by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at
both termini.
[0118] In certain preferred embodiments, polynucleotide of the
invention comprise, or alternatively, consist of, nucleotide
residues 571-627, 580-627, 590-627, 600-627, 610-627, 571-620,
580-620, 590-620, 600-620, 571-610, 580-610, 590-610, 571-600,
580-600, and/or 571-590 of SEQ ID NO:1.
[0119] In certain other preferred embodiments, polynucleotides of
the invention comprise, or alternatively, consist of nucleotide
residues 1-879, 25-879, 50-879, 75-879, 100-879, 125-879, 150-879,
175-879, 200-879, 225-879, 250-879, 275-879, 300-879, 325-879,
350-879, 375-879, 400-879, 425-879, 450-879, 475-879, 500-879,
525-879, 550-879, 575-879, 600-879, 625-879, 650-879, 675-879,
700-879, 725-879, 750-879, 775-879, 800-879, 825-879, 850-879,
1-850, 25-850, 50-850, 75-850, 100-850, 125-850, 150-850, 175-850,
200-850, 225-850, 250-850, 275-850, 300-850, 325-850, 350-850,
375-850, 400-850, 425-850, 450-850, 475-850, 500-850, 525-850,
550-850, 575-850, 600-850, 625-850, 650-850, 675-850, 700-850,
725-850, 750-850, 775-850, 800-850, 825-850, 1-825, 25-825, 50-825,
75-825, 100-825, 125-825, 150-825, 175-825, 200-825, 225-825,
250-825, 275-825, 300-825, 325-825, 350-825, 375-825, 400-825,
425-825, 450-825, 475-825, 500-825, 525-825, 550-825, 575-825,
600-825, 625-825, 650-825, 675-825, 700-825, 725-825, 750-825,
775-825, 800-825, 1-800, 25-800, 50-800, 75-800, 100-800, 125-800,
150-800, 175-800, 200-800, 225-800, 250-800, 275-800, 300-800,
325-800, 350-800, 375-800, 400-800, 425-800, 450-800, 475-800,
500-800, 525-800, 550-800, 575-800, 600-800, 625-800, 650-800,
675-800, 700-800, 725-800, 750-800, 775-800, 1-775, 25-775, 50-775,
75-775, 100-775, 125-775, 150-775, 175-775, 200-775, 225-775,
250-775, 275-775, 300-775, 325-775, 350-775, 375-775, 400-775,
425-775, 450-775, 475-775, 500-775, 525-775, 550-775, 575-775,
600-775, 625-775, 650-775, 675-775, 700-775, 725-775, 750-775,
1-750, 25-750, 50-750, 75-750, 100-750, 125-750, 150-750, 175-750,
200-750, 225-750, 250-750, 275-750, 300-750, 325-750, 350-750,
375-750, 400-750, 425-750, 450-750, 475-750, 500-750, 525-750,
550-750, 575-750, 600-750, 625-750, 650-750, 675-750, 700-750,
725-750, 1-725, 25-725, 50-725, 75-725, 100-725, 125-725, 150-725,
175-725, 200-725, 225-725, 250-725, 275-725, 300-725, 325-725,
350-725, 375-725, 400-725, 425-725, 450-725, 475-725, 500-725,
525-725, 550-725, 575-725, 600-725, 625-725, 650-725, 675-725,
700-725, 1-700, 25-700, 50-700, 75-700, 100-700, 125-700, 150-700,
175-700, 200-700, 225-700, 250-700, 275-700, 300-700, 325-700,
350-700, 375-700, 400-700, 425-700, 450-700, 475-700, 500-700,
525-700, 550-700, 575-700, 600-700, 625-700, 650-700, 675-700,
1-675, 25-675, 50-675, 75-675, 100-675, 125-675, 150-675, 175-675,
200-675, 225-675, 250-675, 275-675, 300-675, 325-675, 350-675,
375-675, 400-675, 425-675, 450-675, 475-675, 500-675, 525-675,
550-675, 575-675, 600-675, 625-675, 650-675, 1-650, 25-650, 50-650,
75-650, 100-650, 125-650, 150-650, 175-650, 200-650, 225-650,
250-650, 275-650, 300-650, 325-650, 350-650, 375-650, 400-650,
425-650, 450-650, 475-650, 500-650, 525-650, 550-650, 575-650,
600-650, 625-650, 1-625, 25-625, 50-625, 75-625, 100-625, 125-625,
150-625, 175-625, 200-625, 225-625, 250-625, 275-625, 300-625,
325-625, 350-625, 375-625, 400-625, 425-625, 450-625, 475-625,
500-625, 525-625, 550-625, 575-625, 600-625, 1-600, 25-600, 50-600,
75-600, 100-600, 125-600, 150-600, 175-600, 200-600, 225-600,
250-600, 275-600, 300-600, 325-600, 350-600, 375-600, 400-600,
425-600, 450-600, 475-600, 500-600, 525-600, 550-600, 575-600,
1-575, 25-575, 50-575, 75-575, 100-575, 125-575, 150-575, 175-575,
200-575, 225-575, 250-575, 275-575, 300-575, 325-575, 350-575,
375-575, 400-575, 425-575, 450-575, 475-575, 500-575, 525-575,
550-575, 1-550, 25-550, 50-550, 75-550, 100-550, 125-550, 150-550,
175-550, 200-550, 225-550, 250-550, 275-550, 300-550, 325-550,
350-550, 375-550, 400-550, 425-550, 450-550, 475-550, 500-550,
525-550, 1-525, 25-525, 50-525, 75-525, 100-525, 125-525, 150-525,
175-525, 200-525, 225-525, 250-525, 275-525, 300-525, 325-525,
350-525, 375-525, 400-525, 425-525, 450-525, 475-525, 500-525,
1-500, 25-500, 50-500, 75-500, 100-500, 125-500, 150-500, 175-500,
200-500, 225-500, 250-500, 275-500, 300-500, 325-500, 350-500,
375-500, 400-500, 425-500, 450-500, 475-500, 1-475, 25-475, 50-475,
75-475, 100-475, 125-475, 150-475, 175-475, 200-475, 225-475,
250-475, 275-475, 300-475, 325-475, 350-475, 375-475, 400-475,
425-475, 450-475, 1-450, 25-450, 50-450, 75-450, 100-450, 125-450,
150-450, 175-450, 200-450, 225-450, 250-450, 275-450, 300-450,
325-450, 350-450, 375-450, 400-450, 425-450, 1-425, 25-425, 50-425,
75-425, 100-425, 125-425, 150-425, 175-425, 200-425, 225-425,
250-425, 275-425, 300-425, 325-425, 350-425, 375-425, 400-425,
1-400, 25-400, 50-400, 75-400, 100-400, 125-400, 150-400, 175-400,
200-400, 225-400, 250-400, 275-400, 300-400, 325-400, 350-400,
375-400, 1-375, 25-375, 50-375, 75-375, 100-375, 125-375, 150-375,
175-375, 200-375, 225-375, 250-375, 275-375, 300-375, 325-375,
350-375, 1-350, 25-350, 50-350, 75-350, 100-350, 125-350, 150-350,
175-350, 200-350, 225-350, 250-350, 275-350, 300-350, 325-350,
1-325, 25-325, 50-325, 75-325, 100-325, 125-325, 150-325, 175-325,
200-325, 225-325, 250-325, 275-325, 300-325, 1-300, 25-300, 50-300,
75-300, 100-300, 125-300, 150-300, 175-300, 200-300, 225-300,
250-300, 275-300, 1-275, 25-275, 50-275, 75-275, 100-275, 125-275,
150-275, 175-275, 200-275, 225-275, 250-275, 1-250, 25-250, 50-250,
75-250, 100-250, 125-250, 150-250, 175-250, 200-250, 225-250,
1-225, 25-225, 50-225, 75-225, 100-225, 125-225, 150-225, 175-225,
200-225, 1-200, 25-200, 50-200, 75-200, 100-200, 125-200, 150-200,
175-200, 1-175, 25-175, 50-175, 75-175, 100-175, 125-175, 150-175,
1-150, 25-150, 50-150, 75-150, 100-150, 125-150, 1-125, 25-125,
50-125, 75-125, 100-125, 1-100, 25-100, 50-100, 75-100, 1-75,
25-75, 50-75, 1-50, 25-50, and/or 1-25 of SEQ ID NO:18.
[0120] In certain additional preferred embodiments, polynucleotides
of the invention comprise, or alternatively, consist of nucleotide
residues 400-627, 425-627, 450-627, 475-627, 500-627, 525-627,
550-627, 575-627, 600-627, 400-600, 425-600, 450-600, 475-600,
500-600, 525-600, 550-600, 575-600, 400-575, 425-575, 450-575,
475-575, 500-575, 525-575, 550-575, 400-550, 425-550, 450-550,
475-550, 500-550, 525-550, 400-500, 425-500, 450-500, 475-500,
400-475, 425-475, 450-475, 400-450, 425-450, 571-800, 600-800,
625-800, 650-800, 675-800, 700-800, 725-800, 750-800, 775-800,
571-775, 600-775, 625-775, 650-775, 675-775, 700-775, 725-775,
750-775, 571-750, 600-750, 625-750, 650-750, 675-750, 700-750,
725-750, 571-725, 600-725, 625-725, 650-725, 675-725, 700-725,
571-700, 600-700, 625-700, 650-700, 675-700, 571-675, 600-675,
625-675, 650-675, 571-650, 600-650, 625-650, 571-625, 600-625,
and/or 571-600 of SEQ ID NO:1.
[0121] In additional preferred embodiments, polynucleotides of the
invention comprise, or alternatively, consist of nucleotide
residues 147-500, 147-450, 147-400, 147-350, 200-500, 200-450,
200-400, 200-350, 250-500, 250-450, 250-400, 250-350, 300-500,
300-450, 300-400, 300-350, 350-750, 350-700, 350-650, 350-600,
350-550, 400-750, 400-700, 400-650, 400-600, 400-550, 425-750,
425-700, 425-650, 425-600, 425-550, 450-1020, 450-1001, 450-950,
450-900, 450-850, 450-800, 450-775, 500-1001, 500-950, 500-900,
500-850, 500-800, 500-775, 550-1001, 550-950, 550-900, 550-850,
550-800, 550-775, 600-1001, 600-950, 600-900, 600-850, 600-800,
600-775, 650-1001, 650-950, 650-900, 650-850, 650-800, 650-775,
700-1001, 700-950, 700-900, 700-850, 700-800, 700-775, 825-1082,
850-1082, 875-1082, 900-1082, 925-1082, 950-1082, 975-1082,
1000-1082, 1025-1082, and/or 1050-1082 of SEQ ID NO:1.
[0122] Preferably, the polynucleotide fragments of the invention
encode a polypeptide which demonstrates a Neutrokine-alpha and/or
Neutrokine-alphaSV functional activity. By a polypeptide
demonstrating "functional activity" is meant, a polypeptide capable
of displaying one or more known functional activities associated
with a full-length and/or secreted Neutrokine-alpha polypeptide
and/or Neutrokine-alphaSV polypeptide. Such functional activities
include, but are not limited to, biological activity (e.g., ability
to stimulate B cell proliferation, survival, differentiation,
and/or activation), antigenicity (ability to bind or compete with a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide for binding
to an anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibody], immunogenicity (ability to generate antibody which binds
to a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide),
ability to form multimers (as described below in the
"Neutrokine-alpha Polypeptides" section) with Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides of the invention, ability to
form heteromultimers (as described below in the "Neutrokine-alpha
Polypeptides" section) with APRIL polypeptides (e.g., SEQ ID NO:20
or SEQ ID NO:47; PCT International Publication Number WO97/33902;
GenBank Accession No. AF046888 (nucleotide) and AAC6132 (protein);
J. Exp. Med. 188(6):1185-1190), ability to bind to a receptor or
ligand (e.g., transmembrane activator and CAML interactor (TACI,
GenBank accession number AAC51790), BAFF-R (GenBank Acession Number
NP 443177) and B-cell maturation antigen (BCMA, GenBank accession
number NP.sub.--001183)) for a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide, and ability to stimulate a
Neutrokine-alpha and/or Neutrokine-alphaSV receptor signalling
cascade (e.g., to activate calcium-modulator and cyclophilin ligand
("CAML"), calcineurin, nuclear factor of activated T cells
transcription factor ("NF-AT"), nuclear factor-kappa B ("NF-kappa
B"), activator protein-1 (AP-1), SRF, extracellular-signal
regulated kinase 1 (ERK-1), polo like kinases (PLK), ELF-1, high
mobility group I (HMG-I), and/or high mobility group Y
(HMG-Y)).
[0123] In additional specific embodiments, the polynucleotide
fragments of the invention encode a polypeptide comprising, or
alternatively, consisting of the predicted intracellular domain
(amino acids 1 to 46 of SEQ ID NO:2), the predicted transmembrane
domain (amino acids 47 to 72 of SEQ ID NO:2), the predicted
extracellular domain (amino acids 73 to 285 of SEQ ID NO:2), or the
predicted TNF conserved domain (amino acids 191 to 284 of SEQ ID
NO:2) of Neutrokine-alpha. In additional embodiments, the
polynucleotide fragments of the invention encode a polypeptide
comprising, or alternatively, consisting of any combination of 1,
2, 3, or all 4 of the above recited domains. Polypeptides encoded
by these polynucleotides are also encompassed by the invention.
[0124] In additional specific embodiments, the polynucleotide
fragments of the invention encode a polypeptide comprising, or
alternatively, consisting of the predicted intracellular domain
(amino acids 1 to 46 of SEQ ID NO:19), the predicted transmembrane
domain (amino acids 47 to 72 of SEQ ID NO:19), the predicted
extracellular domain (amino acids 73 to 266 of SEQ ID NO:19), or
the predicted TNF conserved domain (amino acids 172 to 265 of SEQ
ID NO:19) of Neutrokine-alphaSV. In additional embodiments, the
polynucleotide fragments of the invention encode a polypeptide
comprising, or alternatively, consisting of any combination of 1,
2, 3, or all 4 of the above recited domains. Polypeptides encoded
by these polynucleotides are also encompassed by the invention.
[0125] In another embodiment, polynucleotide fragments of the
invention comprise, or alternatively consist of, polynucleotides
which encode an amino acid sequence selected from residues Met-1 to
Lys-113, Leu-114 to Thr-141, Ile-142 to Lys-160, Gly-161 to
Gln-198, Val-199 to Ala-248, and Gly-250 to Leu-285 of SEQ ID NO:2.
Moreover, polynucleotides that encode any combination of two,
three, four, five or more of these amino acid sequences are also
encompassed by the invention. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0126] In another embodiment, polynucleotide fragments of the
invention comprise, or alternatively consist of, polynucleotides
which encode an amino acid sequence selected from residues Met-1 to
Lys 113, Leu-114 to Thr-141, Gly-142 to Gln-179, Val-180 to
Ala-229, and Gly-230 to Leu-266 of SEQ ID NO:19. Moreover,
polynucleotides that encode any combination of two, three, four,
five or more of these amino acid sequences are also encompassed by
the invention. Polypeptides encoded by these polynucleotides are
also encompassed by the invention.
[0127] In another embodiment, polynucleotide fragments of the
invention comprise, or alternatively consist of, polynucleotides
which encode an amino acid sequence selected from residues Met-1 to
Lys-106, Leu-107 to Thr-134, Glu-135 to Asn-165, Ile-167 to
Lys-184, Gly-185 to Gln-224, Val-225 to Ala-272, and Gly-273 to
Leu-309 of SEQ ID NO:39. Moreover, polynucleotides that encode any
combination of two, three, four, five or more of these amino acid
sequences are also encompassed by the invention. Polypeptides
encoded by these polynucleotides are also encompassed by the
invention.
[0128] In another embodiment, polynucleotide fragments of the
invention comprise, or alternatively consist of, polynucleotides
which encode an amino acid sequence selected from residues Tyr-1 to
Lys-47, Leu-48 to Thr-75, Ile-76 to Lys-94, Gly-95 to Gln-132,
Val-133 to Ala-182, and Gly-183 to Ala-219 of SEQ ID NO:28.
Moreover, polynucleotides that encode any combination of two,
three, four, five or more of these amino acid sequences are also
encompassed by the invention. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0129] In another embodiment, polynucleotide fragments of the
invention comprise, or alternatively consist of, polynucleotides
which encode an amino acid sequence selected from residues Tyr-1 to
Lys-47, Leu-48 to Thr-75, Ile-76 to Lys-94, Gly-95 to Gln-132,
Val-133 to Ala-182, and Gly-183 to Ala-219 of SEQ ID NO:30.
Moreover, polynucleotides that encode any combination of two,
three, four, five or more of these amino acid sequences are also
encompassed by the invention. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0130] In another embodiment, the polynucleotides of the invention
comprise, or alternatively consist of, the sequence shown in SEQ ID
NO:21. The sequence shown as SEQ ID NO:21 encodes a polypeptide
consisting of an initiating methionine residue linked to residues
Ala-134 through Leu-285 of the Neutrokine-alpha polypeptide
sequence shown as SEQ ID NO:2. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0131] In certain additional preferred embodiments, polynucleotides
of the invention comprise, or alternatively, consist of nucleotide
residues 1-459, 15-459, 30-459, 45-459, 60-459, 75-459, 90-459,
105-459, 120-459, 135-459, 150-459, 165-459, 180-459, 195-459,
210-459, 225-459, 240-459, 255-459, 270-459, 285-459, 300-459,
315-459, 330-459, 345-459, 360-459, 375-459, 390-459, 405-459,
420-459, 435-459, 450-459, 1-450, 15-450, 30-450, 45-450, 60-450,
75-450, 90-450, 105-450, 120-450, 135-450, 150-450, 165-450,
180-450, 195-450, 210-450, 225-450, 240-450, 255-450, 270-450,
285-450, 300-450, 315-450, 330-450, 345-450, 360-450, 375-450,
390-450, 405-450, 420-450, 435-450, 1-435, 15-435, 30-435, 45-435,
60-435, 75-435, 90-435, 105-435, 120-435, 135-435, 150-435,
165-435, 180-435, 195-435, 210-435, 225-435, 240-435, 255-435,
270-435, 285-435, 300-435, 315-435, 330-435, 345-435, 360-435,
375-435, 390-435, 405-435, 420-435, 1-420, 15-420, 30-420, 45-420,
60-420, 75-420, 90-420, 105-420, 120-420, 135-420, 150-420,
165-420, 180-420, 195-420, 210-420, 225-420, 240-420, 255-420,
270-420, 285-420, 300-420, 315-420, 330-420, 345-420, 360-420,
375-420, 390-420, 405-420, 1-405, 15-405, 30-405, 45-405, 60-405,
75-405, 90-405, 105-405, 120-405, 135-405, 150-405, 165-405,
180-405, 195-405, 210-405, 225-405, 240-405, 255-405, 270-405,
285-405, 300-405, 315-405, 330-405, 345-405, 360-405, 375-405,
390-405, 1-390, 15-390, 30-390, 45-390, 60-390, 75-390, 90-390,
105-390, 120-390, 135-390, 150-390, 165-390, 180-390, 195-390,
210-390, 225-390, 240-390, 255-390, 270-390, 285-390, 300-390,
315-390, 330-390, 345-390, 360-390, 375-390, 1-375, 15-375, 30-375,
45-375, 60-375, 75-375, 90-375, 105-375, 120-375, 135-375, 150-375,
165-375, 180-375, 195-375, 210-375, 225-375, 240-375, 255-375,
270-375, 285-375, 300-375, 315-375, 330-375, 345-375, 360-375,
1-360, 15-360, 30-360, 45-360, 60-360, 75-360, 90-360, 105-360,
120-360, 135-360, 150-360, 165-360, 180-360, 195-360, 210-360,
225-360, 240-360, 255-360, 270-360, 285-360, 300-360, 315-360,
330-360, 345-360, 1-345, 15-345, 30-345, 45-345, 60-345, 75-345,
90-345, 105-345, 120-345, 135-345, 150-345, 165-345, 180-345,
195-345, 210-345, 225-345, 240-345, 255-345, 270-345, 285-345,
300-345, 315-345, 330-345, 1-330, 15-330, 30-330, 45-330, 60-330,
75-330, 90-330, 105-330, 120-330, 135-330, 150-330, 165-330,
180-330, 195-330, 210-330, 225-330, 240-330, 255-330, 270-330,
285-330, 300-330, 315-330, 1-315, 15-315, 30-315, 45-315, 60-315,
75-315, 90-315, 105-315, 120-315, 135-315, 150-315, 165-315,
180-315, 195-315, 210-315, 225-315, 240-315, 255-315, 270-315,
285-315, 300-315, 1-300, 15-300, 30-300, 45-300, 60-300, 75-300,
90-300, 105-300, 120-300, 135-300, 150-300, 165-300, 180-300,
195-300, 210-300, 225-300, 240-300, 255-300, 270-300, 285-300,
1-285, 15-285, 30-285, 45-285, 60-285, 75-285, 90-285, 105-285,
120-285, 135-285, 150-285, 165-285, 180-285, 195-285, 210-285,
225-285, 240-285, 255-285, 270-285, 1-270, 15-270, 30-270, 45-270,
60-270, 75-270, 90-270, 105-270, 120-270, 135-270, 150-270,
165-270, 180-270, 195-270, 210-270, 225-270, 240-270, 255-270,
1-255, 15-255, 30-255, 45-255, 60-255, 75-255, 90-255, 105-255,
120-255, 135-255, 150-255, 165-255, 180-255, 195-255, 210-255,
225-255, 240-255, 1-240, 15-240, 30-240, 45-240, 60-240, 75-240,
90-240, 105-240, 120-240, 135-240, 150-240, 165-240, 180-240,
195-240, 210-240, 225-240, 1-225, 15-225, 30-225, 45-225, 60-225,
75-225, 90-225, 105-225, 120-225, 135-225, 150-225, 165-225,
180-225, 195-225, 210-225, 1-210, 15-210, 30-210, 45-210, 60-210,
75-210, 90-210, 105-210, 120-210, 135-210, 150-210, 165-210,
180-210, 195-210, 1-195, 15-195, 30-195, 45-195, 60-195, 75-195,
90-195, 105-195, 120-195, 135-195, 150-195, 165-195, 180-195,
1-180, 15-180, 30-180, 45-180, 60-180, 75-180, 90-180, 105-180,
120-180, 135-180, 150-180, 165-180, 1-165, 15-165, 30-165, 45-165,
60-165, 75-165, 90-165, 105-165, 120-165, 135-165, 150-165, 1-150,
15-150, 30-150, 45-150, 60-150, 75-150, 90-150, 105-150, 120-150,
135-150, 1-135, 15-135, 30-135, 45-135, 60-135, 75-135, 90-135,
105-135, 120-135, 1-120, 15-120, 30-120, 45-120, 60-120, 75-120,
90-120, 105-120, 1-105, 15-105, 30-105, 45-105, 60-105, 75-105,
90-105, 1-90, 15-90, 30-90, 45-90, 60-90, 75-90, 1-75, 15-75,
30-75, 45-75, 60-75, 1-60, 15-60, 30-60, 45-60, 1-45, 15-45, 30-45,
1-30, and/or 15-30 of SEQ ID NO:21. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0132] Accordingly, specific embodiments of the invention are
directed to polynucleotides encoding polypeptides which comprise,
or alternatively consist of, the amino acid sequence of beta
pleated sheet region A, A', B, B', C, D, E, F, G, or H disclosed in
FIGS. 7A-1 and 7A-2 and described in Example 6. Additional
embodiments of the invention are directed to polynucleotides
encoding Neutrokine-alpha polypeptides which comprise, or
alternatively consist of, any combination of 1, 2, 3, 4, 5, 6, 7,
8, 9 or all 10 of beta pleated sheet regions A-H disclosed in FIGS.
7A-1 and 7A-2 and described in Example 6. Additional preferred
embodiments of the invention are directed to polypeptides which
comprise, or alternatively consist of, the Neutrokine-alpha amino
acid sequence of beta pleated sheet region A, A', B, B', C, D, E,
F, G, or H disclosed in FIGS. 7A-1 and 7A-2 and described in
Example 6. Additional embodiments of the invention are directed
Neutrokine-alpha polypeptides which comprise, or alternatively
consist of, any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10
of beta pleated sheet regions A through H disclosed in FIGS. 7A-1
and 7A-2 and described in Example 6.
[0133] In certain other preferred embodiments, polynucleotides of
the invention comprise, or alternatively consist of, nucleotide
residues 34-57, 118-123, 133-141, 151-159, 175-216, 232-255,
280-315, 328-357, 370-393, and/or 430-456 of SEQ ID NO:21.
Polypeptides encoded by these polynucleotides are also encompassed
by the invention. These polynucleotide and polypeptide fragments
correspond to the predicted beta-pleated sheet regions shown in
FIGS. 7A-1 and 7A-2. In certain embodiments, polynucleotides of the
invention comprise, or alternatively consist of, a polynucleotide
sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
polynucleotide sequence encoding one, two, three, four, five, six,
seven, eight, nine or ten of the beta-pleated sheet regions
described above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these polynucleotide sequences
are also encompassed by the invention. In another embodiment, the
invention provides an isolated nucleic acid molecule comprising a
polynucleotide which hybridizes under stringent hybridization
conditions to one, two, three, four, five, six, seven, eight, nine
or ten of the beta-pleated sheet polynucleotides of the invention
described above. The meaning of the phrase "stringent conditions"
as used herein is described infra.
[0134] In further preferred embodiments, polynucleotides of the
invention comprise, or alternatively consist of, nucleotide
residues 576-599, 660-665, 675-683, 693-701, 717-758, 774-803,
822-857, 870-899, 912-935, and/or 972-998 of SEQ ID NO:1.
Polypeptides encoded by these polynucleotide fragments are also
encompassed by the invention. These polynucleotide and polypeptide
fragments correspond to the predicted beta-pleated sheet regions
shown in FIGS. 7A-1 and 7A-2.
[0135] In additional preferred embodiments, polynucleotides of the
invention comprise, or alternatively consist of, nucleotide
residues 457-462, 472-480, 490-498, 514-555, 571-600, 619-654,
667-696, 699-732, and/or 769-795 of SEQ ID NO:18. Polypeptides
encoded by these polynucleotide fragments are also encompassed by
the invention. These polynucleotide and polypeptide fragments
correspond to the predicted beta-pleated sheet regions shown in
FIGS. 7A-1 and 7A-2.
[0136] In yet further preferred embodiments, polynucleotides of the
invention comprise, or alternatively consist of, nucleotide
residues 124-129, 139-147, 157-165, 181-222, 238-267, 286-321,
334-363, 376-399, and/or 436-462 of SEQ ID NO:22. Polypeptides
encoded by these polynucleotide fragments are also encompassed by
the invention. These polynucleotide and polypeptide fragments
correspond to the predicted beta-pleated sheet regions shown in
FIGS. 7A-1 and 7A-2. Polypeptides comprising, or alternatively,
consisting of the amino acid sequence of any combination of one,
two, three, four, five, six, seven, eight, nine, ten, or all of
these regions are encompassed by the invention.
[0137] The relative positions of several intron/exon boundaries
were determined for the mouse Neutrokine-alpha (SEQ ID NO:39) based
on sequence analysis of mouse genomic DNA. The apparent second exon
from the 5' end of the mouse Neutrokine-alpha genomic clone
(preliminarily designated "Exon 2") consists of Tyr-187 to Gln-222
of the sequence shown in SEQ ID NO:39. The apparent third exon from
the 5' end of the mouse Neutrokine-alpha genomic clone
(preliminarily designated "Exon 3") comprises Val-223 to Gly-273 of
the sequence shown in SEQ ID NO:39.
[0138] Thus, in one embodiment, the invention provides
polynucleotides encoding polypeptides comprising, or alternatively
consisting of, the amino acid sequence of residues Tyr-187 to
Gln-222 of SEQ ID NO:39. The present invention is also directed to
nucleic acid molecules comprising, or alternatively, consisting of,
a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to the polynucleotide sequence encoding the
mouse Neutrokine-alpha polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention.
[0139] In another embodiment, the invention provides
polynucleotides encoding polypeptides comprising, or alternatively
consisting of, the amino acid sequence of residues Val-223 to
Gly-273 of SEQ ID NO:39. The present invention is also directed to
nucleic acid molecules comprising, or alternatively, consisting of,
a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to the polynucleotide sequence encoding the
mouse Neutrokine-alpha polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention.
[0140] Moreover, the relative positions of the corresponding
intron/exon boundaries were determined for human Neutrokine-alpha
(SEQ ID NO:1 and SEQ ID NO:2) based on an alignment of the
sequences of mouse and human Neutrokine-alpha polypeptides. The
apparent second exon from the 5' end of human Neutrokine-alpha
(also preliminarily designated "Exon 2") consists of, Tyr-163 to
Gln-198 of the sequence shown in SEQ ID NO:2. The apparent third
exon from the 5' end of human Neutrokine-alpha (also preliminarily
designated "Exon 3") consists of, Val-199 to Gly-249 of the
sequence shown in SEQ ID NO:2.
[0141] Thus, in one embodiment, the invention provides
polynucleotides encoding polypeptides comprising, or alternatively
consisting of, the amino acid sequence of residues Tyr-163 to
Gln-198 of SEQ ID NO:2. The present invention is also directed to
nucleic acid molecules comprising, or alternatively, consisting of,
a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to the polynucleotide sequence encoding the
Neutrokine-alpha polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention.
[0142] In another embodiment, the invention provides
polynucleotides encoding polypeptides comprising, or alternatively
consisting of, the amino acid sequence of residues Val-199 to
Gly-249 of SEQ ID NO:2. The present invention is also directed to
nucleic acid molecules comprising, or alternatively, consisting of,
a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to the polynucleotide sequence encoding the
Neutrokine-alpha polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention. The functional activity of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides, and
fragments, variants derivatives, and analogs thereof, can be
assayed by various methods as described herein and as are well
known in the art.
[0143] For example, in one embodiment where one is assaying for the
ability to bind or compete with full-length Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide for binding to anti-Neutrokine-alpha
and/or anti-Neutrokine-alphaSV antibody or binding to
Neutrokine-alpha receptor(s) and/or Neutrokine-alphaSV receptor(s)
on B cells, various immunoassays known in the art can be used,
including but not limited to, competitive and non-competitive assay
systems using techniques such as radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation
assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present
invention.
[0144] In another embodiment, where a Neutrokine-alpha and/or
Neutrokine-alphaSV ligand is identified, or the ability of a
polypeptide fragment, variant or derivative of the invention to
multimerize is being evaluated, binding can be assayed, e.g., by
means well-known in the art, such as, for example, reducing and
non-reducing gel chromatography, protein affinity chromatography,
and affinity blotting. See generally, Phizicky, E., et al., 1995,
Microbiol. Rev. 59:94-123. In another embodiment, physiological
correlates of Neutrokine-alpha and/or Neutrokine-alphaSV binding to
its substrates (signal transduction) can be assayed.
[0145] In addition, assays described herein (see e,g., Examples 6
and 7) and otherwise known in the art may routinely be applied to
measure the ability of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides and fragments, variants derivatives and analogs
thereof to elicit Neutrokine-alpha and/or Neutrokine-alphaSV
related biological activity (e.g., to stimulate, or alternatively
to inhibit (in the case of Neutrokine-alpha and/or
Neutrokine-alphaSV antagonists) signalling mediated by
Neutrokine-alpha and/or Neutrokine-alphaSV; to stimulate, or
alternatively to inhibit B cell proliferation, differentiation
and/or activation; and/or to increase or decrease B cell survival
in vitro or in vivo).
[0146] Other methods will be known to the skilled artisan and are
within the scope of the invention.
[0147] In additional embodiments, the polynucleotides of the
invention encode polypeptides comprising, or alternatively
consisting of, functional attributes of Neutrokine-alpha and
Neutrokine-alphaSV. Preferred embodiments of the invention in this
regard include fragments that comprise, or alternatively consist
of, alpha-helix and alpha-helix forming regions ("alpha-regions"),
beta-sheet and beta-sheet forming regions ("beta-regions"), turn
and turn-forming regions ("turn-regions"), coil and coil-forming
regions ("coil-regions"), hydrophilic regions, hydrophobic regions,
alpha amphipathic regions, beta amphipathic regions, flexible
regions, surface-forming regions and high antigenic index regions
of Neutrokine-alpha and Neutrokine-alphaSV polypeptides.
[0148] It is believed one or more of the beta pleated sheet regions
of Neutrokine-alpha disclosed in FIGS. 7A-1 and 7A-2 is important
for dimerization and also for interactions between Neutrokine-alpha
and its ligands.
[0149] Certain preferred regions in this regard are set out in FIG.
3 (Table I). The data presented in FIG. 3 and that presented in
Table I, merely present a different format of the same results
obtained when the amino acid sequence of SEQ ID NO:2 is analyzed
using the default parameters of the DNA*STAR computer
algorithm.
[0150] The above-mentioned preferred regions set out in FIG. 3 and
in Table I include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequence set out in FIGS. 1A and 1B. As set out in FIG. 3 and in
Table I, such preferred regions include Garnier-Robson
alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman alpha-regions, beta-regions, and coil-regions,
Kyte-Doolittle hydrophilic regions and hydrophobic regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz
flexible regions, Emini surface-forming regions and Jameson-Wolf
regions of high antigenic index. Among highly preferred
polynucleotides in this regard are those that encode polypeptides
comprising, or alternatively consisting of, regions of
Neutrokine-alpha and/or Neutrokine-alphaSV that combine several
structural features, such as several (e.g., 1, 2, 3 or 4) of the
features set out above. Polypeptides encoded by the polynucleotides
are also encompassed by the invention.
[0151] Additionally, the data presented in columns VIII, IX, XIII,
and XIV of Table I can routinely be used to determine regions of
Neutrokine-alpha which exhibit a high degree of potential for
antigenicity (column VIII of Table I represents hydrophilicity
according to Kyte-Doolittle; column IX of Table I represents
hydrophobicity according to Hopp-Woods; column XIII of Table I
represents antigenic index according to Jameson-Wolf; and column
XIV of Table I represents surface probability according to Emini).
Regions of high antigenicity are determined from the data presented
in columns VIII, IX, XIII, and/or IV by choosing values which
represent regions of the polypeptide which are likely to be exposed
on the surface of the polypeptide in an environment in which
antigen recognition may occur in the process of initiation of an
immune response. The data presented in FIG. 6 can also routinely be
presented in a similar tabular format by simply examining the amino
acid sequence disclosed in FIG. 6 (SEQ ID NO:19) using the modules
and algorithms of the DNA*STAR set on default parameters. As above,
the amino acid sequence presented in FIG. 6 can also be used to
determine regions of Neutrokine-alpha which exhibit a high degree
of potential for antigenicity whether presented as a Figure (as in
FIG. 6) or a table (as in Table I).
TABLE-US-00001 TABLE I Res Position I II III IV V VI VII VIII IX X
XI XII XIII XIV Met 1 A . . . . . . 0.73 -0.71 . . . 0.95 1.39 Asp
2 A . . . . T . 1.12 -0.66 * . . 1.15 1.56 Asp 3 A . . . . T . 1.62
-1.09 * . . 1.15 2.12 Ser 4 A . . . . T . 2.01 -1.51 . . . 1.15
4.19 Thr 5 A . . . . T . 2.40 -2.13 . . F 1.30 4.35 Glu 6 A A . . .
. . 2.70 -1.73 * * F 0.90 4.51 Arg 7 A A . . . . . 2.81 -1.34 * * F
0.90 4.51 Glu 8 A A . . . . . 2.00 -1.73 * * F 0.90 6.12 Gln 9 A A
. . . . . 1.99 -1.53 * * F 0.90 2.91 Ser 10 A . . B . . . 2.00
-1.04 * * F 0.90 2.15 Arg 11 A . . B . . . 1.33 -0.66 * * F 0.90
1.66 Leu 12 A . . B . . . 0.41 -0.09 * * F 0.45 0.51 Thr 13 A . . B
. . . 0.46 0.20 * * F -0.15 0.32 Ser 14 A A . . . . . 0.50 -0.19 *
* . 0.30 0.32 Cys 15 A A . . . . . 0.91 -0.19 * * . 0.30 0.78 Leu
16 A A . . . . . 0.80 -0.87 * * F 0.90 1.06 Lys 17 A A . . . . .
1.61 -1.36 . * F 0.90 1.37 Lys 18 A A . . . . . 1.32 -1.74 . * F
0.90 4.44 Arg 19 A A . . . . . 1.67 -1.70 . * F 0.90 5.33 Glu 20 A
A . . . . . 1.52 -2.39 . * F 0.90 5.33 Glu 21 A A . . . . . 2.38
-1.70 . * F 0.90 2.20 Met 22 A A . . . . . 2.33 -1.70 . * F 0.90
2.24 Lys 23 A A . . . . . 1.62 -1.70 * * F 0.90 2.24 Leu 24 A A . .
. . . 0.66 -1.13 * * F 0.75 0.69 Lys 25 A A . . . . . 0.36 -0.49 .
* F 0.45 0.52 Glu 26 A A . B . . . -0.53 -0.71 * * . 0.60 0.35 Cys
27 A A . B . . . -0.74 -0.03 * * . 0.30 0.30 Val 28 A A . B . . .
-1.00 -0.03 * * . 0.30 0.12 Ser 29 A A . B . . . -0.08 0.40 * * .
-0.30 0.11 Ile 30 A . . B . . . -0.08 0.40 * * . -0.30 0.40 Leu 31
A . . B . . . -0.08 -0.17 * . . 0.45 1.08 Pro 32 . . . B . . C 0.29
-0.81 * . F 1.10 1.39 Arg 33 . . . . T . . 0.93 -0.81 . * F 1.50
2.66 Lys 34 . . . . T . . 0.93 -1.07 . . F 1.84 4.98 Glu 35 . . . .
. . C 0.97 -1.37 * * F 1.98 4.32 Ser 36 . . . . . T C 1.89 -1.16 *
* F 2.52 1.64 Pro 37 . . . . . T C 1.80 -1.16 * * F 2.86 1.60 Ser
38 . . . . T T . 1.39 -0.77 * . F 3.40 1.24 Val 39 A . . . . T .
1.39 -0.39 . * F 2.36 1.24 Arg 40 A . . . . . . 1.39 -0.77 * * F
2.46 1.60 Ser 41 A . . . . . . 1.34 -1.20 * * F 2.46 2.00 Ser 42 .
. . . T T . 1.60 -1.16 . * F 3.06 2.67 Lys 43 . . . . T T . 1.09
-1.80 . * F 3.06 2.72 Asp 44 . . . . T T . 1.13 -1.11 * * F 3.40
1.67 Gly 45 A . . . . T . 0.43 -0.81 * * F 2.66 1.03 Lys 46 A A . .
. . . 0.14 -0.70 . . F 1.77 0.52 Leu 47 A A . . . . . 0.13 -0.20 *
. . 0.98 0.31 Leu 48 A A . . . . . -0.72 0.29 * . . 0.04 0.46 Ala
49 A A . . . . . -1.53 0.54 . * . -0.60 0.19 Ala 50 A A . . . . .
-2.00 1.23 . . . -0.60 0.19 Thr 51 A A . . . . . -2.63 1.23 . . .
-0.60 0.19 Leu 52 A A . . . . . -2.63 1.04 . . . -0.60 0.19 Leu 53
A A . . . . . -2.63 1.23 . . . -0.60 0.15 Leu 54 A A . . . . .
-2.34 1.41 . . . -0.60 0.09 Ala 55 A A . . . . . -2.42 1.31 . . .
-0.60 0.14 Leu 56 A A . . . . . -2.78 1.20 . . . -0.60 0.09 Leu 57
A . . . . T . -2.78 1.09 . . . -0.20 0.06 Ser 58 A . . . . T .
-2.28 1.09 . . . -0.20 0.05 Cys 59 A . . . . T . -2.32 1.07 . . .
-0.20 0.09 Cys 60 A . . . . T . -2.59 1.03 . . . -0.20 0.08 Leu 61
. . B B . . . -2.08 0.99 . . . -0.60 0.04 Thr 62 . . B B . . .
-1.97 0.99 . . . -0.60 0.11 Val 63 . . B B . . . -1.91 1.20 . . .
-0.60 0.17 Val 64 . . B B . . . -1.24 1.39 . . . -0.60 0.33 Ser 65
. . B B . . . -1.43 1.10 . . . -0.60 0.40 Phe 66 A . . B . . .
-1.21 1.26 . . . -0.60 0.40 Tyr 67 A . . B . . . -1.49 1.11 . . .
-0.60 0.54 Gln 68 A . . B . . . -1.44 0.97 . . . -0.60 0.41 Val 69
A . . B . . . -0.59 1.27 . . . -0.60 0.39 Ala 70 A . . B . . .
-0.63 0.89 . . . -0.60 0.43 Ala 71 A . . B . . . 0.07 0.56 . * .
-0.60 0.25 Leu 72 A . . . . T . -0.50 0.16 . * . 0.10 0.55 Gln 73 A
. . . . T . -1.09 0.20 . . F 0.25 0.45 Gly 74 A . . . . T . -0.53
0.20 . . F 0.25 0.45 Asp 75 A . . . . T . -0.76 0.09 . * F 0.25
0.73 Leu 76 A A . . . . . -0.06 0.09 . * F -0.15 0.35 Ala 77 A A .
. . . . 0.17 -0.31 . * . 0.30 0.69 Ser 78 A A . . . . . 0.17 -0.24
. * . 0.30 0.42 Leu 79 A A . . . . . -0.30 -0.24 . * . 0.30 0.88
Arg 80 A A . . . . . -0.30 -0.24 . * . 0.30 0.72 Ala 81 A A . . . .
. 0.17 -0.34 . * . 0.30 0.93 Glu 82 A A . . . . . 0.72 -0.30 . * .
0.45 1.11 Leu 83 A A . . . . . 0.99 -0.49 . * . 0.30 0.77 Gln 84 A
A . . . . . 1.21 0.01 . * . -0.15 1.04 Gly 85 A A . . . . . 1.10
0.01 * * . -0.30 0.61 His 86 A A . . . . . 1.73 0.01 * * . -0.15
1.27 His 87 A A . . . . . 0.92 -0.67 . * . 0.75 1.47 Ala 88 A A . .
. . . 1.52 -0.39 . * . 0.45 1.22 Glu 89 A A . . . . . 0.93 -0.39 .
. . 0.45 1.39 Lys 90 A A . . . . . 0.93 -0.39 * . F 0.60 1.03 Leu
91 A . . . . T . 0.38 -0.46 * . . 0.85 1.01 Pro 92 A . . . . T .
0.07 -0.46 . . . 0.70 0.59 Ala 93 A . . . . T . 0.07 -0.03 . . .
0.70 0.29 Gly 94 A . . . . T . -0.14 0.47 . . . -0.20 0.36 Ala 95 A
. . . . . . -0.14 0.21 . * . -0.10 0.36 Gly 96 A . . . . . . 0.08
-0.21 . . F 0.65 0.71 Ala 97 A . . . . . . -0.06 -0.21 . . F 0.65
0.72 Pro 98 A . . . . . . -0.28 -0.21 . * F 0.65 0.71 Lys 99 A A .
. . . . 0.07 -0.03 . . F 0.45 0.59 Ala 100 A A . . . . . 0.66 -0.46
. . F 0.60 1.01 Gly 101 A A . . . . . 0.41 -0.96 . . F 0.90 1.13
Leu 102 A A . . . . . 0.79 -0.89 . . F 0.75 0.57 Glu 103 A A . . .
. . 0.41 -0.46 * . F 0.45 0.88 Glu 104 A A . . . . . -0.49 -0.46 *
. F 0.45 0.89 Ala 105 A A . . . . . -0.21 -0.24 . . . 0.30 0.81 Pro
106 A A . . . . . -0.46 -0.44 . . . 0.30 0.67 Ala 107 A A . . . . .
0.01 0.06 . . . -0.30 0.39 Val 108 A A . . . . . -0.80 0.49 . * .
-0.60 0.38 Thr 109 A A . . . . . -0.76 0.67 . * . -0.60 0.20 Ala
110 A A . . . . . -1.06 0.24 * * . -0.30 0.40 Gly 111 A A . . . . .
-1.54 0.43 * * . -0.60 0.38 Leu 112 A A . . . . . -0.96 0.57 * * .
-0.60 0.23 Lys 113 . A B . . . . -0.31 0.09 * * . -0.30 0.39 Ile
114 . A B . . . . -0.21 0.01 * . . -0.30 0.61 Phe 115 . A B . . . .
-0.21 0.01 * . . 0.15 1.15 Glu 116 . A . . . . C -0.08 -0.17 * . F
1.25 0.58 Pro 117 . A . . . . C 0.39 0.26 * * F 1.10 1.28 Pro 118 .
. . . . . C 0.34 -0.00 . . F 2.20 1.47 Ala 119 . . . . . T C 0.89
-0.79 . * F 3.00 1.47 Pro 120 . . . . . T C 1.59 -0.36 . * F 2.25
0.94 Gly 121 . . . . T T . 1.29 -0.39 . * F 2.15 0.98 Glu 122 . . .
. T T . 1.20 -0.43 . . F 2.00 1.30 Gly 123 . . . . . . C 1.41 -0.54
. . F 1.60 1.12 Asn 124 . . . . . T C 2.00 -0.57 . . F 1.50 1.97
Ser 125 . . . . . T C 1.91 -0.60 . * F 1.50 1.82 Ser 126 . . . . .
T C 2.37 -0.21 . * F 1.54 2.47 Gln 127 . . . . . T C 2.37 -0.64 . *
F 2.18 3.01 Asn 128 . . . . . . C 2.76 -0.64 . . F 2.32 3.61 Ser
129 . . . . . T C 2.87 -1.03 . . F 2.86 5.39 Arg 130 . . . . T T .
2.58 -1.41 * . F 3.40 6.09 Asn 131 . . . . T T . 2.02 -1.31 * . F
3.06 3.83 Lys 132 . . . . T T . 2.02 -1.07 * . F 2.72 2.12 Arg 133
. . . . T . . 1.68 -1.06 * . F 2.18 1.88 Ala 134 . . . . . . C 1.77
-0.63 * . F 1.64 1.15 Val 135 . . . . . . C 1.66 -0.60 * . F 1.49
0.89 Gln 136 . . . . . . C 1.66 -0.60 * . F 1.83 0.79 Gly 137 . . .
. . T C 1.30 -0.60 * . F 2.52 1.35 Pro 138 . . . . . T C 0.33 -0.61
* . F 2.86 2.63 Glu 139 . . . . T T . 0.61 -0.61 * . F 3.40 1.13
Glu 140 A . . . . T . 1.47 -0.53 * . F 2.66 1.64 Thr 141 A . . . .
. . 1.47 -0.56 . . F 2.12 1.84 Val 142 A . . . . . . 1.14 -0.99 . .
F 1.78 1.77 Thr 143 A . . . . T . 0.54 -0.41 . . F 1.19 0.55 Gln
144 A . . . . T . 0.54 0.27 * . F 0.25 0.31 Asp 145 A . . . . T .
-0.27 0.19 * . F 0.25 0.73 Cys 146 A . . . . T . -0.84 0.23 * . .
0.10 0.42 Leu 147 A A . . . . . -0.58 0.43 * . . -0.60 0.17 Gln 148
A A . . . . . -0.27 0.53 * . . -0.60 0.10 Leu 149 A A . . . . .
-0.57 0.53 * * . -0.30 0.32 Ile 150 A A . . . . . -0.57 0.34 * . .
0.30 0.52 Ala 151 . A . . . . C -0.21 -0.34 . * . 1.40 0.52 Asp 152
. . . . T T . 0.39 -0.26 . * F 2.45 0.91 Ser 153 . . . . . T C 0.08
-0.51 . . F 3.00 2.00 Glu 154 . . . . . T C -0.00 -0.71 . . F 2.70
2.86 Thr 155 . . . . . T C 0.89 -0.53 * . F 2.40 1.20 Pro 156 . . .
B . . C 1.52 -0.13 * . F 1.56 1.55 Thr 157 . . . B T . . 1.18 -0.51
* . F 1.92 1.79 Ile 158 A . . B . . . 1.18 -0.09 . . F 1.08 1.23
Gln 159 . . . . T T . 0.93 -0.19 . . F 2.04 1.07 Lys 160 . . . . T
T . 0.93 0.14 * . F 1.60 1.16 Gly 161 . . . . T T . 0.44 0.14 * . F
1.44 2.38 Ser 162 . . . . T T . -0.10 0.24 * . F 1.28 1.19 Tyr 163
. . . B T . . 0.58 0.49 * . . 0.12 0.44 Thr 164 . . B B . . . 0.29
0.91 * . . -0.44 0.69 Phe 165 . . B B . . . -0.57 1.40 * . . -0.60
0.54 Val 166 . . B B . . . -1.03 1.70 . . . -0.60 0.29 Pro 167 . .
B B . . . -1.03 1.63 . . . -0.60 0.16 Trp 168 A . . B . . . -1.49
1.53 . * . -0.60 0.25 Leu 169 A . . B . . . -1.13 1.53 * . . -0.60
0.29 Leu 170 A . . B . . . -0.32 0.89 * . . -0.30 0.38 Ter 171 A .
. . . . . 0.19 0.46 * . . 0.20 0.71 Phe 172 . . . . T . . 0.10
-0.03 * . . 1.80 0.85 Lys 173 . . . . T T . -0.20 -0.33 * . F 2.60
1.38 Arg 174 . . . . . T C -0.20 -0.51 . . F 3.00 1.04 Gly 175 . .
. . . T C 0.61 -0.21 . . F 2.25 0.99 Ser 176 A . . . . T . 0.91
-1.00 * . F 2.05 0.86 Ala 177 A A . . . . . 1.66 -1.00 * . F 1.35
0.76 Leu 178 A A . . . . . 1.61 -1.00 . . F 1.20 1.54 Glu 179 A A .
. . . . 1.50 -1.43 . . F 0.90 1.98 Glu 180 A A . . . . . 1.89 -1.41
* . F 0.90 3.16 Lys 181 A A . . . . . 1.30 -1.91 * . F 0.90 7.66
Glu 182 A A . . . . . 1.08 -1.91 . . F 0.90 3.10 Asn 183 A A . . .
. . 1.03 -1.23 * * F 0.90 1.48 Lys 184 A A . . . . . 1.08 -0.59 * .
F 0.75 0.55 Ile 185 A A . . . . . 1.08 -0.59 * * . 0.60 0.63 Leu
186 A A . . . . . 0.72 -0.59 * * . 0.60 0.68 Val 187 A A . . . . .
0.38 -0.50 . * . 0.30 0.49 Lys 188 A A . . . . . 0.13 -0.07 * * F
0.45 0.69 Glu 189 A . . . . T . -0.61 0.00 * * F 0.40 1.32 Thr 190
. . . . T T . -0.42 0.10 . * F 0.80 1.54 Gly 191 . . . . T T .
-0.50 0.24 * . F 0.65 0.67 Tyr 192 . . . . T T . 0.11 0.93 * * .
0.20 0.27 Phe 193 . . B B . . . -0.28 1.69 . . . -0.60 0.29 Phe 194
. . B B . . . -0.28 1.63 . * . -0.60 0.29 Ile 195 . . B B . . .
-0.82 1.60 . . . -0.60 0.32 Tyr 196 . . B B . . . -1.29 1.49 . . .
-0.60 0.28 Gly 197 . . . B T . . -1.29 1.39 . . . -0.20 0.26 Gln
198 . . . B T . . -0.90 1.36 . . . -0.20 0.59 Val 199 . . . B . . C
-0.20 1.16 . . . -0.40 0.54 Leu 200 . . . B . . C 0.73 0.40 . . .
-0.10 0.92 Tyr 201 . . . . T T . 0.67 -0.03 . . . 1.25 1.06 Thr 202
. . . . T T . 0.77 0.06 . . F 0.80 2.06 Asp 203 . . . . T T . 0.18
0.17 . . F 0.80 3.91 Lys 204 A . . . . T . 0.43 -0.01 . . F 1.00
2.52 Thr 205 A A . . . . . 0.90 -0.16 . . F 0.60 1.73 Tyr 206 A A .
. . . . 1.11 -0.21 . . . 0.45 1.03 Ala 207 A A . . . . . 0.61 0.29
. . . -0.30 0.70 Met 208 A A . . . . . -0.28 0.97 . . . -0.60 0.40
Gly 209 A A . B . . . -0.32 1.17 * . . -0.60 0.18 His 210 A A . B .
. . 0.10 0.81 * . . -0.60 0.31 Leu 211 A A . B . . . 0.39 0.31 . .
. -0.30 0.61 Ile 212 A A . B . . . 1.02 -0.30 . . . 0.45 1.22 Gln
213 A A . B . . . 0.77 -0.73 . * . 0.75 1.80 Arg 214 A A . B . . .
1.08 -0.59 . * F 0.90 1.62 Lys 215 A A . B . . . 0.26 -0.77 * * F
0.90 3.14 Lys 216 A A . B . . . 0.37 -0.81 . * F 0.90 1.35 Val 217
. A B B . . . 0.91 -0.43 * * . 0.30 0.60 His 218 . A B B . . . 0.91
-0.00 . * . 0.30 0.29 Val 219 . A B B . . . 0.80 -0.00 * * . 0.30
0.25 Phe 220 . . B B . . . -0.06 -0.00 * . . 0.30 0.57 Gly 221 A .
. B . . . -0.40 0.04 . * . -0.30 0.35 Asp 222 A . . . . . . -0.36
-0.07 * . . 0.50 0.63 Glu 223 A . . . . . . -1.18 -0.03 * . . 0.50
0.60 Leu 224 A . . B . . . -0.63 -0.17 . . . 0.30 0.45 Ser 225 A .
. B . . . -0.74 -0.11 . . . 0.30 0.39 Leu 226 A . . B . . . -1.10
0.57 . * . -0.60 0.18 Val 227 A . . B . . . -0.99 1.36 . * . -0.60
0.19 Thr 228 A . . B . . . -1.66 0.67 * * . -0.60 0.28 Leu 229 A .
. B . . . -1.73 0.86 * . . -0.60 0.18 Phe 230 A . . B . . . -1.43
0.86 * . . -0.60 0.17 Arg 231 A . . B . . . -0.62 0.61 * . . -0.60
0.21 Cys 232 . . . B T . . -0.37 0.53 * . . -0.20 0.41 Ile 233 . .
. B T . . -0.27 0.46 * . . -0.20 0.46 Gln 234 . . . B T . . 0.54
0.10 * . . 0.10 0.37 Asn 235 . . . B . . C 0.93 0.10 * . . 0.05
1.19 Met 236 . . . B . . C 0.01 0.01 * . F 0.20 2.44 Pro 237 . . .
B . . C 0.47 0.01 * . F 0.44 1.16 Glu 238 . . . . T . . 1.36 0.04 *
. F 1.08 1.12 Thr 239 . . . . . . C 1.36 0.04 * . F 1.12 1.82 Leu
240 . . . . . . C 1.06 -0.17 * . F 1.96 1.89 Pro 241 . . . . T . .
0.99 -0.21 . . F 2.40 1.46 Asn 242 . . . . T . . 0.96 0.36 . . F
1.41 0.54 Asn 243 . . . . T T . 0.66 0.63 . . F 1.22 1.03 Ser 244 .
. . . T T . 0.38 0.33 . . F 1.13 0.89 Cys 245 . . . . T T . 0.84
0.40 . . . 0.74 0.56
Tyr 246 . . . . T T . 0.17 0.43 . . . 0.20 0.35 Ser 247 A . . . . .
. -0.42 0.71 . . . -0.40 0.18 Ala 248 A A . . . . . -0.38 0.83 . .
. -0.60 0.34 Gly 249 A A . . . . . -0.89 0.26 . . . -0.30 0.43 Ile
250 A A . . . . . -0.22 0.19 * . . -0.30 0.27 Ala 251 A A . . . . .
0.02 -0.20 * . . 0.30 0.46 Lys 252 A A . . . . . -0.02 -0.70 . . .
0.60 0.80 Leu 253 A A . . . . . 0.57 -0.70 . . F 0.90 1.13 Glu 254
A A . . . . . 0.91 -1.39 . . F 0.90 1.87 Glu 255 A A . . . . . 0.99
-1.89 . . F 0.90 1.62 Gly 256 A A . . . . . 1.58 -1.20 . * F 0.90
1.62 Asp 257 A A . . . . . 0.72 -1.49 . * F 0.90 1.62 Glu 258 A A .
. . . . 0.94 -0.80 * * F 0.75 0.77 Leu 259 A A . . . . . 0.06 -0.30
* * . 0.30 0.79 Gln 260 A A . . . . . -0.16 -0.04 * . . 0.30 0.33
Leu 261 A A . . . . . 0.30 0.39 * . . -0.30 0.30 Ala 262 A A . . .
. . 0.30 0.39 * . . -0.30 0.70 Ile 263 A A . . . . . 0.30 -0.30 . *
. 0.30 0.70 Pro 264 A . . . . T . 0.52 -0.30 . * F 1.00 1.37 Arg
265 A . . . . T . 0.52 -0.49 . * F 1.00 1.37 Glu 266 A . . . . T .
0.44 -0.59 * * F 1.30 3.38 Asn 267 A . . . . T . 0.73 -0.59 * * F
1.30 1.53 Ala 268 A . . . . . . 0.81 -0.63 * * . 0.95 1.05 Gln 269
A . . . . . . 1.02 0.06 * * . -0.10 0.50 Ile 270 A . . . . . . 0.57
0.06 . * . 0.15 0.52 Ser 271 . . . . . . C 0.57 0.09 . * . 0.60
0.51 Leu 272 . . . . . . C -0.29 -0.41 . * F 1.60 0.49 Asp 273 . .
. . T T . -0.01 -0.17 . * F 2.25 0.52 Gly 274 . . . . T T . -0.71
-0.37 . * F 2.50 0.56 Asp 275 . . . . T T . -0.52 0.03 . * F 1.65
0.59 Val 276 A . . . . T . -0.57 0.13 . * F 1.00 0.30 Thr 277 A . .
B . . . -0.34 0.56 . * . -0.10 0.30 Phe 278 A . . B . . . -1.16
0.63 . * . -0.35 0.18 Phe 279 A . . B . . . -0.77 1.31 . * . -0.60
0.20 Gly 280 A A . . . . . -1.58 0.67 . * . -0.60 0.28 Ala 281 A A
. . . . . -1.53 0.87 . * . -0.60 0.27 Leu 282 A A . . . . . -1.61
0.77 * . . -0.60 0.26 Lys 283 A A . . . . . -1.30 0.41 * . . -0.60
0.33 Leu 284 A A . . . . . -0.99 0.41 . . . -0.60 0.42 Leu 285 A A
. . . . . -1.03 0.34 * . . -0.30 0.65
[0152] Additional preferred nucleic acid fragments of the present
invention include nucleic acid molecules comprising, or
alternatively, consisting of a sequence encoding one or more
epitope-bearing portions of Neutrokine-alpha. In particular, such
nucleic acid fragments of the present invention include nucleic
acid molecules comprising, or alternatively consisting of, a
sequence encoding a polypeptide selected from: from about Phe-115
to about Leu-147, from about Ile-150 to about Tyr-163, from about
Ser-171 to about Phe-194, from about Glu-223 to about Tyr-246, and
from about Ser-271 to about Phe-278, of the amino acid sequence of
SEQ ID NO:2. In this context, "about" means the particularly
recited ranges and ranges larger or smaller by several, a few, 5,
4, 3, 2 or 1 amino acid residues at either or both the amino- and
carboxy-termini. Polypeptides encoded by these nucleic acid
molecules are also encompassed by the invention. Polypeptide
fragments which bear antigenic epitopes of the Neutrokine-alpha may
be easily determined by one of skill in the art using the
above-described analysis of the Jameson-Wolf antigenic index, as
shown in FIG. 3. Methods for determining other such epitope-bearing
portions of Neutrokine-alpha are described in detail below.
[0153] Additional preferred nucleic acid fragments of the present
invention include nucleic acid molecules comprising, or
alternatively consisting of a sequence encoding one or more
epitope-bearing portions of Neutrokine-alphaSV. In particular, such
nucleic acid fragments of the present invention include nucleic
acid molecules comprising, or alternatively consisting of a
sequence encoding a polypeptide selected from about Pro-32 to about
Leu-47, from about Glu-116 to about Ser-143, from about Phe-153 to
about Tyr-173, from about Pro-218 to about Tyr-227, from about
Ser-252 to about Thr-258, from about Ala-232 to about Gln-241; from
about Ile-244 to about Ala-249; and from about Ser-252 to about
Val-257, of the amino acid sequence of SEQ ID NO:19. In this
context, "about" means the particularly recited ranges and ranges
larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid
residues at either or both the amino- and carboxy-termini.
Polypeptides encoded by these nucleic acid molecules are also
encompassed by the invention. Polypeptide fragments which bear
antigenic epitopes of the Neutrokine-alpha may be easily determined
by one of skill in the art using the above-described analysis of
the Jameson-Wolf antigenic index. Methods for determining other
such epitope-bearing portions of Neutrokine-alphaSV are described
in detail below.
[0154] In specific embodiments, the polynucleotides of the
invention are less than 100,000 kb, 50,000 kb, 10,000 kb, 1,000 kb,
500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125
kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb,
7.5 kb, or 5 kb in length.
[0155] In further embodiments, polynucleotides of the invention
comprise at least 15, at least 30, at least 50, at least 100, or at
least 250, at least 500, or at least 1000 contiguous nucleotides of
Neutrokine-alpha coding sequence, but consist of less than or equal
to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb,
30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that
flanks the 5' or 3' coding nucleotide set forth in FIGS. 1A and 1B
(SEQ ID NO:1) or FIGS. 5A and 5B (SEQ ID NO:18). In further
embodiments, polynucleotides of the invention comprise at least 15,
at least 30, at least 50, at least 100, or at least 250, at least
500, or at least 1000 contiguous nucleotides of Neutrokine-alpha
coding sequence, but do not comprise all or a portion of any
Neutrokine-alpha intron. In another embodiment, the nucleic acid
comprising Neutrokine-alpha coding sequence does not contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the
Neutrokine-alpha gene in the genome). In other embodiments, the
polynucleotides of the invention do not contain the coding sequence
of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2,
or 1 genomic flanking gene(s).
[0156] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the sequence complementary to the
coding and/or noncoding sequence depicted in FIGS. 1A and 1B (SEQ
ID NO:1), the sequence of the cDNA clone contained in the deposit
having ATCC accession no. 97768, the sequence complementary to the
coding sequence and/or noncoding sequence depicted in FIGS. 5A and
5B (SEQ ID NO:18), the sequence of the cDNA clone contained in the
deposit having ATCC accession no. 203518, the sequence
complementary to the coding sequence and/or noncoding sequence
(i.e., transcribed, untranslated) depicted in SEQ ID NO:21, the
sequence complementary to the coding sequence and/or noncoding
sequence depicted in SEQ ID NO:22, the sequence complementary to
the coding sequence and/or noncoding sequence depicted in SEQ ID
NO:27, the sequence complementary to the coding sequence and/or
noncoding sequence depicted in SEQ ID NO:29, the sequence
complementary to the coding sequence and/or noncoding sequence
depicted in SEQ ID NO:37, or fragments (such as, for example, the
open reading frame or a fragment thereof) of these sequences, as
described herein. By "stringent hybridization conditions" is
intended overnight incubation at 42.degree. C. in a solution
comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0157] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 40, 50, or
60) nucleotides, and even more preferably about any integer in the
range of 30-70 or 80-150 nucleotides, or the entire length of the
reference polynucleotide. These have uses, which include, but are
not limited to, diagnostic probes and primers as discussed above
and in more detail below. By a portion of a polynucleotide of "at
least about 20 nt in length," for example, is intended to include
the particularly recited ranges, larger or smaller by several (i.e.
5, 4, 3, 2, 1, or 0) amino acids, at either extreme or at both
extremes of the nucleotide sequence of the reference polynucleotide
(e.g., the sequence of one or both of the deposited cDNAs, the
complementary strand of the nucleotide sequence shown in FIGS. 1A
and 1B (SEQ ID NO:1), the complementary strand of the nucleotide
sequence shown in FIGS. 5A and 5B (SEQ ID NO:18), the complementary
strand of the nucleotide sequence shown in SEQ ID NO:21, the
complementary strand of the nucleotide sequence shown in SEQ ID
NO:22, the complementary strand of the nucleotide sequence shown in
SEQ ID NO:27, the complementary strand of the nucleotide sequence
shown in SEQ ID NO:29, and/or the complementary strand of the
nucleotide sequence shown in SEQ ID NO:37). Of course, a
polynucleotide which hybridizes only to a poly A sequence (such as
the 3' terminal poly (A) tract of the Neutrokine-alpha cDNA shown
in FIGS. 1A and 1B (SEQ ID NO:1), the 3' terminal poly(A) tract of
the Neutrokine-alphaSV cDNA shown in FIGS. 5A and 5B (SEQ ID NO:18)
or the 3' terminal poly(A) tract of the Neutrokine-alphaSV cDNA
shown in SEQ ID NO:22), or to a complementary stretch of T (or U)
residues, would not be included in a polynucleotide of the
invention used to hybridize to a portion of a nucleic acid of the
invention, since such a polynucleotide would hybridize to any
nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone generated using oligo dT as a primer).
[0158] As indicated, nucleic acid molecules of the present
invention which encode a Neutrokine-alpha polypeptide or a
Neutrokine-alphaSV polypeptide may include, but are not limited to,
polynucleotides encoding the amino acid sequence of the respective
extracellular domains of the polypeptides, by themselves; and the
coding sequence for the extracellular domains of the respective
polypeptides and additional sequences, such as those encoding the
intracellular and transmembrane domain sequences, or a pre-, or
pro- or prepro-protein sequence; the coding sequence of the
respective extracellular domains of the polypeptides, with or
without the aforementioned additional coding sequences.
[0159] Also encoded by nucleic acids of the invention are the above
protein sequences together with additional, non-coding sequences,
including for example, but not limited to, introns and non-coding
5' and 3' sequences, such as the transcribed, non-translated
sequences that play a role in transcription, mRNA processing,
including splicing and polyadenylation signals, for example,
ribosome binding and stability of mRNA; an additional coding
sequence which codes for additional amino acids, such as those
which provide additional functionalities.
[0160] Thus, the sequence encoding the polypeptide may be fused to
a marker sequence, such as a sequence encoding a peptide which
facilitates purification of the fused polypeptide. In certain
preferred embodiments of this embodiment of the invention, the
marker amino acid sequence is a hexa-histidine peptide, such as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. The
"HA" tag is another peptide useful for purification which
corresponds to an epitope derived from the influenza hemagglutinin
protein, which has been described by Wilson et al., Cell 37: 767
(1984). As discussed below, other such fusion proteins include the
Neutrokine-alpha or the Neutrokine-alphaSV polypeptides fused to Fc
at the N- or C-terminus.
[0161] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the Neutrokine-alpha or
Neutrokine-alphaSV polypeptides of SEQ ID NO:2. Variants may occur
naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques, which include, but are not limited to
oligonucleotide mediated mutagenesis, alanine scanning, PCR
mutagenesis, site directed mutagenesis (see e.g., Carter et al.,
Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids
Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells et al.,
Gene 34:315 (1985)), restriction selection mutagenesis (see e.g.,
Wells er al., Philos. Trans. R. Soc. London SerA 317:415
(1986)).
[0162] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides or portions thereof. Also especially preferred in this
regard are conservative substitutions.
[0163] Additional embodiments of the invention are directed to
isolated nucleic acid molecules comprising a polynucleotide which
encodes the amino acid sequence of a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide (e.g., a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide fragment described herein) having an
amino acid sequence which contains at least one conservative amino
acid substitution, but not more than 50 conservative amino acid
substitutions, even more preferably, not more than 40 conservative
amino acid substitutions, still more preferably, not more than 30
conservative amino acid substitutions, and still even more
preferably, not more than 20 conservative amino acid substitutions,
10-20 conservative amino acid substitutions, 5-10 conservative
amino acid substitutions, 1-5 conservative amino acid
substitutions, 3-5 conservative amino acid substitutions, or 1-3
conservative amino acid substitutions. Of course, in order of
ever-increasing preference, it is highly preferable for a
polynucleotide which encodes the amino acid sequence of a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide to have an
amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5,
4, 3, 2 or 1 conservative amino acid substitutions.
[0164] Further embodiments include an isolated nucleic acid
molecule comprising, or alternatively consisting of, a
polynucleotide having a nucleotide sequence at least 80%, 85%, or
90% identical, and more preferably at least 95%, 96%, 97%, 98% or
99% identical to a polynucleotide selected from the group
consisting of: (a) a nucleotide sequence encoding the
Neutrokine-alpha polypeptide having the complete amino acid
sequence in FIGS. 1A and 1B (i.e., positions 1 to 285 of SEQ ID
NO:2); (b) a nucleotide sequence encoding the Neutrokine-alpha
polypeptide having the complete amino acid sequence in SEQ ID NO:2
excepting the N-terminal methionine (i.e., positions 2 to 285 of
SEQ ID NO:2); (c) a fragment of the polypeptide of (b) having
Neutrokine-alpha functional activity (e.g., antigenic or biological
activity); (d) a nucleotide sequence encoding the predicted
extracellular domain of the Neutrokine-alpha polypeptide having the
amino acid sequence at positions 73-285 in FIGS. 1A and 1B (SEQ ID
NO:2); (e) a nucleotide sequence encoding the Neutrokine-alpha
polypeptide having the amino acid sequence at positions 134-285 in
FIGS. 1A and 1B (SEQ ID NO:2); (f) a nucleotide sequence encoding
the Neutrokine-alpha polypeptide having the complete amino acid
sequence encoded by the cDNA clone contained in the deposit having
ATCC accession number 97768; (g) a nucleotide sequence encoding the
extracellular domain of the Neutrokine-alpha polypeptide having the
amino acid sequence encoded by the cDNA contained in the deposit
having ATCC accession number 97768; and (h) a nucleotide sequence
complementary to any of the nucleotide sequences in (a), (b), (c),
(d), (e), (f), (g), or (h) above. The present invention also
encompasses the above polynucleotide sequences fused to a
heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotides and nucleic acid molecules are also encompassed by
the invention.
[0165] Highly preferred embodiments of the invention are directed
to nucleic acid molecules comprising, or alternatively consisting
of a polynucleotide having a nucleotide sequence at least 80%, 85%,
90% identical and more preferably at least 95%, 96%, 97%, 98%, 99%
or 100% identical to a polynucleotide sequence encoding the
Neutrokine-alpha polypeptide having the amino acid sequence at
positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2). Preferred
embodiments of the invention are directed to nucleic acid molecules
comprising, or alternatively consisting of a polynucleotide having
a nucleotide sequence at least 90% identical to a polynucleotide
sequence encoding the Neutrokine-alpha polypeptide having the amino
acid sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID
NO:2). More preferred embodiments of the invention are directed to
nucleic acid molecules comprising, or alternatively consisting of a
polynucleotide having a nucleotide sequence at least 95% identical
to a polynucleotide sequence encoding the Neutrokine-alpha
polypeptide having the amino acid sequence at positions 134-285 in
FIGS. 1A and 1B (SEQ ID NO:2). More preferred embodiments of the
invention are directed to nucleic acid molecules comprising, or
alternatively consisting of a polynucleotide having a nucleotide
sequence at least 96% identical to a polynucleotide sequence
encoding the Neutrokine-alpha polypeptide having the amino acid
sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2).
[0166] Additionally, more preferred embodiments of the invention
are directed to nucleic acid molecules comprising, or alternatively
consisting of a polynucleotide having a nucleotide sequence at
least 97% to a polynucleotide sequence encoding the
Neutrokine-alpha polypeptide having the amino acid sequence at
positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2). Additionally,
more preferred embodiments of the invention are directed to nucleic
acid molecules comprising, or alternatively consisting of a
polynucleotide having a nucleotide sequence at least 98% to a
polynucleotide sequence encoding the Neutrokine-alpha polypeptide
having the amino acid sequence at positions 134-285 in FIGS. 1A and
1B (SEQ ID NO:2). Additionally, more preferred embodiments of the
invention are directed to nucleic acid molecules comprising, or
alternatively consisting of a polynucleotide having a nucleotide
sequence at least 99% identical to a polynucleotide sequence
encoding the Neutrokine-alpha polypeptide having the amino acid
sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2).
[0167] A further embodiment of the invention relates to an isolated
nucleic acid molecule comprising a polynucleotide which encodes the
amino acid sequence of a Neutrokine-alphaSV polypeptide (e.g., a
Neutrokine-alphaSV polypeptide fragment described herein) having an
amino acid sequence which contains at least one conservative amino
acid substitution, but not more than 50 conservative amino acid
substitutions, even more preferably, not more than 40 conservative
amino acid substitutions, still more preferably not more than 30
conservative amino acid substitutions, and still even more
preferably not more than 20 conservative amino acid substitutions.
Of course, in order of ever-increasing preference, it is highly
preferable for a polynucleotide which encodes the amino acid
sequence of a Neutrokine-alpha polypeptide to have an amino acid
sequence which contains not more than 7-10, 5-10, 3-7, 3-5, 2-5,
1-5, 1-3, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid
substitutions.
[0168] Further embodiments include an isolated nucleic acid
molecule comprising, or alternatively, consisting of a
polynucleotide having a nucleotide sequence at least 80%, 85% or
90% identical, and more preferably at least 95%, 96%, 97%, 98% or
99% identical to a polynucleotide selected from the group
consisting of: (a) a nucleotide sequence encoding the
Neutrokine-alphaSV polypeptide having the complete amino acid
sequence in FIGS. 5A and 5B (i.e., positions 1 to 266 of SEQ ID
NO:19); (b) a nucleotide sequence encoding the Neutrokine-alphaSV
polypeptide having the complete amino acid sequence in SEQ ID NO:19
excepting the N-terminal methionine (i.e., positions 2 to 266 of
SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted
extracellular domain of the Neutrokine-alphaSV polypeptide having
the amino acid sequence at positions 73-266 in FIGS. 5A and 5B (SEQ
ID NO:19); (d) a nucleotide sequence encoding the
Neutrokine-alphaSV polypeptide having the complete amino acid
sequence encoded by the cDNA clone contained in the deposit having
ATCC accession number 203518; (e) a nucleotide sequence encoding
the extracellular domain of the Neutrokine-alphaSV polypeptide
having the amino acid sequence encoded by the cDNA clone contained
in the deposit having ATCC accession number 203518; and (f) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d) or (e), above.
[0169] Further, the invention includes a polynucleotide comprising,
or alternatively, consisting of, a sequence at least 90%, or at
least 95%, identical to any portion of at least about 10 contiguous
nucleotides, about 20 contiguous nucleotides, about 25 contiguous
nucleotides, or about 30 contiguous nucleotides, preferably at
least about 40 nucleotides, or at least about 50 nucleotides, of
the sequence from nucleotide 1 to nucleotide 1082 in FIGS. 1A and
1B (SEQ ID NO:1), preferably excluding the nucleotide sequences
determined from the above-listed 4 cDNA clones and the nucleotide
sequences from nucleotide 797 to 1082, 810 to 1082, and 346 to 542.
The invention also includes a polynucleotide comprising, or
alternatively consisting of, a sequence at least 90%, or at least
95%, identical to any portion of at least about 10 contiguous
nucleotides, about 20 contiguous nucleotides, about 25 contiguous
nucleotides, or about 30 contiguous nucleotides, preferably at
least about 40 nucleotides, or at least about 50 nucleotides, of
the sequence in FIGS. 5A and 5B (SEQ ID NO:18), preferably
excluding the nucleotide sequences determined from the above-listed
4 cDNA clones. The invention also includes a polynucleotide
comprising, or alternatively consisting of a sequence at least 90%,
or at least 95%, identical to any portion of at least about 10
contiguous nucleotides, about 20 contiguous nucleotides, about 25
contiguous nucleotides, or about 30 contiguous nucleotides,
preferably at least about 40 nucleotides, or at least about 50
nucleotides, of the sequence in SEQ ID NO:21, preferably excluding
the nucleotide sequences determined from the above-listed 4 cDNA
clones. The invention also includes a polynucleotide comprising a
sequence at least 90%, or at least 95%, identical to any portion of
at least about 10 contiguous nucleotides, about 20 contiguous
nucleotides, about 25 contiguous nucleotides, or about 30
contiguous nucleotides, preferably at least about 40 nucleotides,
or at least about 50 nucleotides, of the sequence in SEQ ID NO:22,
preferably excluding the nucleotide sequences determined from the
above-listed 4 cDNA clones. The invention also includes a
polynucleotide comprising a sequence at least 90%, or at least 95%,
identical to any portion of at least about 10 contiguous
nucleotides, about 20 contiguous nucleotides, about 25 contiguous
nucleotides, or about 30 contiguous nucleotides, preferably at
least about 40 nucleotides, or at least about 50 nucleotides, of
the sequence in SEQ ID NO:27, preferably excluding the nucleotide
sequences determined from the above-listed 4 cDNA clones. The
invention also includes a polynucleotide comprising a sequence at
least 90%, or at least 95%, identical to any portion of at least
about 10 contiguous nucleotides, about 20 contiguous nucleotides,
about 25 contiguous nucleotides, or about 30 contiguous
nucleotides, preferably at least about 40 nucleotides, or at least
about 50 nucleotides, of the sequence in SEQ ID NO:29, preferably
excluding the nucleotide sequences determined from the above-listed
4 cDNA clones. The invention also includes a polynucleotide
comprising a sequence at least 90%, or at least 95%, identical to
any portion of at least about 10 contiguous nucleotides, about 20
contiguous nucleotides, about 25 contiguous nucleotides, or about
30 contiguous nucleotides, preferably at least about 40
nucleotides, or at least about 50 nucleotides, of the sequence in
SEQ ID NO:37, preferably excluding the nucleotide sequences
determined from the above-listed 4 cDNA clones. In this context
"about" includes the particularly recited ranges, larger or smaller
by several (i.e. 5, 4, 3, 2 or 1) amino acids, at either extreme or
at both extremes.
[0170] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
is intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide
sequence may include up to five mismatches per each 100 nucleotides
of the reference nucleotide sequence encoding the Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These mutations of the reference sequence may
occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence. The reference (query) sequence may be the entire
nucleotide sequence encoding Neutrokine-alpha or
Neutrokine-alphaSV, as shown in FIGS. 1A and 1B (SEQ ID NO:1) and
FIGS. 5A and 5B (SEQ ID NO:18), respectively, or any
Neutrokine-alpha such as, for example, the Neutrokine-alpha
polynucleotides shown as SEQ ID NOs:21, 22, 27, 29, or 37, or any
Neutrokine-alpha or Neutrokine-alphaSV polynucleotide fragment as
described herein.
[0171] As a practical matter, whether any particular nucleic acid
molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to, for instance, the nucleotide sequences shown in FIGS.
1A and 1B, or the nucleotide sequences shown in FIGS. 5A and 5B, or
to the nucleotides sequence of the deposited cDNA clones, or to any
Neutrokine-alpha polynucleotide such as, for example, the
Neutrokine-alpha polynucleotides shown as SEQ ID NOs:21, 22, 27,
29, or 37, or fragments thereof, can be determined conventionally
using known computer programs such as the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive,
Madison, Wis. 53711). Bestfit uses the local homology algorithm of
Smith and Waterman to find the best segment of homology between two
sequences (Advances in Applied Mathematics 2:482-489 (1981)). When
using Bestfit or any other sequence alignment program to determine
whether a particular sequence is, for instance, 95% identical to a
reference sequence according to the present invention, the
parameters are set, of course, such that the percentage of identity
is calculated over the full length of the reference nucleotide
sequence and that gaps in homology of up to 5% of the total number
of nucleotides in the reference sequence are allowed.
[0172] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245
(1990)). In a sequence alignment the query and subject sequences
are both DNA sequences. An RNA sequence can be compared by
converting U's to T's. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB
alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject
nucleotide sequence, whichever is shorter. According to this
embodiment, if the subject sequence is shorter than the query
sequence because of 5' or 3' deletions, not because of internal
deletions, a manual correction is made to the results to take into
consideration the fact that the FASTDB program does not account for
5' and 3' truncations of the subject sequence when calculating
percent identity. For subject sequences truncated at the 5' or 3'
ends, relative to the query sequence, the percent identity is
corrected by calculating the number of bases of the query sequence
that are 5' and 3' of the subject sequence, which are not
matched/aligned, as a percent of the total bases of the query
sequence. A determination of whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of this
embodiment. Only bases outside the 5' and 3' bases of the subject
sequence, as displayed by the FASTDB alignment, which are not
matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score. For
example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the
5' end of the subject sequence and therefore, the FASTDB alignment
does not show a matched/alignment of the first 10 bases at 5' end.
The 10 unpaired bases represent 10% of the sequence (number of
bases at the 5' and 3' ends not matched/total number of bases in
the query sequence) so 10% is subtracted from the percent identity
score calculated by the FASTDB program. If the remaining 90 bases
were perfectly matched the final percent identity would be 90%. In
another example, a 90 base subject sequence is compared with a 100
base query sequence. This time the deletions are internal deletions
so that there are no bases on the 5' or 3' of the subject sequence
which are not matched/aligned with the query. In this case the
percent identity calculated by FASTDB is not manually corrected.
Once again, only bases 5' and 3' of the subject sequence which are
not matched/aligned with the query sequence are manually corrected
for. No other manual corrections are made for the purposes of this
embodiment.
[0173] The present application is directed to nucleic acid
molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the nucleic acid sequences (i.e., polynucleotides)
disclosed herein (e.g., those disclosed in FIGS. 1A and 1B (SEQ ID
NO:1) or to the nucleic acid sequence of the deposited cDNAs),
irrespective of whether they encode a polypeptide having
Neutrokine-alpha and/or Neutrokine-alphaSV functional activity
(e.g., biological activity). In addition, the present application
is also directed to nucleic acid molecules at least 80%, 85%, 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown in FIGS. 5A and 5B (SEQ ID NO:18) or to the nucleic
acid sequence of the deposited cDNA, irrespective of whether they
encode a polypeptide having Neutrokine-alphaSV activity. Moreover,
the present application is also directed to nucleic acid molecules
at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identical to
the nucleic acid sequence shown in SEQ ID NOs:21, 22, 27, 29, or
37, irrespective of whether they encode a polypeptide having
Neutrokine-alpha activity. This is because even where a particular
nucleic acid molecule does not encode a polypeptide having
Neutrokine-alpha and/or Neutrokine-alphaSV activity, one of skill
in the art would still know how to use the nucleic acid molecule,
for instance, as a hybridization probe or a polymerase chain
reaction (PCR) primer. Uses of the nucleic acid molecules of the
present invention that do not encode a polypeptide having
Neutrokine-alpha and/or Neutrokine-alphaSV activity include, inter
alia, (1) isolating the Neutrokine-alpha and/or Neutrokine-alphaSV
gene or allelic variants thereof in a cDNA library; (2) in situ
hybridization (e.g., "FISH") to metaphase chromosomal spreads to
provide precise chromosomal location of the Neutrokine-alpha and/or
Neutrokine-alphaSV gene, as described in Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
(1988); and Northern Blot analysis for detecting Neutrokine-alpha
and/or Neutrokine-alphaSV mRNA expression in specific tissues.
[0174] Preferred, however, are nucleic acid molecules having
sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the nucleic acid sequences disclosed herein (e.g., the
nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) and the
nucleic acid sequence of the deposited cDNAs, or fragments
thereof), which do, in fact, encode a polypeptide having
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide functional
activity (e.g., biological activity). Also preferred are nucleic
acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%,
96%, 97%, 98% or 99% identical to the nucleic acid sequence shown
in FIGS. 5A and 5B (SEQ ID NO:18) or to the nucleic acid sequence
of the deposited cDNA which do, in fact, encode a polypeptide
having Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
functional activity (e.g., biological activity). Also preferred are
nucleic acid molecules having sequences at least 80%, 85%, 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown SEQ ID NOs:21, 22, 27, 29, or 37, which do, in fact,
encode a polypeptide having Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide functional activity (e.g.,
biological activity).
[0175] By "a polypeptide having Neutrokine-alpha polypeptide
functional activity" (e.g., biological activity) and "a polypeptide
having Neutrokine-alphaSV polypeptide functional activity" (e.g.,
biological activity) are intended polypeptides exhibiting activity
similar, but not necessarily identical, to an activity of the
extracellular domain or the full-length Neutrokine-alpha or
Neutrokine-alphaSV polypeptides of the invention, as measured in a
particular functional assay (e.g., immunological or biological
assay). For example, Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide functional activity can be measured by the ability of a
polypeptide sequence described herein to form multimers (e.g.,
homodimers and homotrimers) with the complete Neutrokine-alpha
and/or Neutrokine-alphaSV or extracellular domain of
Neutrokine-alpha and/or Neutrokine-alphaSV, and to bind a
Neutrokine-alpha and/or Neutrokine-alphaSV ligand. Additionally,
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide functional
activity can be measured by the ability of a polypeptide sequence
described herein to form heteromultimers with APRIL (e.g., SEQ ID
NO:20 and SEQ ID NO:47) or APRIL fragments or variants, especially
the extracellular soluble domain of APRIL (e.g., amino acids
105-250 of SEQ ID NO:47). Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide functional activity can be also be
measured by determining the ability of a polypeptide of the
invention to induce lymphocyte (e.g., B cell) proliferation,
differentiation or activation and/or to extend B cell survival.
These functional assays can be routinely performed using techniques
described herein (e.g., see Example 6) and otherwise known in the
art. Additionally, Neutrokine-alpha or Neutrokine-alphaSV
polypeptides of the present invention modulate cell proliferation,
cytotoxicity, cell survival and cell death. An in vitro cell
proliferation, cytotoxicity, cell survival, and cell death assay
for measuring the effect of a protein on certain cells can be
performed by using reagents well known and commonly available in
the art for detecting cell replication and/or death. For instance,
numerous such assays for TNF-related protein activities are
described in the various references in this disclosure. Briefly, an
example of such an assay involves collecting human or animal (e.g.,
mouse) cells and mixing with (1) transfected host cell-supernatant
containing Neutrokine-alpha protein (or a candidate polypeptide) or
(2) nontransfected host cell-supernatant control, and measuring the
effect on cell numbers or viability after incubation of certain
period of time. Such cell proliferation and/or survival modulation
activities as can be measured in this type of assay are useful for
treating tumor, tumor metastasis, infections, autoimmune diseases,
inflammation and other immune-related diseases.
[0176] Neutrokine-alpha modulates cell proliferation and
differentiation in a dose-dependent manner in the above-described
assay. Accordingly, it is preferred that "a polypeptide having
Neutrokine-alpha polypeptide functional activity" (e.g., biological
activity) includes polypeptides that also exhibit any of the same
cell modulatory (particularly immunomodulatory) activities in the
above-described assays in a dose-dependent manner. Although the
degree of dose-dependent activity need not be identical to that of
the Neutrokine-alpha polypeptides, preferably, "a polypeptide
having Neutrokine-alpha polypeptide functional activity" will
exhibit substantially similar dose-dependence in a given activity
as compared to the Neutrokine-alpha polypeptides (i.e., the
candidate polypeptide will exhibit greater activity or not more
than about 25-fold less and, preferably, not more than about
tenfold less activity relative to the reference Neutrokine-alpha
polypeptides).
[0177] In certain preferred embodiments, "a polypeptide having
Neutrokine-alpha polypeptide functional activity" (e.g., biological
activity) and "a polypeptide having Neutrokine-alphaSV polypeptide
functional activity" (e.g., biological activity) includes
polypeptides that also exhibit any of the same B cell (or other
cell type) modulatory (particularly immunomodulatory) activities
described in FIGS. 8A, 8B, 8C, 9A, 9B, 10A, 10B, 10C, 10D, 10E,
10F, 11A, 11B, 11C, 11D, 11E, and 11F and in Example 6.
[0178] Like other members of TNF family, Neutrokine-alpha exhibits
activity on leukocytes including, for example, monocytes,
lymphocytes (e.g., B cells) and neutrophils. For this reason
Neutrokine-alpha is active in directing the proliferation,
differentiation and migration of these cell types. Such activity is
useful for immune enhancement or suppression, myeloprotection, stem
cell mobilization, acute and chronic inflammatory control and
treatment of leukemia. Assays for measuring such activity are known
in the art. For example, see Peters et al., Immun. Today 17:273
(1996); Young et al., J. Exp. Med. 182:1111 (1995); Caux et al.,
Nature 390:258 (1992); and Santiago-Schwarz et al., Adv. Exp. Med.
Biol. 378:7 (1995).
[0179] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
nucleic acid sequence contained in cDNA clone deposited in ATCC
accession no. 97768, or the nucleic acid sequence shown in FIGS. 1A
and 1B (SEQ ID NO:1), or fragments thereof, will encode a
polypeptide "having Neutrokine-alpha polypeptide functional
activity" (e.g., biological activity). One of ordinary skill in the
art will also immediately recognize that a large number of the
nucleic acid molecules having a sequence at least 80%, 85%, 90%,
92%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence contained in cDNA clone deposited in ATCC accession no.
203518 or the nucleic acid sequence shown in FIGS. 5A and 5B (SEQ
ID NO:18) will encode a polypeptide "having Neutrokine-alphaSV
polypeptide functional activity" (e.g., biological activity). In
fact, since degenerate variants of these nucleotide sequences all
encode the same polypeptide, this will be clear to the skilled
artisan even without performing the above described comparison
assay. It will be further recognized in the art that, for such
nucleic acid molecules that are not degenerate variants, a
reasonable number will also encode a polypeptide having
Neutrokine-alpha and/or Neutrokine-alphaSV activity. This is
because the skilled artisan is fully aware of amino acid
substitutions that are either less likely or not likely to
significantly effect protein function (e.g., replacing one
aliphatic amino acid with a second aliphatic amino acid), as
further described below.
[0180] Similarly, polynucleotides encoding polypeptides which
contain all or some portion of the region V-142 through K-160 of
SEQ ID NO:2 are likely to be valuable diagnostic and therapeutic
polynucleotides with regard to detecting and/or altering expression
of either Neutrokine-alpha or Neutrokine-alphaSV polynucleotides.
In addition, polynucleotides which span the junction of amino acid
residues T-141 and G-142 of the Neutrokine-alphaSV polypeptide
shown in SEQ ID NO:19 (in between which the V-142 through K-160
amino acid sequence of Neutrokine-alpha is apparently inserted),
are also likely to be useful both diagnostically and
therapeutically. Such T-141/G-142 spanning polynucleotides will
exhibit a much higher likelihood of hybridization with
Neutrokine-alphaSV polynucleotides than with Neutrokine-alpha
polynucleotides. A partial, non-limiting, non-exclusive list of
such Neutrokine-alphaSV polypeptides which are encoded by
polynucleotides of the invention includes polypeptides comprising,
or alternatively consisting of, an amino acid sequence selected
from the following: G-121 through E-163; E-122 through E-163; G-123
through E-163; N-124 through E-163; S-125 through E-163; S-126
through E-163; Q-127 through E-163; N-128 through E-163; S-129
through E-163; R-130 through E-163; N-131 through E-163; K-132
through E-163; R-133 through E-163; A-134 through E-163; V-135
through E-163; Q-136 through E-163; G-137 through E-163; P-138
through E-163; E-139 through E-163; E-140 through E-163; T-141
through E-163; G-142 through E-163; S-143 through E-163; Y-144
through E-163; T-145 through E-163; F-146 through E-163; V-147
through E-163; P-148 through E-163; W-149 through E-163; L-150
through E-163; L-151 through E-163; S-152 through E-163; F-153
through E-163; K-154 through E-163; R-155 through E-163; G-156
through E-163; S-157 through E-163; A-158 through E-163; L-159
through E-163; E-160 through E-163; E-161 through E-163; K-162
through E-163; G-121 through K-162; G-121 through E-161; G-121
through E-160; G-121 through L-159; G-121 through A-158; G-121
through S-157; G-121 through G-156; G-121 through R-155; G-121
through K-154; G-121 through F-153; G-121 through S-152; G-121
through L-151; G-121 through L-150; G-121 through W-149; G-121
through P-148; G-121 through V-147; G-121 through F-146; G-121
through T-145; G-121 through Y-144; G-121 through S-143; G-121
through G-142; G-121 through T-141; G-121 through E-140; G-121
through E-139; G-121 through P-138; G-121 through G-137; G-121
through Q-136; G-121 through V-135; G-121 through A-134; G-121
through R-133; G-121 through K-132; G-121 through N-131; G-121
through R-130; G-121 through S-129; G-121 through N-128; G-121
through Q-127; through S-126; G-121 through S-125; G-121 through
N-124; G-121 through G-123; and G-121 through E-122 of SEQ ID
NO:19. Polypeptides encoded by these polynucleotides are also
encompassed by the invention.
Vectors and Host Cells
[0181] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, or
which are otherwise engineered to produce the polypeptides of the
invention, and the production of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides, or fragments thereof, by
recombinant or synthetic techniques.
[0182] In one embodiment, the polynucleotides of the invention are
joined to a vector (e.g., a cloning or expression vector). The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells. The
polynucleotides may be joined to a vector containing a selectable
marker for propagation in a host. Introduction of the vector
construct into the host cell can be effected by techniques known in
the art which include, but are not limited to, calcium phosphate
transfection, DEAE-dextran mediated transfection, cationic
lipid-mediated transfection, electroporation, transduction,
infection or other methods. Such methods are described in many
standard laboratory manuals, such as Davis et al., Basic Methods In
Molecular Biology (1986).
[0183] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), a-factor, acid phosphatase, or heat shock proteins,
among others. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, for example,
stabilization or simplified purification of expressed recombinant
product.
[0184] In one embodiment, the DNA of the invention is operatively
associated with an appropriate heterologous regulatory element
(e.g., promoter or enhancer), such as, the phage lambda PL
promoter, the E. coli lac, trp, phoA, and tac promoters, the SV40
early and late promoters and promoters of retroviral LTRs, to name
a few. Other suitable promoters will be known to the skilled
artisan.
[0185] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae
or Pichia pastoris (ATCC Accession No. 201178)); insect cells such
as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
CHO, COS, 293 and Bowes melanoma cells; and plant cells.
Appropriate culture mediums and conditions for the above-described
host cells are known in the art.
[0186] The host cell can be a higher eukaryotic cell, such as a
mammalian cell (e.g., a human derived cell), or a lower eukaryotic
cell, such as a yeast cell, or the host cell can be a prokaryotic
cell, such as a bacterial cell. The host strain may be chosen which
modulates the expression of the inserted gene sequences, or
modifies and processes the gene product in the specific fashion
desired. Expression from certain promoters can be elevated in the
presence of certain inducers; thus expression of the genetically
engineered polypeptide may be controlled. Furthermore, different
host cells have characteristics and specific mechanisms for the
translational and post-translational processing and modification
(e.g., phosphorylation, cleavage) of proteins. Appropriate cell
lines can be chosen to ensure the desired modifications and
processing of the foreign protein expressed. Selection of
appropriate vectors and promoters for expression in a host cell is
a well-known procedure and the requisite techniques for expression
vector construction, introduction of the vector into the host and
expression in the host are routine skills in the art.
[0187] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium, and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice. As a
representative, but nonlimiting example, useful expression vectors
for bacterial use can comprise a selectable marker and bacterial
origin of replication derived from commercially available plasmids
comprising genetic elements of the well-known cloning vector pBR322
(ATCC 37017). Such commercial vectors include, for example,
pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1
(Promega Biotec, Madison, Wis., USA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the
structural sequence to be expressed. Among vectors preferred for
use in bacteria include pHE4-5 (ATCC Accession No. 209311; and
variations thereof), pQE70, pQE60 and pQE-9, available from QIAGEN,
Inc., supra; pBS vectors, Phagescript vectors, Bluescript vectors,
pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from
Pharmacia. Preferred expression vectors for use in yeast systems
include, but are not limited to, pYES2, pYD1, pTEF1/Zeo, pYES2/GS,
pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1,
pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen,
Carlsbad, Calif.). Among preferred eukaryotic vectors are pWLNEO,
pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL (available from Pharmacia). Other suitable
vectors will be readily apparent to the skilled artisan.
[0188] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period. Cells are typically harvested by
centrifugation, disrupted by physical or chemical means, and the
resulting crude extract retained for further purification.
[0189] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well know to those skilled in the art.
[0190] In one embodiment, the yeast Pichia pastoris is used to
express Neutrokine-alpha protein in a eukaryotic system. Pichia
pastoris is a methylotrophic yeast which can metabolize methanol as
its sole carbon source. A main step in the methanol metabolization
pathway is the oxidation of methanol to formaldehyde using O2. This
reaction is catalyzed by the enzyme alcohol oxidase. In order to
metabolize methanol as its sole carbon source, Pichia pastoris must
generate high levels of alcohol oxidase due, in part, to the
relatively low affinity of alcohol oxidase for O2. Consequently, in
a growth medium depending on methanol as a main carbon source, the
promoter region of one of the two alcohol oxidase genes (AOX1) is
highly active. In the presence of methanol, alcohol oxidase
produced from the AOX1 gene comprises up to approximately 30% of
the total soluble protein in Pichia pastoris. See, Ellis, S. B., et
al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast
5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.
15:3859-76 (1987). Thus, a heterologous coding sequence, such as,
for example, a Neutrokine-alpha or Neutrokine-alphaSV
polynucleotide of the present invention, under the transcriptional
regulation of all or part of the AOX1 regulatory sequence is
expressed at exceptionally high levels in Pichia yeast grown in the
presence of methanol.
[0191] In one example, the plasmid vector pPIC9K is used to express
DNA encoding a Neutrokine-alpha or Neutrokine-alphaSV polypeptide
of the invention, as set forth herein, in a Pichea yeast system
essentially as described in "Pichia Protocols: Methods in Molecular
Biology," D. R. Higgins and J. Cregg, eds. The Humana Press,
Totowa, N.J., 1998. This expression vector allows expression and
secretion of a Neutrokine-alpha or Neutrokine-alphaSV protein of
the invention by virtue of the strong AOX1 promoter linked to the
Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide
(i.e., leader) located upstream of a multiple cloning site.
[0192] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815,
as one skilled in the art would readily appreciate, as long as the
proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG as required.
[0193] In one embodiment, high-level expression of a heterologous
coding sequence, such as, for example, a Neutrokine-alpha or
Neutrokine-alphaSV polynucleotide of the present invention, may be
achieved by cloning the heterologous polynucleotide of the
invention into an expression vector such as, for example, pGAPZ or
pGAPZalpha, and growing the yeast culture in the absence of
methanol.
[0194] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples including the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0195] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman (Cell 23:175 (1981)), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0196] In a specific embodiment, constructs designed to express a
portion of the extracellular domain of the Neutrokine-alpha (e.g.,
amino acid residues Ala-134 through Leu-285) are preferred. One of
skill in the art would be able to use the polynucleotide and
polypeptide sequences provided as SEQ ID NO:1 and SEQ ID NO:2,
respectively, or SEQ ID NO:18 and SEQ ID NO:19, respectively, to
design polynucleotide primers to generate such an expression
construct.
[0197] In another embodiment, constructs designed to express the
entire predicted extracellular domain of the Neutrokine-alpha
(i.e., amino acid residues Gln-73 through Leu-285) are preferred.
One of skill in the art would be able to use the polynucleotide and
polypeptide sequences provided as SEQ ID NO:1 and SEQ ID NO:2,
respectively, or SEQ ID NO:18 and SEQ ID NO:19, respectively, to
design polynucleotide primers to generate such an expression
construct.
[0198] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g.,
Neutrokine-alpha coding sequence), and/or to include genetic
material (e.g., heterologous polynucleotide sequences) that is
operably associated with Neutrokine-alpha polynucleotides of the
invention, and which activates, alters, and/or amplifies endogenous
Neutrokine-alpha polynucleotides. For example, techniques known in
the art may be used to operably associate heterologous control
regions (e.g., promoter and/or enhancer) and endogenous
Neutrokine-alpha polynucleotide sequences via homologous
recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24,
1997; International Publication No. WO 96/29411, published Sep. 26,
1996; International Publication No. WO 94/12650, published Aug. 4,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989), the
disclosures of each of which are incorporated by reference in their
entireties).
[0199] The host cells described infra can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, cell-free translation systems can also be
employed to produce the polypeptides of the invention using RNAs
derived from the DNA constructs of the present invention.
[0200] The polypeptide of the invention may be expressed or
synthesized in a modified form, such as a fusion protein
(comprising the polypeptide joined via a peptide bond to a
heterologous protein sequence (of a different protein)), and may
include not only secretion signals, but also additional
heterologous functional regions. Such a fusion protein can be made
by ligating polynucleotides of the invention and the desired
nucleic acid sequence encoding the desired amino acid sequence to
each other, by methods known in the art, in the proper reading
frame, and expressing the fusion protein product by methods known
in the art. Alternatively, such a fusion protein can be made by
protein synthetic techniques, e.g., by use of a peptide
synthesizer. Thus, for instance, a region of additional amino
acids, particularly charged amino acids, may be added to the
N-terminus of the polypeptide to improve stability and persistence
in the host cell, during purification, or during subsequent
handling and storage. Also, peptide moieties may be added to the
polypeptide to facilitate purification. Such regions may be removed
prior to final preparation of the polypeptide. The addition of
peptide moieties to polypeptides to engender secretion or
excretion, to improve stability and to facilitate purification,
among others, are familiar and routine techniques in the art.
[0201] In one embodiment, polynucleotides encoding Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides of the invention may be
fused to signal sequences which will direct the localization of a
protein of the invention to particular compartments of a
prokaryotic or eukaryotic cell and/or direct the secretion of a
protein of the invention from a prokaryotic or eukaryotic cell. For
example, in E. coli, one may wish to direct the expression of the
protein to the periplasmic space. Examples of signal sequences or
proteins (or fragments thereof) to which the polypeptides of the
invention may be fused in order to direct the expression of the
polypeptide to the periplasmic space of bacteria include, but are
not limited to, the pelB signal sequence, the maltose binding
protein (MBP) signal sequence, MBP, the ompA signal sequence, the
signal sequence of the periplasmic E. coli heat-labile enterotoxin
B-subunit, and the signal sequence of alkaline phosphatase. Several
vectors are commercially available for the construction of fusion
proteins which will direct the localization of a protein, such as
the pMAL series of vectors (particularly the pMAL-p series)
available from New England Biolabs. In a specific embodiment,
polynucleotides encoding Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the invention may be fused to the pelB pectate
lyase signal sequence to increase the efficiency of expression and
purification of such polypeptides in Gram-negative bacteria. See,
U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are
herein incorporated by reference in their entireties.
[0202] Examples of signal peptides that may be fused to a
polypeptide of the invention in order to direct its secretion in
mammalian cells include, but are not limited to, the MPIF-1 signal
sequence (amino acids 1-21 of GenBank Accession number AAB51134),
the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID
NO:45), and a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG,
SEQ ID NO:46). A suitable signal sequence that may be used in
conjunction with baculoviral expression systems is the gp67 signal
sequence, (amino acids 1-19 of GenBank Accession Number
AAA72759).
[0203] A preferred fusion protein comprises a heterologous region
from immunoglobulin that is useful to stabilize and purify
proteins. For example, EP-A-O 464 533 (Canadian counterpart
2045869) discloses fusion proteins comprising various portions of
constant region of immunoglobulin molecules together with another
human protein or part thereof. In many cases, the Fc part in a
fusion protein is thoroughly advantageous for use in therapy and
diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5 has been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and
K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0204] Polypeptides of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the polypeptides of the
present invention may be glycosylated or may be non-glycosylated.
In addition, polypeptides of the invention may also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes.
[0205] Polypeptides of the invention can be chemically synthesized
using techniques known in the art (e.g., see Creighton, 1983,
Proteins: Structures and Molecular Principles, W.H. Freeman &
Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310:105-111).
For example, a peptide corresponding to a fragment of the complete
Neutrokine-alpha or Neutrokine-alphaSV polypeptides of the
invention can be synthesized by use of a peptide synthesizer.
Furthermore, if desired, nonclassical amino acids or chemical amino
acid analogs can be introduced as a substitution or addition into
the Neutrokine-alpha or Neutrokine-alphaSV polynucleotide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4-diaminobutyric acid,
a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric
acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid, 3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino
acids such as b-methyl amino acids, Ca-methyl amino acids,
Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0206] The invention encompasses Neutrokine-alpha or
Neutrokine-alphaSV polypeptides which are differentially modified
during or after translation, e.g., by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to an
antibody molecule or other cellular ligand, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including but not limited, to specific chemical cleavage by
cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
NaBH.sub.4, acetylation, formylation, oxidation, reduction,
metabolic synthesis in the presence of tunicamycin, etc.
[0207] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, radioisotopic or affinity label to allow for detection
and isolation of the protein.
[0208] Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, glucose oxidase or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include biotin, umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and acquorin; and examples of suitable radioactive
material include a radioactive metal ion, e.g., alpha-emitters such
as, for example, .sup.213Bi, or other radioisotopes such as, for
example, iodine (.sup.131I, .sup.125I, .sup.123I, .sup.121I),
carbon (.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.115mIn, .sup.113mIn, .sup.112In, .sup.111In), and technetium
(.sup.99Tc, .sup.99mTc), thallium (.sup.201Ti), gallium (.sup.68Ga,
.sup.67Ga), palladium (.sup.103Pd), molybdenum (.sup.99Mo), xenon
(.sup.133Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu,
.sup.159Gd, .sup.149 Pm, .sup.140La, .sup.175Yb, .sup.166Ho,
.sup.90Y, .sup.47Sc, .sup.186 Re, .sup.42Pr, .sup.105 Rh,
.sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P,
.sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se,
.sup.113Sn, and .sup.117Tin.
[0209] In specific embodiments, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention may be labeled
with Europium. For example, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention may be labelled
with Europium using the DELFIA Eu-labeling kit (catalog# 1244-302,
Perkin Elmer Life Sciences, Boston, Mass.) following manufacturer's
instructions.
[0210] In specific embodiments, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention are attached to
macrocyclic chelators useful for conjugating radiometal ions,
including but not limited to, .sup.111In, .sup.177Lu, .sup.90Y,
.sup.166Ho, and .sup.153Sm, to polypeptides. In a preferred
embodiment, the radiometal ion associated with the macrocyclic
chelators attached to Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the invention is .sup.111In. In another preferred
embodiment, the radiometal ion associated with the macrocyclic
chelator attached to Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the invention is .sup.90Y. In specific embodiments,
the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide of the
invention via a linker molecule. Examples of linker molecules
useful for conjugating DOTA to a polypeptide are commonly known in
the art--see, for example, DeNardo et al., Clin Cancer Res.
4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7,
1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999
which are hereby incorporated by reference in their entirety. In
addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose
chelating agents that may be conjugated to antibodies, and methods
for making and using them, are hereby incorporated by reference in
their entireties. Though U.S. Pat. Nos. 5,652,361 and 5,756,065
focus on conjugating chelating agents to antibodies, one skilled in
the art could readily adapt the method disclosed therein in order
to conjugate chelating agents to other polypeptides.
[0211] In one embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention may be labeled
with biotin. In other related embodiments, biotinylated
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the
invention may be used, for example, as an imaging agent or as a
means of identifying one or more Neutrokine-alpha and/or
Neutrokine-alphaSV receptor(s) or other coreceptor or coligand
molecules.
[0212] Also provided by the invention are chemically modified
derivatives of Neutrokine-alpha or Neutrokine-alphaSV which may
provide additional advantages such as increased solubility,
stability and in vivo or in vitro circulating time of the
polypeptide, or decreased immunogenicity (see U.S. Pat. No.
4,179,337). The chemical moieties for derivitization may be
selected from water soluble polymers such as polyethylene glycol,
ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0213] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about
200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000,
18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,
50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,
90,000, 95,000, or 100,000 kDa.
[0214] As noted above, the polyethylene glycol may have a branched
structure. Branched polyethylene glycols are described, for
example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl.
Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are
incorporated herein by reference.
[0215] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include, for example, lysine
residues and the N-terminal amino acid residues; those having a
free carboxyl group may include aspartic acid residues, glutamic
acid residues, and the C-terminal amino acid residue. Sulfhydryl
groups may also be used as a reactive group for attaching the
polyethylene glycol molecules. Preferred for therapeutic purposes
is attachment at an amino group, such as attachment at the
N-terminus or lysine group.
[0216] As suggested above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to a proteins via
covalent bonds to lysine, histidine, aspartic acid, glutamic acid,
or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid
residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the protein or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the protein.
[0217] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration, one
may select from a variety of polyethylene glycol molecules (by
molecular weight, branching, etc.), the proportion of polyethylene
glycol molecules to protein (or peptide) molecules in the reaction
mix, the type of pegylation reaction to be performed, and the
method of obtaining the selected N-terminally pegylated protein.
The method of obtaining the N-terminally pegylated preparation
(i.e., separating this moiety from other monopegylated moieties if
necessary) may be by purification of the N-terminally pegylated
material from a population of pegylated protein molecules.
Selective proteins chemically modified at the N-terminus
modification may be accomplished by reductive alkylation which
exploits differential reactivity of different types of primary
amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate
reaction conditions, substantially selective derivatization of the
protein at the N-terminus with a carbonyl group containing polymer
is achieved.
[0218] As indicated above, pegylation of the proteins of the
invention may be accomplished by any number of means. For example,
polyethylene glycol may be attached to the protein either directly
or by an intervening linker. Linkerless systems for attaching
polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et
al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No.
4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466,
the disclosures of each of which are incorporated herein by
reference.
[0219] One system for attaching polyethylene glycol directly to
amino acid residues of proteins without an intervening linker
employs tresylated MPEG, which is produced by the modification of
monomethoxy polyethylene glycol (MPEG) using tresylchloride
(ClSO.sub.2CH.sub.2CF.sub.3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine
groups of the protein. Thus, the invention includes
protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule
having a 2,2,2-trifluoroethane sulphonyl group.
[0220] Polyethylene glycol can also be attached to proteins using a
number of different intervening linkers. For example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by
reference, discloses urethane linkers for connecting polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein
the polyethylene glycol is attached to the protein by a linker can
also be produced by reaction of proteins with compounds such as
MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,
MPEG-p-nitrophenolcarbonate, and various MPEG-succinate
derivatives. A number additional polyethylene glycol derivatives
and reaction chemistries for attaching polyethylene glycol to
proteins are described in WO 98/32466, the entire disclosure of
which is incorporated herein by reference. Pegylated protein
products produced using the reaction chemistries set out herein are
included within the scope of the invention.
[0221] The number of polyethylene glycol moieties attached to each
protein of the invention (i.e., the degree of substitution) may
also vary. For example, the pegylated proteins of the invention may
be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, or more polyethylene glycol molecules. Similarly, the
average degree of substitution within ranges such as 1-3, 2-4, 3-5,
4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16,
15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per
protein molecule. Methods for determining the degree of
substitution are discussed, for example, in Delgado et al., Crit.
Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[0222] The Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides
can be recovered and purified by known methods which include, but
are not limited to, ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography ("HPLC") is employed for
purification.
Neutrokine-Alpha Polypeptides
[0223] The Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides
of the invention may be in monomers or multimers (i.e., dimers,
trimers, tetramers and higher multimers). Accordingly, the present
invention relates to monomers and multimers of the Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides of the invention, their
preparation, and compositions (preferably, pharmaceutical
compositions) containing them. In specific embodiments, the
polypeptides of the invention are monomers, dimers, trimers or
tetramers. In additional embodiments, the multimers of the
invention are at least dimers, at least trimers, or at least
tetramers.
[0224] In a nonexclusive embodiment, the multimers of the invention
comprise at least one Neutrokine-alpha-human serum albumin fusion
protein as described herein. In another nonexclusive embodiment,
the multimers of the invention are trimeric and comprise one, two
or three Neutrokine-alpha-human serum albumin fusion proteins as
described herein.
[0225] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the invention (including Neutrokine-alpha and/or
Neutrokine-alphaSV fragments, variants, and fusion proteins, as
described herein). These homomers may contain Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides having identical or
different amino acid sequences. In a specific embodiment, a homomer
of the invention is a multimer containing only Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides having an identical amino
acid sequence. In another specific embodiment, a homomer of the
invention is a multimer containing Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides having different amino acid
sequences. In specific embodiments, the multimer of the invention
is a homodimer (e.g., containing Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides having identical or different amino
acid sequences) or a homotrimer (e.g., containing Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides having identical or
different amino acid sequences). In a preferred embodiment, the
multimer of the invention is a homotrimer. In additional
embodiments, the homomeric multimer of the invention is at least a
homodimer, at least a homotrimer, or at least a homotetramer.
[0226] As used herein, the term heteromer refers to a multimer
containing heterologous polypeptides (i.e., polypeptides of a
different protein) in addition to the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention. In a specific
embodiment, the multimer of the invention is a heterodimer, a
heterotrimer, or a heterotetramer. In additional embodiments, the
heteromeric multimer of the invention is at least a heterodimer, at
least a heterotrimer, or at least a heterotetramer. In highly
preferred embodiments, the heteromeric multimer of the invention is
a heterotrimer comprising both Neutrokine alpha-polypeptides and
APRIL polypeptides (e.g., SEQ ID NO:20 or SEQ ID NO:47; PCT
International Publication Number WO97/33902; GenBank Accession No.
AF046888 (nucleotide) and AAC6132 (protein); J. Exp. Med.
188(6):1185-1190). In other highly preferred embodiments, the
heteromeric multimer of the invention is a heterotrimer consisting
of one Neutrokine alpha-polypeptide and two APRIL polypeptides. In
other highly preferred embodiments, the heteromeric multimer of the
invention is a heterotrimer consisting of two Neutrokine
alpha-polypeptides and one APRIL polypeptide.
[0227] In a further nonexclusive embodiment, the heteromers of the
invention comprise CD40 ligand polypeptide sequence(s), or
biologically active fragment(s) or variant(s) thereof.
[0228] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides of the invention. Such
covalent associations may involve one or more amino acid residues
contained in the polypeptide sequence (e.g., that recited in SEQ ID
NO:2 or SEQ ID NO:19, or contained in the polypeptide encoded by
the clones deposited in connection with this application). In one
instance, the covalent associations are cross-linking between
cysteine residues located within the polypeptide sequences which
interact in the native (i.e., naturally occurring) polypeptide. In
another instance, the covalent associations are the consequence of
chemical or recombinant manipulation. Alternatively, such covalent
associations may involve one or more amino acid residues contained
in the heterologous polypeptide sequence in a Neutrokine-alpha
and/or Neutrokine-alphaSV fusion protein. In one example, covalent
associations are between the heterologous sequence contained in a
fusion protein of the invention (see, e.g., U.S. Pat. No.
5,478,925). In a specific example, the covalent associations are
between the heterologous sequence contained in a
Neutrokine-alpha-Fc and/or Neutrokine-alphaSV-Fc fusion protein of
the invention (as described herein). In another specific example,
covalent associations of fusion proteins of the invention are
between heterologous polypeptide sequence from another TNF family
ligand/receptor member that is capable of forming covalently
associated multimers, such as for example, osteoprotegerin (see,
e.g., International Publication No. WO 98/49305, the contents of
which are herein incorporated by reference in its entirety). In
another specific example, covalent associations of fusion proteins
of the invention are between heterologous polypeptide sequence from
CD40L, or a soluble fragment thereof. In another embodiment, two or
more Neutrokine-alpha and/or Neutrokine-alpha polypeptides of the
invention are joined through synthetic linkers (e.g., peptide,
carbohydrate or soluble polymer linkers). Examples include those
peptide linkers described in U.S. Pat. No. 5,073,627 (hereby
incorporated by reference). Proteins comprising multiple
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides separated
by peptide linkers may be produced using conventional recombinant
DNA technology.
[0229] Another method for preparing multimer Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides of the invention involves
use of Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides
fused to a leucine zipper or isoleucine zipper polypeptide
sequence. Leucine zipper or isoleucine zipper domains are
polypeptides that promote multimerization of the proteins in which
they are found. Leucine zippers were originally identified in
several DNA-binding proteins (Landschulz et al., Science 240:1759,
(1988)), and have since been found in a variety of different
proteins. Among the known leucine zippers or isoleucine zippers are
naturally occurring peptides and derivatives thereof that dimerize
or trimerize. Examples of leucine zipper domains suitable for
producing soluble multimeric Neutrokine-alpha and/or
Neutrokine-alphaSV proteins are those described in PCT application
WO 94/10308, hereby incorporated by reference. Recombinant fusion
proteins comprising a soluble Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide fused to a peptide that dimerizes or
trimerizes in solution are expressed in suitable host cells, and
the resulting soluble multimeric Neutrokine-alpha and/or
Neutrokine-alphaSV is recovered from the culture supernatant using
techniques known in the art.
[0230] Certain members of the TNF family of proteins are believed
to exist in trimeric form (Beutler and Huffel, Science 264:667,
1994; Banner et al., Cell 73:431, 1993). Thus, trimeric
Neutrokine-alpha and/or Neutrokine-alphaSV may offer the advantage
of enhanced biological activity. Preferred leucine zipper moieties
are those that preferentially form trimers. One example is a
leucine zipper derived from lung surfactant protein D (SPD), as
described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in
U.S. patent application Ser. No. 08/446,922, hereby incorporated by
reference. Other peptides derived from naturally occurring trimeric
proteins may be employed in preparing trimeric Neutrokine-alpha
and/or Neutrokine-alphaSV.
[0231] In another example, proteins of the invention are associated
by interactions between the Flag.RTM. polypeptide sequence
contained in Flag.RTM.-Neutrokine alpha or
Flag.RTM.-Neutrokine-alphaSV fusion proteins of the invention. In a
further embodiment, proteins of the invention are associated by
interactions between the heterologous polypeptide sequence
contained in Flag.RTM.-Neutrokine-alpha or
Flag.RTM.-Neutrokine-alphaSV fusion proteins of the invention and
anti-Flag.RTM. antibody.
[0232] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Further, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[0233] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain and which can be incorporated by membrane
reconstitution techniques into liposomes (see, e.g., U.S. Pat. No.
5,478,925, which is herein incorporated by reference in its
entirety).
[0234] In one embodiment, the invention provides an isolated
Neutrokine-alpha polypeptide having the amino acid sequence encoded
by the cDNA clone contained in ATCC No. 97768, or the amino acid
sequence in FIGS. 1A and 1B (SEQ ID NO:2), or a polypeptide
comprising a portion (i.e., a fragment) of the above polypeptides.
In another embodiment, the invention provides an isolated
Neutrokine-alphaSV polypeptide having the amino acid encoded by the
cDNA clone contained in ATCC No. 203518, or the amino acid sequence
in FIGS. 5A and 5B (SEQ ID NO:19), or a polypeptide comprising a
portion (i.e, fragment) of the above polypeptides.
[0235] Polypeptide fragments of the present invention include
polypeptides comprising or alternatively, consisting of, an amino
acid sequence contained in SEQ ID NO:2, encoded by the cDNA
contained in the plasmid having ATCC accession number 97768, or
encoded by nucleic acids which hybridize (e.g., under stringent
hybridization conditions) to the nucleotide sequence contained in
the deposited clone, or the complementary strand of the nucleotide
sequence shown in FIGS. 1A-B (SEQ ID NO:1.
[0236] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:19, encoded by the cDNA
contained in the plasmid having ATCC accession number 203518, or
encoded by nucleic acids which hybridize (e.g., under stringent
hybridization conditions) to the nucleotide sequence contained in
the deposited clone, or the complementary strand of the nucleotide
sequence shown in FIGS. 5A-B (SEQ ID NO:18).
[0237] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence encoded by nucleic acids which hybridize (e.g.,
under hybridization conditions described herein) to the
complementary strand of the nucleotide sequence shown in SEQ ID
NO:21.
[0238] Polypeptide fragments of the present invention also include
polypeptides comprising or alternatively, consisting of, an amino
acid sequence contained in SEQ ID NO:23, or encoded by nucleic
acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of the nucleotide
sequence shown in SEQ ID NO:22.
[0239] In addition, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:28, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of the nucleotide
sequence shown in SEQ ID NO:27.
[0240] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:30, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of the nucleotide
sequence shown in SEQ ID NO:29.
[0241] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:38, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of the nucleotide
sequence shown in SEQ ID NO:37.
[0242] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:39, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of a nucleotide
sequence encoding the polypeptide of SEQ ID NO:39.
[0243] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:40, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of a nucleotide
sequence encoding the polypeptide of SEQ ID NO:40.
[0244] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:41, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of a nucleotide
sequence encoding the polypeptide of SEQ ID NO:41.
[0245] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:42, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of a nucleotide
sequence encoding the polypeptide of SEQ ID NO:42.
[0246] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:43, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of a nucleotide
sequence encoding the polypeptide of SEQ ID NO:43.
[0247] Additionally, polypeptide fragments of the present invention
include polypeptides comprising or alternatively, consisting of, an
amino acid sequence contained in SEQ ID NO:44, or encoded by
nucleic acids which hybridize (e.g., under hybridization conditions
described herein) to the complementary strand of a nucleotide
sequence encoding the polypeptide of SEQ ID NO:44.
[0248] Polypeptide fragments of the present invention include
polypeptides comprising or alternatively, consisting of, an amino
acid sequence contained in SEQ ID NO:2, encoded by the cDNA
contained in the deposited clone, or encoded by nucleic acids which
hybridize (e.g., under stringent hybridization conditions) to the
nucleotide sequence contained in the deposited clone, or shown in
FIGS. 1A and 1B (SEQ ID NO:1) or the complementary strand thereto.
Protein fragments may be "free-standing," or comprised within a
larger polypeptide of which the fragment forms a part or region,
most preferably as a single continuous region. Representative
examples of polypeptide fragments of the invention, include, for
example, fragments that comprise or alternatively, consist of from
about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to
200, 201 to 250, and/or 251 to 285 of SEQ ID NO:2. Moreover,
polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in
length.
[0249] In specific embodiments, polypeptide fragments of the
invention comprise, or alternatively consist of, amino acid
residues: 1-46, 31-44, 47-72, 73-285, 73-83, 94-102, 148-152,
166-181, 185-209, 210-221, 226-237, 244-249, 253-265, and/or
277-284, as depicted in FIGS. 1A and 1B (SEQ ID NO:2).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0250] It will be recognized by one of ordinary skill in the art
that mutations targeted to regions of a Neutrokine-alpha
polypeptide of the invention which encompass the nineteen amino
acid residue insertion which is not found in the Neutrokine-alphaSV
polypeptide sequence (i.e., amino acid residues Val-142 through
Lys-160 of the sequence presented in FIGS. 1A and 1B and in SEQ ID
NO:2) may affect the observed biological activities of the
Neutrokine-alpha polypeptide. More specifically, a partial,
non-limiting and non-exclusive list of such residues of the
Neutrokine-alpha polypeptide sequence which may be targeted for
mutation includes the following amino acid residues of the
Neutrokine-alpha polypeptide sequence as shown in SEQ ID NO:2:
V-142; T-143; Q-144; D-145; C-146; L-147; Q-148; L-149; I-150;
A-151; D-152; S-153; E-154; T-155; P-156; T-157; I-158; Q-159; and
K-160. Polynucleotides encoding Neutrokine-alpha polypeptides which
have one or more mutations in the region from V-142 through K-160
of SEQ ID NO:2 are contemplated. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0251] Polypeptide fragments may be "free-standing," or comprised
within a larger polypeptide of which the fragment forms a part or
region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments that comprise or alternatively,
consist of from about amino acid residues: 1 to 15, 16-30, 31-46,
47-55, 56-72, 73-104, 105-163, 163-188, 186-210 and 210-284 of the
amino acid sequence disclosed in SEQ ID NO:2. Additional
representative examples of polypeptide fragments of the invention,
include, for example, fragments that comprise or alternatively,
consist of from about amino acid residues: 1 to 143, 1-150, 47-143,
47-150, 73-143, 73-150, 100-150, 140-145, 142-148, 140-150,
140-200, 140-225, and 140-266 of the amino acid sequence disclosed
in SEQ ID NO:19. Moreover, polypeptide fragments can be at least
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
175 or 200 amino acids in length. In this context, "about" means
the particularly recited ranges and ranges larger or smaller by
several, a few, 5, 4, 3, 2 or 1 amino acid residues at either or
both the amino- and carboxy-termini. Polynucleotides encoding these
polypeptide fragments are also encompassed by the invention.
[0252] Additional preferred embodiments encompass polypeptide
fragments comprising, or alternatively consisting of, the predicted
intracellular domain of Neutrokine-alpha (amino acid residues 1-46
of SEQ ID NO:2), the predicted transmembrane domain of
Neutrokine-alpha (amino acid residues 47-72 of SEQ ID NO:2), the
predicted extracellular domain of Neutrokine-alpha (amino acid
residues 73-285 of SEQ ID NO:2), the predicted TNF conserved domain
of Neutrokine-alpha (amino acids 191 to 284 of SEQ ID NO:2), and a
polypeptide comprising, or alternatively, consisting of the
predicted intracellular domain fused to the predicted extracellular
domain of Neutrokine-alpha (amino acid residues 1-46 fused to amino
acid residues 73-285 of SEQ ID NO:2). Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0253] Further additional preferred embodiments encompass
polypeptide fragments comprising, or alternatively consisting of,
the predicted intracellular domain of Neutrokine-alphaSV (amino
acid residues 1-46 of SEQ ID NO:19), the predicted transmembrane
domain of Neutrokine-alphaSV (amino acid residues 47-72 of SEQ ID
NO:19), the predicted extracellular domain of Neutrokine-alphaSV
(amino acid residues 73-266 of SEQ ID NO:19), the predicted TNF
conserved domain of Neutrokine-alphaSV (amino acids 172 to 265 of
SEQ ID NO:19), and a polypeptide comprising, or alternatively,
consisting of the predicted intracellular domain fused to the
predicted extracellular domain of Neutrokine-alphaSV (amino acid
residues 1-46 fused to amino acid residues 73-266 of SEQ ID NO:19).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0254] Certain additional embodiments of the invention encompass
polypeptide fragments comprising, or alternatively consisting of,
the predicted beta-pleated sheet regions identified in FIGS. 7A-1
and 7A-2. These polypeptide fragments of the invention comprise, or
alternatively consist of, amino acid residues Gln-144 to Ala-151,
Phe-172 to Lys-173, Ala-177 to Glu-179, Asn-183 to Ile-185, Gly-191
to Lys-204, His-210 to Val-219, Leu-226 to Pro-237, Asn-242 to
Ala-251, Gly-256 to Ile-263 and/or Val-276 to Leu-284 of SEQ ID
NO:2. In another, nonexclusive embodiment, these polypeptide
fragments of the invention also comprise, or alternatively consist
of, amino acid residues Phe-153 to Lys-154, Ala-158 to Glu-160,
Asn-164 to Ile-166, Gly-172 to Lys-185, His-191 to Val-200, Leu-207
to Pro-218, Asn-223 to Ala-232, Gly-237 to Ile-244 and/or Val-257
to Leu-265 of SEQ ID NO:19; and amino acid residues Phe-42 to
Lys-43, Ala-47 to Glu-49, Asn-53 to Ile-55, Gly-61 to Pro-74,
His-80 to Val-89, Leu-96 to Pro-107, Asn-112 to Ala-121, Gly-126 to
Ile-133 and/or Asp-146 to Leu-154 of SEQ ID NO:23. In further
nonexclusive embodiments, these polypeptide fragments of the
invention also comprise, or alternatively consist of, amino acid
residues Gln-78 to Ala-85; Phe-106 to Lys-107, Ala-111 to Glu-113,
Asn-117 to Ile-119, Gly-125 to Lys-138, His-144 to Val-153, Leu-160
to Pro-171, Asn-176 to Ala-185, Gly-190 to Ile-197 and/or Val-210
to Leu-218 of SEQ ID NO:28; and amino acid residues Gln-78 to
Ala-85; Phe-106 to Lys-107, Ala-111 to Glu-113, Asn-117 to Ile-119,
Gly-125 to Lys-138, His-144 to Val-153, Leu-160 to Pro-171, Asn-176
to Ala-185, Gly-190 to Ile-197 and/or Val-210 to Leu-218 of SEQ ID
NO:30. Polynucleotides encoding these polypeptide fragments are
also provided.
[0255] A partial, non-limiting, and exemplary list of polypeptides
of the invention which comprise, or alternatively consist of,
combinations of amino acid sequences of the invention includes, for
example, [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to
[Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to
[Val-199 to Ala-248] fused to [Gly-249 to Leu-285] of SEQ ID NO:2;
or [Met-1 to Lys-113] fused to [Val-142 to Lys-160] fused to
[Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to
[Gly-249 to Leu-285] of SEQ ID NO:2; or [Met-1 to Lys-113] fused to
[Leu-114 to Thr-141] fused to [Val-142 to Lys-160] fused to
[Gly-161 to Gln-198] fused to [Gly-249 to Leu-285] of SEQ ID NO:2.
Other combinations may include the polypeptide fragments in an
order other than that recited above (e.g., [Leu-114 to Thr-141]
fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-285] fused
to [Val-142 to Lys-160] of SEQ ID NO:2). Other combinations may
also include heterologous polypeptide fragments as described herein
and/or other polypeptides or polypeptide fragments of the present
invention (e.g., [Met-1 to Lys-113] fused to [Leu-114 to Thr-141]
fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused
to [Gly-249 to Leu-285] of SEQ ID NO:2 fused to a FLAG tag; or
[Met-1 to Lys-113] of SEQ ID NO:2 fused to [Leu-114 to Thr-141] of
SEQ ID NO:2 fused to [Glu-135 to Asn-165] of SEQ ID NO:39 fused to
[Val-142 to Lys-160] of SEQ ID NO:2 fused to [Gly-161 to Gln-198]
of SEQ ID NO:2 fused to [Val-199 to Ala-248] of SEQ ID NO:2 fused
to [Gly-249 to Leu-285] of SEQ ID NO:2). Polynucleotides encoding
any of these polypeptides are encompassed by the invention.
[0256] An additional partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to
[Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to
[Gly-230 to Leu-266] of SEQ ID NO:19; [Met-1 to Lys-113] fused to
[Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to
[Gly-230 to Leu-266] of SEQ ID NO:19; or [Met-1 to Lys-113] fused
to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to
[Gly-230 to Leu-266] of SEQ ID NO:19. Other combinations may
include the polypeptide fragments in an order other than that
recited above (e.g., [Leu-114 to Thr-141] fused to [Val-180 to
Ala-229] fused to [Gly-230 to Leu-266] fused to [Gly-142 to
Gln-179] of SEQ ID NO:19). Other combinations may also include
heterologous polypeptide fragments as described herein and/or other
polypeptides or polypeptide fragments of the present invention
(e.g., [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to
[Gly-142 to Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID NO:19
fused to a FLAG tag or, [Met-1 to Lys-113] of SEQ ID NO:19 fused to
[Leu-114 to Thr-141] of SEQ ID NO:19 fused to [Glu-135 to Asn-165]
of SEQ ID NO:39 fused to [Gly-142 to Gln-179] of SEQ ID NO:19 fused
to [Val-180 to Ala-229] of SEQ ID NO:19 fused to [Gly-230 to
Leu-266] of SEQ ID NO:19). Polynucleotides encoding any of these
polypeptides are encompassed by the invention.
[0257] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Met-1 to Lys-106] fused to [Leu-107 to Thr-134] fused to
[Ile-166 to Lys-184] fused to [Gly-185 to Gln-222] fused to
[Val-223 to Ala-272] fused to [Gly-273 to Leu-309] of SEQ ID NO:39;
[Met-1 to Lys-106] fused to [Glu-135 to Asn-165] fused to [Ile-166
to Lys-184] fused to [Gly-185 to Gln-222] fused to [Val-223 to
Ala-272] fused to [Gly-273 to Leu-309] of SEQ ID NO:39; or [Met-1
to Lys-106] fused to [Leu-107 to Thr-134] fused to [Glu-135 to
Asn-165] fused to [Ile-166 to Lys-184] fused to [Gly-185 to
Gln-222] fused to [Gly-273 to Leu-309] of SEQ ID NO:39. Other
combinations may include the polypeptide fragments in an order
other than that recited above (e.g., [Met-1 to Lys-106] fused to
[Gly-185 to Gln-222] fused to [Ile-166 to Lys-184] fused to
[Val-223 to Ala-272] fused to [Leu-107 to Thr-134] fused to
[Gly-273 to Leu-309] of SEQ ID NO:39). Other combinations may also
include heterologous polypeptide fragments as described herein
and/or other polypeptides or polypeptide fragments of the present
invention (e.g., [Met-1 to Lys-106] fused to [Glu-135 to Asn-165]
fused to [Ile-166 to Lys-184] fused to [Gly-185 to Gln-222] fused
to [Val-223 to Ala-272] fused to [Gly-273 to Leu-309] of SEQ ID
NO:39 fused to a FLAG tag). Polynucleotides encoding any of these
polypeptides are encompassed by the invention.
[0258] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Met-1 to Lys-106] fused to [Leu-107 to Thr-134] fused to
[Glu-135 to Asn-165] fused to [Ile-166 to Pro-180] fused to
[Ala-181 to Gln-202] fused to [Val-203 to Ala-252] fused to
[Gly-253 to Leu-289] of SEQ ID NO:38; [Met-1 to Lys-106] fused to
[Leu-107 to Thr-134] fused to [Ile-166 to Pr0-180] fused to
[Ala-181 to Gln-202] fused to [Val-203 to Ala-252] fused to
[Gly-253 to Leu-289] of SEQ ID NO:38; [Met-1 to Lys-106] fused to
[Leu-107 to Thr-134] fused to [Glu-135 to Asn-165] fused [Ala-181
to Gln-202] fused to [Val-203 to Ala-252] fused to [Gly-253 to
Leu-289] of SEQ ID NO:38; [Met-1 to Lys-106] fused to [Leu-107 to
Thr-134] fused to [Ala-181 to Gln-202] fused to [Val-203 to
Ala-252] fused to [Gly-253 to Leu-289] of SEQ ID NO:38; Other
combinations may include the polypeptide fragments in an order
other than that recited above (e.g., [Met-1 to Lys-106] fused to
[Ala-181 to Gln-202] fused to [Ile-166 to Pro-180] fused to
[Val-203 to Ala-252] fused to [Leu-107 to Thr-134] fused to
[Gly-253 to Leu-289] of SEQ ID NO:38). Other combinations may also
include heterologous polypeptide fragments as described herein
and/or other polypeptides or polypeptide fragments of the present
invention (e.g., [Met-1 to Lys-106] fused to [Glu-135 to Asn-165]
fused to [Ile-166 to Pro-180] fused to [Ala-181 to Gln-202] fused
to [Val-203 to Ala-252] fused to [Gly-253 to Leu-289] of SEQ ID
NO:38 fused to a FLAG tag). Polynucleotides encoding any of these
polypeptides are encompassed by the invention.
[0259] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Met-1 to Lys-106] fused to [Leu-107 to Thr-134] fused to
[Glu-135 to Asn-165] fused to [Arg-166 to Gln-203] fused to
[Val-204 to Ala-253] fused to [Gly-254 to Leu-290] of SEQ ID NO:40;
[Met-1 to Lys-106] fused [Glu-135 to Asn-165] fused to [Arg-166 to
Gln-203] fused to [Val-204 to Ala-253] fused to [Gly-254 to
Leu-290] of SEQ ID NO:40; [Met-1 to Lys-106] fused to [Leu-107 to
Thr-134] fused to [Arg-166 to Gln-203] fused to [Val-204 to
Ala-253] fused to [Gly-254 to Leu-290] of SEQ ID NO:40; or [Met-1
to Lys-106] fused to [Leu-107 to Thr-134] fused to [Glu-135 to
Asn-165] fused to [Arg-166 to Gln-203] fused to [Gly-254 to
Leu-290] of SEQ ID NO:40. Other combinations may include the
polypeptide fragments in an order other than that recited above
(e.g., [Met-1 to Lys-106] fused to [Arg-166 to Gln-203] fused to
[Val-204 to Ala-253] fused to [Leu-107 to Thr-134] fused to
[Gly-254 to Leu-290] of SEQ ID NO:40). Other combinations may also
include heterologous polypeptide fragments as described herein
and/or other polypeptides or polypeptide fragments of the present
invention (e.g., [Met-1 to Lys-106] fused to [Glu-135 to Asn-165]
fused to [Arg-166 to Gln-202] fused to [Val-204 to Ala-253] fused
to [Gly-254 to Leu-290] of SEQ ID NO:38 fused to a FLAG tag).
Polynucleotides encoding any of these polypeptides are encompassed
by the invention.
[0260] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Tyr-1 to Lys-47] fused to [Leu-48 to Thr-75] fused to
[Val-76 to Lys-94] fused to [Gly-95 to Gln-132] fused to [Val-133
to Ala-182] fused to [Gly-183 to Leu-219] of SEQ ID NO:28; [Tyr-1
to Lys-47] fused to [Leu-48 to Thr-75] fused to [Val-76 to Lys-94]
fused to [Val-133 to Ala-182] of SEQ ID NO:28; or [Tyr-1 to Lys-47]
fused to [Val-76 to Lys-94] fused to [Val-133 to Ala-182] fused to
[Gly-183 to Leu-219] of SEQ ID NO:28. Other combinations may
include the polypeptide fragments in an order other than that
recited above (e.g., [Tyr-1 to Lys-47] fused to [Gly-183 to
Leu-219] fused to [Val-133 to Ala-182] fused to [Leu-48 to Thr-75]
of SEQ ID NO:28). Other combinations may also include heterologous
polypeptide fragments as described herein and/or other polypeptides
or polypeptide fragments of the present invention (e.g., [Leu-48 to
Thr-75] fused to [Val-76 to Lys-94] fused to [Gly-95 to Gln-132]
fused to [Val-133 to Ala-182] of SEQ ID NO:28 fused to an Fc
receptor tag). Polynucleotides encoding any of these polypeptides
are encompassed by the invention.
[0261] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Tyr-1 to Lys-47] fused to [Leu-48 to Thr-75] fused to
[Val-76 to Lys-94] fused to [Gly-95 to Gln-132] fused to [Val-133
to Ala-182] fused to [Gly-183 to Leu-219] of SEQ ID NO:30; [Tyr-1
to Lys-47] fused to [Leu-48 to Thr-75] fused to [Val-76 to Lys-94]
fused to [Val-133 to Ala-182] of SEQ ID NO:30; or [Tyr-1 to Lys-47]
fused to [Val-76 to Lys-94] fused to [Val-133 to Ala-182] fused to
[Gly-183 to Leu-219] of SEQ ID NO:30. Other combinations may
include the polypeptide fragments in an order other than that
recited above (e.g., [Tyr-1 to Lys-47] fused to [Gly-183 to
Leu-219] fused to [Val-133 to Ala-182] fused to [Leu-48 to Thr-75]
of SEQ ID NO:30). Other combinations may also include heterologous
polypeptide fragments as described herein and/or other polypeptides
or polypeptide fragments of the present invention (e.g., [Leu-48 to
Thr-75] fused to [Val-76 to Lys-94] fused to [Gly-95 to Gln-132]
fused to [Val-133 to Ala-182] of SEQ ID NO:30 fused to an Fc
receptor tag). Polynucleotides encoding any of these polypeptides
are encompassed by the invention.
[0262] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Ala-1 to Thr-8] fused to [Val-9 to Lys-27] fused to
[Gly-28 to Gln-65] fused to [Val-66 to Ala-115] fused to [Gly-116
to Leu-152] of SEQ ID NO:41; [Ala-1 to Thr-8] fused to [Gly-28 to
Gln-65] fused to [Val-66 to Ala-115] fused to [Gly-116 to Leu-152]
of SEQ ID NO:41; [Ala-1 to Thr-8] fused to [Val-9 to Lys-27] fused
to [Gly-28 to Gln-65] fused to [Gly-116 to Leu-152] of SEQ ID
NO:41; Other combinations may include the polypeptide fragments in
an order other than that recited above (e.g [Ala-1 to Thr-8] fused
to [Gly-116 to Leu-152] fused to [Val-66 to Ala-115] fused to
[Val-9 to Lys-27] of SEQ ID NO:41). Other combinations may also
include heterologous polypeptide fragments as described herein
and/or other polypeptides or polypeptide fragments of the present
invention (e.g., [Ala-1 to Thr-8] fused to [Val-9 to Lys-27] fused
to [Gly-28 to Gln-65] fused to [Val-66 to Ala-115] fused to
[Gly-116 to Leu-152] of SEQ ID NO:41 fused to an Fc receptor tag).
Polynucleotides encoding any of these polypeptides are encompassed
by the invention.
[0263] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Ala-1 to Thr-8] fused to [Glu-9 to Thr-40] fused to
[Arg-41 to Gln-78] fused to [Val-79 to Ala-128] fused to [Gly-129
to Leu-165] of SEQ ID NO:42; [Ala-1 to Thr-8] fused to [Arg-41 to
Gln-78] fused to [Val-79 to Ala-128] fused to [Gly-129 to Leu-165]
of SEQ ID NO:42; [Ala-1 to Thr-8] fused to [Glu-9 to Thr-40] fused
to [Arg-41 to Gln-78] fused to [Gly-129 to Leu-165] of SEQ ID NO:4.
Other combinations may include the polypeptide fragments in an
order other than that recited above (e.g [Ala-1 to Thr-8] fused to
[Gly-129 to Leu-165] fused to [Val-79 to Ala-128] fused to [Arg-41
to Gln-78] fused to [Glu-9 to Thr-40] of SEQ ID NO:42). Other
combinations may also include heterologous polypeptide fragments as
described herein and/or other polypeptides or polypeptide fragments
of the present invention (e.g., [Ala-1 to Thr-8] fused to [Glu-9 to
Thr-40] fused to [Arg-41 to Gln-78] fused to [Val-79 to Ala-128]
fused to [Gly-129 to Leu-165] of SEQ ID NO:42 fused to an Fc
receptor tag). Polynucleotides encoding any of these polypeptides
are encompassed by the invention.
[0264] A further partial, non-limiting, and exemplary list of
polypeptides of the invention which comprise, or alternatively
consist of, combinations of amino acid sequences includes, for
example, [Ala-1 to Thr-8] fused to [Glu-9 to Thr-40] fused to
[Ile-41 to Lys-59] fused to [Gly-60 to Gln-97] fused to [Val-98 to
Ala-147] fused to [Gly-148 to Leu-184] of SEQ ID NO:43; [Ala-1 to
Thr-8] fused [Gly-60 to Gln-97] fused to [Gly-148 to Leu-184] of
SEQ ID NO:43; [Ala-1 to Thr-8] fused to [Glu-9 to Thr-40] fused to
[Gly-60 to Gln-97] fused to [Val-98 to Ala-147] fused to [Gly-148
to Leu-184] of SEQ ID NO:43; [Ala-1 to Thr-8] fused to [Ile-41 to
Lys-59] fused to [Gly-60 to Gln-97] fused to [Val-98 to Ala-147]
fused to [Gly-148 to Leu-184] of SEQ ID NO:43; or [Ala-1 to Thr-8]
fused to [Glu-9 to Thr-40] fused to [Ile-41 to Lys-59] fused to
[Gly-60 to Gln-97] fused to [Gly-148 to Leu-184] of SEQ ID NO:43;
Other combinations may include the polypeptide fragments in an
order other than that recited above (e.g., [Ala-1 to Thr-8] fused
to [Gly-148 to Leu-184] fused to [Val-98 to Ala-147] fused to
[Ile-41 to Lys-59] fused to [Glu-9 to Thr-40] fused to [Gly-60 to
Gln-97] of SEQ ID NO:43). Other combinations may also include
heterologous polypeptide fragments as described herein and/or other
polypeptides or polypeptide fragments of the present invention
(e.g., [Ala-1 to Thr-8] fused to [Glu-9 to Thr-40] fused to [Ile-41
to Lys-59] fused to [Val-98 to Ala-147] fused to [Gly-148 to
Leu-184] of SEQ ID NO:43 fused to an Fc receptor tag).
Polynucleotides encoding any of these polypeptides are encompassed
by the invention.
[0265] Additional embodiments of the invention encompass
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide fragments
comprising, or alternatively consisting of, functional regions of
polypeptides of the invention, such as the Garnier-Robson
alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman alpha-regions, beta-regions, and coil-regions,
Kyte-Doolittle hydrophilic regions and hydrophobic regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz
flexible regions, Emini surface-forming regions and Jameson-Wolf
regions of high antigenic index set out in FIGS. 3 and 6 and in
Table I and as described herein. In a preferred embodiment, the
polypeptide fragments of the invention are antigenic. The data
presented in columns VIII, IX, XIII, and XIV of Table I can be used
to routinely determine regions of Neutrokine-alpha which exhibit a
high degree of potential for antigenicity. Regions of high
antigenicity are determined from the data presented in columns
VIII, IX, XIII, and/or IV by choosing values which represent
regions of the polypeptide which are likely to be exposed on the
surface of the polypeptide in an environment in which antigen
recognition may occur in the process of initiation of an immune
response. Among highly preferred fragments of the invention are
those that comprise regions of Neutrokine-alpha and/or
Neutrokine-alphaSV that combine several structural features, such
as several (e.g., 1, 2, 3 or 4) of the features set out above.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0266] In another embodiment, the invention provides a polypeptide
comprising, or alternatively consisting of, an epitope-bearing
portion of a polypeptide of the invention. Polynucleotides encoding
these polypeptides are also encompassed by the invention. The
epitope of this polypeptide portion is an immunogenic or antigenic
epitope of a polypeptide of the invention. An "immunogenic epitope"
is defined as a part of a protein that elicits an antibody response
when the whole protein is the immunogen. On the other hand, a
region of a protein molecule to which an antibody can bind is
defined as an "antigenic epitope." The number of immunogenic
epitopes of a protein generally is less than the number of
antigenic epitopes. See, for instance, Geysen et al., Proc. Natl.
Acad. Sci. USA 81:3998-4002 (1983).
[0267] As to the selection of polypeptides bearing an antigenic
epitope (i.e., that contain a region of a protein molecule to which
an antibody can bind), it is well known in that art that relatively
short synthetic peptides that mimic part of a protein sequence are
routinely capable of eliciting an antiserum that reacts with the
partially mimicked protein. See, for instance, Sutcliffe, J. G.,
Shinnick, T. M., Green, N. and Learner, R. A. (1983) "Antibodies
that react with predetermined sites on proteins", Science,
219:660-666. Peptides capable of eliciting protein-reactive sera
are frequently represented in the primary sequence of a protein,
can be characterized by a set of simple chemical rules, and are
confined neither to immunodominant regions of intact proteins
(i.e., immunogenic epitopes) nor to the amino or carboxyl
terminals. Antigenic epitope-bearing peptides and polypeptides of
the invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. See, for instance, Wilson et al., Cell 37:767-778
(1984) at 777.
[0268] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least 4, at least 5,
at least 6, at least 7, more preferably at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 20, at least 25, at least 30, at least 40, at
least 50, and, most preferably, between about 15 to about 30 amino
acids contained within the amino acid sequence of a polypeptide of
the invention. Preferred polypeptides comprising immunogenic or
antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in
length. Additional non-exclusive preferred antigenic epitopes
include the antigenic epitopes disclosed herein, as well as
portions thereof.
[0269] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate Neutrokine-alpha- and/or
Neutrokine-alphaSV-specific antibodies include: a polypeptide
comprising, or alternatively consisting of, amino acid residues
from about Phe-115 to about Leu-147 in FIGS. 1A and 1B (SEQ ID
NO:2); a polypeptide comprising, or alternatively consisting of,
amino acid residues from about Ile-150 to about Tyr-163 in FIGS. 1A
and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively
consisting of, amino acid residues from about Ser-171 to about
Phe-194 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising,
or alternatively consisting of, amino acid residues from about
Glu-223 to about Tyr-246 in FIGS. 1A and 1B (SEQ ID NO:2); and a
polypeptide comprising, or alternatively consisting of, amino acid
residues from about Ser-271 to about Phe-278 in FIGS. 1A and 1B
(SEQ ID NO:2). In this context, "about" means the particularly
recited ranges and ranges larger or smaller by several, a few, 5,
4, 3, 2 or 1 amino acid residues at either or both the amino- and
carboxy-termini. These polypeptide fragments have been determined
to bear antigenic epitopes of the Neutrokine-alpha polypeptide by
the analysis of the Jameson-Wolf antigenic index, as shown in FIG.
3 and Table I, above.
[0270] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate Neutrokine-alpha- and/or
Neutrokine-alphaSV-specific antibodies include: a polypeptide
comprising, or alternatively consisting of, amino acid residues
from about Pro-32 to about Leu-47 in FIGS. 5A and 5B (SEQ ID
NO:19); a polypeptide comprising, or alternatively consisting of,
amino acid residues from about Glu-116 to about Ser-143 in FIGS. 5A
and 5B (SEQ ID NO:19); a polypeptide comprising, or alternatively
consisting of, amino acid residues from about Phe-153 to about
Tyr-173 in FIGS. 5A and 5B (SEQ ID NO:19); a polypeptide
comprising, or alternatively consisting of, amino acid residues
from about Pro-218 to about Tyr-227 in FIGS. 5A and 5B (SEQ ID
NO:19); a polypeptide comprising, or alternatively consisting of,
amino acid residues from about Ala-232 to about Gln-241 in FIGS. 5A
and 5B (SEQ ID NO:19); a polypeptide comprising, or alternatively
consisting of, amino acid residues from about Ile-244 to about
Ala-249 in FIGS. 5A and 5B (SEQ ID NO:19); and a polypeptide
comprising, or alternatively consisting of, amino acid residues
from about Ser-252 to about Val-257 in FIGS. 5A and 5B (SEQ ID
NO:19). In this context, "about" means the particularly recited
ranges and ranges larger or smaller by several, a few, 5, 4, 3, 2
or 1 amino acid residues at either or both the amino- and
carboxy-termini. Polynucleotides encoding these polypeptides are
also encompassed by the invention. These polypeptide fragments have
been determined to bear antigenic epitopes of the
Neutrokine-alphaSV polypeptide by the analysis of the Jameson-Wolf
antigenic index, as shown in FIG. 6 and a tabular representation of
the data presented in FIG. 6 generated by the Protean component of
the DNA*STAR computer program (as set forth above).
[0271] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. See, e.g.,
Houghten, R. A. (1985) General method for the rapid solid-phase
synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids. Proc. Natl. Acad. Sci. USA 82:5131-5135; this "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986).
[0272] Epitope-bearing peptides and polypeptides of the invention
have uses that include, but are not limited to, to induce
antibodies according to methods well known in the art. See, for
instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M.
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et
al., J. Gen. Virol. 66:2347-2354 (1985). Immunogenic
epitope-bearing peptides of the invention, i.e., those parts of a
protein that elicit an antibody response when the whole protein is
the immunogen, are identified according to methods known in the
art. See, for instance, Geysen et al., supra. Further still, U.S.
Pat. No. 5,194,392 to Geysen (1990) describes a general method of
detecting or determining the sequence of monomers (amino acids or
other compounds) which is a topological equivalent of the epitope
(i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996)
on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0273] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence of SEQ ID NO:2, or an epitope of the
polypeptide sequence encoded by a polynucleotide sequence contained
in ATCC deposit No. 97768, or encoded by a polynucleotide that
hybridizes to the complement of the sequence of SEQ ID NO:1 or the
cDNA sequence contained in ATCC deposit No. 97768 (e.g., under
hybridization conditions described herein). The present invention
further encompasses polynucleotide sequences comprising, or
alternatively consisting of, a sequence encoding an epitope of a
polypeptide sequence of the invention (such as, for example, the
sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the
complementary strand of a polynucleotide sequence encoding an
epitope of the invention, and polynucleotide sequences which
hybridize to the complementary strand (e.g., under hybridization
conditions described herein).
[0274] The present invention also encompasses polypeptides
comprising, or alternatively consisting of, an epitope of the
polypeptide having an amino acid sequence of SEQ ID NO:19, or an
epitope of the polypeptide sequence encoded by a polynucleotide
sequence contained in ATCC deposit No. 203518, or encoded by a
polynucleotide that hybridizes to the complement of the sequence of
SEQ ID NO:18 or the cDNA sequence contained in ATCC deposit No.
203518 (e.g., under hybridization conditions described herein). The
present invention further encompasses polynucleotide sequences
comprising, or alternatively consisting of, a sequence encoding an
epitope of a polypeptide sequence of the invention (such as, for
example, the sequence disclosed in SEQ ID NO:18), polynucleotide
sequences of the complementary strand of a polynucleotide sequence
encoding an epitope of the invention, and polynucleotide sequences
which hybridize to the complementary strand (e.g., under
hybridization conditions described herein).
[0275] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope," as used herein, is defined
as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method
well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but
does not necessarily exclude cross-reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[0276] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0277] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, and, most
preferably, between about 15 to about 30 amino acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, or 100 amino acid residues in length. Additional
non-exclusive preferred antigenic epitopes include the antigenic
epitopes disclosed herein, as well as portions thereof. Antigenic
epitopes are useful, for example, to raise antibodies, including
monoclonal antibodies, that specifically bind the epitope.
Preferred antigenic epitopes include the antigenic epitopes
disclosed herein, as well as any combination of two, three, four,
five or more of these antigenic epitopes. Antigenic epitopes can be
used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666 (1983)).
[0278] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes
include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic
epitopes. The polypeptides comprising one or more immunogenic
epitopes may be presented for eliciting an antibody response
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse), or, if the polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide
may be presented without a carrier. However, immunogenic epitopes
comprising as few as 8 to 10 amino acids have been shown to be
sufficient to raise antibodies capable of binding to, at the very
least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
[0279] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 micrograms of peptide or carrier
protein and Freund's adjuvant or any other adjuvant known for
stimulating an immune response. Several booster injections may be
needed, for instance, at intervals of about two weeks, to provide a
useful titer of anti-peptide antibody which can be detected, for
example, by ELISA assay using free peptide adsorbed to a solid
surface. The titer of anti-peptide antibodies in serum from an
immunized animal may be increased by selection of anti-peptide
antibodies, for instance, by adsorption to the peptide on a solid
support and elution of the selected antibodies according to methods
well known in the art.
[0280] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof), or albumin (including but not
limited to recombinant human albumin or fragments or variants
thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999,
EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16,
1998, herein incorporated by reference in their entirety)),
resulting in chimeric polypeptides. Such fusion proteins may
facilitate purification and may increase half-life in vivo. This
has been shown for chimeric proteins consisting of the first two
domains of the human CD4-polypeptide and various domains of the
constant regions of the heavy or light chains of mammalian
immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature,
331:84-86 (1988). Enhanced delivery of an antigen across the
epithelial barrier to the immune system has been demonstrated for
antigens (e.g., insulin) conjugated to an FcRn binding partner such
as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and
WO 99/04813). IgG Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG portion desulfide bonds have also
been found to be more efficient in binding and neutralizing other
molecules than monomeric polypeptides or fragments thereof alone.
See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).
Nucleic acids encoding the above epitopes can also be recombined
with a gene of interest as an epitope tag (e.g., the hemagglutinin
("HA") tag or flag tag) to aid in detection and purification of the
expressed polypeptide. For example, a system described by Janknecht
et al. allows for the ready purification of non-denatured fusion
proteins expressed in human cell lines (Janknecht et al., 1991,
Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene
of interest is subcloned into a vaccinia recombination plasmid such
that the open reading frame of the gene is translationally fused to
an amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix-binding domain for the fusion protein. Extracts
from cells infected with the recombinant vaccinia virus are loaded
onto Ni.sup.2+ nitriloacetic acid-agarose column and
histidine-tagged proteins can be selectively eluted with
imidazole-containing buffers.
[0281] In another embodiment, the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the present invention and the
epitope-bearing fragments thereof are fused with a heterologous
antigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleic
acid). In specific embodiments, the heterologous antigen is an
immunogen.
[0282] In a more specific embodiment, the heterologous antigen is
the gp120 protein of HIV, or a fragment thereof. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0283] In another embodiment, the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the present invention and the
epitope-bearing fragments thereof are fused with polypeptide
sequences of another TNF ligand family member (or biologically
active fragments or variants thereof). In a specific embodiment,
the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the
present invention are fused with a CD40L polypeptide sequence. In a
preferred embodiment, the CD40L polypeptide sequence is
soluble.
[0284] The techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of
Neutrokine-alpha and/or Neutrokine-alphaSV thereby effectively
generating agonists and antagonists of Neutrokine-alpha and/or
Neutrokine-alphaSV. See generally, U.S. Pat. Nos. 5,605,793,
5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A.,
et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.
Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J.
Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R.
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference). In one
embodiment, alteration of Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides and corresponding polypeptides
may be achieved by DNA shuffling. DNA shuffling involves the
assembly of two or more DNA segments into a desired
Neutrokine-alpha and/or Neutrokine-alphaSV molecule by homologous,
or site-specific, recombination. In another embodiment,
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides and
corresponding polypeptides may be altered by being subjected to
random mutagenesis by error-prone PCR, random nucleotide insertion
or other methods prior to recombination. In another embodiment, one
or more components, motifs, sections, parts, domains, fragments,
etc., of Neutrokine-alpha and/or Neutrokine-alphaSV may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules. In
preferred embodiments, the heterologous molecules are, for example,
TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta),
LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL,
CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International
Publication No. WO 96/14328), AIM-I (International Publication No.
WO 97/33899), AIM-II (International Publication No. WO 97/34911),
APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha
(International Publication No. WO 98/07880), OPG, OX40, and nerve
growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and
4-IBB, TR2 (International Publication No. WO 96/34095), DR3
(International Publication No. WO 97/33904), DR4 (International
Publication No. WO 98/32856), TR5 (International Publication No. WO
98/30693), TR6 (International Publication No. WO 98/30694), TR7
(International Publication No. WO 98/41629), TRANK, TR9
(International Publication No. WO 98/56892), TR10 (International
Publication No. WO 98/54202), 312C2 (International Publication No.
WO 98/06842), TR12, CAD, and v-FLIP. In further embodiments, the
heterologous molecules are any member of the TNF family.
[0285] In a preferred embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention (including
biologically active fragments or variants thereof), are fused with
soluble CD40L polypeptides, or biologically active fragments or
variants thereof.
[0286] In another preferred embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention (including
biologically active fragments or variants thereof), are fused with
soluble APRIL polypeptides (e.g., SEQ ID NO:20 or SEQ ID NO:47), or
biologically active fragments or variants thereof.
[0287] To improve or alter the characteristics of Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides, protein engineering may be
employed. Recombinant DNA technology known to those skilled in the
art can be used to create novel mutant proteins or "muteins
including single or multiple amino acid substitutions, deletions,
additions or fusion proteins. Such modified polypeptides can show,
e.g., enhanced activity or increased stability. In addition, they
may be purified in higher yields and show better solubility than
the corresponding natural polypeptide, at least under certain
purification and storage conditions. For instance, for many
proteins, including the extracellular domain or the mature form(s)
of a secreted protein, it is known in the art that one or more
amino acids may be deleted from the N-terminus or C-terminus
without substantial loss of biological function. For instance, Ron
et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF
proteins that had heparin binding activity even if 3, 8, or 27
amino-terminal amino acid residues were missing.
[0288] In the present case, since the protein of the invention is a
member of the TNF polypeptide family, deletions of N-terminal amino
acids up to the Gly (G) residue at position 191 in FIGS. 1A and 1B
(SEQ ID NO:2) may retain some biological activity such as, for
example, the ability to stimulate lymphocyte (e.g., B cell)
proliferation, differentiation, and/or activation, and cytotoxicity
to appropriate target cells. Polypeptides having further N-terminal
deletions including the Gly (G) residue would not be expected to
retain biological activities because it is known that this residue
in TNF-related polypeptides is in the beginning of the conserved
domain required for biological activities. However, even if
deletion of one or more amino acids from the N-terminus of a
protein results in modification or loss of one or more biological
functions of the protein, other functional activities may still be
retained. Thus, the ability of the shortened protein to induce
and/or bind to antibodies which recognize the complete or
extracellular domain of the protein generally will be retained when
less than the majority of the residues of the complete or
extracellular domain of the protein are removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art.
[0289] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the Neutrokine-alpha shown
in FIGS. 1A and 1B (SEQ ID NO:2), up to the glycine residue at
position 191 (Gly-191 residue from the amino terminus), and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising, or
alternatively consisting of, the amino acid sequence of residues
n.sup.1-285 of SEQ ID NO:2, where n.sup.1 is an integer in the
range of the amino acid position of amino acid residues 2-190 of
the amino acid sequence in SEQ ID NO:2. Polynucleotides encoding
these polypeptides are also encompassed by the invention. More in
particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, an amino
acid sequence selected from the group consisting of residues 2-285,
3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285, 11-285,
12-285, 13-285, 14-285, 15-285, 16-285, 17-285, 18-285, 19-285,
20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285,
28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285,
36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285,
44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285,
52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285,
60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285,
68-285, 69-285, 70-285, 71-285, 72-285, 73-285, 74-285, 75-285,
76-285, 77-285, 78-285, 79-285, 80-285, 81-285, 82-285, 83-285,
84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-285, 91-285,
92-285, 93-285, 94-285, 95-285, 96-285, 97-285, 98-285, 99-285,
100-285, 101-285, 102-285, 103-285, 104-285, 105-285, 106-285,
107-285, 108-285, 109-285, 110-285, 111-285, 112-285, 113-285,
114-285, 115-285, 116-285, 117-285, 118-285, 119-285, 120-285,
121-285, 122-285, 123-285, 124-285, 125-285, 126-285, 127-285,
128-285, 129-285, 130-285, 131-285, 132-285, 133-285, 134-285,
135-285, 136-285, 137-285, 138-285, 139-285, 140-285, 141-285,
142-285, 143-285, 144-285, 145-285, 146-285, 147-285, 148-285,
149-285, 150-285, 151-285, 152-285, 153-285, 154-285, 155-285,
156-285, 157-285, 158-285, 159-285, 160-285, 161-285, 162-285,
163-285, 164-285, 165-285, 166-285, 167-285, 168-285, 169-285,
170-285, 171-285, 172-285, 173-285, 174-285, 175-285, 176-285,
177-285, 178-285, 179-285, 180-285, 181-285, 182-285, 183-285,
184-285, 185-285, 186-285, 187-285, 188-285, 189-285, and 190-285
of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are
also encompassed by the invention. The present invention is also
directed to nucleic acid molecules comprising, or alternatively,
consisting of, a polynucleotide sequence at least 80%, 85%, 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide
sequence encoding the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides described above. The present invention also
encompasses the above polynucleotide sequences fused to a
heterologous polynucleotide sequence. Polypeptides encoded by these
nucleic acids and/or polynucleotide sequences are also encompassed
by the invention, as are polypeptides comprising, or alternatively
consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%,
95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
described above, and polynucleotides that encode such
polypeptides.
[0290] Furthermore, since the predicted extracellular domain of the
Neutrokine-alpha polypeptides of the invention may itself elicit
biological activity, deletions of N- and C-terminal amino acid
residues from the predicted extracellular region of the polypeptide
(spanning positions Gln-73 to Leu-285 of SEQ ID NO:2) may retain
some biological activity such as, for example, ligand binding,
stimulation of lymphocyte (e.g., B cell) proliferation,
differentiation, and/or activation, and modulation of cell
replication or modulation of target cell activities. However, even
if deletion of one or more amino acids from the N-terminus of the
predicted extracellular domain of a Neutrokine-alpha polypeptide
results in modification or loss of one or more biological functions
of the polypeptide, other functional activities may still be
retained. Thus, the ability of the shortened polypeptides to induce
and/or bind to antibodies which recognize the complete or mature or
extracellular domains of the polypeptides generally will be
retained when less than the majority of the residues of the
complete or mature or extracellular domains of the polypeptides are
removed from the N-terminus. Whether a particular polypeptide
lacking N-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0291] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of Neutrokine-alpha shown in
SEQ ID NO:2, up to the glycine residue at position number 280, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising, or
alternatively consisting of, the amino acid sequence of residues
n.sup.2-285 of SEQ ID NO:2, where n.sup.2 is an integer in the
range of the amino acid position of amino acid residues 73-280 in
SEQ ID NO:2, and 73 is the position of the first residue from the
N-terminus of the predicted extracellular domain of the
Neutrokine-alpha polypeptide (disclosed in SEQ ID NO:2).
Polynucleotides encoding these polypeptides are also encompassed by
the invention. More in particular, in certain embodiments, the
invention provides polynucleotides encoding polypeptides
comprising, or alternatively consisting of, an amino acid sequence
selected from the group consisting of residues of Q-73 to L-285;
G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to
L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285;
L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to
L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285;
P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to
L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285;
G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285;
A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285;
T-109 to L-285; A-111 to L-285; G-111 to L-285; L-112 to L-285;
K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285;
P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285;
G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285;
S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285;
S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285;
R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285;
G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285;
T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285;
D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285;
L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285;
S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285;
T-157 to L-285; 1-158 to L-285; Q-159 to L-285; K-160 to L-285;
G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285;
F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285;
L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285;
K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285;
A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285;
K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285;
I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285;
E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285;
F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285;
G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285;
Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285;
T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285;
G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285;
Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285;
V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285;
G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285;
S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285;
L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285;
I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285;
P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285;
P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285;
C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285;
G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285;
L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285;
D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285;
L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285;
R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285;
Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285;
D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285;
T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-280 to L-285
of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are
also encompassed by the invention. The present invention is also
directed to nucleic acid molecules comprising, or alternatively,
consisting of, a polynucleotide sequence at least 80%, 85%, 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide
sequence encoding the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides described above. The present invention also
encompasses the above polynucleotide sequences fused to a
heterologous polynucleotide sequence. Polypeptides encoded by these
nucleic acids and/or polynucleotide sequences are also encompassed
by the invention, as are polypeptides comprising, or alternatively
consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%,
95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
described above, and polynucleotides that encode such
polypeptides.
[0292] Highly preferred embodiments of the invention are directed
to nucleic acid molecules comprising, or alternatively consisting
of a polynucleotide having a nucleotide sequence at least 80%, 85%,
90% identical and more preferably at least 95%, 96%, 97%, 98%, 99%
or 100% identical to a polynucleotide sequence encoding the
Neutrokine-alpha polypeptide having the amino acid sequence at
positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2). Preferred
embodiments of the invention are directed to nucleic acid molecules
comprising, or alternatively consisting of a polynucleotide having
a nucleotide sequence at least 90% identical to a polynucleotide
sequence encoding the Neutrokine-alpha polypeptide having the amino
acid sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID
NO:2). More preferred embodiments of the invention are directed to
nucleic acid molecules comprising, or alternatively consisting of a
polynucleotide having a nucleotide sequence at least 95% identical
to a polynucleotide sequence encoding the Neutrokine-alpha
polypeptide having the amino acid sequence at positions 134-285 in
FIGS. 1A and 1B (SEQ ID NO:2). More preferred embodiments of the
invention are directed to nucleic acid molecules comprising, or
alternatively consisting of a polynucleotide having a nucleotide
sequence at least 96% identical to a polynucleotide sequence
encoding the Neutrokine-alpha polypeptide having the amino acid
sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2).
[0293] Additionally, more preferred embodiments of the invention
are directed to nucleic acid molecules comprising, or alternatively
consisting of a polynucleotide having a nucleotide sequence at
least 97% to a polynucleotide sequence encoding the
Neutrokine-alpha polypeptide having the amino acid sequence at
positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2). Additionally,
more preferred embodiments of the invention are directed to nucleic
acid molecules comprising, or alternatively consisting of a
polynucleotide having a nucleotide sequence at least 98% to a
polynucleotide sequence encoding the Neutrokine-alpha polypeptide
having the amino acid sequence at positions 134-285 in FIGS. 1A and
1B (SEQ ID NO:2). Additionally, more preferred embodiments of the
invention are directed to nucleic acid molecules comprising, or
alternatively consisting of a polynucleotide having a nucleotide
sequence at least 99% identical to a polynucleotide sequence
encoding the Neutrokine-alpha polypeptide having the amino acid
sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID NO:2).
[0294] In specific embodiments, a polypeptide comprising, or
alternatively consisting of, one of the following N-terminally
deleted polypeptide fragments of Neutrokine-alpha and/or
Neutrokine-alphaSV are preferred: amino acid residues Ala-71
through Leu-285, amino acid residues Ala-81 through Leu-285, amino
acid residues Leu-112 through Leu-285, amino acid residues Ala-134
through Leu-285, amino acid residues Leu-147 through Leu-285, and
amino acid residues Gly-161 through Leu-285 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0295] Similarly, many examples of biologically functional
C-terminal deletion muteins are known. For instance, Interferon
gamma shows up to ten times higher activities by deleting 8-10
amino acid residues from the carboxy terminus of the protein
(Dobeli et al., J. Biotechnology 7:199-216 (1988). Since the
present protein is a member of the TNF polypeptide family,
deletions of C-terminal amino acids up to the leucine residue at
position 284 are expected to retain most if not all biological
activity such as, for example, ligand binding, the ability to
stimulate lymphocyte (e.g., B cell) proliferation, differentiation,
and/or activation, and modulation of cell replication. Polypeptides
having deletions of up to about 10 additional C-terminal residues
(i.e., up to the glycine residue at position 274) also may retain
some activity such as receptor binding, although such polypeptides
would lack a portion of the conserved TNF domain which extends to
about Leu-284 of SEQ ID NO:2. However, even if deletion of one or
more amino acids from the C-terminus of a protein results in
modification or loss of one or more biological functions of the
protein, other functional activities may still be retained. Thus,
the ability of the shortened protein to induce and/or bind to
antibodies which recognize the complete or mature protein generally
will be retained when less than the majority of the residues of the
complete or mature protein are removed from the C-terminus. Whether
a particular polypeptide lacking C-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art.
[0296] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the Neutrokine-alpha
polypeptide shown in FIGS. 1A and 1B (SEQ ID NO:2), up to the
glycine residue at position 274 (Gly-274) and polynucleotides
encoding such polypeptides. In particular, the present invention
provides polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues 1-m.sup.1 of the amino acid
sequence in SEQ ID NO:2, where m.sup.1 is any integer in the range
of the amino acid position of amino acid residues 274-284 in SEQ ID
NO:2. Polynucleotides encoding these polypeptides are also
encompassed by the invention. More in particular, the invention
provides polynucleotides encoding polypeptides comprising, or
alternatively consisting of, an amino acid sequence selected from
the group consisting of residues 1-274, 1-275, 1-276, 1-277, 1-278,
1-279, 1-280, 1-281, 1-282, 1-283 and 1-284 of SEQ ID NO:2.
Polypeptides encoded by these polynucleotides are also encompassed
by the invention. The present invention is also directed to nucleic
acid molecules comprising, or alternatively, consisting of, a
polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or 99% identical to the polynucleotide sequence encoding the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides described
above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these nucleic acids and/or
polynucleotide sequences are also encompassed by the invention, as
are polypeptides comprising, or alternatively consisting of, an
amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that encode such polypeptides.
[0297] Also provided are polypeptides comprising, or alternatively
consisting of, one or more amino acids deleted from both the amino
and the carboxyl termini, which may be described generally as
having residues n.sup.1-m.sup.1 of SEQ ID NO:2, where n.sup.1 and
m.sup.1 are integers as defined above. Also included are a
nucleotide sequence encoding a polypeptide comprising, or
alternatively consisting of, a portion of the complete
Neutrokine-alpha amino acid sequence encoded by the deposited cDNA
clone contained in ATCC Accession No. 97768 where this portion
excludes from 1 to 190 amino acids from the amino terminus or from
1 to 11 amino acids from the C-terminus of the complete amino acid
sequence (or any combination of these N-terminal and C-terminal
deletions) encoded by the cDNA clone in the deposited plasmid.
Polynucleotides encoding all of the above deletion polypeptides are
encompassed by the invention.
[0298] Similarly, deletions of C-terminal amino acid residues of
the predicted extracellular domain of Neutrokine-alpha up to the
leucine residue at position 79 of SEQ ID NO:2 may retain some
biological activity, such as, for example, ligand binding,
stimulation of lymphocyte (e.g., B cell) proliferation,
differentiation, and/or activation, and modulation of cell
replication or modulation of target cell activities. Polypeptides
having further C-terminal deletions including Leu-79 of SEQ ID NO:2
would not be expected to retain biological activities.
[0299] However, even if deletion of one or more amino acids from
the C-terminus of a polypeptide results in modification or loss of
one or more biological functions of the polypeptide, other
functional activities may still be retained. Thus, the ability of
the shortened polypeptide to induce and/or bind to antibodies which
recognize the complete, mature or extracellular forms of the
polypeptide generally will be retained when less than the majority
of the residues of the complete, mature or extracellular forms of
the polypeptide are removed from the C-terminus. Whether a
particular polypeptide lacking C-terminal residues of the predicted
extracellular domain retains such immunologic activities can
readily be determined by routine methods described herein and
otherwise known in the art.
[0300] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the predicted extracellular
domain of Neutrokine-alpha polypeptide shown in SEQ ID NO:2, up to
the leucine residue at position 79 of SEQ ID NO:2, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising, or
alternatively consisting of, the amino acid sequence of residues
73-m of the amino acid sequence in SEQ ID NO:2, where m.sup.2 is
any integer in the range of the amino acid position of amino acid
residues 79 to 285 in the amino acid sequence in SEQ ID NO:2, and
residue 78 is the position of the first residue at the C-terminus
of the predicted extracellular domain of the Neutrokine-alpha
polypeptide (disclosed in SEQ ID NO:2). Polypeptides encoded by
these polynucleotides are also encompassed by the invention. More
in particular, in certain embodiments, the invention provides
polynucleotides encoding polypeptides comprising, or alternatively
consisting of, an amino acid sequence selected from the group
consisting of residues Q-73 to Leu-285; Q-73 to L-284; Q-73 to
K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279;
Q-73 to F-278; Q-73 to T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to
G-274; Q-73 to D-273; Q-73 to L-272; Q-73 to S-271; Q-73 to I-270;
Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-73 to E-266; Q-73 to
R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-261;
Q-73 to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to
G-256; Q-73 to E-255; Q-73 to E-254; Q-73 to L-253; Q-73 to K-252;
Q-73 to A-251; Q-73 to I-250; Q-73 to G-249; Q-73 to A-248; Q-73 to
S-247; Q-73 to Y-246; Q-73 to C-245; Q-73 to S-244; Q-73 to N-243;
Q-73 to N-242; Q-73 to P-241; Q-73 to L-240; Q-73 to T-239; Q-73 to
E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234;
Q-73 to I-233; Q-73 to C-232; Q-73 to R-231; Q-73 to F-230; Q-73 to
L-229; Q-73 to T-228; Q-73 to V-227; Q-73 to L-226; Q-73 to S-225;
Q-73 to L-224; Q-73 to E-223; Q-73 to D-222; Q-73 to G-221; Q-73 to
F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-73 to K-216;
Q-73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to
L-211; Q-73 to H-210; Q-73 to G-209; Q-73 to M-208; Q-73 to A-207;
Q-73 to Y-206; Q-73 to T-205; Q-73 to K-204; Q-73 to D-203; Q-73 to
T-202; Q-73 to Y-201; Q-73 to L-200; Q-73 to V-199; Q-73 to Q-198;
Q-73 to G-197; Q-73 to Y-196; Q-73 to I-195; Q-73 to F-194; Q-73 to
F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-189;
Q-73 to K-188; Q-73 to V-187; Q-73 to L-186; Q-73 to I-185; Q-73 to
K-184; Q-73 to N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180;
Q-73 to E-179; Q-73 to L-178; Q-73 to A-177; Q-73 to S-176; Q-73 to
G-175; Q-73 to R-174; Q-73 to K-173; Q-73 to F-172; Q-73 to S-171;
Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-167; Q-73 to
V-166; Q-73 to F-165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162;
Q-73 to G-161; Q-73 to K-160; Q-73 to Q-159; Q-73 to I-158; Q-73 to
T-157; Q-73 to P-156; Q-73 to T-155; Q-73 to E-154; Q-73 to S-153;
Q-73 to D-152; Q-73 to A-151; Q-73 to I-150; Q-73 to L-149; Q-73 to
Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-145; Q-73 to Q-144;
Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E-140; Q-73 to
E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135;
Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to
R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126;
Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to
G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117;
Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to
L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108;
Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to
E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99;
Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to
G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73
to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85;
Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to
R-80; and Q-73 to L-79 of SEQ ID NO:2. Polypeptides encoded by
these polynucleotides are also encompassed by the invention. The
present invention is also directed to nucleic acid molecules
comprising, or alternatively, consisting of, a polynucleotide
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the polynucleotide sequence encoding the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides described
above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these nucleic acids and/or
polynucleotide sequences are also encompassed by the invention, as
are polypeptides comprising, or alternatively consisting of, an
amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that encode such polypeptides.
[0301] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
the predicted extracellular domain of Neutrokine-alpha, which may
be described generally as having residues n.sup.2-m.sup.2 of SEQ ID
NO:2 where n.sup.2 and m.sup.2 are integers as defined above.
[0302] In another embodiment, a nucleotide sequence encoding a
polypeptide consisting of a portion of the extracellular domain of
the Neutrokine-alpha amino acid sequence encoded by the cDNA
plasmid contained in the deposit having ATCC accession no. 97768,
where this portion excludes from 1 to about 206 amino acids from
the amino terminus of the extracellular domain of the amino acid
sequence encoded by the cDNA plasmid contained in the deposit
having ATCC accession no. 97768, or from 1 to about 206 amino acids
from the carboxy terminus of the extracellular domain of the amino
acid sequence encoded by the cDNA plasmid contained in the deposit
having ATCC accession no. 97768, or any combination of the above
amino terminal and carboxy terminal deletions, of the entire
extracellular domain of the amino acid sequence encoded by the cDNA
plasmid contained in the deposit having ATCC accession no.
97768.
[0303] As mentioned above, even if deletion of one or more amino
acids from the N-terminus of a polypeptide results in modification
or loss of one or more functional activities (e.g., biological
activity) of the polypeptide, other functions or biological
activities may still be retained. Thus, the ability of a shortened
Neutrokine-alpha mutein to induce and/or bind to antibodies which
recognize the full-length or mature forms or the extracellular
domain of the polypeptide generally will be retained when less than
the majority of the residues of the full-length or mature or
extracellular domain of the polypeptide are removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal
residues of a complete polypeptide retains such immunologic
activities can readily be determined by routine methods described
herein and otherwise known in the art. It is not unlikely that a
Neutrokine-alpha mutein with a large number of deleted N-terminal
amino acid residues may retain some functional (e.g., biological or
immunogenic) activities. In fact, peptides composed of as few as
six Neutrokine-alpha amino acid residues may often evoke an immune
response.
[0304] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the predicted full-length amino acid sequence of the
Neutrokine-alpha shown in SEQ ID NO:2, up to the glycine residue at
position number 280 of the sequence shown SEQ ID NO:2 and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues n.sup.3-285 of the sequence shown in SEQ ID
NO:2, where n.sup.3 is an integer in the range of the amino acid
position of amino acid residues 1 to 280 of the amino acid sequence
in SEQ ID NO:2.
[0305] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the group consisting of
residues of D-2 to L-285; D-3 to L-285; S-4 to L-285; T-5 to L-285;
E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to
L-285; R-11 to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285;
C-15 to L-285; L-16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to
L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K-23 to L-285;
L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to
L-285; S-29 to L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285;
R-33 to L-285; K-34 to L-285; E-35 to L-285; S-36 to L-285; P-37 to
L-285; S-38 to L-285; V-39 to L-285; R-40 to L-285; S-41 to L-285;
S-42 to L-285; K-43 to L-285; D-44 to L-285; G-45 to L-285; K-46 to
L-285; L-47 to L-285; L-48 to L-285; A-49 to L-285; A-50 to L-285;
T-51 to L-285; L-52 to L-285; L-53 to L-285; L-54 to L-285; A-55 to
L-285; L-56 to L-285; L-57 to L-285; S-58 to L-285; C-59 to L-285;
C-60 to L-285; L-61 to L-285; T-62 to L-285; V-63 to L-285; V-64 to
L-285; S-65 to L-285; F-66 to L-285; Y-67 to L-285; Q-68 to L-285;
V-69 to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to
L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285;
S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to
L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285;
H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to
L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285;
G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100
to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to
L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to
L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to
L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to
L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to
L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to
L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to
L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to
L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to
L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to
L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to
L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to
L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to
L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to
L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to
L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to
L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to
L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to
L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to
L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to
L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to
L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to
L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to
L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to
L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to
L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to
L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to
L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to
L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to
L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to
L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to
L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to
L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to
L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to
L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to
L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to
L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to
L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to
L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to
L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to
L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to
L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to
L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to
L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to
L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-280 to
L-285 of SEQ ID NO:2. The present application is also directed to
nucleic acid molecules comprising, or alternatively, consisting of,
a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%,
97%, 98% or 99% identical to the polynucleotide sequence encoding
the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides
described above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these nucleic acids and/or
polynucleotide sequences are also encompassed by the invention, as
are polypeptides comprising an amino acid sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino
acid sequence described above, and polynucleotides that encode such
polypeptides.
[0306] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification or loss of one or more functional activities (e.g.,
biological activity) of the protein, other functional activities
may still be retained. Thus, the ability of a shortened
Neutrokine-alpha mutein to induce and/or bind to antibodies which
recognize the complete or mature form or the extracellular domain
of the polypeptide generally will be retained when less than the
majority of the residues of the complete or mature form or the
extracellular domain of the polypeptide are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete polypeptide retains such immunologic
activities can readily be determined by routine methods described
herein and otherwise known in the art. It is not unlikely that a
Neutrokine-alpha mutein with a large number of deleted C-terminal
amino acid residues may retain some functional (e.g., biological or
immunogenic) activities. In fact, peptides composed of as few as
six Neutrokine-alpha amino acid residues may often evoke an immune
response.
[0307] Accordingly, the present invention further provides in
another embodiment, polypeptides having one or more residues
deleted from the carboxy terminus of the amino acid sequence of the
Neutrokine-alpha shown in SEQ ID NO:2, up to the glutamic acid
residue at position number 6, and polynucleotides encoding such
polypeptides. In particular, the present invention provides
polypeptides comprising the amino acid sequence of residues
1-m.sup.3 of SEQ ID NO:2, where m.sup.3 is an integer in the range
of the amino acid position of amino acid residues 6-284 of the
amino acid sequence in SEQ ID NO:2.
[0308] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the group consisting of
residues M-1 to L-284; M-1 to K-283; M-1 to L-282; M-1 to A-281;
M-1 to G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; M-1 to
V-276; M-1 to D-275; M-1 to G-274; M-1 to D-273; M-1 to L-272; M-1
to S-271; M-1 to I-270; M-1 to Q-269; M-1 to A-268; M-1 to N-267;
M-1 to E-266; M-1 to R-265; M-1 to P-264; M-1 to I-263; M-1 to
A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258; M-1
to D-257; M-1 to G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253;
M-1 to K-252; M-1 to A-251; M-1 to I-250; M-1 to G-249; M-1 to
A-248; M-1 to S-247; M-1 to Y-246; M-1 to C-245; M-1 to S-244; M-1
to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T-239;
M-1 to E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to
Q-234; M-1 to I-233; M-1 to C-232; M-1 to R-231; M-1 to F-230; M-1
to L-229; M-1 to T-228; M-1 to V-227; M-1 to L-226; M-1 to S-225;
M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-1 to
F-220; M-1 to V-219; M-1 to H-218; M-1 to V-217; M-1 to K-216; M-1
to K-215; M-1 to R-214; M-1 to Q-213; M-1 to I-212; M-1 to L-211;
M-1 to H-210; M-1 to G-209; M-1 to M-208; M-1 to A-207; M-1 to
Y-206; M-1 to T-205; M-1 to K-204; M-1 to D-203; M-1 to T-202; M-1
to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197;
M-1 to Y-196; M-1 to I-195; M-1 to F-194; M-1 to F-193; M-1 to
Y-192; M-1 to G-191; M-1 to T-190; M-1 to E-189; M-1 to K-188; M-1
to V-187; M-1 to L-186; M-1 to I-185; M-1 to K-184; M-1 to N-183;
M-1 to E-182; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-1 to
L-178; M-1 to A-177; M-1 to S-176; M-1 to G-175; M-1 to R-174; M-1
to K-173; M-1 to F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169;
M-1 to W-168; M-1 to P-167; M-1 to V-166; M-1 to F-165; M-1 to
T-164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-1 to K-160; M-1
to Q-159; M-1 to I-158; M-1 to T-157; M-1 to P-156; M-1 to T-155;
M-1 to E-154; M-1 to S-153; M-1 to D-152; M-1 to A-151; M-1 to
I-150; M-1 to L-149; M-1 to Q-148; M-1 to L-147; M-1 to C-146; M-1
to D-145; M-1 to Q-144; M-1 to T-143; M-1 to V-142; M-1 to T-141;
M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to
Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1
to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127;
M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to
E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1
to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113;
M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to
V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1
to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99;
M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94;
M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89;
M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84;
M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79;
M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74;
M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69;
M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64;
M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59;
M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54;
M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49;
M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44;
M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39;
M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34;
M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29;
M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24;
M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19;
M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14;
M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1
to E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID NO:2. The present
application is also directed to nucleic acid molecules comprising,
or alternatively, consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the
polynucleotide sequence encoding the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention, as are polypeptides comprising an
amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that encode such polypeptides.
[0309] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
a Neutrokine-alpha polypeptide, which may be described generally as
having residues n.sup.3-m.sup.3 of SEQ ID NO:2, where n.sup.3 and
m.sup.3 are integers as defined above.
[0310] Furthermore, since the predicted extracellular domain of the
Neutrokine-alphaSV polypeptides of the invention may itself elicit
functional activity (e.g., biological activity), deletions of N-
and C-terminal amino acid residues from the predicted extracellular
region of the polypeptide at positions Gln-73 to Leu-266 of SEQ ID
NO:19 may retain some functional activity, such as, for example,
ligand binding, to stimulation of lymphocyte (e.g., B cell)
proliferation, differentiation, and/or activation, modulation of
cell replication, modulation of target cell activities and/or
immunogenicity. However, even if deletion of one or more amino
acids from the N-terminus of the predicted extracellular domain of
a Neutrokine-alphaSV polypeptide results in modification or loss of
one or more functional activities of the polypeptide, other
functional activities may still be retained. Thus, the ability of
the shortened polypeptides to induce and/or bind to antibodies
which recognize the complete or mature or extracellular domains of
the polypeptides generally will be retained when less than the
majority of the residues of the complete or mature or extracellular
domains of the polypeptides are removed from the N-terminus.
Whether a particular polypeptide lacking N-terminal residues of a
complete polypeptide retains such immunologic activities can
readily be determined by routine methods described herein and
otherwise known in the art.
[0311] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of Neutrokine-alphaSV shown in
SEQ ID NO:19, up to the glycine residue at position number 261, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues n.sup.4-266 of SEQ ID NO:19, where n.sup.4 is
an integer in the range of the amino acid position of amino acid
residues 73-261 of the amino acid sequence in SEQ ID NO:19, and 261
is the position of the first residue from the N-terminus of the
predicted extracellular domain Neutrokine-alphaSV polypeptide
(shown in SEQ ID NO:19).
[0312] More in particular, in certain embodiments, the invention
provides polynucleotides encoding polypeptides comprising, or
alternatively consisting of, an amino acid sequence selected from
the group consisting of residues of Q-73 to L-266; G-74 to L-266;
D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to
L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266;
Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to
L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266;
A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to
L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to
L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to
L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to
L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to
L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to
L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to
L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to
L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to
L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to
L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to
L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to
L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to
L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to
L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to
L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to
L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to
L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to
L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to
L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to
L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to
L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to
L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to
L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to
L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to
L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to
L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to
L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to
L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to
L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to
L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to
L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to
L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to
L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to
L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to
L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to
L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to
L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to
L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to
L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to
L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to
L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; and G-261 to
L-266 of SEQ ID NO:19. The present application is also directed to
nucleic acid molecules comprising, or alternatively, consisting of,
a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%,
97%, 98% or 99% identical to the polynucleotide sequence encoding
the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides
described above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these nucleic acids and/or
polynucleotide sequences are also encompassed by the invention, as
are polypeptides comprising an amino acid sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino
acid sequence described above, and polynucleotides that encode such
polypeptides.
[0313] Similarly, deletions of C-terminal amino acid residues of
the predicted extracellular domain of Neutrokine-alphaSV up to the
leucine residue at position 79 of SEQ ID NO:19 may retain some
functional activity, such as, for example, ligand binding, the
ability to stimulate lymphocyte (e.g., B cell) proliferation,
differentiation, and/or activation, modulation of cell replication,
modulation of target cell activities and/or immunogenicity.
Polypeptides having further C-terminal deletions including Leu-79
of SEQ ID NO:19 would not be expected to retain biological
activities.
[0314] However, even if deletion of one or more amino acids from
the C-terminus of a polypeptide results in modification or loss of
one or more functional activities (e.g., biological activity) of
the polypeptide, other functional activities may still be retained.
Thus, the ability of the shortened polypeptide to induce and/or
bind to antibodies which recognize the complete, mature or
extracellular forms of the polypeptide generally will be retained
when less than the majority of the residues of the complete, mature
or extracellular forms of the polypeptide are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of the predicted extracellular domain retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0315] Accordingly, the present invention further provides
polypeptides having one or more residues from the carboxy terminus
of the amino acid sequence of the predicted extracellular domain of
Neutrokine-alphaSV shown in SEQ ID NO:19, up to the leucine residue
at position 79 of SEQ ID NO:19, and polynucleotides encoding such
polypeptides. In particular, the present invention provides
polypeptides having the amino acid sequence of residues 73-m.sup.4
of the amino acid sequence in SEQ ID NO:19, where m.sup.4 is any
integer in the range of the amino acid position of amino acid
residues 79-265 of the amino acid sequence in SEQ ID NO:19.
[0316] More in particular, in certain embodiments, the invention
provides polynucleotides encoding polypeptides comprising, or
alternatively consisting of, an amino acid sequence selected from
the group consisting of residues Q-73 to L-265; Q-73 to K-264; Q-73
to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to
F-259; Q-73 to T-258; Q-73 to V-257; Q-73 to D-256; Q-73 to G-255;
Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; Q-73 to I-251; Q-73 to
Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246;
Q-73 to P-245; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-73 to
Q-241; Q-73 to L-240; Q-73 to E-239; Q-73 to D-238; Q-73 to G-237;
Q-73 to E-236; Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to
A-232; Q-73 to I-231; Q-73 to G-230; Q-73 to A-229; Q-73 to S-228;
Q-73 to Y-227; Q-73 to C-226; Q-73 to S-225; Q-73 to N-224; Q-73 to
N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-73 to E-219;
Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; Q-73 to
I-214; Q-73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210;
Q-73 to T-209; Q-73 to V-208; Q-73 to L-207; Q-73 to S-206; Q-73 to
L-205; Q-73 to E-204; Q-73 to D-203; Q-73 to G-202; Q-73 to F-201;
Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-73 to K-197; Q-73 to
K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-192;
Q-73 to H-191; Q-73 to G-190; Q-73 to Q-7389; Q-73 to A-188; Q-73
to Y-187; Q-73 to T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to
T-183; Q-73 to Y-182; Q-73 to L-181; Q-73 to V-180; Q-73 to Q-179;
Q-73 to G-178; Q-73 to Y-177; Q-73 to I-176; Q-73 to F-175; Q-73 to
F-174; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-170;
Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to
K-165; Q-73 to N-164; Q-73 to E-163; Q-73 to K-162; Q-73 to E-161;
Q-73 to E-160; Q-73 to L-159; Q-73 to A-158; Q-73 to S-157; Q-73 to
G-156; Q-73 to R-155; Q-73 to K-154; Q-73 to F-153; Q-73 to S-152;
Q-73 to L-151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-148; Q-73 to
V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143;
Q-73 to G-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to
P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134;
Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to
S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125;
Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to
P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116;
Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to
G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107;
Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to
L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98;
Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to
A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73
to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84;
Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to
L-79; and Q-73 to S-78 of SEQ ID NO:19. The present application is
also directed to nucleic acid molecules comprising, or
alternatively, consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the
polynucleotide sequence encoding the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention, as are polypeptides comprising an
amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that encode such polypeptides.
[0317] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
the predicted extracellular domain of Neutrokine-alphaSV, which may
be described generally as having residues n.sup.4-m.sup.4 of SEQ ID
NO:19 where n.sup.4 and m.sup.4 are integers as defined above.
[0318] In another embodiment, a nucleotide sequence encoding a
polypeptide consisting of a portion of the extracellular domain of
the Neutrokine-alphaSV amino acid sequence encoded by the cDNA
clone contained in the deposit having ATCC Accession No. 203518,
where this portion excludes from 1 to about 260 amino acids from
the amino terminus of the extracellular domain of the amino acid
sequence encoded by cDNA clone contained in the deposit having ATCC
Accession No. 203518, or from 1 to about 187 amino acids from the
carboxy terminus of the extracellular domain of the amino acid
sequence encoded by cDNA clone contained in the deposit having ATCC
Accession No. 203518, or any combination of the above amino
terminal and carboxy terminal deletions, of the entire
extracellular domain of the amino acid sequence encoded by the cDNA
clone contained in the deposit having ATCC Accession No.
203518.
[0319] As mentioned above, even if deletion of one or more amino
acids from the N-terminus of a polypeptide results in modification
or loss of one or more functional activities (e.g., biological
activity) of the polypeptide, other functional activities may still
be retained. Thus, the ability of a shortened Neutrokine-alphaSV
mutein to induce and/or bind to antibodies which recognize the
full-length or mature forms or the extracellular domain of the
polypeptide generally will be retained when less than the majority
of the residues of the full-length or mature or extracellular
domain of the polypeptide are removed from the N-terminus. Whether
a particular polypeptide lacking N-terminal residues of a complete
polypeptide retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art. It is not unlikely that a Neutrokine-alphaSV mutein
with a large number of deleted N-terminal amino acid residues may
retain functional (e.g., immunogenic) activities. In fact, peptides
composed of as few as six Neutrokine-alphaSV amino acid residues
may often evoke an immune response.
[0320] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the predicted full-length amino acid sequence of the
Neutrokine-alphaSV shown in SEQ ID NO:19, up to the glycine residue
at position number 261 of the sequence shown SEQ ID NO:19 and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues n.sup.5-266 of the sequence shown in SEQ ID
NO:19, where n.sup.5 is an integer in the range of the amino acid
position of amino acid residues 1 to 261 of the amino acid sequence
in SEQ ID NO:19.
[0321] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the group consisting of
residues of D-2 to L-266; D-3 to L-266; S-4 to L-266; T-5 to L-266;
E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to
L-266; R-11 to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266;
C-15 to L-266; L-16 to L-266; K-17 to L-266; K-18 to L-266; R-19 to
L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266;
L-24 to L-266; K-25 to L-266; E-26 to L-266; C-27 to L-266; V-28 to
L-266; S-29 to L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266;
R-33 to L-266; K-34 to L-266; E-35 to L-266; S-36 to L-266; P-37 to
L-266; S-38 to L-266; V-39 to L-266; R-40 to L-266; S-41 to L-266;
S-42 to L-266; K-43 to L-266; D-44 to L-266; G-45 to L-266; K-46 to
L-266; L-47 to L-266; L-48 to L-266; A-49 to L-266; A-50 to L-266;
T-51 to L-266; L-52 to L-266; L-53 to L-266; L-54 to L-266; A-55 to
L-266; L-56 to L-266; L-57 to L-266; S-58 to L-266; C-59 to L-266;
C-60 to L-266; L-61 to L-266; T-62 to L-266; V-63 to L-266; V-64 to
L-266; S-65 to L-266; F-66 to L-266; Y-67 to L-266; Q-68 to L-266;
V-69 to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to
L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266;
S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to
L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266;
H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to
L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266;
G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100
to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to
L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to
L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to
L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to
L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to
L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to
L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to
L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to
L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to
L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to
L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to
L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to
L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to
L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to
L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to
L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to
L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to
L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to
L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to
L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to
L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to
L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to
L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to
L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to
L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to
L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to
L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to
L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to
L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to
L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to
L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to
L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to
L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to
L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to
L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to
L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to
L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to
L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to
L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to
L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to
L-266; and G-261 to L-266 of SEQ ID NO:19. The present application
is also directed to nucleic acid molecules comprising, or
alternatively, consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the
polynucleotide sequence encoding the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention, as are polypeptides comprising an
amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that encode such polypeptides.
[0322] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification or loss of one or more functional activities (e.g.,
biological activities) of the protein, other functional activities
may still be retained. Thus, the ability of a shortened
Neutrokine-alphaSV mutein to induce and/or bind to antibodies which
recognize the complete or mature form or the extracellular domain
of the polypeptide generally will be retained when less than the
majority of the residues of the complete or mature form or the
extracellular domain of the polypeptide are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete polypeptide retains such immunologic
activities can readily be determined by routine methods described
herein and otherwise known in the art. It is not unlikely that a
Neutrokine-alphaSV mutein with a large number of deleted C-terminal
amino acid residues may retain some functional (e.g., immunogenic)
activities. In fact, peptides composed of as few as six
Neutrokine-alphaSV amino acid residues may often evoke an immune
response.
[0323] Accordingly, the present invention further provides in
another embodiment, polypeptides having one or more residues
deleted from the carboxy terminus of the amino acid sequence of the
Neutrokine-alphaSV shown in SEQ ID NO:19, up to the glutamic acid
residue at position number 6, and polynucleotides encoding such
polypeptides. In particular, the present invention provides
polypeptides comprising the amino acid sequence of residues
1-m.sup.5 of SEQ ID NO:19, where m.sup.5 is an integer in the range
of the amino acid position of amino acid residues 6 to 265 in the
amino acid sequence of SEQ ID NO:19.
[0324] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the group consisting of
residues M-1 to L-265; M-1 to K-264; M-1 to L-263; M-1 to A-262;
M-1 to G-261; M-1 to F-260; M-1 to F-259; M-1 to T-258; M-1 to
V-257; M-1 to D-256; M-1 to G-255; M-1 to D-254; M-1 to L-253; M-1
to S-252; M-1 to I-251; M-1 to Q-250; M-1 to A-249; M-1 to N-248;
M-1 to E-247; M-1 to R-246; M-1 to P-245; M-1 to I-244; M-1 to
A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1
to D-238; M-1 to G-237; M-1 to E-236; M-1 to E-235; M-1 to L-234;
M-1 to K-233; M-1 to A-232; M-1 to I-231; M-1 to G-230; M-1 to
A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226; M-1 to S-225; M-1
to N-224; M-1 to N-223; M-1 to P-222; M-1 to L-221; M-1 to T-220;
M-1 to E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to
Q-215; M-1 to I-214; M-1 to C-213; M-1 to R-212; M-1 to F-211; M-1
to L-210; M-1 to T-209; M-1 to V-208; M-1 to L-207; M-1 to S-206;
M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-1 to
F-201; M-1 to V-200; M-1 to H-199; M-1 to V-198; M-1 to K-197; M-1
to K-196; M-1 to R-195; M-1 to Q-194; M-1 to I-193; M-1 to L-192;
M-1 to H-191; M-1 to G-190; M-1 to M-189; M-1 to A-188; M-1 to
Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-1 to T-183; M-1
to Y-182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178;
M-1 to Y-177; M-1 to I-176; M-1 to F-175; M-1 to F-174; M-1 to
Y-173; M-1 to G-172; M-1 to T-171; M-1 to E-170; M-1 to K-169; M-1
to V-168; M-1 to L-167; M-1 to I-166; M-1 to K-165; M-1 to N-164;
M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-1 to
L-159; M-1 to A-158; M-1 to S-157; M-1 to G-156; M-1 to R-155; M-1
to K-154; M-1 to F-153; M-1 to S-152; M-1 to L-151; M-1 to L-150;
M-1 to W-149; M-1 to P-148; M-1 to V-147; M-1 to F-146; M-1 to
T-145; M-1 to Y-144; M-1 to S-143; M-1 to G-142; M-1 to T-141; M-1
to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136;
M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to
N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1
to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122;
M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to
P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1
to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108;
M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to
E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1
to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to
A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to
A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to
L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to
S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to
Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to
Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to
V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to
S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to
L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to
L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to
K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to
S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to
R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to
V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to
K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to
K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to
T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to
E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID NO:19. The present
application is also directed to nucleic acid molecules comprising,
or alternatively, consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the
polynucleotide sequence encoding the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides described above. The present
invention also encompasses the above polynucleotide sequences fused
to a heterologous polynucleotide sequence. Polypeptides encoded by
these nucleic acids and/or polynucleotide sequences are also
encompassed by the invention, as are polypeptides comprising an
amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that encode such polypeptides.
[0325] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
a Neutrokine-alphaSV polypeptide, which may be described generally
as having residues n.sup.5-m.sup.5 of SEQ ID NO:19, where n.sup.5
and m.sup.5 are integers as defined above.
[0326] In additional embodiments, the present invention provides
polypeptides comprising the amino acid sequence of residues
134-m.sup.6 of SEQ ID NO:2, where m.sup.6 is an integer from 140 to
285, corresponding to the position of the amino acid residue in SEQ
ID NO:2. For example, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the group consisting of
residues A-134 to Leu-285; A-134 to L-284; A-134 to K-283; A-134 to
L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to
F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to
G-274; A-134 to D-273; A-134 to L-272; A-134 to S-271; A-134 to
I-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to
E-266; A-134 to R-265; A-134 to P-264; A-134 to I-263; A-134 to
A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-259; A-134 to
E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to
E-254; A-134 to L-253; A-134 to K-252; A-134 to A-251; A-134 to
I-250; A-134 to G-249; A-134 to A-248; A-134 to S-247; A-134 to
Y-246; A-134 to C-245; A-134 to S-244; A-134 to N-243; A-134 to
N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134 to
E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; A-134 to
Q-234; A-134 to I-233; A-134 to C-232; A-134 to R-231; A-134 to
F-230; A-134 to L-229; A-134 to T-228; A-134 to V-227; A-134 to
L-226; A-134 to S-225; A-134 to L-224; A-134 to E-223; A-134 to
D-222; A-134 to G-221; A-134 to F-220; A-134 to V-219; A-134 to
H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to
R-214; A-134 to Q-213; A-134 to I-212; A-134 to L-211; A-134 to
H-210; A-134 to G-209; A-134 to M-208; A-134 to A-207; A-134 to
Y-206; A-134 to T-205; A-134 to K-204; A-134 to D-203; A-134 to
T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134 to
Q-198; A-134 to G-197; A-134 to Y-196; A-134 to I-195; A-134 to
F-194; A-134 to F-193; A-134 to Y-192; A-134 to G-191; A-134 to
T-190; A-134 to E-189; A-134 to K-188; A-134 to V-187; A-134 to
L-186; A-134 to I-185; A-134 to K-184; A-134 to N-183; A-134 to
E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134 to
L-178; A-134 to A-177; A-134 to S-176; A-134 to G-175; A-134 to
R-174; A-134 to K-173; A-134 to F-172; A-134 to S-171; A-134 to
L-170; A-134 to L-169; A-134 to W-168; A-134 to P-167; A-134 to
V-166; A-134 to F-165; A-134 to T-164; A-134 to Y-163; A-134 to
S-162; A-134 to G-161; A-134 to K-160; A-134 to Q-159; A-134 to
I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to
E-154; A-134 to S-153; A-134 to D-152; A-134 to A-151; A-134 to
I-150; A-134 to L-149; A-134 to Q-148; A-134 to L-147; A-134 to
C-146; A-134 to D-145; A-134 to Q-144; A-134 to T-143; A-134 to
V-142; A-134 to T-141; and A-134 to E-140 of SEQ ID NO:2. The
present application is also directed to nucleic acid molecules
comprising, or alternatively, consisting of, a polynucleotide
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the polynucleotide sequence encoding the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides described
above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these nucleic acids and/or
polynucleotide sequences are also encompassed by the invention, as
are polypeptides comprising an amino acid sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino
acid sequence described above, and polynucleotides that encode such
polypeptides.
[0327] Additional preferred polypeptide fragments of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group consisting of residues: M-1 to C-15; D-2 to
L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to
E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to
E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17
to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35;
M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to
R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31
to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49;
S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to
L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-45
to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A-49 to V-63;
A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to
Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59
to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77;
V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to
E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73
to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91;
S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to
G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87
to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to
A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109;
G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100
to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to
P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to
E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to
S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to
R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to
A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to
P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to
V-142; S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to
C-146; R-133 to L-147; A-134 to Q-148; V-135 to L-149; Q-136 to
I-150; G-137 to A-151; P-138 to D-152; E-139 to S-153; E-140 to
E-154; T-141 to T-155; V-142 to P-156; T-143 to T-157; Q-144 to
I-158; D-145 to Q-159; C-146 to K-160; L-147 to G-161; Q-148 to
S-162; L-149 to Y-163; I-150 to T-164; A-151 to F-165; D-152 to
V-166; S-153 to P-167; E-154 to W-168; T-155 to L-169; P-156 to
L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to
R-174; G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to
L-178; F-165 to E-179; V-166 to E-180; P-167 to K-181; W-168 to
E-182; L-169 to N-183; L-170 to K-184; S-171 to I-185; F-172 to
L-186; K-173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to
T-190; A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to
F-194; K-181 to I-195; E-182 to Y-196; N-183 to G-197; K-184 to
Q-198; I-185 to V-199; L-186 to L-200; V-187 to Y-201; K-188 to
T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205; Y-192 to
Y-206; F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to
H-210; G-197 to L-211; Q-198 to I-212; V-199 to Q-213; L-200 to
R-214; Y-201 to K-215; T-202 to K-216; D-203 to V-217; K-204 to
H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-208 to
D-222; G-209 to E-223; H-210 to L-224; L-211 to S-225; I-212 to
L-226; Q-213 to V-227; R-214 to T-228; K-215 to L-229; K-216 to
F-230; V-217 to R-231; H-218 to C-232; V-219 to I-233; F-220 to
Q-234; G-221 to N-235; D-222 to M-236; E-223 to P-237; L-224 to
E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to
N-242; L-229 to N-243; F-230 to S-244; R-231 to C-245; C-232 to
Y-246; I-233 to S-247; Q-234 to A-248; N-235 to G-249; M-236 to
I-250; P-237 to A-251; E-238 to K-252; T-239 to L-253; L-240 to
E-254; P-241 to E-255; N-242 to G-256; N-243 to D-257; S-244 to
E-258; C-245 to L-259; Y-246 to Q-260; S-247 to L-261; A-248 to
A-262; G-249 to I-263; I-250 to P-264; A-251 to R-265; K-252 to
E-266; L-253 to N-267; E-254 to A-268; E-255 to Q-269; G-256 to
I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to
G-274; L-261 to D-275; A-262 to V-276; 1-263 to T-277; P-264 to
F-278; R-265 to F-279; E-266 to G-280; N-267 to A-281; A-268 to
L-282; Q-269 to K-283; I-270 to L-284; and S-271 to L-285 of SEQ ID
NO:2. Preferably, these polypeptide fragments have one or more
functional activities (e.g., biological activity, antigenicity, and
immunogenicity) of Neutrokine-alpha and/or Neutrokine-alpha SV
polypeptides of the invention and may be used, for example, to
generate or screen for antibodies, as described further below. The
present invention is also directed to polypeptides comprising, or
alternatively, consisting of, an amino acid sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence described above. The present invention also encompasses
the above amino acid sequences fused to a heterologous amino acid
sequence as described herein. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0328] Additional preferred polypeptide fragments of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group consisting of residues: M-1 to C-15; D-2 to
L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to
E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to
E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17
to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35;
M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to
R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31
to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49;
S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to
L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-45
to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A-49 to V-63;
A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to
Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59
to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77;
V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to
E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73
to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91;
S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to
G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87
to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to
A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109;
G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100
to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to
P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to
E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to
S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to
R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to
A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to
P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to
G-142; S-129 to S-143; R-130 to Y-144; N-131 to T-145; K-132 to
F-146; R-133 to V-147; A-134 to P-148; V-135 to W-149; Q-136 to
L-150; G-137 to L-151; P-138 to S-152; E-139 to F-153; E-140 to
K-154; T-141 to R-155; G-142 to G-156; S-143 to S-157; Y-144 to
A-158; T-145 to L-159; F-146 to E-160; V-147 to E-161; P-148 to
K-162; W-149 to E-163; L-150 to N-164; L-151 to K-165; S-152 to
I-166; F-153 to L-167; K-154 to V-168; R-155 to K-169; G-156 to
E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-160 to
F-174; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to
G-178; K-165 to Q-179; I-166 to V-180; L-167 to L-181; V-168 to
Y-182; K-169 to T-183; E-170 to D-184; T-171 to K-185; G-172 to
T-186; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-176 to
G-190; Y-177 to H-191; G-178 to L-192; Q-179 to I-193; V-180 to
Q-194; L-181 to R-195; Y-182 to K-196; T-183 to K-197; D-184 to
V-198; K-185 to H-199; T-186 to V-200; Y-187 to F-201; A-188 to
G-202; M-189 to D-203; G-190 to E-204; H-191 to L-205; L-192 to
S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to
L-210; K-197 to F-211; V-198 to R-212; H-199 to C-213; V-200 to
I-214; F-201 to Q-215; G-202 to N-216; D-203 to M-217; E-204 to
P-218; L-205 to E-219; S-206 to T-220; L-207 to L-221; V-208 to
P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to
C-226; C-213 to Y-227; I-214 to S-228; Q-215 to A-229; N-216 to
G-230; M-217 to I-231; P-218 to A-232; E-219 to K-233; T-220 to
L-234; L-221 to E-235; P-222 to E-236; N-223 to G-237; N-224 to
D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; S-228 to
L-242; A-229 to A-243; G-230 to I-244; I-231 to P-245; A-232 to
R-246; K-233 to E-247; L-234 to N-248; E-235 to A-249; E-236 to
Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253; L-240 to
D-254; Q-241 to G-255; L-242 to D-256; A-243 to V-257; I-244 to
T-258; P-245 to F-259; R-246 to F-260; E-247 to G-261; N-248 to
A-262; A-249 to L-263; Q-250 to K-264; I-251 to L-265; and S-252 to
L-266 of SEQ ID NO:19. Preferably, these polypeptide fragments have
one or more functional activities (e.g., biological activity,
antigenicity, and immunogenicity) of Neutrokine-alpha and/or
Neutrokine-alpha SV polypeptides of the invention and may be used,
for example, to generate or screen for antibodies, as described
further below. The present invention is also directed to
polypeptides comprising, or alternatively, consisting of, an amino
acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
99% identical to an amino acid sequence described above. The
present invention also encompasses the above amino acid sequences
fused to a heterologous amino acid sequence as described herein.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0329] Additional preferred polypeptide fragments of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group consisting of residues: M-1 to F-15; D-2 to
C-16; E-3 to S-17; S-4 to E-18; A-5 to K-19; K-6 to G-20; T-7 to
E-21; L-8 to D-22; P-9 to M-23; P-10 to K-24; P-11 to V-25; C-12 to
G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to I-30; S-17
to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35;
D-22 to E-36; M-23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to
F-40; Y-27 to G-41; D-28 to I-42; P-29 to C-43; I-30 to R-44; T-31
to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35 to L-49;
E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F-40 to
L-54; G-41 to L-55; I-42 to A-56; C-43 to L-57; R-44 to L-58; D-45
to S-59; G-46 to S-60; R-47 to S-61; L-48 to F-62; L-49 to T-63;
A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67; L-54 to
Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59
to L-73; S-60 to Q-74; S-61 to A-75; F-62 to D-76; T-63 to L-77;
A-64 to M-78; M-65 to N-79; S-66 to L-80; L-67 to R-81; Y-68 to
M-82; Q-69 to E-83; L-70 to L-84; A-71 to Q-85; A-72 to S-86; L-73
to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91;
M-78 to T-92; N-79 to P-93; L-80 to A-94; R-81 to A-95; M-82 to
A-96; E-83 to G-97; L-84 to A-98; Q-85 to P-99; S-86 to E-100; Y-87
to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-104; A-91 to
V-105; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-95 to T-109;
A-96 to P-110; G-97 to A-111; A-98 to A-112; P-99 to P-113; E-100
to R-114; L-101 to P-115; T-102 to H-116; A-103 to N-117; G-104 to
S-118; V-105 to S-119; K-106 to R-120; L-107 to G-121; L-108 to
H-122; T-109 to R-123; P-110 to N-124; A-111 to R-125; A-112 to
R-126; P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to
G-130; N-117 to P-131; S-118 to E-132; S-119 to E-133; R-120 to
T-134; G-121 to E-135; H-122 to Q-136; R-123 to D-137; N-124 to
V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to
A-142; Q-129 to P-143; G-130 to P-144; P-131 to A-145; E-132 to
P-146; E-133 to C-147; T-134 to L-148; E-135 to P-149; Q-136 to
G-150; D-137 to C-151; V-138 to R-152; D-139 to H-153; L-140 to
S-154; S-141 to Q-155; A-142 to H-156; P-143 to D-157; P-144 to
D-158; A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to
N-162; P-149 to L-163; G-150 to R-164; C-151 to N-165; R-152 to
I-166; H-153 to I-167; S-154 to Q-168; Q-155 to D-169; H-156 to
C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160 to
I-174; M-161 to A-175; N-162 to D-176; L-163 to S-177; R-164 to
D-178; N-165 to T-179; I-166 to P-180; I-167 to A-181; Q-168 to
L-182; D-169 to E-183; C-170 to E-184; L-171 to K-185; Q-172 to
E-186; L-173 to N-187; I-174 to K-188; A-175 to I-189; D-176 to
V-190; S-177 to V-191; D-178 to R-192; T-179 to Q-193; P-180 to
T-194; A-181 to G-195; L-182 to Y-196; E-183 to F-197; E-184 to
F-198; K-185 to I-199; E-186 to Y-200; N-187 to S-201; K-188 to
Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-192 to
T-206; Q-193 to D-207; T-194 to P-208; G-195 to I-209; Y-196 to
F-210; F-197 to A-211; F-198 to M-212; I-199 to G-213; Y-200 to
H-214; S-201 to V-215; Q-202 to I-216; V-203 to Q-217; L-204 to
R-218; Y-205 to K-219; T-206 to K-220; D-207 to V-221; P-208 to
H-222; I-209 to V-223; F-210 to F-224; A-211 to G-225; M-212 to
D-226; G-213 to E-227; H-214 to L-228; V-215 to S-229; I-216 to
L-230; Q-217 to V-231; R-218 to T-232; K-219 to L-233; K-220 to
F-234; V-221 to R-235; H-222 to C-236; V-223 to I-237; F-224 to
Q-238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to
K-242; S-229 to T-243; L-230 to L-244; V-231 to P-245; T-232 to
N-246; L-233 to N-247; F-234 to S-248; R-235 to C-249; C-236 to
Y-250; I-237 to S-251; Q-238 to A-252; N-239 to G-253; M-240 to
I-254; P-241 to A-255; K-242 to R-256; T-243 to L-257; L-244 to
E-258; P-245 to E-259; N-246 to G-260; N-247 to D-261; S-248 to
E-262; C-249 to I-263; Y-250 to Q-264; S-251 to L-265; A-252 to
A-266; G-253 to I-267; I-254 to P-268; A-255 to R-269; R-256 to
E-270; L-257 to N-271; E-258 to A-272; E-259 to Q-273; G-260 to
I-274; D-261 to S-275; E-262 to R-276; I-263 to N-277; Q-264 to
G-278; L-265 to D-279; A-266 to D-280; I-267 to T-281; P-268 to
F-282; R-269 to F-283; E-270 to G-284; N-271 to A-285; A-272 to
L-286; Q-273 to K-287; I-274 to L-288; and S-275 to L-289 of SEQ ID
NO:38. Preferably, these polypeptide fragments have one or more
functional activities (e.g., biological activity, antigenicity, and
immunogenicity) of Neutrokine-alpha and/or Neutrokine-alpha SV
polypeptides of the invention and may be used, for example, to
generate or screen for antibodies, as described further below. The
present invention is also directed to polypeptides comprising, or
alternatively, consisting of, an amino acid sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence described above. The present invention also encompasses
the above amino acid sequences fused to a heterologous amino acid
sequence as described herein. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0330] It will be recognized by one of ordinary skill in the art
that some amino acid sequences of the Neutrokine-alpha and
Neutrokine-alphaSV polypeptides can be varied without significant
effect of the structure or function of the polypeptide. If such
differences in sequence are contemplated, it should be remembered
that there will be critical areas on the polypeptide which
determine activity.
[0331] Thus, the invention further includes variations of the
Neutrokine-alpha polypeptide which show Neutrokine-alpha
polypeptide functional activity (e.g., biological activity) or
which include regions of Neutrokine-alpha polypeptide such as the
polypeptide fragments described herein. The invention also includes
variations of the Neutrokine-alphaSV polypeptide which show
Neutrokine-alphaSV polypeptide functional activity (e.g.,
biological activity) or which include regions of Neutrokine-alphaSV
polypeptide such as the polypeptide fragments described herein.
Such mutants include deletions, insertions, inversions, repeats,
and type substitutions selected according to general rules known in
the art so as have little effect on activity. For example, guidance
concerning how to make phenotypically silent amino acid
substitutions is provided in Bowie, J. U. et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main approaches for studying the
tolerance of an amino acid sequence to change. The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality.
[0332] As the authors state, these studies have revealed that
proteins are surprisingly tolerant of amino acid substitutions. The
authors further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described in Bowie, J.
U. et al., supra, and the references cited therein. Typically seen
as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0333] Thus, the fragment, derivative or analog of the polypeptide
of FIGS. 1A and 1B (SEQ ID NO:2), or that encoded by the deposited
cDNA plasmid, may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid
residues includes a substituent group, or (iii) one in which the
extracellular domain of the polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or (iv) one in
which the additional amino acids are fused to the extracellular
domain of the polypeptide, such as an IgG Fc fusion region peptide
or leader or secretory sequence or a sequence which is employed for
purification of the extracellular domain of the polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0334] Furthermore, the fragment, derivative or analog of the
polypeptide of FIGS. 5A and 5B (SEQ ID NO:19), or that encoded by
the deposited cDNA plasmid, may be (i) one in which one or more of
the amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code, or (ii) one in which one or more
of the amino acid residues includes a substituent group, or (iii)
one in which the extracellular domain of the polypeptide is fused
with another compound, such as a compound to increase the half-life
of the polypeptide (for example, polyethylene glycol), or (iv) one
in which the additional amino acids are fused to the extracellular
domain of the polypeptide, such as, a soluble biologically active
fragment of another TNF ligand family member (e.g., CD40 Ligand),
an IgG Fc fusion region peptide or leader or secretory sequence or
a sequence which is employed for purification of the extracellular
domain of the polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0335] Thus, the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the present invention may include one or more amino
acid substitutions, deletions or additions, either from natural
mutations or human manipulation. As indicated, changes are
preferably of a minor nature, such as conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein (see Table II).
TABLE-US-00002 TABLE II Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0336] In one embodiment of the invention, polypeptide comprises,
or alternatively consists of, the amino acid sequence of a
Neutrokine-alpha or Neutrokine-alphaSV polypeptide having an amino
acid sequence which contains at least one conservative amino acid
substitution, but not more than 50 conservative amino acid
substitutions, even more preferably, not more than 40 conservative
amino acid substitutions, still more preferably, not more than 30
conservative amino acid substitutions, and still even more
preferably, not more than 20 conservative amino acid substitutions.
Of course, in order of ever-increasing preference, it is highly
preferable for a peptide or polypeptide to have an amino acid
sequence which comprises the amino acid sequence of a
Neutrokine-alpha polypeptide, which contains at least one, but not
more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid
substitutions.
[0337] For example, site directed changes at the amino acid level
of Neutrokine-alpha can be made by replacing a particular amino
acid with a conservative substitution. Preferred conservative
substitution mutations of the Neutrokine-alpha amino acid sequence
provided in SEQ ID NO:2 include: M1 replaced with A, G, I, L, S, T,
or V; D2 replaced with E; D3 replaced with E; S4 replaced with A,
G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6
replaced with D; R7 replaced with H, or K; E8 replaced with D; Q9
replaced with N; S10 replaced with A, G, I, L, T, M, or V; R11
replaced with H, or K; L12 replaced with A, G, I, S, T, M, or V;
T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G,
I, L, T, M, or V; L16 replaced with A, G, I, S, T, M, or V; K17
replaced with H, or R; K18 replaced with H, or R; R19 replaced with
H, or K; E20 replaced with D; E21 replaced with D; M22 replaced
with A, G, I, L, S, T, or V; K23 replaced with H, or R; L24
replaced with A, G, I, S, T, M, or V; K25 replaced with H, or R;
E26 replaced with D; V28 replaced with A, G, I, L, S, T, or M; S29
replaced with A, G, I, L, T, M, or V; 130 replaced with A, G, L, S,
T, M, or V; L31 replaced with A, G, I, S, T, M, or V; R33 replaced
with H, or K; K34 replaced with H, or R; E35 replaced with D; S36
replaced with A, G, I, L, T, M, or V; S38 replaced with A, G, I, L,
T, M, or V; V39 replaced with A, G, I, L, S, T, or M; R40 replaced
with H, or K; S41 replaced with A, G, I, L, T, M, or V; S42
replaced with A, G, I, L, T, M, or V; K43 replaced with H, or R;
D44 replaced with E; G45 replaced with A, I, L, S, T, M, or V; K46
replaced with H, or R; L47 replaced with A, G, I, S, T, M, or V;
L48 replaced with A, G, I, S, T, M, or V; A49 replaced with G, I,
L, S, T, M, or V; A50 replaced with G, I, L, S, T, M, or V; T51
replaced with A, G, I, L, S, M, or V; L52 replaced with A, G, I, S,
T, M, or V; L53 replaced with A, G, I, S, T, M, or V; L54 replaced
with A, G, I, S, T, M, or V; A55 replaced with G, I, L, S, T, M, or
V; L56 replaced with A, G, I, S, T, M, or V; L57 replaced with A,
G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or V; L61
replaced with A, G, I, S, T, M, or V; T62 replaced with A, G, I, L,
S, M, or V; V63 replaced with A, G, I, L, S, T, or M; V64 replaced
with A, G, I, L, S, T, or M; S65 replaced with A, G, I, L, T, M, or
V; F66 replaced with W, or Y; Y67 replaced with F, or W; Q68
replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70
replaced with G, I, L, S, T, M, or V; A71 replaced with G, I, L, S,
T, M, or V; L72 replaced with A, G, I, S, T, M, or V; Q73 replaced
with N; G74 replaced with A, I, L, S, T, M, or V; D75 replaced with
E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G,
I, L, S, T, M, or V; S78 replaced with A, G, I, L, T, M, or V; L79
replaced with A, G, I, S, T, M, or V; R80 replaced with H, or K;
A81 replaced with G, I, L, S, T, M, or V; E82 replaced with D; L83
replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85
replaced with A, I, L, S, T, M, or V; H86 replaced with K, or R;
H87 replaced with K, or R; A88 replaced with G, I, L, S, T, M, or
V; E89 replaced with D; K90 replaced with H, or R; L91 replaced
with A, G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or
V; G94 replaced with A, I, L, S, T, M, or V; A95 replaced with G,
I, L, S, T, M, or V; G96 replaced with A, I, L, S, T, M, or V; A97
replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R;
A100 replaced with G, I, L, S, T, M, or V; G101 replaced with A, I,
L, S, T, M, or V; L102 replaced with A, G, I, S, T, M, or V; E103
replaced with D; E104 replaced with D; A105 replaced with G, I, L,
S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108
replaced with A, G, I, L, S, T, or M; T109 replaced with A, G, I,
L, S, M, or V; A110 replaced with G, I, L, S, T, M, or V; G111
replaced with A, I, L, S, T, M, or V; L112 replaced with A, G, I,
S, T, M, or V; K113 replaced with H, or R; I114 replaced with A, G,
L, S, T, M, or V; F115 replaced with W, or Y; E116 replaced with D;
A119 replaced with G, I, L, S, T, M, or V; G121 replaced with A, I,
L, S, T, M, or V; E122 replaced with D; G123 replaced with A, I, L,
S, T, M, or V; N124 replaced with Q; S125 replaced with A, G, I, L,
T, M, or V; S126 replaced with A, G, I, L, T, M, or V; Q127
replaced with N; N128 replaced with Q; S129 replaced with A, G, I,
L, T, M, or V; R130 replaced with H, or K; N131 replaced with Q;
K132 replaced with H, or R; R133 replaced with H, or K; A134
replaced with G, I, L, S, T, M, or V; V135 replaced with A, G, I,
L, S, T, or M; Q136 replaced with N; G137 replaced with A, I, L, S,
T, M, or V; E139 replaced with D; E140 replaced with D; T141
replaced with A, G, I, L, S, M, or V; V142 replaced with A, G, I,
L, S, T, or M; T143 replaced with A, G, I, L, S, M, or V; Q144
replaced with N; D145 replaced with E; L147 replaced with A, G, I,
S, T, M, or V; Q148 replaced with N; L149 replaced with A, G, I, S,
T, M, or V; I150 replaced with A, G, L, S, T, M, or V; A151
replaced with G, I, L, S, T, M, or V; D152 replaced with E; S153
replaced with A, G, I, L, T, M, or V; E154 replaced with D; T155
replaced with A, G, I, L, S, M, or V; T157 replaced with A, G, I,
L, S, M, or V; I158 replaced with A, G, L, S, T, M, or V; Q159
replaced with N; K160 replaced with H, or R; G161 replaced with A,
I, L, S, T, M, or V; S162 replaced with A, G, I, L, T, M, or V;
Y163 replaced with F, or W; T164 replaced with A, G, I, L, S, M, or
V; F165 replaced with W, or Y; V166 replaced with A, G, I, L, S, T,
or M; W168 replaced with F, or Y; L169 replaced with A, G, I, S, T,
M, or V; L170 replaced with A, G, I, S, T, M, or V; S171 replaced
with A, G, I, L, T, M, or V; F172 replaced with W, or Y; K173
replaced with H, or R; R174 replaced with H, or K; G175 replaced
with A, I, L, S, T, M, or V; S176 replaced with A, G, I, L, T, M,
or V; A177 replaced with G, I, L, S, T, M, or V; L178 replaced with
A, G, I, S, T, M, or V; E179 replaced with D; E180 replaced with D;
K181 replaced with H, or R; E182 replaced with D; N183 replaced
with Q; K184 replaced with H, or R; I185 replaced with A, G, L, S,
T, M, or V; L186 replaced with A, G, I, S, T, M, or V; V187
replaced with A, G, I, L, S, T, or M; K188 replaced with H, or R;
E189 replaced with D; T190 replaced with A, G, I, L, S, M, or V;
G191 replaced with A, I, L, S, T, M, or V; Y192 replaced with F, or
W; F193 replaced with W, or Y; F194 replaced with W, or Y; I195
replaced with A, G, L, S, T, M, or V; Y196 replaced with F, or W;
G197 replaced with A, I, L, S, T, M, or V; Q198 replaced with N;
V199 replaced with A, G, I, L, S, T, or M; L200 replaced with A, G,
I, S, T, M, or V; Y201 replaced with F, or W; T202 replaced with A,
G, I, L, S, M, or V; D203 replaced with E; K204 replaced with H, or
R; T205 replaced with A, G, I, L, S, M, or V; Y206 replaced with F,
or W; A207 replaced with G, I, L, S, T, M, or V; M208 replaced with
A, G, I, L, S, T, or V; G209 replaced with A, I, L, S, T, M, or V;
H210 replaced with K, or R; L211 replaced with A, G, I, S, T, M, or
V; I212 replaced with A, G, L, S, T, M, or V; Q213 replaced with N;
R214 replaced with H, or K; K215 replaced with H, or R; K216
replaced with H, or R; V217 replaced with A, G, I, L, S, T, or M;
H218 replaced with K, or R; V219 replaced with A, G, I, L, S, T, or
M; F220 replaced with W, or Y; G221 replaced with A, I, L, S, T, M,
or V; D222 replaced with E; E223 replaced with D; L224 replaced
with A, G, I, S, T, M, or V; S225 replaced with A, G, I, L, T, M,
or V; L226 replaced with A, G, I, S, T, M, or V; V227 replaced with
A, G, I, L, S, T, or M; T228 replaced with A, G, I, L, S, M, or V;
L229 replaced with A, G, I, S, T, M, or V; F230 replaced with W, or
Y; R231 replaced with H, or K; I233 replaced with A, G, L, S, T, M,
or V; Q234 replaced with N; N235 replaced with Q; M236 replaced
with A, G, I, L, S, T, or V; E238 replaced with D; T239 replaced
with A, G, I, L, S, M, or V; L240 replaced with A, G, I, S, T, M,
or V; N242 replaced with Q; N243 replaced with Q; S244 replaced
with A, G, I, L, T, M, or V; Y246 replaced with F, or W; S247
replaced with A, G, I, L, T, M, or V; A248 replaced with G, I, L,
S, T, M, or V; G249 replaced with A, I, L, S, T, M, or V; I250
replaced with A, G, L, S, T, M, or V; A251 replaced with G, I, L,
S, T, M, or V; K252 replaced with H, or R; L253 replaced with A, G,
I, S, T, M, or V; E254 replaced with D; E255 replaced with D; G256
replaced with A, I, L, S, T, M, or V; D257 replaced with E; E258
replaced with D; L259 replaced with A, G, I, S, T, M, or V; Q260
replaced with N; L261 replaced with A, G, I, S, T, M, or V; A262
replaced with G, I, L, S, T, M, or V; I263 replaced with A, G, L,
S, T, M, or V; R265 replaced with H, or K; E266 replaced with D;
N267 replaced with Q; A268 replaced with G, I, L, S, T, M, or V;
Q269 replaced with N; I270 replaced with A, G, L, S, T, M, or V;
S271 replaced with A, G, I, L, T, M, or V; L272 replaced with A, G,
I, S, T, M, or V; D273 replaced with E; G274 replaced with A, I, L,
S, T, M, or V; D275 replaced with E; V276 replaced with A, G, I, L,
S, T, or M; T277 replaced with A, G, I, L, S, M, or V; F278
replaced with W, or Y; F279 replaced with W, or Y; G280 replaced
with A, I, L, S, T, M, or V; A281 replaced with G, I, L, S, T, M,
or V; L282 replaced with A, G, I, S, T, M, or V; K283 replaced with
H, or R; L284 replaced with A, G, I, S, T, M, or V; and/or L285
replaced with A, G, I, S, T, M, or V. Polynucleotides encoding
these polypeptides are also encompassed by the invention. The
resulting Neutrokine-alpha proteins of the invention may be
routinely screened for Neutrokine-alpha and/or Neutrokine-alphaSV
functional activity and/or physical properties (such as, for
example, enhanced or reduced stability and/or solubility).
Preferably, the resulting proteins of the invention have an
increased and/or a decreased Neutrokine-alpha and/or
Neutrokine-alphaSV functional activity. More preferably, the
resulting Neutrokine-alpha and/or Neutrokine-alphaSV proteins of
the invention have more than one increased and/or decreased
Neutrokine-alpha and/or Neutrokine-alpha SV functional activity
and/or physical property.
[0338] In another embodiment, site directed changes at the amino
acid level of Neutrokine-alphaSV can be made by replacing a
particular amino acid with a conservative substitution. Preferred
conservative substitution mutations of the Neutrokine-alphaSV amino
acid sequence provided in SEQ ID NO:19 include: M1 replaced with A,
G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E; S4
replaced with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L,
S, M, or V; E6 replaced with D; R7 replaced with H, or K; E8
replaced with D; Q9 replaced with N; S10 replaced with A, G, I, L,
T, M, or V; R11 replaced with H, or K; L12 replaced with A, G, I,
S, T, M, or V; T13 replaced with A, G, I, L, S, M, or V; S14
replaced with A, G, I, L, T, M, or V; L16 replaced with A, G, I, S,
T, M, or V; K17 replaced with H, or R; K18 replaced with H, or R;
R19 replaced with H, or K; E20 replaced with D; E21 replaced with
D; M22 replaced with A, G, I, L, S, T, or V; K23 replaced with H,
or R; L24 replaced with A, G, I, S, T, M, or V; K25 replaced with
H, or R; E26 replaced with D; V28 replaced with A, G, I, L, S, T,
or M; S29 replaced with A, G, I, L, T, M, or V; 130 replaced with
A, G, L, S, T, M, or V; L31 replaced with A, G, I, S, T, M, or V;
R33 replaced with H, or K; K34 replaced with H, or R; E35 replaced
with D; S36 replaced with A, G, I, L, T, M, or V; S38 replaced with
A, G, I, L, T, M, or V; V39 replaced with A, G, I, L, S, T, or M;
R40 replaced with H, or K; S41 replaced with A, G, I, L, T, M, or
V; S42 replaced with A, G, I, L, T, M, or V; K43 replaced with H,
or R; D44 replaced with E; G45 replaced with A, I, L, S, T, M, or
V; K46 replaced with H, or R; L47 replaced with A, G, I, S, T, M,
or V; L48 replaced with A, G, I, S, T, M, or V; A49 replaced with
G, I, L, S, T, M, or V; A50 replaced with G, I, L, S, T, M, or V;
T51 replaced with A, G, I, L, S, M, or V; L52 replaced with A, G,
I, S, T, M, or V; L53 replaced with A, G, I, S, T, M, or V; L54
replaced with A, G, I, S, T, M, or V; A55 replaced with G, I, L, S,
T, M, or V; L56 replaced with A, G, I, S, T, M, or V; L57 replaced
with A, G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or
V; L61 replaced with A, G, I, S, T, M, or V; T62 replaced with A,
G, I, L, S, M, or V; V63 replaced with A, G, I, L, S, T, or M; V64
replaced with A, G, I, L, S, T, or M; S65 replaced with A, G, I, L,
T, M, or V; F66 replaced with W, or Y; Y67 replaced with F, or W;
Q68 replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70
replaced with G, I, L, S, T, M, or V; A71 replaced with G, I, L, S,
T, M, or V; L72 replaced with A, G, I, S, T, M, or V; Q73 replaced
with N; G74 replaced with A, I, L, S, T, M, or V; D75 replaced with
E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G,
I, L, S, T, M, or V; S78 replaced with A, G, I, L, T, M, or V; L79
replaced with A, G, I, S, T, M, or V; R80 replaced with H, or K;
A81 replaced with G, I, L, S, T, M, or V; E82 replaced with D; L83
replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85
replaced with A, I, L, S, T, M, or V; H86 replaced with K, or R;
H87 replaced with K, or R; A88 replaced with G, I, L, S, T, M, or
V; E89 replaced with D; K90 replaced with H, or R; L91 replaced
with A, G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or
V; G94 replaced with A, I, L, S, T, M, or V; A95 replaced with G,
I, L, S, T, M, or V; G96 replaced with A, I, L, S, T, M, or V; A97
replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R;
A100 replaced with G, I, L, S, T, M, or V; G101 replaced with A, I,
L, S, T, M, or V; L102 replaced with A, G, I, S, T, M, or V; E103
replaced with D; E104 replaced with D; A105 replaced with G, I, L,
S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108
replaced with A, G, I, L, S, T, or M; T109 replaced with A, G, I,
L, S, M, or V; A110 replaced with G, I, L, S, T, M, or V; G111
replaced with A, I, L, S, T, M, or V; L112 replaced with A, G, I,
S, T, M, or V; K113 replaced with H, or R; I114 replaced with A, G,
L, S, T, M, or V; F115 replaced with W, or Y; E116 replaced with D;
A119 replaced with G, I, L, S, T, M, or V; G121 replaced with A, I,
L, S, T, M, or V; E122 replaced with D; G123 replaced with A, I, L,
S, T, M, or V; N124 replaced with Q; S125 replaced with A, G, I, L,
T, M, or V; S126 replaced with A, G, I, L, T, M, or V; Q127
replaced with N; N128 replaced with Q; S129 replaced with A, G, I,
L, T, M, or V; R130 replaced with H, or K; N131 replaced with Q;
K132 replaced with H, or R; R133 replaced with H, or K; A134
replaced with G, I, L, S, T, M, or V; V135 replaced with A, G, I,
L, S, T, or M; Q136 replaced with N; G137 replaced with A, I, L, S,
T, M, or V; E139 replaced with D; E140 replaced with D; T141
replaced with A, G, I, L, S, M, or V; G142 replaced with A, I, L,
S, T, M, or V; S143 replaced with A, G, I, L, T, M, or V; Y144
replaced with F, or W; T145 replaced with A, G, I, L, S, M, or V;
F146 replaced with W, or Y; V147 replaced with A, G, I, L, S, T, or
M; W149 replaced with F, or Y; L150 replaced with A, G, I, S, T, M,
or V; L151 replaced with A, G, I, S, T, M, or V; S152 replaced with
A, G, I, L, T, M, or V; F153 replaced with W, or Y; K154 replaced
with H, or R; R155 replaced with H, or K; G156 replaced with A, I,
L, S, T, M, or V; S157 replaced with A, G, I, L, T, M, or V; A158
replaced with G, I, L, S, T, M, or V; L159 replaced with A, G, I,
S, T, M, or V; E160 replaced with D; E161 replaced with D; K162
replaced with H, or R; E163 replaced with D; N164 replaced with Q;
K165 replaced with H, or R; I166 replaced with A, G, L, S, T, M, or
V; L167 replaced with A, G, I, S, T, M, or V; V168 replaced with A,
G, I, L, S, T, or M; K169 replaced with H, or R; E170 replaced with
D; T171 replaced with A, G, I, L, S, M, or V; G172 replaced with A,
I, L, S, T, M, or V; Y173 replaced with F, or W; F174 replaced with
W, or Y; F175 replaced with W, or Y; I176 replaced with A, G, L, S,
T, M, or V; Y177 replaced with F, or W; G178 replaced with A, I, L,
S, T, M, or V; Q179 replaced with N; V180 replaced with A, G, I, L,
S, T, or M; L181 replaced with A, G, I, S, T, M, or V; Y182
replaced with F, or W; T183 replaced with A, G, I, L, S, M, or V;
D184 replaced with E; K185 replaced with H, or R; T186 replaced
with A, G, I, L, S, M, or V; Y187 replaced with F, or W; A188
replaced with G, I, L, S, T, M, or V; M189 replaced with A, G, I,
L, S, T, or V; G190 replaced with A, I, L, S, T, M, or V; H191
replaced with K, or R; L192 replaced with A, G, I, S, T, M, or V;
I193 replaced with A, G, L, S, T, M, or V; Q194 replaced with N;
R195 replaced with H, or K; K196 replaced with H, or R; K197
replaced with H, or R; V198 replaced with A, G, I, L, S, T, or M;
H199 replaced with K, or R; V200 replaced with A, G, I, L, S, T, or
M; F201 replaced with W, or Y; G202 replaced with A, I, L, S, T, M,
or V; D203 replaced with E; E204 replaced with D; L205 replaced
with A, G, I, S, T, M, or V; S206 replaced with A, G, I, L, T, M,
or V; L207 replaced with A, G, I, S, T, M, or V; V208 replaced with
A, G, I, L, S, T, or M; T209 replaced with A, G, I, L, S, M, or V;
L210 replaced with A, G, I, S, T, M, or V; F211 replaced with W, or
Y; R212 replaced with H, or K; I214 replaced with A, G, L, S, T, M,
or V; Q215 replaced with N; N216 replaced with Q; M217 replaced
with A, G, I, L, S, T, or V; E219 replaced with D; T220 replaced
with A, G, I, L, S, M, or V; L221 replaced with A, G, I, S, T, M,
or V; N223 replaced with Q; N224 replaced with Q; S225 replaced
with A, G, I, L, T, M, or V; Y227 replaced with F, or W; S228
replaced with A, G, I, L, T, M, or V; A229 replaced with G, I, L,
S, T, M, or V; G230 replaced with A, I, L, S, T, M, or V; I231
replaced with A, G, L, S, T, M, or V; A232 replaced with G, I, L,
S, T, M, or V; K233 replaced with H, or R; L234 replaced with A, G,
I, S, T, M, or V; E235 replaced with D; E236 replaced with D; G237
replaced with A, I, L, S, T, M, or V; D238 replaced with E; E239
replaced with D; L240 replaced with A, G, I, S, T, M, or V; Q241
replaced with N; L242 replaced with A, G, I, S, T, M, or V; A243
replaced with G, I, L, S, T, M, or V; 1244 replaced with A, G, L,
S, T, M, or V; R246 replaced with H, or K; E247 replaced with D;
N248 replaced with Q; A249 replaced with G, I, L, S, T, M, or V;
Q250 replaced with N; I251 replaced with A, G, L, S, T, M, or V;
S252 replaced with A, G, I, L, T, M, or V; L253 replaced with A, G,
I, S, T, M, or V; D254 replaced with E; G255 replaced with A, I, L,
S, T, M, or V; D256 replaced with E; V257 replaced with A, G, I, L,
S, T, or M; T258 replaced with A, G, I, L, S, M, or V; F259
replaced with W, or Y; F260 replaced with W, or Y; G261 replaced
with A, I, L, S, T, M, or V; A262 replaced with G, I, L, S, T, M,
or V; L263 replaced with A, G, I, S, T, M, or V; K264 replaced with
H, or R; L265 replaced with A, G, I, S, T, M, or V; and/or L266
replaced with A, G, I, S, T, M, or V. Polynucleotides encoding
these polypeptides are also encompassed by the invention. The
resulting Neutrokine-alpha proteins of the invention may be
routinely screened for Neutrokine-alpha and/or Neutrokine-alphaSV
functional activity and/or physical properties (such as, for
example, enhanced or reduced stability and/or solubility).
Preferably, the resulting proteins of the invention have an
increased and/or a decreased Neutrokine-alpha and/or
Neutrokine-alphaSV functional activity. More preferably, the
resulting Neutrokine-alpha and/or Neutrokine-alphaSV proteins of
the invention have more than one increased and/or decreased
Neutrokine-alpha and/or Neutrokine-alpha SV functional activity
and/or physical property.
[0339] In another embodiment, site directed changes at the amino
acid level of Neutrokine-alpha can be made by replacing a
particular amino acid with a conservative substitution. Preferred
conservative substitution mutations of the Neutrokine-alpha amino
acid sequence provided in SEQ ID NO:23 include: R1 replaced with H,
or K; V2 replaced with A, G, I, L, S, T, or M; V3 replaced with A,
G, I, L, S, T, or M; D4 replaced with E; L5 replaced with A, G, I,
S, T, M, or V; S6 replaced with A, G, I, L, T, M, or V; A7 replaced
with G, I, L, S, T, M, or V; A10 replaced with G, I, L, S, T, M, or
V; L13 replaced with A, G, I, S, T, M, or V; G15 replaced with A,
I, L, S, T, M, or V; R17 replaced with H, or K; H18 replaced with
K, or R; S19 replaced with A, G, I, L, T, M, or V; Q20 replaced
with N; H21 replaced with K, or R; D22 replaced with E; D23
replaced with E; N24 replaced with Q; G25 replaced with A, I, L, S,
T, M, or V; M26 replaced with A, G, I, L, S, T, or V; N27 replaced
with Q; L28 replaced with A, G, I, S, T, M, or V; R29 replaced with
H, or K; N30 replaced with Q; R31 replaced with H, or K; T32
replaced with A, G, I, L, S, M, or V; Y33 replaced with F, or W;
T34 replaced with A, G, I, L, S, M, or V; F35 replaced with W, or
Y; V36 replaced with A, G, I, L, S, T, or M; W38 replaced with F,
or Y; L39 replaced with A, G, I, S, T, M, or V; L40 replaced with
A, G, I, S, T, M, or V; S41 replaced with A, G, I, L, T, M, or V;
F42 replaced with W, or Y; K43 replaced with H, or R; R44 replaced
with H, or K; G45 replaced with A, I, L, S, T, M, or V; N46
replaced with Q; A47 replaced with G, I, L, S, T, M, or V; L48
replaced with A, G, I, S, T, M, or V; E49 replaced with D; E50
replaced with D; K51 replaced with H, or R; E52 replaced with D;
N53 replaced with Q; K54 replaced with H, or R; I55 replaced with
A, G, L, S, T, M, or V; V56 replaced with A, G, I, L, S, T, or M;
V57 replaced with A, G, I, L, S, T, or M; R58 replaced with H, or
K; Q59 replaced with N; T60 replaced with A, G, I, L, S, M, or V;
G61 replaced with A, I, L, S, T, M, or V; Y62 replaced with F, or
W; F63 replaced with W, or Y; F64 replaced with W, or Y; I65
replaced with A, G, L, S, T, M, or V; Y66 replaced with F, or W;
S67 replaced with A, G, I, L, T, M, or V; Q68 replaced with N; V69
replaced with A, G, I, L, S, T, or M; L70 replaced with A, G, I, S,
T, M, or V; Y71 replaced with F, or W; T72 replaced with A, G, I,
L, S, M, or V; D73 replaced with E; I75 replaced with A, G, L, S,
T, M, or V; F76 replaced with W, or Y; A77 replaced with G, I, L,
S, T, M, or V; M78 replaced with A, G, I, L, S, T, or V; G79
replaced with A, I, L, S, T, M, or V; H80 replaced with K, or R;
V81 replaced with A, G, I, L, S, T, or M; I82 replaced with A, G,
L, S, T, M, or V; Q83 replaced with N; R84 replaced with H, or K;
K85 replaced with H, or R; K86 replaced with H, or R; V87 replaced
with A, G, I, L, S, T, or M; H88 replaced with K, or R; V89
replaced with A, G, I, L, S, T, or M; F90 replaced with W, or Y;
G91 replaced with A, I, L, S, T, M, or V; D92 replaced with E; E93
replaced with D; L94 replaced with A, G, I, S, T, M, or V; S95
replaced with A, G, I, L, T, M, or V; L96 replaced with A, G, I, S,
T, M, or V; V97 replaced with A, G, I, L, S, T, or M; T98 replaced
with A, G, I, L, S, M, or V; L99 replaced with A, G, I, S, T, M, or
V; F100 replaced with W, or Y; R10 replaced with H, or K; I103
replaced with A, G, L, S, T, M, or V; Q104 replaced with N; N105
replaced with Q; M106 replaced with A, G, I, L, S, T, or V; K108
replaced with H, or R; T109 replaced with A, G, I, L, S, M, or V;
L110 replaced with A, G, I, S, T, M, or V; N112 replaced with Q;
N113 replaced with Q; S114 replaced with A, G, I, L, T, M, or V;
Y116 replaced with F, or W; S117 replaced with A, G, I, L, T, M, or
V; A118 replaced with G, I, L, S, T, M, or V; G119 replaced with A,
I, L, S, T, M, or V; I120 replaced with A, G, L, S, T, M, or V;
A121 replaced with G, I, L, S, T, M, or V; R122 replaced with H, or
K; L123 replaced with A, G, I, S, T, M, or V; E124 replaced with D;
E125 replaced with D; G126 replaced with A, I, L, S, T, M, or V;
D127 replaced with E; E128 replaced with D; I129 replaced with A,
G, L, S, T, M, or V; Q130 replaced with N; L131 replaced with A, G,
I, S, T, M, or V; A132 replaced with G, I, L, S, T, M, or V; I133
replaced with A, G, L, S, T, M, or V; R135 replaced with H, or K;
E136 replaced with D; N137 replaced with Q; A138 replaced with G,
I, L, S, T, M, or V; Q139 replaced with N; I140 replaced with A, G,
L, S, T, M, or V; S141 replaced with A, G, I, L, T, M, or V; R142
replaced with H, or K; N143 replaced with Q; G144 replaced with A,
I, L, S, T, M, or V; D145 replaced with E; D146 replaced with E;
T147 replaced with A, G, I, L, S, M, or V; F148 replaced with W, or
Y; F149 replaced with W, or Y; G150 replaced with A, I, L, S, T, M,
or V; A151 replaced with G, I, L, S, T, M, or V; L152 replaced with
A, G, I, S, T, M, or V; K153 replaced with H, or R; L154 replaced
with A, G, I, S, T, M, or V; and/or L155 replaced with A, G, I, S,
T, M, or V. Polynucleotides encoding these polypeptides are also
encompassed by the invention. The resulting Neutrokine-alpha
proteins of the invention may be routinely screened for
Neutrokine-alpha and/or Neutrokine-alphaSV functional activity
and/or physical properties (such as, for example, enhanced or
reduced stability and/or solubility). Preferably, the resulting
proteins of the invention have an increased and/or a decreased
Neutrokine-alpha and/or Neutrokine-alphaSV functional activity.
More preferably, the resulting Neutrokine-alpha and/or
Neutrokine-alphaSV proteins of the invention have more than one
increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha
SV functional activity and/or physical property.
[0340] In another embodiment, site directed changes at the amino
acid level of Neutrokine-alpha can be made by replacing a
particular amino acid with a conservative substitution. Preferred
conservative substitution mutations of the Neutrokine-alpha amino
acid sequence provided in SEQ ID NO:38 include: M1 replaced with A,
G, I, L, S, T, or V; D2 replaced with E; E3 replaced with D; S4
replaced with A, G, I, L, T, M, or V; A5 replaced with G, I, L, S,
T, M, or V; K6 replaced with H, or R; T7 replaced with A, G, I, L,
S, M, or V; L8 replaced with A, G, I, S, T, M, or V; L13 replaced
with A, G, I, S, T, M, or V; F15 replaced with W, or Y; S17
replaced with A, G, I, L, T, M, or V; E18 replaced with D; K19
replaced with H, or R; G20 replaced with A, I, L, S, T, M, or V;
E21 replaced with D; D22 replaced with E; M23 replaced with A, G,
I, L, S, T, or V; K24 replaced with H, or R; V25 replaced with A,
G, I, L, S, T, or M; G26 replaced with A, I, L, S, T, M, or V; Y27
replaced with F, or W; D28 replaced with E; I30 replaced with A, G,
L, S, T, M, or V; T31 replaced with A, G, I, L, S, M, or V; Q33
replaced with N; K34 replaced with H, or R; E35 replaced with D;
E36 replaced with D; G37 replaced with A, I, L, S, T, M, or V; A38
replaced with G, I, L, S, T, M, or V; W39 replaced with F, or Y;
F40 replaced with W, or Y; G41 replaced with A, I, L, S, T, M, or
V; I42 replaced with A, G, L, S, T, M, or V; R44 replaced with H,
or K; D45 replaced with E; G46 replaced with A, I, L, S, T, M, or
V; R47 replaced with H, or K; L48 replaced with A, G, I, S, T, M,
or V; L49 replaced with A, G, I, S, T, M, or V; A50 replaced with
G, I, L, S, T, M, or V; A51 replaced with G, I, L, S, T, M, or V;
T52 replaced with A, G, I, L, S, M, or V; L53 replaced with A, G,
I, S, T, M, or V; L54 replaced with A, G, I, S, T, M, or V; L55
replaced with A, G, I, S, T, M, or V; A56 replaced with G, I, L, S,
T, M, or V; L57 replaced with A, G, I, S, T, M, or V; L58 replaced
with A, G, I, S, T, M, or V; S59 replaced with A, G, I, L, T, M, or
V; S60 replaced with A, G, I, L, T, M, or V; S61 replaced with A,
G, I, L, T, M, or V; F62 replaced with W, or Y; T63 replaced with
A, G, I, L, S, M, or V; A64 replaced with G, I, L, S, T, M, or V;
M65 replaced with A, G, I, L, S, T, or V; S66 replaced with A, G,
I, L, T, M, or V; L67 replaced with A, G, I, S, T, M, or V; Y68
replaced with F, or W; Q69 replaced with N; L70 replaced with A, G,
I, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; A72
replaced with G, I, L, S, T, M, or V; L73 replaced with A, G, I, S,
T, M, or V; Q74 replaced with N; A75 replaced with G, I, L, S, T,
M, or V; D76 replaced with E; L77 replaced with A, G, I, S, T, M,
or V; M78 replaced with A, G, I, L, S, T, or V; N79 replaced with
Q; L80 replaced with A, G, I, S, T, M, or V; R81 replaced with H,
or K; M82 replaced with A, G, I, L, S, T, or V; E83 replaced with
D; L84 replaced with A, G, I, S, T, M, or V; Q85 replaced with N;
S86 replaced with A, G, I, L, T, M, or V; Y87 replaced with F, or
W; R88 replaced with H, or K; G89 replaced with A, I, L, S, T, M,
or V; S90 replaced with A, G, I, L, T, M, or V; A91 replaced with
G, I, L, S, T, M, or V; T92 replaced with A, G, I, L, S, M, or V;
A94 replaced with G, I, L, S, T, M, or V; A95 replaced with G, I,
L, S, T, M, or V; A96 replaced with G, I, L, S, T, M, or V; G97
replaced with A, I, L, S, T, M, or V; A98 replaced with G, I, L, S,
T, M, or V; E100 replaced with D; L101 replaced with A, G, I, S, T,
M, or V; T102 replaced with A, G, I, L, S, M, or V; A103 replaced
with G, I, L, S, T, M, or V; G104 replaced with A, I, L, S, T, M,
or V; V105 replaced with A, G, I, L, S, T, or M; K106 replaced with
H, or R; L107 replaced with A, G, I, S, T, M, or V; L108 replaced
with A, G, I, S, T, M, or V; T109 replaced with A, G, I, L, S, M,
or V; A111 replaced with G, I, L, S, T, M, or V; A112 replaced with
G, I, L, S, T, M, or V; R114 replaced with H, or K; H116 replaced
with K, or R; N117 replaced with Q; S118 replaced with A, G, I, L,
T, M, or V; S119 replaced with A, G, I, L, T, M, or V; R120
replaced with H, or K; G121 replaced with A, I, L, S, T, M, or V;
H122 replaced with K, or R; R123 replaced with H, or K; N124
replaced with Q; R125 replaced with H, or K; R126 replaced with H,
or K; A127 replaced with G, I, L, S, T, M, or V; F128 replaced with
W, or Y; Q129 replaced with N; G130 replaced with A, I, L, S, T, M,
or V; E132 replaced with D; E133 replaced with D; T134 replaced
with A, G, I, L, S, M, or V; E135 replaced with D; Q136 replaced
with N; D137 replaced with E; V138 replaced with A, G, I, L, S, T,
or M; D139 replaced with E; L140 replaced with A, G, I, S, T, M, or
V; S141 replaced with A, G, I, L, T, M, or V; A142 replaced with G,
I, L, S, T, M, or V; A145 replaced with G, I, L, S, T, M, or V;
L148 replaced with A, G, I, S, T, M, or V; G150 replaced with A, I,
L, S, T, M, or V; R152 replaced with H, or K; H153 replaced with K,
or R; S154 replaced with A, G, I, L, T, M, or V; Q155 replaced with
N; H156 replaced with K, or R; D157 replaced with E; D158 replaced
with E; N159 replaced with Q; G160 replaced with A, I, L, S, T, M,
or V; M161 replaced with A, G, I, L, S, T, or V; N162 replaced with
Q; L163 replaced with A, G, I, S, T, M, or V; R164 replaced with H,
or K; N165 replaced with Q; I166 replaced with A, G, L, S, T, M, or
V; I167 replaced with A, G, L, S, T, M, or V; Q168 replaced with N;
D169 replaced with E; L171 replaced with A, G, I, S, T, M, or V;
Q172 replaced with N; L173 replaced with A, G, I, S, T, M, or V;
I174 replaced with A, G, L, S, T, M, or V; A175 replaced with G, I,
L, S, T, M, or V; D176 replaced with E; S177 replaced with A, G, I,
L, T, M, or V; D178 replaced with E; T179 replaced with A, G, I, L,
S, M, or V; A181 replaced with G, I, L, S, T, M, or V; L182
replaced with A, G, I, S, T, M, or V; E183 replaced with D; E184
replaced with D; K185 replaced with H, or R; E186 replaced with D;
N187 replaced with Q; K188 replaced with H, or R; I189 replaced
with A, G, L, S, T, M, or V; V190 replaced with A, G, I, L, S, T,
or M; V191 replaced with A, G, I, L, S, T, or M; R192 replaced with
H, or K; Q193 replaced with N; T194 replaced with A, G, I, L, S, M,
or V; G195 replaced with A, I, L, S, T, M, or V; Y196 replaced with
F, or W; F197 replaced with W, or Y; F198 replaced with W, or Y;
I199 replaced with A, G, L, S, T, M, or V; Y200 replaced with F, or
W; S201 replaced with A, G, I, L, T, M, or V; Q202 replaced with N;
V203 replaced with A, G, I, L, S, T, or M; L204 replaced with A, G,
I, S, T, M, or V; Y205 replaced with F, or W; T206 replaced with A,
G, I, L, S, M, or V; D207 replaced with E; I209 replaced with A, G,
L, S, T, M, or V; F210 replaced with W, or Y; A211 replaced with G,
I, L, S, T, M, or V; M212 replaced with A, G, I, L, S, T, or V;
G213 replaced with A, I, L, S, T, M, or V; H214 replaced with K, or
R; V215 replaced with A, G, I, L, S, T, or M; I216 replaced with A,
G, L, S, T, M, or V; Q217 replaced with N; R218 replaced with H, or
K; K219 replaced with H, or R; K220 replaced with H, or R; V221
replaced with A, G, I, L, S, T, or M; H222 replaced with K, or R;
V223 replaced with A, G, I, L, S, T, or M; F224 replaced with W, or
Y; G225 replaced with A, I, L, S, T, M, or V; D226 replaced with E;
E227 replaced with D; L228 replaced with A, G, I, S, T, M, or V;
S229 replaced with A, G, I, L, T, M, or V; L230 replaced with A, G,
I, S, T, M, or V; V231 replaced with A, G, I, L, S, T, or M; T232
replaced with A, G, I, L, S, M, or V; L233 replaced with A, G, I,
S, T, M, or V; F234 replaced with W, or Y; R235 replaced with H, or
K; I237 replaced with A, G, L, S, T, M, or V; Q238 replaced with N;
N239 replaced with Q; M240 replaced with A, G, I, L, S, T, or V;
K242 replaced with H, or R; T243 replaced with A, G, I, L, S, M, or
V; L244 replaced with A, G, I, S, T, M, or V; N246 replaced with Q;
N247 replaced with Q; S248 replaced with A, G, I, L, T, M, or V;
Y250 replaced with F, or W; S251 replaced with A, G, I, L, T, M, or
V; A252 replaced with G, I, L, S, T, M, or V; G253 replaced with A,
I, L, S, T, M, or V; I254 replaced with A, G, L, S, T, M, or V;
A255 replaced with G, I, L, S, T, M, or V; R256 replaced with H, or
K; L257 replaced with A, G, I, S, T, M, or V; E258 replaced with D;
E259 replaced with D; G260 replaced with A, I, L, S, T, M, or V;
D261 replaced with E; E262 replaced with D; I263 replaced with A,
G, L, S, T, M, or V; Q264 replaced with N; L265 replaced with A, G,
I, S, T, M, or V; A266 replaced with G, I, L, S, T, M, or V; I267
replaced with A, G, L, S, T, M, or V; R269 replaced with H, or K;
E270 replaced with D; N271 replaced with Q; A272 replaced with G,
I, L, S, T, M, or V; Q273 replaced with N; I274 replaced with A, G,
L, S, T, M, or V; S275 replaced with A, G, I, L, T, M, or V; R276
replaced with H, or K; N277 replaced with Q; G278 replaced with A,
I, L, S, T, M, or V; D279 replaced with E; D280 replaced with E;
T281 replaced with A, G, I, L, S, M, or V; F282 replaced with W, or
Y; F283 replaced with W, or Y; G284 replaced with A, I, L, S, T, M,
or V; A285 replaced with G, I, L, S, T, M, or V; L286 replaced with
A, G, I, S, T, M, or V; K287 replaced with H, or R; L288 replaced
with A, G, I, S, T, M, or V; and/or L289 replaced with A, G, I, S,
T, M, or V. Polynucleotides encoding these polypeptides are also
encompassed by the invention. The resulting Neutrokine-alpha
proteins of the invention may be routinely screened for
Neutrokine-alpha and/or Neutrokine-alphaSV functional activity
and/or physical properties (such as, for example, enhanced or
reduced stability and/or solubility). Preferably, the resulting
proteins of the invention have an increased and/or a decreased
Neutrokine-alpha and/or Neutrokine-alphaSV functional activity.
More preferably, the resulting Neutrokine-alpha and/or
Neutrokine-alphaSV proteins of the invention have more than one
increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha
SV functional activity and/or physical property.
[0341] Amino acids in the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the present invention that are
essential for function can be identified by methods known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for functional activity, such ligand binding and the ability
to stimulate lymphocyte (e.g., B cell) as, for example,
proliferation, differentiation, and/or activation.
[0342] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).
In another embodiment, the invention provides for polypeptides
having amino acid sequences containing non-conservative
substitutions of the amino acid sequence provided in SEQ ID NO:2.
For example, non-conservative substitutions of the Neutrokine-alpha
protein sequence provided in SEQ ID NO:2 include: M1 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; T5 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; R7 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; E8 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q9 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S10 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; R1 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L12 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; T13 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S14 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; C15 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; L16 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; K17 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; K18 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; R19 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; E20 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E21 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M22 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; K23 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L24 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E26 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C27 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
V28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S29
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I30 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L31 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R33 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K34 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E35
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; S36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P37
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; S38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V39
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R40 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S41
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S42 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; K43 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D44 replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G45
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K46 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L47
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L48 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A49 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; T51 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L52 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L54
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A55 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L56 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S58 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; C59 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; C60 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; L61 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; T62 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; V64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F66
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
Y67 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; Q68 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; V69 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A71
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q73 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G74 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D75 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L76 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A77 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S78 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L79 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; A81 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; E82 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; L83 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q84 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; G85 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; H86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; H87 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; K90 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; L91 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; P92 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; A93 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; G94 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A97
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P98 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
K99 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; A100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G101 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L102
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E103 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E104
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; A105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P106
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; A107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T109
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A110 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G111 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L112 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; K113 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; I114 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; F115 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; E116 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P118 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P120
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; G121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E122 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; G123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N124 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; S125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S126 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q127
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; N128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; S129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; R130 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; N131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; K132 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; R133 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; A134 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; V135 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q136 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; G137 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; P138 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; E139 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; E140 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T141 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; V142 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; T143 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; Q144 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; D145 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; C146 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L147 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; Q148 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L149 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; I150 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A151 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; D152 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; S153 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; E154 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; T155 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P156 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; T157 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; I158 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q159 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; K160 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; G161 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; S162 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; Y163 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; T164 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; F165 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; V166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P167 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; W168 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; L169 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F172
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
K173 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; R174 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; G175 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A177
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L178 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E179 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E180 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K181
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E182 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; N183 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; K184 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; I185 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L186 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V187 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K188 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; E189 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T190 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Y192 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; F193 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; F194 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; I195 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; Y196 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; G197 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Q198 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; V199 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Y201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; T202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D203 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; K204 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T205 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Y206 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; A207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; M208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G209
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H210 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L211
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I212 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q213 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R214 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K215
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K216 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H218 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V219 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F220 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; G221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D222 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; E223 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; L224 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; S225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L226 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V227
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T228 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L229 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F230 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; R231 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C232 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I233
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q234 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N235
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; M236 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P237
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; E238 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T239 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P241 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; N242 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; N243 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; S244 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; C245 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; Y246 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; S247 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; A248 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; G249 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; I250 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A251 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K252
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
L253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E254
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; E255 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; G256 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D257 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E258 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; L259 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q260 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; L261 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; A262 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; I263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P264
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; R265 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E266 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; N267 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; A268 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q269 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; I270 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C;
S271 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L272
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D273 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G274
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D275 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V276
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T277 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F278 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F279 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G280
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A281 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L282 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; K283 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L284 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; and/or L285 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these
polypeptides are also encompassed by the invention. The resulting
Neutrokine-alpha proteins of the invention may be routinely
screened for Neutrokine-alpha and/or Neutrokine-alphaSV functional
activities and/or physical properties (such as, for example,
enhanced or reduced stability and/or solubility) described
throughout the specification and known in the art. Preferably, the
resulting proteins of the invention have an increased and/or a
decreased Neutrokine-alpha and/or Neutrokine-alphaSV functional
activity. More preferably, the resulting Neutrokine-alpha and/or
Neutrokine-alphaSV proteins of the invention have more than one
increased and/or decreased Neutrokine-alpha and/or
Neutrokine-alphaSV functional activity and/or physical
property.
[0344] In an additional embodiment, Neutrokine-alpha polypeptides
of the invention comprise, or alternatively consist of, more than
one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and
50) replaced with the substituted amino acids as described above
(either conservative or nonconservative).
In another embodiment of the invention, non-conservative
substitutions of the Neutrokine-alphaSV protein sequence provided
in SEQ ID NO:19 include: M1 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E6 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; R7 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E8 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; Q9 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; S10 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; R11 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; L12 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; T13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C15
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K17
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; R19 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E20 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; E21 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; M22 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; K23 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; L24 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; K25 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; E26 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; C27 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V28 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; S29 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; I30 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; L31 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P32 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; R33 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; K34 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; E35 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S36 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P37 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S38 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V39 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R40 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; S41 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S42 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; K43 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; D44 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; G45 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; K46 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; L47 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; L48 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; A49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T51
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L52 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A55 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L57 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S58
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C59 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
C60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T62 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V64 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S65 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; F66 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; Y67 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q68 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V69 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A70 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; A71 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; Q73 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; G74 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; D75 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; L76 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; S78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L79
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R80 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A81
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E82 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L83
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q84 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G85
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H86 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H87
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; L91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P92 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; A93 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; G94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A95
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G96 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A97 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P98 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K99 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A100 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G101 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L102 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; E103 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; E104 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A105 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P106 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A107 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; V108 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; T109 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; A110 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; G111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K113
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
I114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F115
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
E116 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; P117 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; P118 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; A119 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; P120 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; G121 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; E122 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G123 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; N124 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; S125 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S126 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; Q127 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; N128 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; S129 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R130 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; N131 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; K132 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R133 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A134 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V135 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; Q136 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G137 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E139 replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E140
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G142
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S143 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y144 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T145 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F146 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V147 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P148 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W149
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
L150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L151
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S152 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F153 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K154 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R155
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
G156 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S157
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A158 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L159 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E160 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E161 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K162 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E163
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; N164 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; K165 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; I166 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L167 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V168 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K169
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E170 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; T171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G172 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y173
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
F174 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; F175 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; I176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Y177 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; G178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q179 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; V180 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y182
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
T183 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D184
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; K185 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; T186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Y187 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; A188 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
M189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G190
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H191 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L192
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I193 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q194 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R195 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K196
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K197 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V198 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H199 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; G202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D203 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; E204 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; L205 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; S206 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V208
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T209 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L210 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F211 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; R212 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C213 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I214
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q215 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N216
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; M217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P218
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; E219 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T220 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P222 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; N223 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; N224 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; S225 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; C226 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; Y227 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; S228 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; A229 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; G230 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; I231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A232 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K233
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
L234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E235
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; E236 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; G237 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D238 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E239 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; L240 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q241 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; L242 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; A243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; I244 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P245
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; R246 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E247 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; N248 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; A249 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q250 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; I251 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; S252 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D254 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; G255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D256 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; V257 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F259
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
F260 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; G261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A262 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L263
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K264 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L265
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L266
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
Polynucleotides encoding these polypeptides are also encompassed by
the invention. The resulting Neutrokine-alpha proteins of the
invention may be routinely screened for Neutrokine-alpha and/or
Neutrokine-alphaSV functional activities and/or physical properties
(such as, for example, enhanced or reduced stability and/or
solubility) described throughout the specification and known in the
art. Preferably, the resulting proteins of the invention have an
increased and/or a decreased Neutrokine-alpha
and/or Neutrokine-alphaSV functional activity. More preferably, the
resulting Neutrokine-alpha and/or Neutrokine-alphaSV proteins of
the invention have more than one increased and/or decreased
Neutrokine-alpha and/or Neutrokine-alphaSV functional activity
and/or physical property.
[0346] In an additional embodiment, Neutrokine-alpha polypeptides
of the invention comprise, or alternatively consist of, more than
one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and
50) replaced with the substituted amino acids as described above
(either conservative or nonconservative).
[0347] For example, preferred non-conservative substitutions of the
Neutrokine-alpha protein sequence provided in SEQ ID NO:23 include:
R1 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; V2 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V3
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D4 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L5
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S6 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A7 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P8 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P9 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A10 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P11 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C12 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
L13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P14
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; G15 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C16
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; R17 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; H18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; S19 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; Q20 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; H21 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; D22 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; D23 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N24 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G25 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; M26 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; N27 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; L28 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; R29 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; N30 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R31 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T32 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y33 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T34 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; F35 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V36 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P37 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W38 replaced with
D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L39 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L40 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S41 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; F42 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; K43 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; R44 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G45 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; N46 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; A47 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; E49 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; E50 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K51 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E52 replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N53
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; K54 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; 155 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V57
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R58 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q59
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; T60 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G61
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y62 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F63
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
F64 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; I65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y66
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
S67 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q68
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; V69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L70
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y71 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T72
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D73 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P74
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; I75 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F76
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M78
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G79 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H80 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V81 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; I82 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; Q83 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; R84 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K85 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K86 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V87
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H88 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V89
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F90 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G91
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D92 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E93
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; L94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S95
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L96 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V97 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; T98 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L99 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; F100 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; R101 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; C102 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; I103 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; Q104 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; N105 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M106 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P107 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K108 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T109
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L110 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P111 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N112
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; N113 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; S114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; C115 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or P; Y116 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; S117 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; A118 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; G119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I120
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A121 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R122 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L123 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E124 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E125 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G126
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D127 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E128
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; I129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q130
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; L131 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A132
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I133 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P134 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R135
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E136 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; N137 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; A138 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; Q139 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; I140 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; S141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
R142 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; N143 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; G144 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; D145 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; D146 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; T147 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; F148 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; F149 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; G150 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; A151 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L152 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K153 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; L154 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
and/or L155 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
Polynucleotides encoding these polypeptides are also encompassed by
the invention. The resulting Neutrokine-alpha proteins of the
invention may be routinely screened for Neutrokine-alpha and/or
Neutrokine-alphaSV functional activities and/or physical properties
(such as, for example, enhanced or reduced stability and/or
solubility) described throughout the specification and known in the
art. Preferably, the resulting proteins of the invention have an
increased and/or a decreased Neutrokine-alpha and/or
Neutrokine-alphaSV functional activity. More preferably, the
resulting Neutrokine-alpha and/or Neutrokine-alphaSV proteins of
the invention have more than one increased and/or decreased
Neutrokine-alpha and/or Neutrokine-alphaSV functional activity
and/or physical property.
[0348] In an additional embodiment, Neutrokine-alpha polypeptides
of the invention comprise, or alternatively consist of, more than
one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and
50) replaced with the substituted amino acids as described above
(either conservative or nonconservative).
For example, preferred non-conservative substitutions of the
Neutrokine-alpha protein sequence provided in SEQ ID NO:38 include:
M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E3
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A5
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K6 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T7
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L8 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P9 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P10 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
P11 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; C12 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or P; L13 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; C14 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; F15 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; C16 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S17 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E18 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K19 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G20 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E21 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D22 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M23
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K24 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V25
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G26 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y27 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D28 replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P29
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; I30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T31
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
Q33 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; K34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; E35 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; E36 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; G37 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A38 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; W39 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; F40 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; G41 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; I42 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; C43 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; R44 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; D45 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G46 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R47 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L49 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A51 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T52
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L54 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L55 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A56 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L57 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S59
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S60 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S61 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; F62 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; T63 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; A64 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; M65 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; S66 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L67 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y68
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
Q69 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; L70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A71 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A72
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L73 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q74 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A75 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D76 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L77 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; M78 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; N79 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; L80 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; R81 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; M82 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; E83 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; L84 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; Q85 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; S86 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; Y87 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; R88 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G89 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S90 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A91 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; T92 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P93 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; A94 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G97
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A98 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P99 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E100 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L101
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T102 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A103 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; G104 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; V105 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; K106 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; L107 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L108 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; T109 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P110 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; A111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P113
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; R114 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; P115 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; H116 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; N117 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; S118 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S119 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; R120 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; G121 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; H122 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; R123 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; N124 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; R125 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R126 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A127 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F128 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q129 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G130
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P131 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
E132 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; E133 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; T134 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; E135 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; Q136 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; D137 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; V138 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; D139 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; L140 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S141 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; A142 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P143 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; P144 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; A145 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; P146 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; C147 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L148 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P149 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G150 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C151 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R152
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
H153 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; S154 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q155 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; H156 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; D157 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; D158 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; N159 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; G160 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; M161 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; N162 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; L163 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; R164 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; N165 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; I166 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; I167 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q168 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; D169 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; C170 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L171 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q172 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L173 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; I174 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A175 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; D176 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; S177 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; D178 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; T179 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; P180 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; A181 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; L182 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; E183 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; E184 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; K185 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E186 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N187 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K188
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
I189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V190
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V191 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R192 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q193 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T194
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G195 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y196 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F197 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F198
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
I199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y200
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
S201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q202
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; V203 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L204
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y205 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T206
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D207 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P208
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; I209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F210 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; A211 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
M212 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G213
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H214 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V215
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I216 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q217 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R218 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K219
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K220 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H222 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V223 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F224 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; G225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D226 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; E227 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; L228 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; S229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L230 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V231
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T232 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L233 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F234 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; R235 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C236 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I237
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q238 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N239
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; M240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P241
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; K242 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; T243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L244 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P245
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; N246 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; N247 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; S248 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; C249 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; Y250 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; S251 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A252 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; G253 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; I254 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R256
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
L257 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E258
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; E259 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; G260 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D261 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E262 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; I263 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q264 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; L265 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; A266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; I267 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P268
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; R269 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E270 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; N271 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A272 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q273 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I274 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S275 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; R276 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N277 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G278 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D279 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D280 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T281
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F282 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F283
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
G284 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A285
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L286 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; K287 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L288 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L289 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
The resulting Neutrokine-alpha proteins of the invention may be
routinely screened for Neutrokine-alpha and/or Neutrokine-alphaSV
functional activities and/or physical properties (such as, for
example, enhanced or reduced stability and/or solubility) described
throughout the specification and known in the art. Preferably, the
resulting proteins of the invention have an increased and/or a
decreased Neutrokine-alpha and/or Neutrokine-alphaSV functional
activity. More preferably, the resulting Neutrokine-alpha and/or
Neutrokine-alphaSV proteins of the invention have more than one
increased and/or decreased Neutrokine-alpha and/or
Neutrokine-alphaSV functional activity and/or physical
property.
[0350] In an additional embodiment, Neutrokine-alpha polypeptides
of the invention comprise, or alternatively consist of, more than
one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and
50) replaced with the substituted amino acids as described above
(either conservative or nonconservative).
[0351] Replacement of amino acids can also change the selectivity
of the binding of a ligand to cell surface receptors. For example,
Ostade et al., Nature 361:266-268 (1993) describes certain
mutations resulting in selective binding of TNF-alpha to only one
of the two known types of TNF receptors. Since Neutrokine-alpha and
Neutrokine-alphaSV are members of the TNF polypeptide family,
mutations similar to those in TNF-alpha are likely to have similar
effects in Neutrokine-alpha and/or Neutrokine-alphaSV.
[0352] Sites that are critical for ligand-receptor binding can also
be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science
255:306-312 (1992)).
[0353] Since Neutrokine-alpha is a member of the TNF-related
protein family, to modulate rather than completely eliminate
functional activities (e.g., biological activities) of
Neutrokine-alpha, mutations may be made in sequences encoding amino
acids in the TNF conserved domain, i.e., in positions Gly-191
through Leu-284 of FIGS. 1A and 1B (SEQ ID NO:2), more preferably
in residues within this region which are not conserved in all, most
or several members of the TNF family (e.g., TNF-alpha, TNF-beta,
LT-beta, and Fas Ligand) (see e.g., FIGS. 2A, 2B, 2C, and 2D). By
making a specific mutation in Neutrokine-alpha in the position
where such a conserved amino acid is typically found in related
TNFs, the Neutrokine-alpha mutein will act as an antagonist, thus
possessing activity for example, which inhibits lymphocyte (e.g., B
cell) proliferation, differentiation, and/or activation.
Accordingly, polypeptides of the present invention include
Neutrokine-alpha mutants. Such Neutrokine-alpha mutants comprise,
or alternatively consist of, fragments, variants or derivatives of
the full-length or preferably the extracellular domain of the
Neutrokine-alpha amino acid sequence shown in FIGS. 1A and 1B (SEQ
ID NO:2). Polynucleotides encoding the above Neutrokine-alpha
mutants are also encompassed by the invention.
[0354] Since Neutrokine-alphaSV is a member of the TNF-related
protein family, to modulate rather than completely eliminate
functional activities (e.g., biological activities) of
Neutrokine-alphaSV, mutations may be made in sequences encoding
amino acids in the TNF conserved domain, i.e., in positions Gly-172
through Leu-265 of FIGS. 5A and 5B (SEQ ID NO:19), more preferably
in residues within this region which are not conserved in all, most
or several members of the TNF family (e.g., TNF-alpha, TNF-beta,
LT-beta, and Fas Ligand) (see e.g., FIGS. 2A 2B, 2C and 2D). By
making a specific mutation in Neutrokine-alphaSV in the position
where such a conserved amino acid is typically found in related
TNFs, the Neutrokine-alphaSV mutein will act as an antagonist, thus
possessing activity for example, which inhibits lymphocyte (e.g., B
cell) proliferation, differentiation, and/or activation.
Accordingly, polypeptides of the present invention include
Neutrokine-alphaSV mutants. Such Neutrokine-alphaSV mutants
comprise, or alternatively consist of, fragments, variants or
derivatives of the full-length or preferably the extracellular
domain of the Neutrokine-alphaSV amino acid sequence shown in FIGS.
5A and 5B (SEQ ID NO:19 Polynucleotides encoding the above
Neutrokine-alpha SV mutants are also encompassed by the
invention.
[0355] In addition, it will be recognized by one of ordinary skill
in the art that mutations targeted to regions of a Neutrokine-alpha
polypeptide of the invention which encompass the nineteen amino
acid residue insertion which is not found in the Neutrokine-alphaSV
polypeptide sequence (i.e., amino acid residues Val-142 through
Lys-160 of the sequence presented in FIGS. 1A and 1B and in SEQ ID
NO:2) may affect the observed functional activities (e.g.,
biological activity) of the Neutrokine-alpha polypeptide. More
specifically, a partial, non-limiting and non-exclusive list of
such residues of the Neutrokine-alpha polypeptide sequence which
may be targeted for mutation includes the following amino acid
residues of the Neutrokine-alpha polypeptide sequence as shown in
SEQ ID NO:2: V-142; T-143; Q-144; D-145; C-146; L-147; Q-148;
L-149; I-150; A-151; D-152; S-153; E-154; T-155; P-156; T-157;
I-158; Q-159; and K-160.
[0356] Recombinant DNA technology known to those skilled in the art
(see, for instance, DNA shuffling supra) can be used to create
novel mutant proteins or muteins including single or multiple amino
acid substitutions, deletions, additions or fusion proteins. Such
modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
[0357] Thus, the invention also encompasses Neutrokine-alpha and/or
Neutrokine-alphaSV derivatives and analogs that have one or more
amino acid residues deleted, added, or substituted to generate
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides that are
better suited for expression, scale up, etc., in the host cells
chosen. For example, cysteine residues can be deleted or
substituted with another amino acid residue in order to eliminate
disulfide bridges; N-linked glycosylation sites can be altered or
eliminated to achieve, for example, expression of a homogeneous
product that is more easily recovered and purified from yeast hosts
which are known to hyperglycosylate N-linked sites. To this end, a
variety of amino acid substitutions at one or both of the first or
third amino acid positions on any one or more of the glycosylation
recognitions sequences in the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention, and/or an amino
acid deletion at the second position of any one or more such
recognition sequences will prevent glycosylation of the
Neutrokine-alpha and/or Neutrokine-alphaSV at the modified
tripeptide sequence (see, e.g., Miyajimo et al., EMBO J.
5(6):1193-1197). By way of non-limiting example, mutation of the
serine at position 244 to alanine either singly or in combination
with mutation of the asparagine at position 242 to glutamine
abolishes glycosylation of the mature soluble form of
Neutrokine-alpha (amino acids 134-285) of SEQ ID NO:2) when
expressed in the yeast Pichea pastoris. A mutant Neutrokine-alpha
polypeptide in which only the asparagine at position 242 is mutated
to glutamine, is still glycosylated when expressed in Pichea
pastoris. In this mutant, the glycosylation event may be due to the
activation or unmasking of an O-linked glycosylation site at serine
244. Similar mutations affecting glycosylation could also be made
in Neutrokine alpha-SV polypeptide, i.e., aspargine-223 to
glutamine and/or serine-224 to alanine of SEQ ID NO:19.
[0358] Additionally, one or more of the amino acid residues of the
polypeptides of the invention (e.g., arginine and lysine residues)
may be deleted or substituted with another residue to eliminate
undesired processing by proteases such as, for example, furins or
kexins. One possible result of such a mutation is that
Neutrokine-alpha polypeptide of the invention is not cleaved and
released from the cell surface.
[0359] In a specific embodiment, Lys-132 and/or Arg-133 of the
Neutrokine-alpha sequence shown in SEQ ID NO:2 is mutated to
another amino acid residue, or deleted altogether, to prevent or
diminish release of the soluble form of Neutrokine-alpha from cells
expressing Neutrokine-alpha. In a more specific embodiment, Lys-132
of the Neutrokine-alpha sequence shown in SEQ ID NO:2 is mutated to
Ala-132. In another, nonexclusive specific embodiment, Arg-133 of
the Neutrokine-alpha sequence shown in SEQ ID NO:2 is mutated to
Ala-133. These mutated proteins, and/or polynucleotides encoding
these proteins have uses such as, for example, in ex vivo therapy
or gene therapy, to engineer cells expressing a Neutrokine-alpha
polypeptide that is retained on the surface of the engineered
cells.
[0360] In a specific embodiment, Cys-146 of the Neutrokine-alpha
sequence shown in SEQ ID NO:2 is mutated to another amino acid
residue, or deleted altogether, for example, to aid preventing or
diminishing oligomerization of the mutant Neutrokine-alpha
polypeptide when expressed in an expression system (essentially as
described in Example 1). In a specific embodiment, Cys-146 is
replaced with a serine amino acid residue. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0361] In another specific embodiment, Cys-232 of the
Neutrokine-alpha sequence shown in SEQ ID NO:2 is mutated to
another amino acid residue, or deleted altogether, for example, to
aid preventing or diminishing oligomerization of the mutant
Neutrokine-alpha polypeptide when expressed in an expression system
(essentially as described in Example 1). In a specific embodiment,
Cys-232 is replaced with a serine amino acid residue.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0362] In yet another specific embodiment, Cys-245 of the
Neutrokine-alpha sequence shown in SEQ ID NO:2 is mutated to
another amino acid residue, or deleted altogether, for example, to
aid preventing or diminishing oligomerization of the mutant
Neutrokine-alpha polypeptide when expressed in an expression system
(essentially as described in Example 1). In a specific embodiment,
Cys-245 is replaced with a serine amino acid residue.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0363] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides can be substantially
purified by the one-step method described in Smith and Johnson,
Gene 67:31-40 (1988).
[0364] The polypeptides of the present invention include the
complete polypeptide encoded by the deposited cDNA (ATCC Deposit
No. 97768) including the intracellular, transmembrane and
extracellular domains of the polypeptide encoded by the deposited
cDNA, the mature soluble polypeptide encoded by the deposited cDNA,
the extracellular domain minus the intracellular and transmembrane
domains of the protein, the complete polypeptide of FIGS. 1A and 1B
(amino acid residues 1-285 of SEQ ID NO:2), the mature soluble
polypeptide of FIGS. 1A and 1B (amino acids 134-285 of SEQ ID
NO:2), the extracellular domain of FIGS. 1A and 1B (amino acid
residues 73-285 of SEQ ID NO:2) minus the intracellular and
transmembrane domains, as well as polypeptides which have at least
80%, 85%, 90% similarity, more preferably at least 95% similarity,
and still more preferably at least 96%, 97%, 98% or 99% similarity
to those described above. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0365] The polypeptides of the present invention also include the
complete polypeptide encoded by the deposited cDNA including the
intracellular, transmembrane and extracellular domains of the
polypeptide encoded by the deposited cDNA (ATCC Deposit No.
203518), the mature soluble polypeptide encoded by the deposited
cDNA, the extracellular domain minus the intracellular and
transmembrane domains of the protein, the complete polypeptide of
FIGS. 5A and 5B (amino acid residues 1-266 of SEQ ID NO:19), the
mature soluble polypeptide of FIGS. 5A and 5B (amino acid residues
134-266 of SEQ ID NO:19), the extracellular domain of FIGS. 5A and
5B (amino acid residues 73-266 of SEQ ID NO:19) minus the
intracellular and transmembrane domains, as well as polypeptides
which have at least 80%, 85%, 90% similarity, more preferably at
least 95% similarity, and still more preferably at least 96%, 97%,
98% or 99% similarity to those described above. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0366] Further polypeptides of the present invention include
polypeptides at least 80%, or at least 85% identical, more
preferably at least 90% or 95% identical, still more preferably at
least 96%, 97%, 98% or 99% identical to the polypeptide encoded by
the deposited cDNA (ATCC Deposit No. 97768) or to the polypeptide
of FIGS. 1A and 1B (SEQ ID NO:2), and also include portions of such
polypeptides with at least 30 amino acids and more preferably at
least 50 amino acids. Polynucleotides encoding these polypeptides
are also encompassed by the invention.
[0367] Further polypeptides of the present invention include
polypeptides at least 80%, or at least 85% identical, more
preferably at least 90% or 95% identical, still more preferably at
least 96%, 97%, 98% or 99% identical to the polypeptide encoded by
the deposited cDNA (ATCC Deposit No. 203518) or to the polypeptide
of FIGS. 5A and 5B (SEQ ID NO:19), and also include portions of
such polypeptides with at least 30 amino acids and more preferably
at least 50 amino acids. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0368] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Advances in
Applied Mathematics 2:482-489, 1981) to find the best segment of
similarity between two sequences.
[0369] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide is intended
that the amino acid sequence of the polypeptide is identical to the
reference sequence except that the polypeptide sequence may include
up to five amino acid alterations per each 100 amino acids of the
reference amino acid of the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to
a reference amino acid sequence, up to 5% of the amino acid
residues in the reference sequence may be deleted or substituted
with another amino acid, or a number of amino acids up to 5% of the
total amino acid residues in the reference sequence may be inserted
into the reference sequence. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
[0370] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID
NO:2), the amino acid sequence encoded by the deposited cDNA clone
HNEDU15 (ATCC Accession No. 97768), or fragments thereof, or, for
instance, to the amino acid sequence shown in FIGS. 5A and 5B (SEQ
ID NO:19), the amino acid sequence encoded by the deposited cDNA
clone HDPMC52 (ATCC Accession No. 203518), or fragments thereof,
can be determined conventionally using known computer programs such
the Bestfit program (Wisconsin Sequence Analysis Package, Version 8
for Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0371] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=1, Window Size=sequence
length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or
the length of the subject amino acid sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence due to N- or C-terminal deletions,
not because of internal deletions, a manual correction is made to
the results to take into consideration the fact that the FASTDB
program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For
subject sequences truncated at the N- and C-termini, relative to
the query sequence, the percent identity is corrected by
calculating the number of residues of the query sequence that are
N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. A determination of
whether a residue is matched/aligned is determined by results of
the FASTDB sequence alignment. This percentage is then subtracted
from the percent identity, calculated by the above FASTDB program
using the specified parameters, to arrive at a final percent
identity score. This final percent identity score is what is used
for the purposes of this embodiment. Only residues to the N- and
C-termini of the subject sequence, which are not matched/aligned
with the query sequence, are considered for the purposes of
manually adjusting the percent identity score. That is, only query
residue positions outside the farthest N- and C-terminal residues
of the subject sequence. For example, a 90 amino acid residue
subject sequence is aligned with a 100 residue query sequence to
determine percent identity. The deletion occurs at the N-terminus
of the subject sequence and therefore, the FASTDB alignment does
not show a matching/alignment of the first 10 residues at the
N-terminus. The 10 unpaired residues represent 10% of the sequence
(number of residues at the N- and C-termini not matched/total
number of residues in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are made for the purposes of this
embodiment.
[0372] The polypeptides of the present invention have uses that
include, but are not limited to, as a molecular weight marker on
SDS-PAGE gels or on molecular sieve gel filtration columns using
methods well known to those skilled in the art. Additionally, as
described in detail below, the polypeptides of the present
invention have uses that include, but are not limited to, raising
polyclonal and monoclonal antibodies, which are useful in assays
for detecting Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide expression as described below or as agonists and
antagonists capable of enhancing or inhibiting Neutrokine-alpha
and/or Neutrokine-alphaSV function. The polypeptides of the
invention also have therapeutic uses as described herein. Further,
such polypeptides can be used in the yeast two-hybrid system to
"capture" Neutrokine-alpha and/or Neutrokine-alphaSV binding
proteins which are also candidate agonists and antagonists
according to the present invention. The yeast two hybrid system is
described in Fields and Song, Nature 340:245-246 (1989).
Transgenics and "Knock-Outs"
[0373] The polypeptides of the invention can also be expressed in
transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,
micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express
polypeptides of the invention in humans, as part of a gene therapy
protocol.
[0374] Any technique known in the art may be used to introduce the
transgene (i.e., polynucleotides of the invention) into animals to
produce the founder lines of transgenic animals. Such techniques
include, but are not limited to, pronuclear microinjection
(Paterson, et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et
al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S.
Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA
82:6148-6152 (1985)), blastocysts or embryos; gene targeting in
embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the
invention using a gene gun (see, e.g., Ulmer et al., Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent stem cells and transferring the stem cells back into
the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell 57:717-723 (1989); etc. For a review of such techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989),
which is incorporated by reference herein in its entirety. See
also, U.S. Pat. No. 5,464,764 (Capecchi, et al., Positive-Negative
Selection Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi,
et al., Cells and Non-Human Organisms Containing Predetermined
Genomic Modifications and Positive-Negative Selection Methods and
Vectors for Making Same); U.S. Pat. No. 4,736,866 (Leder, et al.,
Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner,
et al., Genetic Transformation of Zygotes); each of which is hereby
incorporated by reference in its entirety.
[0375] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides of the invention, for
example, nuclear transfer into enucleated oocytes of nuclei from
cultured embryonic, fetal, or adult cells induced to quiescence
(Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)).
[0376] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
or chimeric animals. The transgene may be integrated as a single
transgene or as multiple copies such as in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lasko et al.
(Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. When it is
desired that the polynucleotide transgene be integrated into the
chromosomal site of the endogenous gene, gene targeting is
preferred. Briefly, when such a technique is to be utilized,
vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via
homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene may also be selectively introduced
into a particular cell type, thus inactivating the endogenous gene
in only that cell type, by following, for example, the teaching of
Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory
sequences required for such a cell-type specific inactivation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art. In addition to expressing
the polypeptide of the present invention in a ubiquitous or tissue
specific manner in transgenic animals, it would also be routine for
one skilled in the art to generate constructs which regulate
expression of the polypeptide by a variety of other means (for
example, developmentally or chemically regulated expression).
[0377] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but
are not limited to, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, reverse
transcriptase-PCR (rt-PCR); and TaqMan PCR. Samples of transgenic
gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the
transgene product.
[0378] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; breeding to place the transgene on a distinct
background that is appropriate for an experimental model of
interest; and breeding of transgenic animals to other animals
bearing a distinct transgene or knockout mutation.
[0379] Transgenic and "knock-out" animals of the invention have
uses which include, but are not limited to, animal model systems
useful in elaborating the biological function of Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides, studying conditions and/or
disorders associated with aberrant Neutrokine-alpha and/or
Neutrokine-alphaSV expression, and in screening for compounds
effective in ameliorating such conditions and/or disorders.
[0380] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the polypeptides of the invention. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
[0381] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, e.g., genetically engineered fibroblasts
can be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each
of which is incorporated by reference herein in its entirety).
[0382] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
Antibodies
[0383] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, polypeptide fragment, or variant of SEQ ID NO:2 and/or
SEQ ID NO:19, and/or an epitope, of the present invention (as
determined by immunoassays well known in the art for assaying
specific antibody-antigen binding). In specific embodiments,
antibodies of the invention bind homomeric, especially
homotrimeric, Neutrokine-alpha polypeptides. In other specific
embodiments, antibodies of the invention bind heteromeric,
especially heterotrimeric, Neutrokine-alpha polypeptides such as a
heterotrimer containing two Neutrokine-alpha polypeptides and one
APRIL polypeptide (e.g., SEQ ID NO:20 or SEQ ID NO:47) or a
heterotrimer containing one Neutrokine-alpha polypeptide and two
APRIL polypeptides.
[0384] In particularly preferred embodiments, the antibodies of the
invention bind homomeric, especially homotrimeric, Neutrokine-alpha
polypeptides, wherein the individual protein components of the
multimers consist of the mature form of Neutrokine alpha (e.g.,
amino acids residues 134-285 of SEQ ID NO:2, or amino acids
residues 134-266 of SEQ ID NO:19.) In other specific embodiments,
antibodies of the invention bind heteromeric, especially
heterotrimeric, Neutrokine-alpha polypeptides such as a
heterotrimer containing two Neutrokine-alpha polypeptides and one
APRIL polypeptide or a heterotrimer containing one Neutrokine-alpha
polypeptide and two APRIL polypeptides, and wherein the individual
protein components of the Neutrokine-alpha heteromer consist of the
mature extracellular soluble portion of either Neutrokine-alpha or
(e.g., amino acids residues 134-285 of SEQ ID NO:2, or amino acids
residues 134-266 of SEQ ID NO:19) or the mature extracellular
soluble portion APRIL (e.g., amino acid residues 105-250 of SEQ ID
NO:47).
[0385] In specific embodiments, the antibodies of the invention
bind conformational epitopes of a Neutrokine-alpha and/or
Neutrokine-alphaSV monomeric protein. In specific embodiments, the
antibodies of the invention bind conformational epitopes of a
Neutrokine-alpha and/or Neutrokine-alphaSV multimeric, especially
trimeric, protein. In other embodiments, antibodies of the
invention bind conformational epitopes that arise from the
juxtaposition of Neutrokine-alpha and/or Neutrokine alpha SV with a
heterologous polypeptide, such as might be present when
Neutrokine-alpha or Neutrokine-alpha SV forms heterotrimers (e.g.,
with APRIL polypeptides (e.g., SEQ ID NO:20 or SEQ ID NO:47)), or
in fusion proteins between Neutrokine alpha and a heterologous
polypeptide.
[0386] Antibodies of the invention include, but are not limited to,
polyclonal, monoclonal, multispecific, human, humanized or chimeric
antibodies, single chain antibodies, Fab fragments, F(ab')
fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies (including, e.g., anti-id
antibodies to antibodies of the invention), and epitope-binding
fragments of any of the above. The term "antibody," as used herein,
refers to immunoglobulin molecules and immunologically active
portions of immunoglobulin molecules, i.e., molecules that contain
an antigen binding site that immunospecifically binds an antigen.
The immunoglobulin molecules of the invention can be of any type
(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
In preferred embodiments, the immunoglobulin is an IgG1 or an IgG4
isotype. Immunoglobulins may have both a heavy and light chain. An
array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be
paired with a light chain of the kappa or lambda forms.
[0387] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat),
donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used herein, "human" antibodies include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulin and that do not
express endogenous immunoglobulins, as described infra and, for
example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0388] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0389] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Antibodies which specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0390] In specific embodiments, antibodies of the invention bind to
polypeptides comprising Phe-115 to Leu-147, Ile-150 to Tyr-163,
Ser-171 to Phe-194, Glu-223 to Tyr-246, and Ser-271 to Phe-278 of
the amino acid sequence of SEQ ID NO:2. In another specific
embodiment, antibodies of the invention bind to polypeptides
consisting of Phe-115 to Leu-147, Ile-150 to Tyr-163, Ser-171 to
Phe-194, Glu-223 to Tyr-246, and Ser-271 to Phe-278 of the amino
acid sequence of SEQ ID NO:2. In a preferred embodiment, antibodies
of the invention bind to a polypeptide comprising Glu-223 to
Tyr-246 of SEQ ID NO:2. In another preferred embodiment, antibodies
of the invention bind to a polypeptide consisting of Glu-223 to
Tyr-246 of SEQ ID NO:2. In a more preferred embodiment, antibodies
of the invention bind to a polypeptide consisting of Phe-230 to
Asn-242 of SEQ ID NO:2. In further preferred, nonexclusive
embodiments, the antibodies of the invention inhibit one or more
biological activities of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the invention through specific binding. In more
preferred embodiments, the antibody of the invention inhibits
Neutrokine-alpha- and/or Neutrokine-alphaSV-mediated B cell
proliferation.
[0391] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
In a specific embodiment, antibodies of the present invention cross
react with APRIL (e.g., SEQ ID NO:20 or SEQ ID NO:47; PCT
International Publication Number WO97/33902; GenBank Accession No.
AF046888 (nucleotide) and AAC6132 (protein); J. Exp. Med.
188(6):1185-1190). In specific embodiments, antibodies of the
present invention cross-react with murine, rat and/or rabbit
homologs of human proteins and the corresponding epitopes thereof.
Antibodies that do not bind polypeptides with less than 95%, less
than 90%, less than 85%, less than 80%, less than 75%, less than
70%, less than 65%, less than 60%, less than 55%, and less than 50%
identity (as calculated using methods known in the art and
described herein) to a polypeptide of the present invention are
also included in the present invention. In a specific embodiment,
the above-described cross-reactivity is with respect to any single
specific antigenic or immunogenic polypeptide, or combination(s) of
2, 3, 4, 5, or more of the specific antigenic and/or immunogenic
polypeptides disclosed herein. Further included in the present
invention are antibodies which bind polypeptides encoded by
polynucleotides which hybridize to a polynucleotide of the present
invention under hybridization conditions (as described herein).
Antibodies of the present invention may also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. In specific embodiments, antibodies of the invention
bind Neutrokine-alpha and/or Neutokine-alphaSV polypeptides, or
fragments or variants thereof, with a dissociation constant or
K.sub.D of less than or equal to 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, or 10.sup.-5 M. More preferably, antibodies of
the invention bind Neutrokine-alpha and/or Neutokine-alphaSV
polypeptides or fragments or variants thereof with a dissociation
constant or K.sub.D less than or equal to 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8M,
or 10.sup.-8 M. Even more preferably, antibodies of the invention
bind Neutrokine-alpha and/or Neutokine-alphaSV polypeptides or
fragments or variants thereof with a dissociation constant or
K.sub.D less than or equal to 5.times.10.sup.-9 M, 10.sup.-9 M,
5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11 M,
10.sup.-11M, 5.times.10.sup.-12 M, 10.sup.-12 M, 5.times.10.sup.-13
M, 5.times.10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15 M, or 10.sup.-15 M. The invention encompasses
antibodies that bind Neutrokine-alpha and/or Neutokine-alphaSV
polypeptides with a dissociation constant or K.sub.D that is within
any one of the ranges that are between each of the individual
recited values.
[0392] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 95%, at least 90%, at
least 85%, at least 80%, at least 75%, at least 70%, at least 60%,
or at least 50%.
[0393] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. Preferably, antibodies of the
present invention bind an antigenic epitope disclosed herein, or a
portion thereof. The invention features both receptor-specific
antibodies and ligand-specific antibodies. The invention also
features receptor-specific antibodies which do not prevent ligand
binding but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody.
[0394] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the peptides of
the invention disclosed herein. The above antibody agonists can be
made using methods known in the art. See, e.g., PCT publication WO
96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood
92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678
(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et
al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci.
111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman et al., Neuron 14(4):755-762 (1995); Muller et al.,
Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine
8(1):14-20 (1996) (which are all incorporated by reference herein
in their entireties).
[0395] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0396] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495;
WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP
396,387.
[0397] The antibodies of the invention include derivatives that are
modified, i.e, by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0398] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0399] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0400] A "monoclonal antibody" may comprise, or alternatively
consist of, two proteins, i.e., a heavy and a light chain.
[0401] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
and are discussed in detail in the Examples (e.g., Example 9). In a
non-limiting example, mice can be immunized with a polypeptide of
the invention or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well-known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0402] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0403] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0404] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen-binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[0405] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0406] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816,397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework region from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[0407] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0408] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0409] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0410] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
Polynucleotides Encoding Antibodies
[0411] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NO:2. In
another preferred embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:19. In
another preferred embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:23. In
another preferred embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:28. In
another preferred embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:30. In
another preferred embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:39. In
another preferred embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:40. In
another embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:41. In
another embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:42. In
another embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:43. In
another embodiment, the antibody binds specifically to a
polypeptide having the amino acid sequence of SEQ ID NO:44.
[0412] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0413] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0414] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0415] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well known in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0416] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0417] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
Methods of Producing Antibodies
[0418] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0419] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody molecule or a heavy or light
chain of an antibody, or portion thereof (preferably containing the
heavy or light chain variable domain), of the invention has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0420] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, or a single chain antibody of the invention, operably
linked to a heterologous promoter. In preferred embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed
below.
[0421] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0422] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0423] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0424] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (E.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0425] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0426] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0427] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIB TECH 11(5):155-215); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0428] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0429] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0430] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0431] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalent and
non-covalent conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. The antibodies may be
specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452 (1991), which are incorporated by
reference in their entireties.
[0432] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341 (1992) (said references incorporated by
reference in their entireties).
[0433] As discussed, supra, the polypeptides corresponding to a
polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may
be fused or conjugated to the above antibody portions to increase
the in vivo half life of the polypeptides or for use in
immunoassays using methods known in the art. Further, the
polypeptides corresponding to SEQ ID NO:2 may be fused or
conjugated to the above antibody portions to facilitate
purification. Also as discussed, supra, the polypeptides
corresponding to a polypeptide, polypeptide fragment, or a variant
of SEQ ID NO:19 may be fused or conjugated to the above antibody
portions to increase the in vivo half life of the polypeptides or
for use in immunoassays using methods known in the art. Moreover,
the polypeptides corresponding to SEQ ID NO:19 may be fused or
conjugated to the above antibody portions to facilitate
purification. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. (EP 394,827; Traunecker et
al., Nature 331:84-86 (1988). The polypeptides of the present
invention fused or conjugated to an antibody having
disulfide-linked dimeric structures (due to the IgG) may also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many
cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and thus can result in, for example, improved
pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
the Fc part after the fusion protein has been expressed, detected,
and purified, would be desired. For example, the Fc portion may
hinder therapy and diagnosis if the fusion protein is used as an
antigen for immunizations. In drug discovery, for example, human
proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0434] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
[0435] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and acquorin; and examples of suitable radioactive
material include iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.201Ti), gallium (.sup.68 Ga, .sup.67Ga), palladium (.sup.103
Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149 Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru,
.sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P, .sup.153Gd,
.sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se, .sup.113Sn, and
117Tin.
[0436] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, .sup.213Bi. In specific
embodiments, antibodies of the invention are attached to
macrocyclic chelators useful for conjugating radiometal ions,
including but not limited to, .sup.111In, .sup.177Lu, .sup.90Y,
.sup.166Ho, and .sup.153Sm, to polypeptides. In preferred
embodiments, the radiometal ion associated with the macrocyclic
chelators attached to antibodies of the invention is .sup.111In. In
preferred embodiments, the radiometal ion associated with the
macrocyclic chelators attached to antibodies of the invention is
.sup.90Y. In specific embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide of the
invention via a linker molecule. Examples of linker molecules
useful for conjugating DOTA to a polypeptide are commonly known in
the art--see, for example, DeNardo et al., Clin Cancer Res.
4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7
(1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999)
which are hereby incorporated by reference in their entirety. In
addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose
chelating agents that may be conjugated to antibodies, and methods
for making and using them, are hereby incorporated by reference in
their entireties.
[0437] A cytotoxin or cytotoxic agent includes any agent that is
detrimental to cells and includes such molecules as small molecule
toxins and enzymatically active toxins of bacterial, fungal, plant,
or animal origin, or fragments thereof. Examples include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide (VP-16), tenoposide, vincristine,
vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine), improsulfan, piposulfan, benzodopa,
carboquone, meturedopa, uredopa, altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide
trimethylolomelamine, chlomaphazine, cholophosphamide,
estramustine, ifosfamide, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard, chlorozotocin, fotemustine,
nimustine, ranimustine, aclacinomysins, azaserine, cactinomycin,
calichearmicin, carabicin, caminomycin, carzinophilin,
chromomycins, detorubicin, 6-diazo-5-oxo-L-norleucine, epirubicin,
esorubicin, idarubicin, marcellomycin, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, quelamycin,
rodorubicin, streptonigrin, tubercidin, ubenimex, zinostatin,
zorubicin, denopterin, pteropterin, trimetrexate, fludarabine,
thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur,
dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU,
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone, aminoglutethimide, mitotane, trilostane, frolinic
acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid,
amsacrine, bestrabucil, bisantrene, edatraxate, defofamine,
demecolcine, diaziquone, elformithine, elliptiniurn acetate,
etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine,
mitoguazone, mopidamol, nitracrine, pentostatin, phenamet,
pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine,
PSKO, razoxane, sizofuran, spirogermanium, tenuazonic acid,
triaziquone, 2, 2',2''-trichlorotriethylamine, urethan, vindesine,
dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,
gacytosine, arabinoside ("Ara-C"), taxoids, e.g. paclitaxel
(TAXOL", Bristol-Myers Squibb Oncology, Princeton, N.J.) doxetaxel
(TAXOTERE", Rh6ne-Poulenc Rorer, Antony, France), gemcitabine,
ifosfamide, vinorelbine, navelbine, novantrone, teniposide,
aminopterin, xeloda, ibandronate, CPT-I 1, topoisomerase inhibitor
RFS 2000, difluoromethylornithine (DMFO), retinoic acid,
esperamicins, capecitabine, and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene, LY
117018, onapristone, toremifene (Fareston), and anti-androgens such
as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin,
and pharmaceutically acceptable salts, acids or derivatives of any
of the above.
[0438] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, alpha-interferon, beta-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), CD40 Ligand, a
thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or
endostatin; or, biological response modifiers such as, for example,
lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
or other growth factors.
[0439] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0440] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody in Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0441] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0442] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
Immunophenotyping
[0443] The antibodies of the invention may be utilized for
immunophenotyping of cell lines and biological samples. The
translation product of the gene of the present invention may be
useful as a cell specific marker, or more specifically as a
cellular marker that is differentially expressed at various stages
of differentiation and/or maturation of particular cell types.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies to screen for cellular
populations expressing the marker(s), and include magnetic
separation using antibody-coated magnetic beads, "panning" with
antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,
Cell, 96:737-49 (1999)).
[0444] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e. minimal residual disease (MRD) in acute leukemic
patients) and "non-self" cells in transplantations to prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for the screening of hematopoietic stem and progenitor cells
capable of undergoing proliferation and/or differentiation, as
might be found in human umbilical cord blood.
Assays For Antibody Binding
[0445] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used, include but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0446] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0447] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
.sup.32P or .sup.125I) diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0448] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0449] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., .sup.3H or .sup.125I) with the antibody of interest
in the presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest conjugated to a labeled
compound (e.g., .sup.3H or .sup.125I) in the presence of increasing
amounts of an unlabeled second antibody.
Therapeutic Uses
[0450] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention and/or a receptor for
the polypeptide of the invention (e.g., transmembrane activator and
CAML interactor (TACI, GenBank accession number AAC51790), BAFF-R
(GenBank Acession Number NP 44.3177) and B-cell maturation antigen
(BCMA, GenBank accession number NP.sub.--001183)), including, but
not limited to, any one or more of the diseases, disorders, or
conditions described herein (e.g., autoimmune diseases, disorders,
or conditions associated with such diseases or disorders,
including, but not limited to, autoimmune hemolytic anemia
(including but not limited to cryoglobinemia or Coombs positive
anemia), autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia purpura, autoimmunocytopenia, autoimmune
neutropenia, hemolytic anemia, antiphospholipid syndrome,
dermatitis (e.g., atopic dermatitis), allergic encephalomyelitis,
myocarditis, relapsing polychondritis, rheumatic heart disease,
Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary
Inflammation, Guillain-Barre Syndrome, diabetes mellitus (e.g.,
Type I diabetes mellitus or insulin dependent diabetes mellitis),
juvenile onset diabetes, and autoimmune inflammatory eye,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis, systemic lupus erythematosus, discoid lupus,
Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as,
for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c)
insulin resistance, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, rheumatoid arthritis, scleroderma with
anti-collagen antibodies, mixed connective tissue disease,
polymyositis/dermatomyositis, pernicious anemia (Addison's
disease), idiopathic Addison's disease, infertility,
glomerulonephritis such as primary glomerulonephritis, IgA
glomerulonephritis, and IgA nephropathy, bullous pemphigoid,
Sjogren's syndrome, diabetes millitus, and adrenergic drug
resistance (including adrenergic drug resistance with asthma or
cystic fibrosis), gluten sensitive enteropathy, dense deposit
disease, chronic active hepatitis, primary biliary cirrhosis, other
endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy
syndrome, urticaria, atopic dermatitis, asthma, inflammatory
myopathies, and other inflammatory, granulamatous, degenerative,
and atrophic disorders) and other disorders such as inflammatory
skin diseases including psoriasis and sclerosis, responses
associated with inflammatory bowel disease (such as Crohn's disease
and ulcerative colitis), respiratory distress syndrome (including
adult respiratory distress syndrome, ARDS), meningitis,
encephalitis, colitis, allergic conditions such as eczema and other
conditions involving infiltration of T cells and chronic
inflammatory responses, atherosclerosis, leukocyte adhesion
deficiency, Reynaud's syndrome, and immune responses associated
with acute and delayed hypersensitivity mediated by cytokines and
T-lymphocytes typically found in tuberculosis, sarcoidosis,
granulomatosis and diseases involving leukocyte diapedesis, central
nervous system (CNS) inflammatory disorder, multiple organ injury
syndrome, antigen-antibody complex mediated diseases,
anti-glomerular basement membrane disease, Lambert-Eaton myasthenic
syndrome, Beheet disease, giant cell arteritis, immune complex
nephritis, IgA nephropathy, IgM polyneuropathies or autoimmune
thrombocytopenia etc.
[0451] In a specific embodiment, antibodies of the invention are
used to treat, inhibit, prognose, diagnose or prevent rheumatoid
arthritis. In a specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent advanced
rheumatoid arthritis.
[0452] In another specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent systemic
lupus erythematosis.
[0453] For example, an antibody, or antibodies, of the present
invention are used to treat patients with clinical diagnosis of
rheumatoid arthritis (RA). The patient treated will not have a B
cell malignancy. Moreover, the patient is optionally further
treated with any one or more agents employed for treating RA such
as salicylate; nonsteroidal anti-inflammatory drugs such as
indomethacin, phenylbutazone, phenylacetic acid derivatives (e.g.
ibuprofen and fenoprofen), naphthalene acetic acids (naproxen),
pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac),
halogenated anthranilic acid (meclofenamate sodium), piroxicam,
zomepirac and diflunisal; antimalarials such as chloroquine; gold
salts; penicillamine; or immunosuppressive agents such as
methotrexate or corticosteroids in dosages known for such drugs or
reduced dosages. Preferably however, the patient is only treated
with an antibody, or antibodies, of the present invention.
Antibodies of the present invention are administered to the RA
patient according to a dosing schedule as described infra, which
may be readily determined by one of ordinary skill in the art. The
primary response is determined by the Paulus index (Paulus et al.
Arthritis Rheum. 33:477-484 (1990)), i.e. improvement in morning
stiffness, number of painful and inflamed joints, erythrocyte
sedimentation (ESR), and at least a 2-point improvement on a
5-point scale of disease severity assessed by patient and by
physician. Administration of an antibody, or antibodies, of the
present invention will alleviate one or more of the symptoms of RA
in the patient treated as described above.
[0454] In a further specific embodiment, antibodies of the
invention are used to treat, inhibit, prognose, diagnose or prevent
hemolytic anemia. For example, patients diagnosed with autoimmune
hemolytic anemia (AIHA), e.g., cryoglobinemia or Coombs positive
anemia, are treated with an antibody, or antibodies, of the present
invention. AIHA is an acquired hemolytic anemia due to
auto-antibodies that react with the patient's red blood cells. The
patient treated will not have a B cell malignancy. Further adjunct
therapies (such as glucocorticoids, prednisone, azathioprine,
cyclophosphamide, vinca-laden platelets or Danazol) may be combined
with the antibody therapy, but preferably the patient is treated
with an antibody, or antibodies, of the present invention as a
single-agent throughout the course of therapy. Antibodies of the
present invention are administered to the hemolytic anemia patient
according to a dosing schedule as described infra, which may be
readily determined by one of ordinary skill in the art. Overall
response rate is determined based upon an improvement in blood
counts, decreased requirement for transfusions, improved hemoglobin
levels and/or a decrease in the evidence of hemolysis as determined
by standard chemical parameters. Administration of an antibody, or
antibodies of the present invention will improve any one or more of
the symptoms of hemolytic anemia in the patient treated as
described above. For example, the patient treated as described
above will show an increase in hemoglobin and an improvement in
chemical parameters of hemolysis or return to normal as measured by
serum lactic dehydrogenase and/or bilirubin.
[0455] In another specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent adult
immune thrombocytopenic purpura. Adult immune thrombocytopenic
purpura (ITP) is a relatively rare hematologic disorder that
constitutes the most common of the immune-mediated cytopenias. The
disease typically presents with severe thrombocytopenia that may be
associated with acute hemorrhage in the presence of normal to
increased megakaryocytes in the bone marrow. Most patients with ITP
have an IgG antibody directed against target antigens on the outer
surface of the platelet membrane, resulting in platelet
sequestration in the spleen and accelerated reticuloendothelial
destruction of platelets (Bussell, J. B. Hematol. Oncol. Clin.
North Am. (4):179 (1990)). A number of therapeutic interventions
have been shown to be effective in the treatment of ITP. Steroids
are generally considered first-line therapy, after which most
patients are candidates for intravenous immunoglobulin (IVIG),
splenectomy, or other medical therapies including vincristine or
immunosuppressive/cytotoxic agents. Up to 80% of patients with ITP
initially respond to a course of steroids, but far fewer have
complete and lasting remissions. Splenectomy has been recommended
as standard second-line therapy for steroid failures, and leads to
prolonged remission in nearly 60% of cases yet may result in
reduced immunity to infection. Splenectomy is a major surgical
procedure that may be associated with substantial morbidity (15%)
and mortality (2%). IVIG has also been used as second line medical
therapy, although only a small proportion of adult patients with
ITP achieve remission. Therapeutic options that would interfere
with the production of autoantibodies by activated B cells without
the associated morbidities that occur with corticosteroids and/or
splenectomy would provide an important treatment approach for a
proportion of patients with ITP. Patients with clinical diagnosis
of ITP are treated with an antibody, or antibodies of the present
invention, optionally in combination with steroid therapy. The
patient treated will not have a B cell malignancy. Antibodies of
the present invention are administered to the RA patient according
to a dosing schedule as described infra, which may be readily
determined by one of ordinary skill in the art. Overall patient
response rate is determined based upon a platelet count determined
on two consecutive occasions two weeks apart following treatments
as described above. See, George et al. "Idiopathic Thrombocytopenic
Purpura: A Practice Guideline Developed by Explicit Methods for The
American Society of Hematology", Blood 88:3-40 (1996), expressly
incorporated herein by reference.
[0456] In other embodiments, antibody agonists of the invention are
be used to treat, inhibit or prevent immunodeficiencies, and/or
disorders, or conditions associated with immunodeficiencies. Such
immunodeficiencies include, but are not limited to, severe combined
immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine
deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia
(XLA), Bruton's disease, congenital agammaglobulinemia, X-linked
infantile agammaglobulinemia, acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.
[0457] In another specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent CVID, or
a subgroup of individuals having CVID.
[0458] In another specific embodiment, antibody agonists of the
invention are used as an adjuvant to stimulate B cell
proliferation, immunoglobulin production, and/or to enhance B cell
survival.
[0459] The treatment and/or prevention of diseases, disorders, or
conditions associated with aberrant expression and/or activity of a
polypeptide of the invention and/or a receptor for the polypeptide
of the invention (e.g., TACI, BCMA or BAFF-R) includes, but is not
limited to, alleviating symptoms associated with those diseases,
disorders or conditions. The antibodies of the invention may also
be used to target and kill cells expressing Neutrokine-alpha on
their surface and/or cells having Neutrokine-alpha bound to their
surface. Antibodies of the invention may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0460] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0461] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0462] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy, anti-tumor
agents, antibiotics, and immunoglobulin). Generally, administration
of products of a species origin or species reactivity (in the case
of antibodies) that is the same species as that of the patient is
preferred. Thus, in a preferred embodiment, human antibodies,
fragments derivatives, analogs, or nucleic acids, are administered
to a human patient for therapy or prophylaxis.
[0463] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10.sup.-5 M, 10.sup.-5 M,
5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M,
5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M,
5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11 M,
10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, and 10.sup.-15 M.
Gene Therapy
[0464] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0465] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0466] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0467] In a preferred embodiment, the compound comprises nucleic
acid sequences encoding an antibody, said nucleic acid sequences
being part of expression vectors that express the antibody or
fragments or chimeric proteins or heavy or light chains thereof in
a suitable host. In particular, such nucleic acid sequences have
promoters operably linked to the antibody coding region, said
promoter being inducible or constitutive, and, optionally,
tissue-specific. In another particular embodiment, nucleic acid
molecules are used in which the antibody coding sequences and any
other desired sequences are flanked by regions that promote
homologous recombination at a desired site in the genome, thus
providing for intrachromosomal expression of the antibody encoding
nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In
specific embodiments, the expressed antibody molecule is a single
chain antibody; alternatively, the nucleic acid sequences include
sequences encoding both the heavy and light chains, or fragments
thereof, of the antibody.
[0468] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0469] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[0470] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding an antibody of the invention are used. For
example, a retroviral vector can be used (see Miller et al., Meth.
Enzymol. 217:581-599 (1993)). These retroviral vectors contain the
components necessary for the correct packaging of the viral genome
and integration into the host cell DNA. The nucleic acid sequences
encoding the antibody to be used in gene therapy are cloned into
one or more vectors, which facilitates delivery of the gene into a
patient. More detail about retroviral vectors can be found in
Boesen et al., Biotherapy 6:291-302 (1994), which describes the use
of a retroviral vector to deliver the mdr1 gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons
and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and
Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
[0471] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[0472] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[0473] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0474] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Clin., Pharmac. Ther.
29:69-92m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0475] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0476] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include, but are not limited to, epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0477] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0478] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985
(1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[0479] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
[0480] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
Therapeutic and/or Prophylactic Administration and Composition
[0481] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred embodiment,
the compound is substantially purified (e.g., substantially free
from substances that limit its effect or produce undesired side
effects). The subject is preferably an animal, including but not
limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0482] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0483] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0484] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as silastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0485] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[0486] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[0487] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0488] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0489] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0490] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0491] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0492] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0493] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0494] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
Diagnosis and Imaging
[0495] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases
and/or disorders associated with the aberrant expression and/or
activity of a polypeptide of the invention. The invention provides
for the detection of aberrant expression of a polypeptide of
interest, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0496] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0497] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting protein gene expression include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.201Ti), gallium (.sup.68 Ga, .sup.67Ga), palladium (.sup.103
Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149 Pm,
.sup.140 La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.86Re, .sup.88Re, .sup.42Pr, .sup.105Rh, .sup.97Ru; luminescent
labels, such as luminol; and fluorescent labels, such as
fluorescein and rhodamine, and biotin.
[0498] In specific embodiments, antibodies of the invention are
attached to macrocyclic chelators useful for conjugating radiometal
ions, including but not limited to, .sup.177Lu, .sup.90Y,
.sup.166Ho, and .sup.153Sm, to polypeptides. In a preferred
embodiment, the radiometal ion associated with the macrocyclic
chelator attached to antibodies of the invention is .sup.111In. In
another preferred embodiments, the radiometal ion associated with
the macrocyclic chelator attached to antibodies of the invention is
.sup.90Y. In specific embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide of the
invention via a linker molecule. Examples of linker molecules
useful for conjugating DOTA to a polypeptide are commonly known in
the art--see, for example, DeNardo et al., Clin Cancer Res.
4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7,
1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999
which are hereby incorporated by reference in their entirety.
[0499] Techniques known in the art may be applied to label proteins
of the invention (including antibodies of the invention). Such
techniques include, but are not limited to, the use of bifunctional
conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631;
5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139;
5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of
each of which are hereby incorporated by reference in its entirety)
and direct coupling reactions (e.g., Bolton-Hunter, Chloramine-T
reaction, and Iodogen.RTM.-based labelling).
[0500] One embodiment of the invention is the detection and
diagnosis of a disease or disorder associated with aberrant
expression of a polypeptide of interest in an animal, preferably a
mammal and most preferably a human. In one embodiment, diagnosis
comprises: (a) administering (for example, parenterally,
subcutaneously, or intraperitoneally) to a subject an effective
amount of a labeled molecule which specifically binds to the
polypeptide of interest; (b) waiting for a time interval following
the administering for permitting the labeled molecule to
preferentially concentrate at sites in the subject where the
polypeptide is expressed (and for unbound labeled molecule to be
cleared to background level); (c) determining background level; and
(d) detecting the labeled molecule in the subject, such that
detection of labeled molecule above the background level indicates
that the subject has a particular disease or disorder associated
with aberrant expression of the polypeptide of interest. Background
level can be determined by various methods including, comparing the
amount of labeled molecule detected to a standard value previously
determined for a particular system. As described herein, specific
embodiments of the invention are directed to the use of the
antibodies of the invention to quantitate or qualitate
concentrations of cells of B cell lineage or cells of monocytic
lineage.
[0501] Also as described herein, antibodies of the invention may be
used to treat, diagnose, or prognose an individual having an
immunodeficiency. In a specific embodiment, antibodies of the
invention are used to treat, diagnose, and/or prognose an
individual having common variable immunodeficiency disease (CVID)
or a subset of this disease. In another embodiment, antibodies of
the invention are used to diagnose, prognose, treat or prevent a
disorder characterized by deficient serium immunoglobulin
production, recurrent infections, and/or immune system
dysfunction.
[0502] Also as described herein, antibodies of the invention may be
used to treat, diagnose, or prognose an individual having an
autoimmune disease or disorder. In a specific embodiment,
antibodies of the invention are used to treat, diagnose, and/or
prognose an individual having systemic lupus erythematosus, or a
subset of the disease. In another specific embodiment, antibodies
of the invention are used to treat, diagnose and/or prognose an
individual having rheumatoid arthritis, or a subset of this
disease.
[0503] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of .sup.99mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which contain the specific protein. In vivo tumor imaging is
described in S. W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor
Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc. (1982).
[0504] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0505] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0506] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0507] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
Kits
[0508] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention comprise two or more antibodies
(monoclonal and/or polyclonal) that recognize the same and/or
different sequences or regions of the polypeptide of the invention.
In another specific embodiment, the kits of the present invention
contain a means for detecting the binding of an antibody to a
polypeptide of interest (e.g., the antibody may be conjugated to a
detectable substrate such as a fluorescent compound, an enzymatic
substrate, a radioactive compound or a luminescent compound, or a
second antibody which recognizes the first antibody may be
conjugated to a detectable substrate).
[0509] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0510] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0511] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0512] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, Mo.).
[0513] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0514] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[0515] The invention further relates to antibodies which act as
agonists or antagonists of the polypeptides of the present
invention. For example, the present invention includes antibodies
which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. Included
are both receptor-specific antibodies and ligand-specific
antibodies. Included are receptor-specific antibodies which do not
prevent ligand binding but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. Also included are
receptor-specific antibodies which both prevent ligand binding and
receptor activation. Likewise, included are neutralizing antibodies
which bind the ligand and prevent binding of the ligand to the
receptor, as well as antibodies which bind the ligand, thereby
preventing receptor activation, but do not prevent the ligand from
binding the receptor. Further included are antibodies which
activate the receptor. These antibodies may act as agonists for
either all or less than all of the biological activities affected
by ligand-mediated receptor activation. The antibodies may be
specified as agonists or antagonists for biological activities
comprising specific activities disclosed herein. Further included
are antibodies that bind to Neutrokine-alpha and/or
Neutrokine-alphaSV irrespective of whether Neutrokine-alpha or
Neutrokine-alphaSV is bound to a Neutrokine-alpha Receptor. These
antibodies act as Neutrokine-alpha and/or Neutrokine-alphaSV
agonists as reflected in an increase in cellular proliferation in
response to binding of Neutrokine-alpha and/or Neutrokine-alphaSV
to a Neutrokine-alpha receptor in the presence of these antibodies.
The above antibody agonists can be made using methods known in the
art. See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng, B. et
al., Blood 92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res.
58(16):3668-3678 (1998); Harrop, J. A. et al., J. Immunol.
161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res.
58(15):3209-3214 (1998); Yoon, D. Y. et al., J. Immunol.
160(7):3170-3179 (1998); Prat, M. et al., J. Cell. Sci.
111(Pt2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard, J. et al., Cytokinde 9(4):233-241
(1997); Carlson, N. G. et al., J. Biol. Chem. 272(17):11295-11301
(1997); Taryman, R. E. et al., Neuron 14(4):755-762 (1995); Muller,
Y. A. et al., Structure 6(9):1153-1167 (1998); Bartunek, P. et al.,
Cytokine 8(1):14-20 (1996) (said references incorporated by
reference in their entireties).
[0516] At least fourteen murine monoclonal antibodies have been
generated against Neutrokine-alpha. These monoclonal antibodies are
designated: 12D6, 2E5, 9B6, 1B8, 5F4, 9A5, 10G12, 11G12, 16B4, 3D4,
16C9, 13D5, 15C10, and 12C5. Preliminary analysis of these
antibodies indicates that each binds Neutrokine-alpha protein in a
Western blot analysis and when Neutrokine-alpha protein is bound to
an ELISA plate. However, further analysis of antibodies 12D6, 2E5,
9B6, 1B8, 5F4, 9A5, 10G12, 11G12, and 16B4 indicates that only the
antibodies designated 12D6, 9B6, 2E5, 10G12, 9A5, and 11G12 bind a
membrane-bound form of Neutrokine-alpha. Thus, a subset of the
monoclonal antibodies generated against Neutrokine-alpha have been
determined to bind only the membrane-bound form of Neutrokine-alpha
(i.e., this subset does not bind the soluble form of
Neutrokine-alpha corresponding to amino acids 134 to 285 of SEQ ID
NO:2), which as discussed herein, is primarily limited to
expression on monocytes and dendritic cells.
[0517] Antibody 9B6 has been found to bind specifically to the
membrane-bound form of Neutrokine-alpha, but not to the soluble
form of Neutrokine-alpha.
[0518] Epitope mapping of antibody 9B6 has indicated that this
antibody binds specifically to an amino acid sequence contained in
amino acid residues from about Ser-171 to about Phe-194 of SEQ ID
NO:2. More particularly, epitope mapping has indicated that
antibody 9B6 binds specifically to a peptide comprising amino acid
residues Lys-173 to Lys-188 of SEQ ID NO:2.
[0519] In contrast, antibodies 16C9 and 15C10 have been found to
bind the soluble form of Neutrokine-alpha (amino acids 134 to 285
of SEQ ID NO:2) and to inhibit Neutrokine-alpha-mediated
proliferation of B cells. See for example, Example 10. The 15C10
antibody has also been found to inhibit binding of Neutrokine-alpha
to its receptor. Epitope mapping of antibody 15C10 has indicated
that this antibody binds specifically to an amino acid sequence
contained in amino acid residues from about Glu-223 to about
Tyr-246 of SEQ ID NO:2. More particularly, epitope mapping has
indicated that antibody 15C10 binds specifically to a peptide
comprising amino acid residues Val-227 to Asn-242 of SEQ ID NO:2.
Antibody 15C10 also binds specifically to a peptide comprising
amino acid residues Phe-230 to Cys-245 of SEQ ID NO:2. It is likely
that the epitope of 15C10 is conformational rather than linear and
that antibody 15C10 may make specific binding contacts with amino
acid residues in the full length Neutrokine-alpha protein outside
of amino acid residues 223-246 of SEQ ID NO:2 as well as within
amino acid residues 223-246.
[0520] Furthermore, competitive binding studies have shown that
antibodies 3D4 and 15C10 bind similar or identical epitopes (see
Example 15).
[0521] As described above, anti-Neutrokine-alpha monoclonal
antibodies have been prepared. Hybridomas producing the antibodies
referred to as 9B6 and 15C10 were deposited with the ATCC located
at 10801 University Boulevard, Manassas, Va. 20110-2209, on Jan.
27, 2000 and were assigned deposit accession numbers PTA-1159 and
PTA-1158, respectively. The ATCC deposits were made pursuant to the
terms of the Budapest Treaty on the international recognition of
the deposit of microorganisms for the purposes of patent procedure.
The amino acid sequence of the VH and VL domains of the 15C10
antibody are shown in SEQ ID NOS: 58 and 59, respectively.
[0522] NS0 cell lines engineered to secrete chimeric forms of
antibodies 3D4 and 15C10 were deposited with the ATCC located at
10801 University Boulevard, Manassas, Va. 20110-2209, at on Oct.
24, 2001 and were assigned deposit accession numbers PTA-3795 and
PTA-3794, respectively. Chimeric antibodies 3D4 and 15C10 contain
murine variable regions and human constant (IgG1 and kappa)
regions. The ATCC deposits were made pursuant to the terms of the
Budapest Treaty on the international recognition of the deposit of
microorganisms for the purposes of patent procedure.
[0523] In one embodiment, the antibodies of the invention have one
or more of the same biological characteristics as one or more of
the antibodies secreted by the deposited cell lines (ATCC accession
numbers PTA-1158, PTA-1159, PTA-3795 and PTA-3794). By "biological
characteristics" is meant, the in vitro or in vivo activities or
properties of the antibodies, such as, for example, the ability to
bind to Neutrokine-alpha (e.g., the polypeptide of SEQ ID NO:2, the
mature form of Neutrokine-alpha, the membrane-bound form of
Neutrokine-alpha, the soluble form of Neutrokine-alpha (amino acids
134 to 285 of SEQ ID NO:2), and an antigenic and/or epitope region
of Neutrokine-alpha), the ability to substantially block
Neutrokine-alpha/Neutrokine-alpha receptor binding, or the ability
to block Neutrokine-alpha mediated biological activity (e.g.,
stimulation of B cell proliferation and immunoglobulin production).
Optionally, the antibodies of the invention will bind to the same
epitope as at least one of the antibodies specifically referred to
herein. Such epitope binding can be routinely determined using
assays known in the art.
[0524] Thus, in one embodiment, the invention provides antibodies
that specifically bind the membrane-bound form of Neutrokine-alpha
and do not bind the soluble form of Neutrokine-alpha. These
antibodies have uses which include, but are not limited to, as
diagnostic probes for identifying and/or isolating monocyte
lineages expressing the membrane bound form of Neutrokine-alpha.
For example, the expression of the membrane bound form of
Neutrokine-alpha is elevated on activated monocytes, and
accordingly, antibodies encompassed by the invention may be used to
detect and/or quantitate levels of activated monocytes.
Additionally, antibodies that only bind the membrane bound form of
Neutrokine-alpha may be used to target toxins to neoplastic,
preneoplastic, and/or other cells that express the membrane bound
form of Neutrokine-alpha (e.g., monocytes and dendritic cells).
[0525] In another embodiment, antibodies of the invention
specifically bind only the soluble form of Neutrokine-alpha (amino
acids 134 to 285 of SEQ ID NO:2). These antibodies have uses which
include, but are not limited to, uses such as diagnostic probes for
assaying soluble Neutrokine-alpha in biological samples, and as
therapeutic agents that target toxins to cells expressing
Neutrokine-alpha receptors (e.g., B cells), and/or to reduce or
block in vitro or in vivo Neutrokine-alpha mediated biological
activity (e.g., stimulation of B cell proliferation and/or
immunoglobulin production).
[0526] The invention also provides for antibodies that specifically
bind both the membrane-bound and soluble form of
Neutrokine-alpha.
[0527] As described above, the invention encompasses antibodies
that inhibit or reduce the ability of Neutrokine-alpha and/or
Neutrokine-alphaSV to bind Neutrokine-alpha receptor and/or
Neutrokine-alphaSV receptor in vitro and/or in vivo. In a specific
embodiment, antibodies of the invention inhibit or reduce the
ability of Neutrokine-alpha and/or Neutrokine-alphaSV to bind
Neutrokine-alpha receptor and/or Neutrokine-alphaSV receptor in
vitro. In another nonexclusive specific embodiment, antibodies of
the invention inhibit or reduce the ability of Neutrokine-alpha
and/or Neutrokine-alphaSV to bind bind Neutrokine-alpha receptor
and/or Neutrokine-alphaSV receptor in vivo. Such inhibition can be
assayed using techniques described herein or otherwise known in the
art.
[0528] The invention also encompasses, antibodies that bind
specifically to Neutrokine-alpha and/or Neutrokine-alphaSV, but do
not inhibit the ability of Neutrokine-alpha and/or
Neutrokine-alphaSV to bind Neutrokine-alpha receptor and/or
Neutrokine-alphaSV receptor in vitro and/or in vivo. In a specific
embodiment, antibodies of the invention do not inhibit or reduce
the ability of Neutrokine-alpha and/or Neutrokine-alphaSV to bind
Neutrokine-alpha receptor and/or Neutrokine-alphaSV receptor in
vitro. In another nonexclusive specific embodiment, antibodies of
the invention do not inhibit or reduce the ability of
Neutrokine-alpha and/or Neutrokine-alphaSV to bind Neutrokine-alpha
receptor and/or Neutrokine-alphaSV receptor in vivo.
[0529] As described above, the invention encompasses antibodies
that inhibit or reduce a Neutrokine-alpha and/or
Neutrokine-alphaSV-mediated biological activity in vitro and/or in
vivo. In a specific embodiment, antibodies of the invention inhibit
or reduce Neutrokine-alpha- and/or Neutrokine-alphaSV-mediated B
cell proliferation in vitro. Such inhibition can be assayed by
routinely modifying B cell proliferation assays described herein or
otherwise known in the art. In another nonexclusive specific
embodiment, antibodies of the invention inhibit or reduce
Neutrokine-alpha- and/or Neutrokine-alphaSV-mediated B cell
proliferation in vivo. In a specific embodiment, the antibody of
the invention is 15C10, or a humanized form thereof. In another
preferred specific embodiment, the antibody is 16C9, or a humanized
form thereof. Thus, in specific embodiments of the invention, a
16C9 and/or 15C10 antibody, or humanized forms thereof, are used to
bind soluble Neutrokine-alpha and/or Neutrokine-alphaSV and/or
agonists and/or antagonists thereof and thereby inhibit (either
partially or completely) B cell proliferation.
[0530] Alternatively, the invention also encompasses, antibodies
that bind specifically to a Neutrokine-alpha and/or
Neutrokine-alphaSV, but do not inhibit or reduce a Neutrokine-alpha
and/or Neutrokine-alphaSV-mediated biological activity in vitro
and/or in vivo (e.g., stimulation of B cell proliferation). In a
specific embodiment, antibodies of the invention do not inhibit or
reduce a Neutrokine-alpha and/or Neutrokine-alphaSV-mediated
biological activity in vitro. In another non-exclusive embodiment,
antibodies of the invention do not inhibit or reduce a
Neutrokine-alpha and/or Neutrokine-alphaSV mediated biological
activity in vivo. In a specific embodiment, the antibody of the
invention is 9B6, or a humanized form thereof.
[0531] As described above, the invention encompasses antibodies
that specifically bind to the same epitope as at least one of the
antibodies specifically referred to herein, in vitro and/or in
vivo.
[0532] In a specific embodiment, the antibodies of the invention
specifically bind to an amino acid sequence contained in amino acid
residues from about Ser-171 to about Phe-194 of SEQ ID NO:2, in
vitro. In another specific, non-exclusive embodiment, the
antibodies of the invention specifically bind to an amino acid
sequence contained in amino acid residues from about Ser-171 to
about Phe-194 of SEQ ID NO:2, in vivo. In another specific,
non-exclusive embodiment, the antibodies of the invention
specifically bind to an amino acid sequence contained in amino acid
residues from Lys-173 to Lys-188 of SEQ ID NO:2, in vitro. In
another specific, non-exclusive embodiment, the antibodies of the
invention specifically bind to an amino acid sequence contained in
amino acid residues from Lys-173 to Lys-188 of SEQ ID NO:2, in
vivo.
[0533] In an additional specific embodiment, the antibodies of the
invention specifically bind to an amino acid sequence contained in
amino acid residues from about Glu-223 to about Tyr-246 of SEQ ID
NO:2, in vitro. In another specific, non-exclusive embodiment, the
antibodies of the invention specifically bind to an amino acid
sequence contained in amino acid residues from about Glu-223 to
about Tyr-246 of SEQ ID NO:2, in vivo. In another specific,
non-exclusive embodiment, the antibodies of the invention
specifically bind to an amino acid sequence contained in amino acid
residues from Val-227 to Asn-242 of SEQ ID NO:2, in vitro. In
another specific, non-exclusive embodiment, the antibodies of the
invention specifically bind to an amino acid sequence contained in
amino acid residues from Val-227 to Asn-242 of SEQ ID NO:2, in
vivo. In another specific, non-exclusive embodiment, the antibodies
of the invention specifically bind to an amino acid sequence
contained in amino acid residues from Phe-230 to Cys-245 of SEQ ID
NO:2, in vitro. In another specific, non-exclusive embodiment, the
antibodies of the invention specifically bind to an amino acid
sequence contained in amino acid residues from Phe-230 to Cys-245
of SEQ ID NO:2, in vivo.
[0534] The invention also provides antibodies that competitively
inhibit the binding of the 9B6 monoclonal antibody produced by the
hybridoma deposited as PTA-1159 to a polypeptide of the invention,
preferably the polypeptide of SEQ ID NO:2, more preferable to a
polypeptide having the amino acid sequence of residues Ser-171 to
Phe-194 of SEQ ID NO:2. Competitive inhibition can be determined by
any method known in the art, for example, using the competitive
binding assays described herein. In preferred embodiments, the
antibody competitively inhibits the binding of 9B6 monoclonal
antibody by at least 95%, at least 90%, at least 85%, at least 80%,
at least 75%, at least 70%, at least 60%, at least 50%, to the
polypeptide of SEQ ID NO:2, or more preferable to a polypeptide
having the amino acid sequence of residues Ser-171 to Phe-194 of
SEQ ID NO:2.
[0535] The invention also provides antibodies that competitively
inhibit the binding of the 15C10 monoclonal antibody produced by
the hybridoma deposited as PTA-1158 to a polypeptide of the
invention, preferably the polypeptide of SEQ ID NO:2, more
preferable to a polypeptide having the amino acid sequence of
residues Glu-223 to Tyr-246 of SEQ ID NO:2. In preferred
embodiments, the antibody competitively inhibits the binding of
15C10 monoclonal antibody by at least 95%, at least 90%, at least
85%, at least 80%, at least 75%, at least 70%, at least 60%, at
least 50%, to the polypeptide of SEQ ID NO:2, or more preferable to
a polypeptide having the amino acid sequence of residues Glu-223 to
Tyr-246 of SEQ ID NO:2.
[0536] Additional embodiments of the invention are directed to the
9B6 antibody and to the hybridoma cell line expressing this
antibody. A hybridoma cell line expressing Antibody 9B6 was
deposited with the ATCC on Jan. 7, 2000 and has been assigned ATCC
Deposit No. PTA-1159. In a preferred embodiment, antibody 9B6 is
humanized.
[0537] Additional embodiments of the invention are directed to the
15C10 antibody and to the hybridoma cell line expressing this
antibody. A hybridoma cell line expressing Antibody 15C10 was
deposited with the ATCC on Jan. 7, 2000 and has been assigned ATCC
Deposit No. PTA-1158. In a preferred embodiment, antibody 15C10 is
humanized.
[0538] In a specific embodiment, the specific antibodies described
above are humanized using techniques described herein or otherwise
known in the art and then used as therapeutics as described
herein.
[0539] In another specific embodiment, any of the antibodies listed
above are used in a soluble form.
[0540] In another specific embodiment, any of the antibodies listed
above are conjugated to a toxin or a label (as described infra).
Such conjugated antibodies are used to kill a particular population
of cells or to quantitate a particular population of cells. In a
preferred embodiment, such conjugated antibodies are used to kill B
cells expressing Neutrokine-alpha receptor on their surface. In
another preferred embodiment, such conjugated antibodies are used
to quantitate B cells expressing Neutrokine-alpha receptor on their
surface. In another preferred embodiment, such conjugated
antibodies are used to kill monocyte cells expressing the
membrane-bound form of Neutrokine-alpha. In another preferred
embodiment, such conjugated antibodies are used to quantitate
monocyte cells expressing the membrane-bound form of
Neutrokine-alpha and/or Neutrokine-alphaSV. In highly preferred
embodiments, such conjugated antibodies that bind the membrane
bound form of Neutokine-alpha and/or Neutrokine-alphaSV are used to
kill Acute Mylegenous Leukemia cells, Chronic Lymphocytic leukemia
cells, Multiple Myeloma cells, Non-Hodgkin's Lymphoma cells, and
Hodgkins's lymphoma cells.
[0541] The antibodies of the invention also have uses as
therapeutics and/or prophylactics which include, but are not
limited to, in activating monocytes or blocking monocyte activation
and/or killing monocyte lineages that express the membrane bound
form of Neutrokine-alpha on their cell surfaces (e.g., to treat,
prevent, and/or diagnose myeloid leukemias, monocyte based
leukemias and lymphomas, monocytosis, monocytopenia, rheumatoid
arthritis, and other diseases or conditions associated with
activated monocytes). In a specific embodiment, the antibodies of
the invention fix complement. In other specific embodiments, as
further described herein, the antibodies of the invention (or
fragments thereof) are associated with heterologous polypeptides or
nucleic acids (e.g. toxins, such as, compounds that bind and
activate endogenous cytotoxic effector systems, and radioisotopes;
and cytotoxic prodrugs).
[0542] In another embodiment, one or more monoclonal antibodies are
produced wherein they recognize or bind Neutrokine-alpha and/or a
mutein thereof, but do not recognize or bind Neutrokine-alphaSV
and/or a mutein thereof. In a related embodiment, one or more
monoclonal antibodies are produced wherein they recognize or bind
Neutrokine-alphaSV and/or a mutein thereof, but do not recognize or
bind Neutrokine-alpha and/or a mutein thereof.
[0543] As discussed above, antibodies to the Neutrokine-alpha
and/or Neutrokine-alpha SV polypeptides of the invention can, in
turn, be utilized to generate anti-idiotype antibodies that "mimic"
the Neutrokine-alpha, using techniques well known to those skilled
in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444
(1989), and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For
example, antibodies which bind to Neutrokine-alpha and/or
Neutrokine-alpha SV and competitively inhibit the Neutrokine-alpha
and/or Neutrokine-alpha SV multimerization and/or binding to ligand
can be used to generate anti-idiotypes that "mimic" the
Neutrokine-alpha TNF multimerization and/or binding domain and, as
a consequence, bind to and neutralize Neutrokine-alpha or
Neutrokine-alpha SV and/or its ligand. Such neutralizing
anti-idiotypes or Fab fragments of such anti-idiotypes can be used
in therapeutic regimens to neutralize Neutrokine-alpha ligand. For
example, such anti-idiotypic antibodies can be used to bind
Neutrokine-alpha and/or Neutrokine-alpha SV, or to bind
Neutrokine-alpha and/or Neutrokine-alpha SV receptors on the
surface of cells of B cell lineage, and thereby block
Neutrokine-alpha and/or Neutrokine-alpha SV mediated B cell
activation, proliferation, and/or differentiation.
Immune System-Related Disorder Diagnosis
[0544] Neutrokine-alpha is expressed in kidney, lung, peripheral
leukocyte, bone marrow, T cell lymphoma, B cell lymphoma, activated
T cells, stomach cancer, smooth muscle, macrophages, and cord blood
tissue, and particularly cells of monocytic lineage. Moreover,
Neutrokine-alphaSV is expressed in primary dendritic cells.
Additionally, Neutrokine-alpha is expressed on the cell surface of
the following non-hematopoietic tumor cell lines. Colon carcinomas
HCT 116 (ATCC Accession No. CCL-247) and HT-29 (ATCC Accession No.
HTB-38); Colon adenocarcinomas Caco-2 (ATCC Accession No. HTB-37),
COLO 201 (ATCC Accession No. CCL-224), and WiDr (ATCC Accession No.
CCL-218); Breast adenocarcinoma MDA-MB-231 (ATCC Accession No.
HTB-26); Bladder squamous carcinoma SCaBER (ATCC Accession No.
HTB-3); Bladder carcinoma HT-1197 (ATCC Accession No. CRL-1473);
Kidney carcinomas A-498 (ATCC Accession No. HTB-44), Caki-1 (ATCC
Accession No. HTB-46), and Caki-2 (ATCC Accession No. HTG-47);
Kidney, Wilms tumor SK-NEP-1 (ATCC Accession No. HTB-48); and
Pancreas carcinomas Hs 766T (ATCC Accession No. HTB-134), MIA
PaCa-2 (ATCC Accession No. CRL-1420), and SU.86.86 (ATCC Accession
No. CRL-1837). For a number of immune system-related disorders,
substantially altered (increased or decreased) levels of
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression can be
detected in immune system tissue or other cells or bodily fluids
(e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken
from an individual having such a disorder, relative to a "standard"
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression level,
that is, the Neutrokine-alpha and/or Neutrokine-alphaSV expression
level in immune system tissues or bodily fluids from an individual
not having the immune system disorder. Thus, the invention provides
a diagnostic method useful during diagnosis of an immune system
disorder, which involves measuring the expression level of the gene
encoding the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
in immune system tissue or other cells or body fluid from an
individual and comparing the measured gene expression level with a
standard Neutrokine-alpha and/or Neutrokine-alphaSV gene expression
level, whereby an increase or decrease in the gene expression level
compared to the standard is indicative of an immune system disorder
or normal activation, proliferation, differentiation, and/or
death.
[0545] In particular, it is believed that certain tissues in
mammals with cancer of cells or tissue of the immune system express
significantly enhanced or reduced levels of the Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide and mRNA encoding the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide when
compared to a corresponding "standard" level. Further, it is
believed that enhanced or depressed levels of the Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide can be detected in certain
body fluids (e.g., sera, plasma, urine, and spinal fluid) or cells
or tissue from mammals with such a cancer when compared to sera
from mammals of the same species not having the cancer.
[0546] For example, as disclosed herein, Neutrokine-alpha is highly
expressed in cells of monocytic lineage. Accordingly,
polynucleotides of the invention (e.g., polynucleotide sequences
complementary to all or a portion of Neutrokine-alpha mRNA and/or
Neutrokine-alphaSV mRNA) and antibodies (and antibody fragments)
directed against the polypeptides of the invention may be used to
quantitate or qualitate concentrations of cells of monocytic
lineage (e.g., monocytic leukemia cells) expressing
Neutrokine-alpha on their cell surfaces. These antibodies
additionally have diagnostic applications in detecting
abnormalities in the level of Neutrokine-alpha gene expression, or
abnormalities in the structure and/or temporal, tissue, cellular,
or subcellular location of Neutrokine-alpha and/or
Neutrokine-alphaSV. These diagnostic assays may be performed in
vivo or in vitro, such as, for example, on blood samples, biopsy
tissue or autopsy tissue.
[0547] Additionally, as disclosed herein, Neutrokine-alpha receptor
is expressed primarily on cells of B cell lineage. Accordingly,
Neutrokine-alpha polypeptides of the invention (including labeled
Neutrokine-alpha polypeptides and Neutrokine-alpha fusion
proteins), and anti-Neutrokine-alpha antibodies (including
anti-Neutrokine-alpha antibody fragments) against the polypeptides
of the invention may be used to quantitate or qualitate
concentrations of cells of B cell lineage (e.g., B cell related
leukemias or lymphomas) expressing Neutrokine-alpha receptor on
their cell surfaces.
[0548] Neutrokine-alpha polypeptides and antibodies additionally
have diagnostic applications in detecting abnormalities in the
level of Neutrokine-alpha receptor gene expression (e.g.,
transmembrane activator and CAML interactor (TACI, GenBank
accession number AAC51790), BAFF-R (GenBank Acession Number NP
443177) and B-cell maturation antigen (BCMA, GenBank accession
number NP.sub.--001183)), or abnormalities in the structure and/or
temporal, tissue, cellular, or subcellular location of
Neutrokine-alpha receptor and/or diagnosing activity/defects in
signalling pathways associated with Neutrokine-alpha. These
diagnostic assays may be performed in vivo or in vitro, such as,
for example, on blood samples or biopsy tissue using techniques
described herein or otherwise known in the art.
[0549] In one embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention are used to treat, prevent, diagnose,
or prognose an individual having an immunodeficiency.
[0550] Immunodeficiencies that may be treated, prevented,
diagnosed, and/or prognosed with the Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention, include, but are not limited to one
or more immunodeficiencies selected from: severe combined
immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine
deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia
(XLA), Bruton's disease, congenital agammaglobulinemia, X-linked
infantile agammaglobulinemia, acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), chronic granulomatous disease,
Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with
hyper IgM, non X-linked immunodeficiency with hyper IgM, selective
IgA deficiency, IgG subclass deficiency (with or without IgA
deficiency), antibody deficiency with normal or elevated Igs,
immunodeficiency with thymoma, Ig heavy chain deletions, kappa
chain deficiency, B cell lymphoproliferative disorder (BLPD),
selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss
type), reticular dysgenesis, neonatal neutropenia, severe
congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia
with immunodeficiency, ataxia-telangiectasia, short limbed
dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.
[0551] According to this embodiment, an individual having an
immunodeficiency expresses aberrantly low levels of
Neutrokine-alpha and/or Neutrokine-alpha SV when compared to an
individual not having an immunodeficiency. Any means described
herein or otherwise known in the art may be applied to detect
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or
polypeptides of the invention (e.g., FACS analysis or ELISA
detection of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the invention and hybridization or PCR detection of
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides of the
invention) and to determine the expression profile of
Neutrokine-alpha and/or Neutrokine-alphaSV, polynucleotides and/or
polypeptides of the invention in a biological sample.
[0552] A biological sample of a person afflicted with an
immunodeficiency is characterized by low levels of expression of
Neutrokine-alpha and/or Neutrokine-alphaSV when compared to that
observed in individuals not having an immunodeficiency. Thus,
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides and/or
polypeptides of the invention, and/or agonists or antagonists
thereof, may be used according to the methods of the invention in
the diagnosis and/or prognosis of an immunodeficiency. For example,
a biological sample obtained from a person suspected of being
afflicted with an immunodeficiency ("the subject") may be analyzed
for the relative expression level(s) of Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides and/or polypeptides of the
invention. The expression level(s) of one or more of these
molecules of the invention is (are) then compared to the expression
level(s) of the same molecules of the invention as expressed in a
person known not to be afflicted with an immunodeficiency. A
significant difference in expression level(s) of Neutrokine-alpha,
and/or Neutrokine-alphaSV, polynucleotides and/or polypeptides of
the invention, and/or agonists and/or antagonists thereof, between
samples obtained from the subject and the control suggests that the
subject is afflicted with an immunodeficiency.
[0553] In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention are used to treat, diagnose and/or
prognose an individual having common variable immunodeficiency
disease ("CVID"; also known as "acquired agammaglobulinemia" and
"acquired hypogammaglobulinemia") or a subset of this disease.
According to this embodiment, an individual having CVID or a subset
of individuals having CVID expresses aberrant levels of
Neutrokine-alpha and/or Neutrokine-alpha Receptor on their B cells
and/or monocytes, when compared to individuals not having CVID. Any
means described herein or otherwise known in the art may be applied
to detect Neutrokine-alpha polynucleotides or polypeptides of the
invention and/or Neutrokine-alpha Receptor polypeptides (e.g., FACS
analysis or ELISA detection of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention and hybridization
or PCR detection of Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides of the invention) and to determine differentially
the expression profile of Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or Neutrokine-alpha receptor polypeptides in a sample
containing at least monocyte cells or some component thereof (e.g.,
RNA) as compared to a sample containing at least B cells or a
component thereof (e.g., RNA). In the instance where a sample
containing at least monocyte cells or some component thereof (e.g.,
RNA) is determined to reflect Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotide or polypeptide expression and a
sample containing at least B cells or a component thereof (e.g.,
RNA) is determined to reflect less than normal levels of
Neutrokine-alpha receptor polynucleotide or polypeptide expression,
the samples may be correlated with the occurrence of CVID (i.e.,
"acquired agammaglobulinemia" or "acquired
hypogammaglobulinemia").
[0554] A subset of persons afflicted with CVID are characterized by
high levels of expression of both Neutrokine-alpha and the
Neutrokine-alpha receptor ("NAR") in peripheral or circulating B
cells when compared to that observed in individuals not having
CVID. In contrast, persons who are not afflicted with CVID are
typically characterized by low levels of Neutrokine-alpha
expression and high levels of NAR expression in peripheral or
circulating B cells. Thus, Neutrokine-alpha, Neutrokine-alphaSV
polypeptides, and/or NAR polypeptides, polynucleotides and/or
polypeptides of the invention, and/or agonists or antagonists
thereof, may be used according to the methods of the invention in
the differential diagnosis of this subset of CVID. For example, a
sample of peripheral B cells obtained from a person suspected of
being afflicted with CVID ("the subject") may be analyzed for the
relative expression level(s) of Neutrokine-alpha,
Neutrokine-alphaSV, and/or NAR polynucleotides and/or polypeptides
of the invention. The expression level(s) of one or more of these
molecules of the invention is (are) then compared to the expression
level(s) of the same molecules of the invention as expressed in a
person known not to be afflicted with CVID ("the control"). A
significant difference in expression level(s) of Neutrokine-alpha
and/or Neutrokine-alphaSV polynucleotides or polypeptides of the
invention, and/or NAR polypeptides, and/or agonists and/or
antagonists thereof, between samples obtained from the subject and
the control suggests that the subject is afflicted with this subset
of CVID.
[0555] Cunningham-Rundles and Bodian followed 248 CVID patients
over a period of 1-25 years and discovered that a number of
associated diseases or conditions appear with increased frequency
in CVID patients (Cunningham-Rundles and Bodian, J. Clin. Immunol.,
92:34-48 (1999) which is herein incorporated by reference in its
entirety.) The most important clinical events include infections,
autoimmunity, inflammatory disorders, marked by gastrointestinal
and granulomatous disease, cancer and hepatitis. Most CVID patients
are at increased risk of recurrent infections particularly of the
respiratory tract. The types of acute and recurring bacterial
infections exhibited in most patients include pneumonia, bronchitis
and sinusitis. Children with CVID have a marked increased risk of
otitis media. Additionally, blood borne infections including
sepsis, meningitis, septic arthritis, and osteomyelitis are seen
with increased frequency in these patients.
[0556] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or agonists or
antagonists thereof (e.g., anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSVantibodies) are used to diagnose, prognose,
treat, or prevent conditions associated with CVID, including, but
not limited to, conditions associated with acute and recurring
infections (e.g., pneumonia, bronchitis, sinusitis, otitis media,
sepsis, meningitis, septic arthritis, and osteomyelitis), chronic
lung disease, autoimmunity, granulomatous disease, lymphoma,
cancers (e.g., cancers of the breast, stomach, colon, mouth,
prostate, lung, vagina, ovary, skin, and melanin forming cells
(i.e. melanoma), inflammatory bowel disease (e.g., Crohn's disease,
ulcerative colitis, and ulcerative proctitis), malabsorption,
Hodgkin's disease, and Waldenstrom's macroglobulinemia.
[0557] In a specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or agonists or
antagonists thereof (e.g., anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSVantibodies) are used to diagnose, prognose,
treat, or prevent a disorder characterized by deficient serum
immunoglobulin production, recurrent infections, and/or immune
system dysfunction. Moreover, Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or agonists or
antagonists thereof (e.g., anti-Neutrokine-alpha and/or
anti-Neutrokine-alphaSV antibodies) may be used to diagnose,
prognose, treat, or prevent infections of the joints, bones, skin,
and/or parotid glands, blood-borne infections (e.g., sepsis,
meningitis, septic arthritis, and/or osteomyelitis), autoimmune
diseases (e.g., those disclosed herein), inflammatory disorders,
and malignancies, and/or any disease or disorder or condition
associated with these infections, diseases, disorders and/or
malignancies) including, but not limited to, CVID, other primary
immune deficiencies, HIV disease, CLL, recurrent bronchitis,
sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis,
meningitis, herpes zoster (e.g., severe herpes zoster), and/or
Pneumocystis carnii.
[0558] In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention are used to treat, diagnose, or
prognose an individual having an autoimmune disease or
disorder.
[0559] Autoimmune diseases or disorders that may be treated,
diagnosed, or prognosed using Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention include, but are not limited to, one
or more of the following: autoimmune hemolytic anemia, autoimmune
neonatal thrombocytopenia, idiopathic thrombocytopenia purpura,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,
dermatitis, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, glomerulonephritis (e.g,
IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary
Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes
mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis,
hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus
erythematosus, Goodpasture's syndrome, Pemphigus, Receptor
autoimmunities such as, for example, (a) Graves' Disease, (b)
Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic
anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis,
scleroderma with anti-collagen antibodies, mixed connective tissue
disease, polymyositis/dermatomyositis, pernicious anemia,
idiopathic Addison's disease, infertility, glomerulonephritis such
as primary glomerulonephritis and IgA nephropathy, bullous
pemphigoid, Sjogren's syndrome, diabetes millitus, and adrenergic
drug resistance (including adrenergic drug resistance with asthma
or cystic fibrosis), chronic active hepatitis, primary biliary
cirrhosis, other endocrine gland failure, vitiligo, vasculitis,
post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma,
inflammatory myopathies, and other inflammatory, granulamatous,
degenerative, and atrophic disorders.
[0560] According to this embodiment, an individual having an
autoimmune disease or disorder expresses aberrantly high levels of
Neutrokine-alpha, Neutrokine-alpha SV, and/or NAR when compared to
an individual not having an autoimmune disease or disorder. Any
means described herein or otherwise known in the art may be applied
to detect Neutrokine-alpha, and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention and/or NAR
polypeptides (e.g., FACS analysis or ELISA detection of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the
invention and hybridization or PCR detection of Neutrokine-alpha
and/or Neutrokine-alphaSV polynucleotides of the invention) and to
determine the expression profile of Neutrokine-alpha and/or
Neutrokine-alphaSV, polynucleotides and/or polypeptides of the
invention and/or NAR polypeptides in a biological sample.
[0561] A biological sample of persons afflicted with an autoimmune
disease or disorder is characterized by high levels of expression
of Neutrokine-alpha, Neutrokine-alphaSV, and/or NAR when compared
to that observed in individuals not having an autoimmune disease or
disorder. Thus, Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides and/or polypeptides of the invention, and/or
agonists or antagonists thereof, may be used according to the
methods of the invention in the diagnosis and/or prognosis of an
autoimmune disease or disorder. For example, a biological sample
obtained from a person suspected of being afflicted with an
autoimmune disease or disorder ("the subject") may be analyzed for
the relative expression level(s) of Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides and/or polypeptides of the
invention and/or NAR polypeptides. The expression level(s) of one
or more of these molecules of the invention is (are) then compared
to the expression level(s) of the same molecules of the invention
as expressed in a person known not to be afflicted with an
autoimmune disease or disorder. A significant difference in
expression level(s) of Neutrokine-alpha, and/or Neutrokine-alphaSV,
polynucleotides and/or polypeptides of the invention, and/or
agonists and/or antagonists thereof, and/or NAR polypeptides
between samples obtained from the subject and the control suggests
that the subject is afflicted with an autoimmune disease or
disorder.
[0562] In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention are used to treat, diagnose, or
prognose an individual having systemic lupus erythematosus or a
subset of this disease. According to this embodiment, an individual
having systemic lupus erythematosus or a subset of individuals
having systemic lupus erythematosus expresses aberrantly high
levels of Neutrokine-alpha and/or Neutrokine-alpha SV when compared
to an individual not having systemic lupus erythematosus or this
subset of systemic lupus erythematosus. Any means described herein
or otherwise known in the art may be applied to detect
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or
polypeptides of the invention (e.g., FACS analysis or ELISA
detection of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides of the invention and hybridization or PCR detection of
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides of the
invention) and to determine the expression profile of
Neutrokine-alpha and/or Neutrokine-alphaSV, polynucleotides and/or
polypeptides of the invention in a biological sample.
[0563] A biological sample of persons afflicted with systemic lupus
erythematosus is characterized by high levels of expression of
Neutrokine-alpha and/or Neutrokine-alphaSV when compared to that
observed in individuals not having systemic lupus erythematosus.
Thus, Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides
and/or polypeptides of the invention, and/or agonists or
antagonists thereof, may be used according to the methods of the
invention in the diagnosis and/or prognosis of systemic lupus
erythematosus or a subset of systemic lupus erythematosus. For
example, a biological sample obtained from a person suspected of
being afflicted with systemic lupus erythematosus ("the subject")
may be analyzed for the relative expression level(s) of
Neutrokine-alpha, and/or Neutrokine-alphaSV polynucleotides and/or
polypeptides of the invention. The expression level(s) of one or
more of these molecules of the invention is (are) then compared to
the expression level(s) of the same molecules of the invention as
expressed in a person known not to be afflicted with systemic lupus
erythematosus. A significant difference in expression level(s) of
Neutrokine-alpha, and/or Neutrokine-alphaSV, polynucleotides and/or
polypeptides of the invention, and/or agonists and/or antagonists
thereof, between samples obtained from the subject and the control
suggests that the subject is afflicted with systemic lupus
erythematosus or a subset thereof.
[0564] Furthermore, there is a direct correlation between the
severity of systemic lupus erythematosus, or a subset of this
disease, and the concentration of Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides (RNA) and/or polypeptides of the
invention. Thus, Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides, (RNA), polypeptides and/or agonists or antagonists
of the invention, may be used according to the methods of the
invention in prognosis of the severity of systemic lupus
erythematosus or a subset of systemic lupus erythematosus. For
example, a biological sample obtained from a person suspected of
being afflicted with systemic lupus erythematosus ("the subject")
may be analyzed for the relative expression level(s) of
Neutrokine-alpha, and/or Neutrokine-alphaSV polynucleotides and/or
polypeptides of the invention. The expression level(s) of one or
more of these molecules of the invention is (are) then compared to
the expression level(s) of the same molecules of the invention as
expressed in a panel of persons known to represent a range in
severities of this disease. According to this method, the match of
expression level with a characterized member of the panel indicates
the severity of the disease.
[0565] Elevated levels of soluble Neutrokine-alpha have been
observed in the serum of patients with Systemic Lupus Erythematosus
(SLE). In comparing the sera of 150 SLE patients with that of 38
control individuals, it was found that most of the SLE patients had
more than 5 ng/ml of serum Neutrokine-alpha, more than 30% of SLE
patients had levels greater than 10 ng/ml, and approximately 10% of
SLE patients had serum Neutrokine-alpha levels greater than 20
ng/ml. In contrast, the majority of normal controls had
Neutrokine-alpha levels less than 5 ng/ml, and less than 10% had
levels higher than 10 ng/ml. The elevated levels of
Neutrokine-alpha protein in sera is present in the soluble form and
has biologic activity as assayed by the ability to stimulate
anti-IgM treated B cells in vitro. SLE patients with more than 15
ng/ml serum Neutrokine-alpha were also found to have elevated
levels of anti-dsDNA antibodies compared to both normal controls
and SLE patients with less than 5 ng/ml of serum Neutrokine-alpha
(unpublished data).
[0566] In addition the serum of two subgroups of patients which
were positive for anti-nuclear antibodies (ANA+) but did not meet
the formal requirements of the American College of Rheumatology
(ACR) for classification of SLE were analyzed for Neutrokine-alpha
levels. The first subgroup of sera was ANA+sera that came from
patients who did not present with the clinical impression of SLE.
This group had only slightly elevated levels of Neutrokine-alpha
(.about.9 ng/ml Neutrokine-alpha). The second subgroup however,
which was ANA+sera from patients who presented with the clinical
impression of SLE, had significantly increased Neutrokine-alpha
levels (.about.15 ng/ml). These results suggest that an elevated
level of Neutrokine-alpha precedes the formal fulfillment of the
ACR criteria. The ACR criteria are described in Tan, E. M., et al,
Arthritis and Rheumatism 25:1271-1277 (1982).
[0567] In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention are used to treat, diagnose, or
prognose an individual having rheumatoid arthritis or a subset of
this disease. According to this embodiment, an individual having
rheumatoid arthritis or a subset of individuals having rheumatoid
arthritis expresses aberrantly high levels of Neutrokine-alpha
and/or Neutrokine-alpha SV when compared to an individual not
having rheumatoid arthritis or this subset of rheumatoid arthritis.
Any means described herein or otherwise known in the art may be
applied to detect Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention (e.g., FACS
analysis or ELISA detection of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention and hybridization
or PCR detection of Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides of the invention) and to determine the expression
profile of Neutrokine-alpha and/or Neutrokine-alphaSV,
polynucleotides and/or polypeptides of the invention in a
biological sample.
[0568] A biological sample of persons afflicted with rheumatoid
arthritis is characterized by high levels of expression of
Neutrokine-alpha and/or Neutrokine-alphaSV when compared to that
observed in individuals not having rheumatoid arthritis. Thus,
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides and/or
polypeptides of the invention, and/or agonists or antagonists
thereof, may be used according to the methods of the invention in
the diagnosis and/or prognosis of rheumatoid arthritis or a subset
of rheumatoid arthritis. For example, a biological sample obtained
from a person suspected of being afflicted with rheumatoid
arthritis ("the subject") may be analyzed for the relative
expression level(s) of Neutrokine-alpha, and/or Neutrokine-alphaSV
polynucleotides and/or polypeptides of the invention. The
expression level(s) of one or more of these molecules of the
invention is (are) then compared to the expression level(s) of the
same molecules of the invention as expressed in a person known not
to be afflicted with rheumatoid arthritis. A significant difference
in expression level(s) of Neutrokine-alpha, and/or
Neutrokine-alphaSV, polynucleotides and/or polypeptides of the
invention, and/or agonists and/or antagonists thereof, between
samples obtained from the subject and the control suggests that the
subject is afflicted with rheumatoid arthritis or a subset
thereof.
[0569] In other specific embodiments, antibodies of the invention
which specifically bind to Neutrokine-alpha and/or
Neutrokine-alphaSV can be used for diagnostic purposes to detect,
diagnose, prognosep or monitor Sjogren's Syndrome or conditions
associated therewith. The invention provides for the detection of
aberrant expression of Neutrokine-alpha and/or Neutrokine-alphaSV
comprising: (a) assaying the expression of Neutrokine-alpha and/or
Neutrokine-alphaSV in a biological sample of an individual using
one or more antibodies of the invention that immunospecifically
binds to Neutrokine-alpha and/or Neutrokine-alphaSV; and (b)
comparing the level of Neutrokine-alpha and/or Neutrokine-alphaSV
with a standard level of Neutrokine-alpha and/or
Neutrokine-alphaSV, e.g., in normal biological samples, whereby an
increase in the assayed level of Neutrokine-alpha and/or
Neutrokine-alphaSV compared to the standard level of
Neutrokine-alpha and/or Neutrokine-alphaSV is indicative of
Sjogren's Syndrome.
[0570] In other specific embodiments, antibodies of the invention
which specifically bind to Neutrokine-alpha and/or
Neutrokine-alphaSV can be used for diagnostic purposes to detect,
diagnose, prognose, or monitor HIV infection or conditions
associated therewith (e.g., AIDS) The invention provides for the
detection of aberrant expression of Neutrokine-alpha and/or
Neutrokine-alphaSV comprising: (a) assaying the expression of
Neutrokine-alpha and/or Neutrokine-alphaSV in a biological sample
of an individual using one or more antibodies of the invention that
immunospecifically binds to Neutrokine-alpha and/or
Neutrokine-alphaSV; and (b) comparing the level of Neutrokine-alpha
and/or Neutrokine-alphaSV with a standard level of Neutrokine-alpha
and/or Neutrokine-alphaSV, e.g., in normal biological samples,
whereby an increase in the assayed level of Neutrokine-alpha and/or
Neutrokine-alphaSV compared to the standard level of
Neutrokine-alpha and/or Neutrokine-alphaSV is indicative of HIV
infection.
[0571] In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides or
Neutrokine-alpha and/or Neutrokine-alphaSV agonists or antagonists
(e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) of the invention are used to treat, diagnose, or
prognose an individual with an immune-based rheumatologic diseases,
including but not limited to, SLE, rheumatoid arthritis, CREST
syndrome (a variant of scleroderma characterized by calcinosis,
Raynaud's phenomenon, esophageal motility disorders, sclerodactyl),
and telangiectasia.), seronegative spondyloarthropathy (SpA),
polymyositis/dermatomyositis, microscopic polyangiitis, hepatitis
C-associated arthritis, Takayasu's arteritis, and undifferentiated
connective tissue disorder. According to this embodiment, an
individual having an immune-based rheumatologic disease or a subset
of individuals having a particular immune-based rheumatologic
disease expresses aberrantly high levels of Neutrokine-alpha and/or
Neutrokine-alpha SV when compared to an individual not having the
particular immune-based rheumatologic disease or this subset of
individuals having the particular immune-based rheumatologic
disease. Any means described herein or otherwise known in the art
may be applied to detect Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention (e.g., FACS
analysis or ELISA detection of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention and hybridization
or PCR detection of Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides of the invention) and to determine the expression
profile of Neutrokine-alpha and/or Neutrokine-alphaSV,
polynucleotides and/or polypeptides of the invention in a
biological sample.
[0572] A biological sample of persons afflicted with an
immune-based rheumatologic disease is characterized by high levels
of expression of Neutrokine-alpha and/or Neutrokine-alphaSV when
compared to that observed in individuals not having an immune-based
rheumatologic disease. Thus, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides and/or polypeptides of the
invention, and/or agonists or antagonists thereof, may be used
according to the methods of the invention in the diagnosis and/or
prognosis of an immune-based rheumatologic disease. For example, a
biological sample obtained from a person suspected of being
afflicted with an immune-based rheumatologic disease ("the
subject") may be analyzed for the relative expression level(s) of
Neutrokine-alpha, and/or Neutrokine-alphaSV polynucleotides and/or
polypeptides of the invention. The expression level(s) of one or
more of these molecules of the invention is (are) then compared to
the expression level(s) of the same molecules of the invention as
expressed in a person known not to be afflicted with an
immune-based rheumatologic disease. A significant difference in
expression level(s) of Neutrokine-alpha, and/or Neutrokine-alphaSV,
polynucleotides and/or polypeptides of the invention, and/or
agonists and/or antagonists thereof, between samples obtained from
the subject and the control suggests that the subject is afflicted
with an immune-based rheumatologic disease.
[0573] It has been observed, that serum Neutrokina-alpha levels
inversely correlate with nephrotic-range proteinuria (>3 gm
proteinuria in a 24 hour urine collection) using a sample of 71 SLE
patients (p=0.019). Proteinuria was determined in 71 SLE patients
within one month of phlebotomy for serum Neutrokine-alpha
determination. Serum Neutrokine-alpha was classified as low,
normal, or high based on the 5.sup.th through 95.sup.th percentiles
for normal controls. Nephrotic-range proteinuria was inversely
correlated with serum Neutrokine-alpha levels. Thus, in specific
embodiments, serum levels of Neutrokine-alpha in individuals
diagnosed with an immune based rheumatologic disease (e.g., SLE,
rheumatoid arthritis, CREST syndrome (a variant of scleroderma
characterized by calcinosis, Raynaud's phenomenon, esophageal
motility disorders, sclerodactyl), and telangiectasia.),
seronegative spondyloarthropathy (SpA),
polymyositis/dermatomyositis, microscopic polyangiitis, hepatitis
C-associated arthritis, Takayasu's arteritis, and undifferentiated
connective tissue disorder) may be used to determine, diagnose,
progonose, or monitor the severity of certain aspects or symptoms
of the disease, such as nephrotic-range proteinuria.
[0574] Thus, the invention provides a diagnostic method useful
during diagnosis of a immune system disorder, including cancers of
this system, and immunodeficiencies and/or autoimmune diseases
which involves measuring the expression level of the gene encoding
the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide in
immune system tissue or other cells or body fluid from an
individual and comparing the measured gene expression level with a
standard Neutrokine-alpha and/or Neutrokine-alphaSV gene expression
level, whereby an increase or decrease in the gene expression level
compared to the standard is indicative of an immune system
disorder.
[0575] Levels of soluble Neutrokine-alpha in the serum of patients
with follicular non-Hodgkin's lymphoma are elevated elevated
compared to levels of soluble neutrokine-alpha in the sera of
healthy individuals. Thus, in a specific embodiment, the invention
provides method of diagnosing non-Hodgkin's lymphoma which involves
measuring the expression level of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides and/or polynucleotides in immune
system tissue or other cells or body fluid from an individual and
comparing the measured gene expression level with a standard
Neutrokine-alpha and/or Neutrokine-alphaSV gene expression level,
whereby an increase in the gene expression level compared to the
standard is indicative of non-Hodgkin's Lymphoma. Other forms of
Non-Hodgkin's lymphoma which may be diagnosed according to the
above method include, but are not limited to, mantle cell lymphoma,
diffuse large cell lymphoma, chronic lymphocytic leukemia, small
lymphocytic leukemia, and marginal zone lymphoma.
[0576] Where a diagnosis of a disorder in the immune system,
including, but not limited to, diagnosis of a tumor, diagnosis of
an immunodeficiency, and/or diagnosis of an autoimmune disease, has
already been made according to conventional methods, the present
invention is useful as a prognostic indicator, whereby patients
exhibiting enhanced or depressed Neutrokine-alpha and/or
Neutrokine-alphaSV gene expression will experience a worse clinical
outcome relative to patients expressing the gene at a level nearer
the standard level.
[0577] By analyzing or determining the expression level of the gene
encoding the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
is intended qualitatively or quantitatively measuring or estimating
the level of the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide or the level of the mRNA encoding the Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide in a first biological sample
either directly (e.g., by determining or estimating absolute
protein level or mRNA level) or relatively (e.g., by comparing to
the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide level or
mRNA level in a second biological sample). Preferably, the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide level or
mRNA level in the first biological sample is measured or estimated
and compared to a standard Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide level or mRNA level, the standard
being taken from a second biological sample obtained from an
individual not having the disorder or being determined by averaging
levels from a population of individuals not having a disorder of
the immune system. As will be appreciated in the art, once a
standard Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
level or mRNA level is known, it can be used repeatedly as a
standard for comparison.
[0578] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide or mRNA. As indicated, biological
samples include body fluids (such as sera, plasma, urine, synovial
fluid and spinal fluid) which contain free extracellular domains of
the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide, immune
system tissue, and other tissue sources found to express complete
or free extracellular domain of the Neutrokine-alpha and/or
Neutrokine-alphaSV or a Neutrokine-alpha and/or Neutrokine-alphaSV
receptor. Methods for obtaining tissue biopsies and body fluids
from mammals are well known in the art. Where the biological sample
is to include mRNA, a tissue biopsy is the preferred source.
[0579] The compounds of the present invention are useful for
diagnosis, prognosis, or treatment of various immune system-related
disorders in mammals, preferably humans. Such disorders include,
but are not limited to tumors (e.g., B cell and monocytic cell
leukemias and lymphomas, See Example) and tumor metastasis,
infections by bacteria, viruses and other parasites,
immunodeficiencies, inflammatory diseases, lymphadenopathy,
autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus
erythamatosus, Sjogren syndrome, mixed connective tissue disease,
and inflammatory myopathies), and graft versus host disease.
[0580] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels
of mRNA encoding the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide are then assayed using any appropriate method. These
include Northern blot analysis, S1 nuclease mapping, the polymerase
chain reaction (PCR), reverse transcription in combination with the
polymerase chain reaction (RT-PCR), and reverse transcription in
combination with the ligase chain reaction (RT-LCR).
[0581] Assaying Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide levels in a biological sample can occur using
antibody-based techniques. For example, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide expression in tissues can be studied
with classical immunohistological methods (Jalkanen, M., et al., J.
Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
and fluorescence activated cell sorting (FACS). Suitable antibody
assay labels are known in the art and include enzyme labels, (e.g.,
glucose oxidase, alkaline phosphatase and horse radish peroxidase)
and radioisotopes, such as iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.20Ti), gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.103
Pd), molybdenum (.sup.99Mo), xenon (.sup.33Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149 Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru;
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[0582] Techniques known in the art may be applied to label
antibodies of the invention. Such techniques include, but are not
limited to, the use of bifunctional conjugating agents (see e.g.,
U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;
5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560; and 5,808,003; the contents of each of which are hereby
incorporated by reference in its entirety) and direct coupling
reactions (e.g., Bolton-Hunter, Chloramine-T reaction, and
Iodogen.RTM.-based labelling).
[0583] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the
Neutrokine-alpha gene (such as, for example, cells of monocytic
lineage) or cells or tissue which are known, or suspected, to
express the Neutrokine-alpha receptor gene (such as, for example,
cells of B cell lineage and the spleen). The protein isolation
methods employed herein may, for example, be such as those
described in Harlow and Lane (Harlow, E. and Lane, D., 1988,
"Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.), which is incorporated herein by
reference in its entirety. The isolated cells can be derived from
cell culture or from a patient. The analysis of cells taken from
culture may be a necessary step in the assessment of cells that
could be used as part of a cell-based gene therapy technique or,
alternatively, to test the effect of compounds on the expression of
the Neutrokine-alpha gene or Neutrokine-alpha receptor gene.
[0584] For example, antibodies, or fragments of antibodies, such as
those described herein, may be used to quantitatively or
qualitatively detect the presence of Neutrokine-alpha gene products
or conserved variants or peptide fragments thereof. This can be
accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody coupled with light
microscopic, flow cytometric, or fluorimetric detection.
[0585] The antibodies (or fragments thereof) or Neutrokine-alpha
polypeptides or polypeptides of the present invention may,
additionally, be employed histologically, as in immunofluorescence,
immunoelectron microscopy or non-immunological assays, for in situ
detection of Neutrokine-alpha gene products or conserved variants
or peptide fragments thereof, or for Neutrokine-alpha binding to
Neutrokine-alpha receptor. In situ detection may be accomplished by
removing a histological specimen from a patient, and applying
thereto a labeled antibody or Neutrokine-alpha polypeptide of the
present invention. The antibody (or fragment) or Neutrokine-alpha
polypeptide is preferably applied by overlaying the labeled
antibody (or fragment) onto a biological sample. Through the use of
such a procedure, it is possible to determine not only the presence
of the Neutrokine-alpha gene product, or conserved variants or
peptide fragments, or Neutrokine-alpha polypeptide binding, but
also its distribution in the examined tissue. Using the present
invention, those of ordinary skill will readily perceive that any
of a wide variety of histological methods (such as staining
procedures) can be modified in order to achieve such in situ
detection.
[0586] Immunoassays and non-immunoassays for Neutrokine-alpha gene
products or conserved variants or peptide fragments thereof will
typically comprise incubating a sample, such as a biological fluid,
a tissue extract, freshly harvested cells, or lysates of cells
which have been incubated in cell culture, in the presence of a
detectably labeled antibody capable of identifying Neutrokine-alpha
gene products or conserved variants or peptide fragments thereof,
and detecting the bound antibody by any of a number of techniques
well-known in the art.
[0587] Immunoassays and non-immunoassays for Neutrokine-alpha
receptor gene products or conserved variants or peptide fragments
thereof will typically comprise incubating a sample, such as a
biological fluid, a tissue extract, freshly harvested cells, or
lysates of cells which have been incubated in cell culture, in the
presence of a detectable or labeled Neutrokine-alpha polypeptide
capable of identifying Neutrokine-alpha receptor gene products or
conserved variants or peptide fragments thereof, and detecting the
bound Neutrokine-alpha polypeptide by any of a number of techniques
well-known in the art.
[0588] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled anti-Neutrokine-alpha antibody or detectable
Neutrokine-alpha polypeptide. The solid phase support may then be
washed with the buffer a second time to remove unbound antibody or
polypeptide. Optionally the antibody is subsequently labeled. The
amount of bound label on solid support may then be detected by
conventional means.
[0589] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0590] The binding activity of a given lot of anti-Neutrokine-alpha
antibody or Neutrokine-alpha polypeptide may be determined
according to well-known methods. Those skilled in the art will be
able to determine operative and optimal assay conditions for each
determination by employing routine experimentation.
[0591] In addition to assaying Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide levels or polynucleotide levels in a
biological sample obtained from an individual, Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides or polynucleotides can also
be detected in vivo by imaging. For example, in one embodiment of
the invention, Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide and/or anti-Neutrokine-alpha antibody is used to image
B cell lymphomas. In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides and/or anti-Neutrokine-alpha
antibodies and/or Neutrokine-alpha polynucleotides of the invention
(e.g., polynucleotides complementary to all or a portion of
Neutrokine-alpha and/or Neutrokine-alphaSV mRNA) is used to image
lymphomas (e.g., monocyte and B cell lymphomas).
[0592] Antibody labels or markers for in vivo imaging of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide include
those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the antibody by labeling of
nutrients for the relevant hybridoma. Where in vivo imaging is used
to detect enhanced levels of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide for diagnosis in humans, it may be
preferable to use human antibodies or "humanized" chimeric
monoclonal antibodies. Such antibodies can be produced using
techniques described herein or otherwise known in the art. For
example methods for producing chimeric antibodies are known in the
art. See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).
[0593] Additionally, any Neutrokine-alpha polypeptide whose
presence can be detected, can be administered. For example,
Neutrokine-alpha polypeptides labeled with a radio-opaque or other
appropriate compound can be administered and visualized in vivo, as
discussed, above for labeled antibodies. Further such
Neutrokine-alpha polypeptides can be utilized for in vitro
diagnostic procedures.
[0594] A Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide-specific antibody or antibody fragment which has been
labeled with an appropriate detectable imaging moiety, such as a
radioisotope (for example, iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.201Ti), gallium (.sup.68 Ga, .sup.67Ga), palladium (.sup.103
Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149 Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru), a
radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined
for immune system disorder. It will be understood in the art that
the size of the subject and the imaging system used will determine
the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the
quantity of radioactivity injected will normally range from about 5
to 20 millicuries of .sup.99mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which contain Neutrokine-alpha protein. In vivo tumor imaging
is described in S. W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor
Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
[0595] With respect to antibodies, one of the ways in which the
anti-Neutrokine-alpha antibody can be detectably labeled is by
linking the same to an enzyme and using the linked product in an
enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked
Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7,
Microbiological Associates Quarterly Publication, Walkersville,
Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J.
E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980,
Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et
al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The
enzyme which is bound to the antibody will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual means.
Enzymes which can be used to detectably label the antibody include,
but are not limited to, malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. Additionally, the detection can be
accomplished by colorimetric methods which employ a chromogenic
substrate for the enzyme. Detection may also be accomplished by
visual comparison of the extent of enzymatic reaction of a
substrate in comparison with similarly prepared standards.
[0596] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect
Neutrokine-alpha through the use of a radioimmunoassay (RIA) (see,
for example, Weintraub, B., Principles of Radioimmunoassays,
Seventh Training Course on Radioligand Assay Techniques, The
Endocrine Society, March, 1986, which is incorporated by reference
herein). The radioactive isotope can be detected by means
including, but not limited to, a gamma counter, a scintillation
counter, or autoradiography.
[0597] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave-length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and
fluorescamine.
[0598] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0599] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0600] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling include, but are not limited to,
luciferin, luciferase and acquorin.
Treatment of Immune System-Related Disorders
[0601] As noted above, Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides and polypeptides, and anti-Neutrokine-alpha
antibodies, are useful for diagnosis of conditions involving
abnormally high or low expression of Neutrokine-alpha and/or
Neutrokine-alphaSV activities. Given the cells and tissues where
Neutrokine-alpha and/or Neutrokine-alphaSV is expressed as well as
the activities modulated by Neutrokine-alpha and/or
Neutrokine-alphaSV, it is readily apparent that a substantially
altered (increased or decreased) level of expression of
Neutrokine-alpha and/or Neutrokine-alphaSV in an individual
compared to the standard or "normal" level produces pathological
conditions related to the bodily system(s) in which
Neutrokine-alpha and/or Neutrokine-alphaSV is expressed and/or is
active.
[0602] It will also be appreciated by one of ordinary skill that,
since the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides
of the invention are members of the TNF family, the extracellular
domains of the respective proteins may be released in soluble form
from the cells which express Neutrokine-alpha and/or
Neutrokine-alphaSV by proteolytic cleavage and therefore, when
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
(particularly a soluble form of the respective extracellular
domains) is added from an exogenous source to cells, tissues or the
body of an individual, the polypeptide will exert its modulating
activities on any of its target cells of that individual. Also,
cells expressing this type II transmembrane protein may be added to
cells, tissues or the body of an individual whereby the added cells
will bind to cells expressing receptor for Neutrokine-alpha and/or
Neutrokine-alphaSV whereby the cells expressing Neutrokine-alpha
and/or Neutrokine-alphaSV can cause responses (e.g., proliferation
or cytotoxicity) in the receptor-bearing target cells.
[0603] In one embodiment, the invention provides a method of
delivering compositions containing the polypeptides of the
invention (e.g., compositions containing Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or anti-Neutrokine-alpha and/or
anti-Neutrokine-alphaSV antibodies associated with heterologous
polypeptides, heterologous nucleic acids, toxins, or prodrugs) to
targeted cells, such as, for example, B cells expressing
Neutrokine-alpha and/or Neutrokine-alphaSV receptor, or monocytes
expressing the cell surface bound form of Neutrokine-alpha and/or
Neutrokine-alphaSV. Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides or anti-Neutrokine-alpha and/or
anti-Neutrokine-alphaSV antibodies of the invention may be
associated with heterologous polypeptides, heterologous nucleic
acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic
and/or covalent interactions.
[0604] In one embodiment, the invention provides a method for the
specific delivery of compositions of the invention to cells by
administering polypeptides of the invention (e.g., Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides or anti-Neutrokine-alpha
and/or anti-Neutrokine-alphaSV antibodies) that are associated with
heterologous polypeptides or nucleic acids. In one example, the
invention provides a method for delivering a therapeutic protein
into the targeted cell. In another example, the invention provides
a method for delivering a single stranded nucleic acid (e.g.,
antisense or ribozymes) or double stranded nucleic acid (e.g., DNA
that can integrate into the cell's genome or replicate episomally
and that can be transcribed) into the targeted cell.
[0605] In another embodiment, the invention provides for a method
of killing cells of hematopoietic origin, comprising, or
alternatively consisting of, contacting Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides with cells of hematopoietic origin.
In specific embodiments, the method of killing cells of
hematopoietic origin, comprises, or alternatively consists of,
administering to an animal in which such killing is desired, a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide in an amount
effective to kill cells of hematopoietic origin. Cells of
hematopoietic origin include, but are not limited to, lymphocytes
(e.g., B cells and T cells), monocytes, macrophages, dendritic
cells, polymorphonuclear leukocytes (e.g., basophils, eosinophils,
neutrophils), mast cells, platelets, erythrocytes and progenitor
cells of these lineages. Cells of hematopoietic origin include, but
are not limited to, healthy and diseased cell as found present in
an animal, preferably a mammal and most preferably a human, or as
isolated from an animal, transformed cells, cell lines derived from
the above listed cell types, and cell cultures derived from the
above listed cell types. Cells of hematopoietic origin may be found
or isolated in, for example, resting, activated or anergic
states.
[0606] In another embodiment, the invention provides a method for
the specific destruction (i.e., killing) of cells (e.g., the
destruction of tumor cells) by administering Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides or Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide conjugates of the invention (e.g.,
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides conjugated
with radioisotopes, toxins, or cytotoxic prodrugs) in which such
destruction of cells is desired. In a specific embodiment, the
invention provides a method for the specific destruction of cells
of B cell lineage (e.g., B cell related leukemias or lymphomas) by
administering Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides in association with toxins or cytotoxic prodrugs prior
to or following bone marrow transplant.
[0607] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering polypeptides of the invention (e.g.,
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides or
anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV antibodies) in
association with toxins or cytotoxic prodrugs.
[0608] In a specific embodiment, the invention provides a method
for the specific destruction of cells of B cell lineage (e.g., B
cell related leukemias or lymphomas such as myeloma, multiple
myeloma and non-Hodgkin's lymphoma) by administering
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides in
association with toxins or cytotoxic prodrugs. In a specific
embodiment, the invention provides a method for the specific
destruction of cells of B cell lineage (e.g., B cell related
leukemias or lymphomas such as myeloma, multiple myeloma and
non-Hodgkin's lymphoma) by administering Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides in association with toxins or
cytotoxic prodrugs prior to, during, or following bone marrow
transplant or stem cell transplant (e.g., autologous or
non-autologous, single or tandem transplant of CD34+stem
cells).
[0609] In another specific embodiment, the invention provides a
method for the specific destruction of cells of monocytic lineage
(e.g., monocytic leukemias or lymphomas) by administering
anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV antibodies in
association with toxins or cytotoxic prodrugs.
[0610] Biodistribution studies (See Example 12) of radiolabeled
Neutrokine-alpha polypeptide (amino acids 134-285 of SEQ ID NO:2)
that had been injected into BALB/c mice demonstrated that
Neutrokine-alpha has high in vivo targeting specificity for
lymphoid tissues such as spleen and lymph nodes. Thus in specific
embodiments, the invention provides a method for the specific
destruction or disablement of lymphoid tissue (e.g., lymph nodes
and spleen) by administering Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha, and/or anti-Neutrokine-alphaSV polypeptides
in association with radioisotopes, toxins or cytotoxic prodrugs. In
preferred embodiments, the lymphoid tissue is not permanently
destroyed, but rather is temporarily disabled, (e.g, cells of
hematopoietic lineage in lymphoid tissues are destroyed/killed
while Neutrokine-alpha, Neutrokine-alphaSV, anti-Neutrokine-alpha,
and/or anti-Neutrokine-alphaSV polypeptides in association with
radioisotopes, toxins or cytotoxic prodrugs are administered, but
these populations recover once administration of Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSV polypeptides in association with
radioisotopes, toxins or cytotoxic prodrugs is stopped.)
[0611] By "toxin" is meant compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins,
modified toxins, catalytic subunits of toxins, cytotoxins
(cytotoxic agents), or any molecules or enzymes not normally
present in or on the surface of a cell that under defined
conditions cause the cell's death. Toxins that may be used
according to the methods of the invention include, but are not
limited to, radioisotopes known in the art, compounds such as, for
example, antibodies (or complement fixing containing portions
thereof) that bind an inherent or induced endogenous cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic
or cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, .sup.213Bi, or other
radioisotopes such as, for example, .sup.103Pd, .sup.133X,
.sup.131I, .sup.68Ge, .sup.57C, .sup.65 Zn, .sup.85Sr, .sup.32P,
.sup.35S, .sup.90Y, .sup.153Sm, .sup.153Gd, .sup.169Yb, .sup.51Cr,
.sup.54Mn, .sup.75Se, .sup.113Sn, Yttrium, Tin, Rhenium, Holmium,
and Rhenium; luminescent labels, such as luminol; and fluorescent
labels, such as fluorescein and rhodamine, and biotin. In specific
embodiments Neutrokine-alpha polypeptides, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibodies or anti-Neutrokine-alphaSV
antibodies of the invention are conjugated or fused to a
polypeptide cytotoxin. An example of a suitable polypeptide
cytotoxin is a ribosome-inactivating protein. Type I
ribosome-inactivating proteins are single-chain proteins, while
type II ribosome-inactivating proteins consist of two nonidentical
subunits (A and B chains) joined by a disulfide bond (for a review,
see Soria et al., Targeted Diagn. Ther. 7:193 (1992)). In one
embodiment, type I ribosome-inactivating proteins that may be used
include, but are not limited to, polypeptides from Saponaria
officinalis (e.g., saporin-1, saporin-2, saporin-3, saporin-6),
Momordica charantia (e.g, momordin), Byronia dioica (e.g., bryodin,
bryodin-2), Trichosanthes kirilowii (e.g., trichosanthin,
trichokirin), Gelonium multiflorum (e.g., gelonin), Phytolacca
americana (e.g., pokeweed antiviral protein, pokeweed antiviral
protein-II, pokeweed antiviral protein-S), Phytolacca dodecandra
(e.g., dodecandrin, Mirabilis antiviral protein).
Ribosome-inactivating proteins are described, for example, by Walsh
et al., U.S. Pat. No. 5,635,384. In specific embodiments, one or
more Neutrokine-alpha polypeptides, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibodies or anti-Neutrokine-alphaSV
antibodies of the invention are conjugated or fused to a fragment
or variant of the ribosome inactivating proteins described above,
particularly when said fragment or variant retains activity.
[0612] In another embodiment, type II ribosome-inactivating
proteins that may be used according to the invention include, but
are not limited to, polypeptides from Ricinus communis (e.g.,
ricin), Abrus precatorius (e.g., abrin), Adenia digitata (e.g.,
modeccin). Since type II ribosome-inactivating proteins include a B
chain that binds galactosides and a toxic A chain that depurinates
adensoine, type II ribosome-inactivating protein conjugates should
include the A chain. Additional ribosome-inactivating proteins that
may be used according to the invention, include but are not limited
to, bouganin, clavin, maize ribosome-inactivating proteins,
Vaccaria pyramidata ribosome-inactivating proteins, nigrine b,
basic nigrine 1, ebuline, racemosine b, luffin-a, luffin-b,
luffin-S, and other ribosome-inactivating proteins known to those
of skill in the art. See, for example, Bolognesi and Stirpe,
International Publication No. WO98/55623, Colnaghi et al.,
International Publication No. WO97/49726, Hey et al., U.S. Pat. No.
5,635,384, Bolognesi and Stirpe, International Publication No.
WO95/07297, Arias et al., International Publication No. WO94/20540,
Watanabe et al., J. Biochem. 106:6 977 (1989); Islam et al., Agric.
Biol. Chem. 55:229 (1991), and Gao et al., FEBS Lett. 347:257
(1994). In specific embodiments, one or more Neutrokine-alpha
polypeptides, Neutrokine-alphaSV, anti-Neutrokine-alpha antibodies
or anti-Neutrokine-alphaSV antibodies of the invention are
conjugated or fused to a fragment or variant of the ribosome
inactivating proteins described above, particularly when said
fragment or variant retains activity.
[0613] Additional ribosome inactivating proteins (RIPs) that may be
conjugated or fused to Neutrokine-alpha polypeptides,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibodies or
anti-Neutrokine-alphaSV antibodies of the invention include, but
are not limited to, Type I Plant RIPs such as Pokeweed antiviral
proteins, Tritin, Gelonin, Momordin, Saporin, Dianthin, and Maize
RIP; Type II Plant RIPs such as Ricin, Abrin, Modecin, Viscumin,
Volkensin, Cinnamomin, Mistletoe lectin I and Luffangulin (6 kda);
Bacterial RIPS such as Shiga toxin, and Shiga-like toxin; and
Fungal RIPS such as alpha-sarcin, mitogillin, and restrictocin. In
specific embodiments, one or more Neutrokine-alpha polypeptides,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibodies or
anti-Neutrokine-alphaSV antibodies of the invention are conjugated
or fused to a fragment or variant of the ribosome inactivating
proteins described above, particularly when said fragment or
variant retains activity.
[0614] Ricin A homologues that may be conjugated or fused to
Neutrokine-alpha polypeptides, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibodies or anti-Neutrokine-alphaSV
antibodies of the invention include, but are not limited to,
polypeptides that have the same amino acid sequence as a protein
selected from the group consisting of: type 2 ribosome-inactivating
protein cinnamomin III precursor, abrin-d precursor from Indian
licorice or fragment thereof, RIP precursor of Sambucus nigra, RIP
bryodin II precursor, alpha-trichosanthin, ribosome-inactivating
protein precursor from Sambucus ebulus, karasurin-B, Trichobakin,
Beta-Luffin, Beta-galactoside specific lectin I A chain (MLA; ML-I
A), lectin chain A isoform 2 from Viscum album subsp. coloratum,
curcin precursor from Jatropha curcas, trichoanguina from snake
gourd, ribosome-inactivating protein IRAb from Iris hollandica,
Momordin, trichoanguin from Trichosanthes cucumerina, momordin II
from Momordica balsamina, gelonin from Gelonium multiflorum,
ribosome inactivating protein RIPm from Polygonatum multiflorum,
ribosome inactivating protein Euserratin 2 precursor from Euphorbia
serrata, alpha-PAP (pokeweed antiviral protein) from Phytolacca
americana, ribosome-inactivating protein gynostemmin II from
Gynostemma pentaphyllum, trichosanthin, ribosome-inactivating
protein 2 from Phytolacca insularis, bouganin from Bougainvillea
spectabilis, antiviral protein from Clerodendrum aculeatum,
anti-viral protein PAP from Phytolacca acinosa, type I RIP
moschatin I, antiviral ribosome inactivating protein from
Chenopodium album, Betavulgin from Beta vulgaris subsp. vulgaris,
ribosome-inactivating protein benincasin from Benincasa hispida,
ribosome-inactivating protein ME1 from Mirabilis expansa,
MAP--MIRABILIS--Four-o'clock (Marvel-of-peru), RIP amaranthin,
amarandin-S from Amaranthus tricolor, ribosome-inactivating protein
from Spinacia oleracea, type 1 RIP musarmin 3 from Muscari, saporin
from Saponaria officinalis, dianthin 30 from Dianthus caryophyllus,
and tritin from Triticum aestivum. In specific embodiments, one or
more Neutrokine-alpha polypeptides, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibodies or anti-Neutrokine-alphaSV
antibodies of the invention are conjugated or fused to a fragment
or variant of the polypeptides described above, particularly when
said fragment or variant retains activity.
[0615] In specific embodiments, one or more Neutrokine-alpha
polypeptides, Neutrokine-alphaSV, anti-Neutrokine-alpha antibodies
or anti-Neutrokine-alphaSV antibodies of the invention are
conjugated or fused to gelonin (for example as described in GenBank
Accession Number P33186, which is herein incorporated by reference.
In specific embodiments, one or more Neutrokine-alpha polypeptides,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibodies or
anti-Neutrokine-alphaSV antibodies of the invention are conjugated
or fused to amino acids 47 to 297 of gelonin as disclosed in
GenBank Accession Number P33186. In other specific embodiments, one
or more Neutrokine-alpha polypeptides, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibodies or anti-Neutrokine-alphaSV
antibodies of the invention are conjugated or fused to amino acids
47 to 297 of gelonin as disclosed in GenBank Accession Number
P33186 with the exception that the Asp-297 is changed to Cys
297.
[0616] Other toxins that may be conjugated or fused to
Neutrokine-alpha polypeptides, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibodies or anti-Neutrokine-alphaSV
antibodies of the invention include, but are not limited to
diptheria toxin, Pseudomonas exotoxin A, Botulinum neurotoxin,
Clostridium perfringens epsilon toxin, Conotoxins, and
Staphylococcal enterotoxins. In specific embodiments, one or more
Neutrokine-alpha polypeptides, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibodies or anti-Neutrokine-alphaSV
antibodies of the invention are conjugated or fused to a fragment
or variant of the polypeptides described above, particularly when
said fragment or variant retains activity.
[0617] Techniques known in the art may be applied to label
polypeptides and antibodies of the invention. Such techniques
include, but are not limited to, the use of bifunctional
conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631;
5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139;
5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of
each of which are hereby incorporated by reference in its entirety)
and direct coupling reactions (e.g., Bolton-Hunter, Chloramine-T
reaction, and Iodogen.RTM. based labeling methods).
[0618] A cytotoxin or cytotoxic agent includes any agent that is
detrimental to cells. Examples include paclitaxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicine, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof. Therapeutic agents include, but are not limited
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0619] By "cytotoxic prodrug" is meant a non-toxic compound that is
converted by an enzyme, normally present in the cell, into a
cytotoxic compound. Cytotoxic prodrugs that may be used according
to the methods of the invention include, but are not limited to,
glutamyl derivatives of benzoic acid mustard alkylating agent,
phosphate derivatives of etoposide or mitomycin C, cytosine
arabinoside, daunorubicin, and phenoxyacetamide derivatives of
doxorubicin.
[0620] In specific embodiments, Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSV polypeptides in association with
radioisotopes, toxins or cytotoxic prodrugs are used to treat or
ameliorate the symptoms of autoimmune diseases. In preferred
embodiments, Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha, and/or anti-Neutrokine-alphaSV polypeptides
in association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of systemic lupus
erythematosus. Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha, and/or anti-Neutrokine-alphaSV polypeptides
in association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of rheumatoid arthritis
including advanced rheumatoid arthritis. In preferred embodiments,
Neutrokine-alpha, Neutrokine-alphaSV, anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSV polypeptides in association with
radioisotopes, toxins or cytotoxic prodrugs are used to treat or
ameliorate the symptoms of idiopathic thrombocytopenic purpura
(ITP).
[0621] In other preferred embodiments Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSV polypeptides in association with
radioisotopes, toxins or cytotoxic prodrugs are used to treat or
ameliorate the symptoms of Sjogren's syndrome. In other preferred
embodiments, Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha, and/or anti-Neutrokine-alphaSV polypeptides
in association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of IgA nephropathy. In
other preferred embodiments, Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha, and/or anti-Neutrokine-alphaSV polypeptides
in association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of Myasthenia gravis. In
preferred embodiments, Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha, and/or anti-Neutrokine-alphaSV polypeptides
in association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of multiple sclerosis. In
still other preferred embodiments, Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSV polypeptides in association with
radioisotopes, toxins or cytotoxic prodrugs are used to treat or
ameliorate the symptoms of vasculitis.
[0622] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing immunoglobulin production
(e.g. IgM, IgG, and/or IgA production), comprising, or
alternatively consisting of, contacting an effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide with cells
of hematopoietic origin, wherein the effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide inhibits or
reduces Neutrokine-alpha and/or Neutrokine-alphaSV mediated
immunoglobulin production. In specific embodiments, the invention
provides methods and compositions for inhibiting or reducing
immunoglobulin production (e.g. IgM, IgG, and/or IgA production) in
response to T cell dependent antigens, comprising, or alternatively
consisting of, contacting an effective amount of Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide with cells of hematopoietic
origin, wherein the effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide inhibits or reduces Neutrokine-alpha
and/or Neutrokine-alphaSV mediated immunoglobulin production in
response to T cell dependent antigens. In specific embodiments, the
invention provides methods and compositions for inhibiting or
reducing immunoglobulin production (e.g. IgM, IgG, and/or IgA
production) in response to T cell independent antigens, comprising,
or alternatively consisting of, contacting an effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide with cells
of hematopoietic origin, wherein the effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide inhibits or
reduces Neutrokine-alpha and/or Neutrokine-alphaSV mediated
immunoglobulin production in response to T cell independent
antigens.
[0623] In another embodiment, the invention provides methods and
compositions for inhibiting or reducing immunoglobulin production
(e.g. IgM, IgG, and/or IgA production), comprising, or
alternatively consisting of, administering to an animal in which
such inhibition or reduction is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide in an amount effective to inhibit or
reduce immunoglobulin production. In another embodiment, the
invention provides methods and compositions for inhibiting or
reducing immunoglobulin production (e.g. IgM, IgG, and/or IgA
production) in response to T cell dependent antigens, comprising,
or alternatively consisting of, administering to an animal in which
such inhibition or reduction is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide in an amount effective to inhibit or
reduce immunoglobulin production in response to T cell dependent
antigens. In another embodiment, the invention provides methods and
compositions for inhibiting or reducing immunoglobulin production
(e.g. IgM, IgG, and/or IgA production) in response to T cell
independent antigens, comprising, or alternatively consisting of,
administering to an animal in which such inhibition or reduction is
desired, a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
in an amount effective to inhibit or reduce immunoglobulin
production in response to T cell independent antigens.
[0624] In another embodiment, the invention provides methods and
compositions for stimulating immunoglobulin production (e.g. IgM,
IgG, and/or IgA production), comprising, or alternatively
consisting of, contacting an effective amount of Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide with cells of hematopoietic
origin, wherein the effective amount of the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide stimulates Neutrokine-alpha and/or
Neutrokine-alphaSV mediated immunoglobulin production. In another
embodiment, the invention provides methods and compositions for
stimulating immunoglobulin production (e.g. IgM, IgG, and/or IgA
production) in response to T cell dependent antigens comprising, or
alternatively consisting of, contacting an effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide with cells
of hematopoietic origin, wherein the effective amount of the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide stimulates
Neutrokine-alpha and/or Neutrokine-alphaSV mediated immunoglobulin
production in response to T cell dependent antigens. In another
embodiment, the invention provides methods and compositions for
stimulating immunoglobulin production (e.g. IgM, IgG, and/or IgA
production) in response to T cell independent antigens comprising,
or alternatively consisting of, contacting an effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide with cells
of hematopoietic origin, wherein the effective amount of the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide stimulates
Neutrokine-alpha and/or Neutrokine-alphaSV mediated immunoglobulin
production in response to T cell independent antigens.
[0625] In another embodiment, the invention provides methods and
compositions for stimulating immunoglobulin production (e.g. IgM,
IgG, and/or IgA production) comprising, or alternatively consisting
of, administering to an animal in which such stimulation is
desired, a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
in an amount effective to stimulate immunoglobulin production. In
another embodiment, the invention provides methods and compositions
for stimulating immunoglobulin production (e.g. IgM, IgG, and/or
IgA production) in response to T cell dependent antigens
comprising, or alternatively consisting of, administering to an
animal in which such stimulation is desired, a Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide in an amount effective to
stimulate immunoglobulin production in response to T cell dependent
antigens.
[0626] In another embodiment, the invention provides methods and
compositions for stimulating immunoglobulin production (e.g. IgM,
IgG, and/or IgA production) in response to T cell independent
antigens comprising, or alternatively consisting of, administering
to an animal in which such stimulation is desired, a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide in an amount
effective to stimulate immunoglobulin production in response to T
cell independent antigens.
[0627] Determination of immunoglobulin levels are most often
performed by comparing the level of immunoglobulin in a sample to a
standard containing a known amount of immunoglobulin using ELISA
assays. Determination of immunoglobulin levels in a given sample,
can readily be determined using ELISA or other method known in the
art.
[0628] Receptors belonging to the TNF receptor (TNFR) super family
(e.g., TACI, BAFF-R and BCMA, receptors to which Neutrokine-alpha
polypeptides can bind) can be classified into two types based on
the presence or absence of a conserved cytoplasmic domain
responsible for apoptosis called a "death domain." TNF receptors
without death domains, such as TNF-R2 HVEM/ATAR, RANK, CD27, CD30,
CD40, and OX40 interact with TNF receptor associated factors (TRAF
1-6) and mediate anti-apoptotic survival and or proliferative
responses via activation of the transcription factor NF-kappaB
(reviewed in Wajant et al., Cytokine and Growth Factor Reviews
10(1):15-26, 1999). TACI, BCMA and BAFF-R do not contain death
domains.
[0629] Investigation of Neutrokine-alpha (which bind TACI, BCMA,
and BAFF-R) induced signaling in human tonsillar B cells
co-stimulated with Staph. Aureus Cowan consistently revealed that
mRNA for ERK-1 and PLK were upregulated by Neutrokine-alpha+SAC
treatment (see Example 11). Polo like kinases (PLK) belong to a sub
family of serine/threonine kinases related to Saccharomyces
cerevisiae cell cycle protein CDC5 (29). The expression of PLK is
induced during G2 and S phase of the cell cycle. PLK is reported to
play a role in cell proliferation (Lee et al., Proc. Natl. Acad.
Sci. 95:9301-9306). The role or extracellular-signal related
kinases (ERK1/2) in cell survival and proliferative effects of
growth factors and other agonists has been extensively studied. The
induced expression of PLK and ERK-1 is consistent with the survival
and proliferative effects of Neutrokine-alpha on B cells.
[0630] Additionally, in some samples of human tonsillar B cells
stimulated with Neutrokine-alpha and SAC, mRNA for CD25
(IL-2Ralpha) was upregulated. Nuclear extracts from Human tonsillar
B cells treated with Neutrokine-alpha and from IM-9 cells treated
with Neutrokine-alpha were able to shift probes from the CD25
promoter region containing sites for NF-kappaB, SRF, ELF-1 and
HMGI/Y in an electromobility shift assay. ELF-1 for example, is a
transcription factor that is part of the ETS family of proteins and
whose expression appears to be restricted to T and B cells. Binding
sites for ELF-1 have been described in the promoters of a number of
proteins that are important in the regulation of the immune
response.
[0631] Thus, Neutrokine-alpha induced signaling has been shown to
be consistent with the activation of cellular activation and
cellular proliferation pathways as well as with cellular signaling
pathways that regulate B cell lifespan. Further, Neutrokine-alpha
and/or Neutrokine-alphaSV treatment of B cells induces cellular
proliferation immunoglobulin secretion, a characteristic of
activated B cells (Moore et al., Science 285:260-263, 1999).
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides may
inhibit, stimulate, or not significantly alter these
Neutrokine-alpha and/or Neutrokine-alphaSV mediated activities.
[0632] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing proliferation of cells of
hematopoietic origin, comprising, or alternatively consisting of,
contacting an effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide with cells of hematopoietic origin,
wherein the effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide inhibits or reduces Neutrokine-alpha
and/or Neutrokine-alphaSV mediated proliferation of cells of
hematopoietic origin. In another embodiment, the invention provides
methods and compositions for inhibiting or reducing reducing
proliferation of cells of hematopoietic origin comprising, or
alternatively consisting of, administering to an animal in which
such inhibition or reduction is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide in an amount effective to inhibit or
reduce B cell proliferation. In preferred embodiments, the cells of
hematopoietic origin are B cells.
[0633] In one embodiment, the invention provides methods and
compositions for stimulating proliferation of cells of
hematopoietic origin, comprising, or alternatively consisting of,
contacting an effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide with cells of hematopoietic origin,
wherein the effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide stimulates Neutrokine-alpha and/or
Neutrokine-alphaSV mediated proliferation of cells of hematopoietic
origin. In another embodiment, the invention provides methods and
compositions for stimulating proliferation of cells of
hematopoietic origin comprising, or alternatively consisting of,
administering to an animal in which such stimulation is desired, a
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide in an amount
effective to stimulate B cell proliferation. In preferred
embodiments, the cells of hematopoietic origin are B cells. B cell
proliferation is most commonly assayed in the art by measuring
tritiated thymidine incorporation (see Examples 6 & 7). This
and other assays are commonly known in the art and could be
routinely adapted for the use of determining the effect of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides on B cell
proliferation.
[0634] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing activation of cells of
hematopoietic origin, comprising, or alternatively consisting of,
contacting an effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide with cells of hematopoietic origin,
wherein the effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide inhibits or reduces Neutrokine-alpha
and/or Neutrokine-alphaSV mediated activation of cells of
hematopoietic origin. In one embodiment, the invention provides
methods and compositions for inhibiting or reducing activation of
cells of hematopoietic origin, comprising, or alternatively
consisting of, administering to an animal in which such inhibition
or reduction is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide in an amount effective to inhibit or
reduce activation of cells of hematopoietic origin. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0635] In one embodiment, the invention provides methods and
compositions for increasing activation of cells of hematopoietic
origin, comprising, or alternatively consisting of, contacting an
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide with cells of hematopoietic origin, wherein the
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide increases Neutrokine-alpha and/or Neutrokine-alphaSV
mediated activation of cells of hematopoietic origin. In one
embodiment, the invention provides methods and compositions for
increasing activation of cells of hematopoietic origin, comprising,
or alternatively consisting of, administering to an animal in which
such increase is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide in an amount effective to increase
activation of cells of hematopoietic origin. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0636] B cell activation can measured in a variety of ways, such as
FACS analysis of activation markers expressed on B cells. B cells
activation markers include, but are not limited to, CD26, CD 28, CD
30, CD 38, CD 39, CD 69, CD70 CD71, CD 77, CD 83, CD126, CDw130,
and B220. Additionally, B cell activation may be measured by
analysis of the activation of signaling molecules involved in B
cell activation. By way of non-limiting example, such analysis may
take the form of analyzing mRNA levels of signaling molecules by
Northern analysis or real time PCR (See Example 11). One can also
measure, for example, the phosphorylation of signaling molecules
using anti-phosphotyrosine antibodies in a Western blot. B cell
activation may also be measured by measuring the calcium levels in
B cells. These and other methods of determining B cell activation
are commonly known in the art and could be routinely adapted for
the use of determining the effect of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides on B cell activation.
[0637] In one embodiment, the invention provides methods and
compositions for decreasing lifespan of cells of hematopoietic
origin, comprising, or alternatively consisting of, contacting an
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide with cells of hematopoietic origin, wherein the
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide inhibits or reduces Neutrokine-alpha and/or
Neutrokine-alphaSV regulated lifespan of cells of hematopoietic
origin. In one embodiment, the invention provides methods and
compositions for decreasing lifespan of cells of hematopoietic
origin, comprising, or alternatively consisting of, administering
to an animal in which such decrease is desired, a Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptide in an amount effective to
decrease lifespan of cells of hematopoietic origin. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0638] In one embodiment, the invention provides methods and
compositions for increasing lifespan of cells of hematopoietic
origin, comprising, or alternatively consisting of, contacting an
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide with cells of hematopoietic origin, wherein the
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide increases Neutrokine-alpha and/or Neutrokine-alphaSV
regulated lifespan of cells of hematopoietic origin. In one
embodiment, the invention provides methods and compositions for
increasing lifespan of cells of hematopoietic origin, comprising,
or alternatively consisting of, administering to an animal in which
such increase is desired, a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide in an amount effective to increase
lifespan of cells of hematopoietic origin. In preferred
embodiments, the cells of hematopoietic origin are B cells.
[0639] B cell life span in vivo may be measured by
5-bromo-2'-deoxyuridine (BrdU) labeling experiments which are well
known to one skilled in the art. BrdU is a thymidine analogue that
gets incorporated into the DNA of dividing cells. Cells containing
BrdU in their DNA can be detected using, for example fluorescently
labeled anti-BrdU antibody and flow cytometry. Briefly, an animal
is injected with BrdU in an amount sufficient to label developing B
cells. Then, a sample of B cells is withdrawn from the animal, for
example, from peripheral blood, and analyzed for the percentage of
cells that contain BrdU. Such an analysis performed at several time
points can be used to calculate the half life of B cells.
Alternatively, B cell survival may be measured in vitro. For
example B cells may be cultured under conditions where
proliferation does not occur, (for example the media should contain
no reagents that crosslink the immunoglobulin receptor, such as
anti-IgM antibodies) for a period of time (usually 2-4 days). At
the end of this time, the percent of surviving cells is determined,
using for instance, the vital dye Trypan Blue, or by staining cells
with propidium iodide or any other agent designed to specifically
stain apoptotic cells and analyzing the percentage of cells stained
using flow cytometry. One could perform this experiment under
several conditions, such as B cells treated with Neutrokine-alpha,
B cells treated with Neutrokine-alpha and/or
Neutrokine-alphaSV-polypeptide complexes, and untreated B cells in
order to determine the effects of Neutrokine-alpha and/or
Neutrokine-alphaSV and Neutrokine-alpha polypeptides on B cells
survival. These and other methods for determining B cell lifespan
are commonly known in the art and could routinely be adapted to
determining the effect of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides on Neutrokine-alpha and/or
Neutrokine-alphaSV regulated B cell lifespan.
[0640] It will be appreciated that conditions caused by a decrease
in the standard or normal level of Neutrokine-alpha and/or
Neutrokine-alphaSV activity in an individual, particularly
disorders of the immune system, can be treated by administration of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide (in the form
of soluble extracellular domain or cells expressing the complete
protein) or agonist. Thus, the invention also provides a method of
treatment of an individual in need of an increased level of
Neutrokine-alpha and/or Neutrokine-alphaSV activity comprising
administering to such an individual a pharmaceutical composition
comprising an amount of an isolated Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide of the invention, or agonist
thereof, effective to increase the Neutrokine-alpha and/or
Neutrokine-alphaSV activity level in such an individual.
[0641] It will also be appreciated that conditions caused by a
increase in the standard or normal level of Neutrokine-alpha and/or
Neutrokine-alphaSV activity in an individual, particularly
disorders of the immune system, can be treated by administration of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides (in the
form of soluble extracellular domain or cells expressing the
complete protein) or antagonist (e.g, an anti-Neutrokine-alpha
antibody). Thus, the invention also provides a method of treatment
of an individual in need of an decreased level of Neutrokine-alpha
and/or Neutrokine-alphaSV activity comprising administering to such
an individual a pharmaceutical composition comprising an amount of
an isolated Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
of the invention, or antagonist thereof, effective to decrease the
Neutrokine-alpha and/or Neutrokine-alphaSV activity level in such
an individual. A non-limiting example of a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide of the invention that can be
administered to an individual in need of an decreased level of
Neutrokine-alpha and/or Neutrokine-alphaSV activity, is a dominant
negative mutant of a Neutrokine-alpha and/or Neutrokine-alphaSV,
which binds to a Neutrokine-alpha and/or Neutrokine-alphaSV
receptor but that does not induce signal transduction.
[0642] Autoantibody production is common to several autoimmune
diseases and contributes to tissue destruction and exacerbation of
disease. Autoantibodies can also lead to the occurrence of immune
complex deposition complications and lead to many symptoms of
systemic lupus erythomatosis, including kidney failure, neuralgic
symptoms and death. Modulating antibody production independent of
cellular response would also be beneficial in many disease states.
B cells have also been shown to play a role in the secretion of
arthritogenic immunoglobulins in rheumatoid arthritis, (Korganow et
al., Immunity 10:451-61, 1999). As such, inhibition of Neutrokine
alpha-mediated antibody production would be beneficial in treatment
of autoimmune diseases such as myasthenia gravis and rheumatoid
arthritis. Compounds of the invention that selectively block or
neutralize the action of B-lymphocytes would be useful for such
purposes. To verify these capabilities in compositions of the
present invention, such compositions are evaluated using assays
known in the art and described herein.
[0643] The invention provides methods employing compositions of the
invention (e.g., Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention and/or agonists
and/or antagonists thereof) for selectively blocking or
neutralizing the actions of B-cells in association with end stage
renal diseases, which may or may not be associated with autoimmune
diseases. Such methods would also be useful for treating
immunologic renal diseases. Such methods would be would be useful
for treating glomerulonephritis associated with diseases such as
membranous nephropathy, IgA nephropathy or Berger's Disease, IgM
nephropathy, Goodpasture's Disease, post-infectious
glomerulonephritis, mesangioproliferative disease, minimal-change
nephrotic syndrome. Such methods would also serve as therapeutic
applications for treating secondary glomerulonephritis or
vasculitis associated with such diseases as lupus, polyarteritis,
Henoch-Schonlein, Scleroderma, HIV-related diseases, amyloidosis or
hemolytic uremic syndrome. The methods of the present invention
would also be useful as part of a therapeutic application for
treating interstitial nephritis or pyelonephritis associated with
chronic pyelonephritis, analgesic abuse, nephrocalcinosis,
nephropathy caused by other agents, nephrolithiasis, or chronic or
acute interstitial nephritis.
[0644] The methods of the present invention also include use of
compositions of the invention in the treatment of hypertensive or
large vessel diseases, including renal artery stenosis or occlusion
and cholesterol emboli or renal emboli.
[0645] The present invention also provides methods for diagnosis
and treatment of renal or urological neoplasms, multiple myelomas,
lymphomas, light chain neuropathy or amyloidosis.
[0646] The invention also provides methods for blocking or
inhibiting activated B cells using compositions of the invention
for the treatment of asthma and other chronic airway diseases such
as bronchitis and emphysema.
[0647] Neutrokine-alpha, and/or Neutrokine-alphaSV polynucleotides
or polypeptides of the invention, or agonists of Neutrokine-alpha,
and/or Neutrokine-alphaSV, can be used in the treatment of
infectious agents. For example, by increasing the immune response,
particularly increasing the proliferation and differentiation of B
cells, infectious diseases may be treated. The immune response may
be increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, Neutrokine-alpha,
and/or Neutrokine-alphaSV polynucleotides or polypeptides, or
agonists of Neutrokine-alpha, and/or Neutrokine-alphaSV, may also
directly inhibit the infectious agent, without necessarily
eliciting an immune response.
[0648] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated by Neutrokine-alpha,
and/or Neutrokine-alphaSV polynucleotides or polypeptides, or
agonists or antagonists of Neutrokine-alpha, and/or
Neutrokine-alphaSV. Examples of viruses, include, but are not
limited to the following DNA and RNA viruses and viral families:
Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue,
EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),
Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B,
and parainfluenza), Papilloma virus, Papovaviridae, Parvoviridae,
Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiolitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia. Neutrokine-alpha, and/or Neutrokine-alphaSV
polynucleotides or polypeptides, or agonists or antagonists of
Neutrokine-alpha, and/or Neutrokine-alphaSV, can be used to treat,
prevent, diagnose, and/or detect any of these symptoms or diseases.
In specific embodiments, Neutrokine alpha polynucleotides,
polypeptides, or agonists are used to treat, prevent, and/or
diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g.,
hepatitis B). In an additional specific embodiment Neutrokine alpha
polynucleotides, polypeptides, or agonists are used to treat
patients nonresponsive to one or more other commercially available
hepatitis vaccines. In a further specific embodiment, Neutrokine
alpha polynucleotides, polypeptides, or agonists are used to treat,
prevent, and/or diagnose AIDS. In an additional specific embodiment
Neutrokine-alpha and/or Neutrokine-alphaSV and/or Neutrokine-alpha
Receptor polynucleotides, polypeptides, agonists, and/or
antagonists are used to treat, prevent, and/or diagnose patients
with cryptosporidiosis.
[0649] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated by Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or agonists or
antagonists of Neutrokine-alpha, and/or Neutrokine-alphaSV,
include, but not limited to, the following Gram-Negative and
Gram-positive bacteria and bacterial families and fungi:
Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),
Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax,
Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia
(e.g., Borrelia burgdorferi, Brucellosis, Candidiasis,
Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,
E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E.
coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella
typhi, and Salmonella paratyphi), Serratia, Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,
Listeria (e.g, Listeria monocytogenes), Mycoplasmatales,
Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,
Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,
Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,
Heamophilus influenza type B), Pasteurella), Pseudomonas,
Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp.,
Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal
(e.g., Streptococcus pneumoniae and Group B Streptococcus). These
bacterial or fungal families can cause the following diseases or
symptoms, including, but not limited to: bacteremia, endocarditis,
eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis,
opportunistic infections (e.g., AIDS related infections),
paronychia, prosthesis-related infections, Reiter's Disease,
respiratory tract infections, such as Whooping Cough or Empyema,
sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid
Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis
(e.g., meningitis types A and B), Chlamydia, Syphilis, Diphtheria,
Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene,
tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually
transmitted diseases, skin diseases (e.g., cellulitis,
dermatocycoses), toxemia, urinary tract infections, wound
infections. Neutrokine-alpha, and/or Neutrokine-alphaSV
polynucleotides or polypeptides, or agonists or antagonists of
Neutrokine-alpha, and/or Neutrokine-alphaSV, can be used to treat,
prevent, diagnose, and/or detect any of these symptoms or diseases.
In specific embodiments, Neutrokine alpha polynucleotides,
polypeptides, or agonists thereof are used to treat, prevent,
and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis
type B.
[0650] Moreover, parasitic agents causing disease or symptoms that
can be treated by Neutrokine-alpha, and/or Neutrokine-alphaSV
polynucleotides or polypeptides, or agonists of Neutrokine-alpha,
and/or Neutrokine-alphaSV, include, but not limited to, the
following families or class: Amebiasis, Babesiosis, Coccidiosis,
Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,
Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,
Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans
(e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium
malariae and Plasmodium ovale). These parasites can cause a variety
of diseases or symptoms, including, but not limited to: Scabies,
Trombiculiasis, eye infections, intestinal disease (e.g.,
dysentery, giardiasis), liver disease, lung disease, opportunistic
infections (e.g., AIDS related), malaria, pregnancy complications,
and toxoplasmosis. Neutrokine-alpha, and/or Neutrokine-alphaSV
polynucleotides or polypeptides, or agonists or antagonists of
Neutrokine-alpha, and/or Neutrokine-alphaSV, can be used to treat,
prevent, diagnose, and/or detect any of these symptoms or diseases.
In specific embodiments, Neutrokine alpha polynucleotides,
polypeptides, or agonists thereof are used to treat, prevent,
and/or diagnose malaria.
[0651] In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, are used to treat,
prevent, and/or diagnose inner ear infection (such as, for example,
otitis media), as well as other infections characterized by
infection with Streptococcus pneumoniae and other pathogenic
organisms.
[0652] In a specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or agonists or
antagonists thereof (e.g., anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSV antibodies) are used to treat or prevent a
disorder characterized by deficient serum immunoglobulin
production, recurrent infections, and/or immune system dysfunction.
Moreover, Neutrokine-alpha, and/or
Neutrokine-alphaSVpolynucleotides or polypeptides, or agonists or
antagonists thereof (e.g., anti-Neutrokine-alpha, and/or
anti-Neutrokine-alphaSV antibodies) may be used to treat or prevent
infections of the joints, bones, skin, and/or parotid glands,
blood-borne infections (e.g., sepsis, meningitis, septic arthritis,
and/or osteomyelitis), autoimmune diseases (e.g., those disclosed
herein), inflammatory disorders, and malignancies, and/or any
disease or disorder or condition associated with these infections,
diseases, disorders and/or malignancies) including, but not limited
to, CVID, other primary immune deficiencies, HIV disease, CLL,
multiple myeloma, recurrent bronchitis, sinusitis, otitis media,
conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster
(e.g., severe herpes zoster), and/or Pneumocystis carnii.
[0653] Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides
or polypeptides of the invention, or agonists or antagonists
thereof, may be used to diagnose, prognose, treat or prevent one or
more of the following diseases or disorders, or conditions
associated therewith: primary immunodeficiencies, immune-mediated
thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g.,
recent bone marrow transplant in adults or children), chronic
B-cell lymphocytic leukemia, HIV infection (e.g., adult or
pediatric HIV infection), chronic inflammatory demyelinating
polyneuropathy, and post-transfusion purpura.
[0654] Additionally, Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention, or agonists or
antagonists thereof, may be used to diagnose, prognose, treat or
prevent one or more of the following diseases, disorders, or
conditions associated therewith, Guillain-Barre syndrome, anemia
(e.g., anemia associated with parvovirus B19, patients with stable
multiple myeloma who are at high risk for infection (e.g.,
recurrent infection), autoimmune hemolytic anemia (e.g., warm-type
autoimmune hemolytic anemia), thrombocytopenia (e.g, neonatal
thrombocytopenia), and immune-mediated neutropenia),
transplantation (e.g, cytamegalovirus (CMV)-negative recipients of
CMV-positive organs), hypogammaglobulinemia (e.g.,
hypogammaglobulinemic neonates with risk factor for infection or
morbidity), epilepsy (e.g, intractable epilepsy), systemic
vasculitic syndromes, myasthenia gravis (e.g, decompensation in
myasthenia gravis), dermatomyositis, and polymyositis.
[0655] Additional preferred embodiments of the invention include,
but are not limited to, the use of Neutrokine-alpha and/or
Neutrokine-alpha SV polypeptides, Neutrokine-alpha and/or
Neutrokine-alpha SV polynucleotides, and functional agonists
thereof, in the following applications:
[0656] Administration to an animal (e.g., mouse, rat, rabbit,
hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse,
cow, sheep, dog, cat, non-human primate, and human, most preferably
human) to boost the immune system to produce increased quantities
of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to
promote or enhance immunoglobulin class switching (e.g., to induce
a B cell express an IgM antibody to class switch to a different
immunoglobulin isotype such as IgG, IgA, or IgE), to induce higher
affinity antibody production (e.g., IgG, IgA, IgM, and IgE, for
instance, by the modulation of the rate or quantity of somatic
hypermutation or by modulation of the process/mechanism of
selection of B cells expressing mutated antibodies), and/or to
increase an immune response. In a specific nonexclusive embodiment,
Neutrokine-alpha polypeptides of the invention, and/or agonists
thereof, are administered to boost the immune system to produce
increased quantities of IgG. In another specific nonexclusive
embodiment, Neutrokine-alpha polypeptides of the invention and/or
agonists thereof, are administered to boost the immune system to
produce increased quantities of IgA. In another specific
nonexclusive embodiment, Neutrokine-alpha polypeptides of the
invention and/or agonists thereof, are administered to boost the
immune system to produce increased quantities of IgM.
[0657] Administration to an animal (including, but not limited to,
those listed above, and also including transgenic animals)
incapable of producing functional endogenous antibody molecules or
having an otherwise compromised endogenous immune system, but which
is capable of producing human immunoglobulin molecules by means of
a reconstituted or partially reconstituted immune system from
another animal (see, e.g., published PCT Application Nos.
WO98/24893, WO/9634096, WO/9633735, and WO/9110741).
[0658] A vaccine adjuvant that enhances immune responsiveness to
specific antigen. In a specific embodiment, the vaccine adjuvant is
a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide described
herein. In another specific embodiment, the vaccine adjuvant is a
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotide described
herein (i.e., the Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotide is a genetic vaccine adjuvant). As discussed herein,
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides may be
administered using techniques known in the art, including but not
limited to, liposomal delivery, recombinant vector delivery,
injection of naked DNA, and gene gun delivery.
[0659] An adjuvant to enhance tumor-specific immune responses.
[0660] An adjuvant to enhance anti-viral immune responses.
Anti-viral immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include, but are not
limited to, virus and virus associated diseases or symptoms
described herein or otherwise known in the art. In specific
embodiments, the compositions of the invention are used as an
adjuvant to enhance an immune response to a virus, disease, or
symptom selected from the group consisting of: AIDS, meningitis,
Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific
embodiment, the compositions of the invention are used as an
adjuvant to enhance an immune response to a virus, disease, or
symptom selected from the group consisting of: HIV/AIDS,
Respiratory syncytial virus, Dengue, Rotavirus, Japanese B
encephalitis, Influenza A and B, Parainfluenza, Measles,
Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever,
Herpes simplex, and yellow fever. In another specific embodiment,
the compositions of the invention are used as an adjuvant to
enhance an immune response to the HIV gp120 antigen.
[0661] An adjuvant to enhance anti-bacterial or anti-fungal immune
responses. Anti-bacterial or anti-fungal immune responses that may
be enhanced using the compositions of the invention as an adjuvant,
include bacteria or fungus and bacteria or fungus associated
diseases or symptoms described herein or otherwise known in the
art. In specific embodiments, the compositions of the invention are
used as an adjuvant to enhance an immune response to a bacteria or
fungus, disease, or symptom selected from the group consisting of:
tetanus, Diphtheria, botulism, and meningitis type B. In another
specific embodiment, the compositions of the invention are used as
an adjuvant to enhance an immune response to a bacteria or fungus,
disease, or symptom selected from the group consisting of: Vibrio
cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella
paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group
B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium
(malaria).
[0662] An adjuvant to enhance anti-parasitic immune responses.
Anti-parasitic immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include parasite and
parasite associated diseases or symptoms described herein or
otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a parasite. In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an
immune response to Plasmodium (malaria).
[0663] As a stimulator of B cell responsiveness to pathogens.
[0664] As an agent that elevates the immune status of an individual
prior to their receipt of immunosuppressive therapies.
[0665] As an agent to induce production of higher affinity
antibodies.
[0666] As an agent to induce class switching of B cells expressing
IgM antibodies.
[0667] As an agent to induce class switching of activated B cells
expressing IgM antibodies.
[0668] As an agent to increase serum immunoglobulin
concentrations.
[0669] As an agent to accelerate recovery of immunocompromised
individuals.
[0670] As an agent to boost immunoresponsiveness among aged
populations.
[0671] As an immune system enhancer prior to, during, or after bone
marrow transplant and/or other transplants (e.g., allogeneic or
xenogeneic organ transplantation). With respect to transplantation,
compositions of the invention may be administered prior to,
concomitant with, and/or after transplantation. In a specific
embodiment, compositions of the invention are administered after
transplantation, prior to the beginning of recovery of T-cell
populations. In another specific embodiment, compositions of the
invention are first administered after transplantation after the
beginning of recovery of T cell populations, but prior to full
recovery of B cell populations.
[0672] As an agent to boost immunoresponsiveness among B cell
immunodeficient individuals, such as, for example, an individual
who has undergone a partial or complete splenectomy. B cell
immunodeficiencies that may be ameliorated or treated by
administering the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides or polynucleotides of the invention, or agonists
thereof, include, but are not limited to, severe combined
immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine
deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia
(XLA), Bruton's disease, congenital agammaglobulinemia, X-linked
infantile agammaglobulinemia, acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.
[0673] As an agent to boost immunoresponsiveness among individuals
having an acquired loss of B cell function. Conditions resulting in
an acquired loss of B cell function that may be ameliorated or
treated by administering the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, include, but are not limited to,
HIV Infection, AIDS, bone marrow transplant, multiple myeloma and B
cell chronic lymphocytic leukemia (CLL).
[0674] Patients with CLL and myeloma are at risk for increased
infections. Thus, one aspect of the present invention provides for
the use of Neutrokine alpha, Neutrokine alphaSV,
anti-Neutrokine-alpha and or anti-Neutrokine alphaSV
polynucleotides and/or polypeptides as an agent to boost
immunoresponsiveness in CLL and myeloma patients.
[0675] As an agent to boost immunoresponsiveness among individuals
having a temporary immune deficiency. Conditions resulting in a
temporary immune deficiency that may be ameliorated or treated by
administering the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides or polynucleotides of the invention, or agonists
thereof, include, but are not limited to, recovery from viral
infections (e.g., influenza), conditions associated with
malnutrition, recovery from infectious mononucleosis, or conditions
associated with stress, recovery from measles, recovery from blood
transfusion, recovery from surgery, and recovery from burns.
[0676] As a regulator of antigen presentation by monocytes,
dendritic cells, and/or B-cells. In one embodiment,
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides (in
soluble, membrane-bound or transmembrane forms) or polynucleotides
enhance antigen presentation or antagonize antigen presentation in
vitro or in vivo. Moreover, in related embodiments, this
enhancement or antagonization of antigen presentation may be useful
in anti-tumor treatment or to modulate the immune system.
[0677] As a mediator of mucosal immune responses. The expression of
Neutrokine-alpha by monocytes and the responsiveness of B cells to
this factor suggests that it may be involved in exchange of signals
between B cells and monocytes or their differentiated progeny. This
activity is in many ways analogous to the CD40-CD154 signaling
between B cells and T cells. Neutrokine-alpha may therefore be an
important regulator of T cell independent immune responses to
environmental pathogens. In particular, the unconventional B cell
populations (CD5+) that are associated with mucosal sites and
responsible for much of the innate immunity in humans may respond
to Neutrokine-alpha thereby enhancing an individual's protective
immune status.
[0678] As an agent to direct an individual's immune system towards
development of a humoral response (i.e. TH2) as opposed to a TH1
cellular response.
[0679] As a means to induce tumor proliferation and thus make it
more susceptible to anti-neoplastic agents. For example, multiple
myeloma is a slowly dividing disease and is thus refractory to
virtually all anti-neoplastic regimens. If these cells were forced
to proliferate more rapidly their susceptibility profile would
likely change.
[0680] As a B cell specific binding protein to which specific
activators or inhibitors of cell growth may be attached. The result
would be to focus the activity of such activators or inhibitors
onto normal, diseased, or neoplastic B cell populations.
[0681] As a means of detecting B-lineage cells by virtue of its
specificity. This application may require labeling the protein with
biotin or other agents (e.g., as described herein) to afford a
means of detection.
[0682] As a stimulator of B cell production in pathologies such as
AIDS, chronic lymphocyte disorder and/or Common Variable
Immunodeficiency.
[0683] As part of a B cell selection device the function of which
is to isolate B cells from a heterogenous mixture of cell types.
Neutrokine-alpha could be coupled to a solid support to which B
cells would then specifically bind. Unbound cells would be washed
out and the bound cells subsequently eluted. A nonlimiting use of
this selection would be to allow purging of tumor cells from, for
example, bone marrow or peripheral blood prior to transplant.
[0684] As a therapy for generation and/or regeneration of lymphoid
tissues following surgery, trauma or genetic defect.
[0685] As a gene-based therapy for genetically inherited disorders
resulting in immuno-incompetence such as observed among SCID
patients.
[0686] As an antigen for the generation of antibodies to inhibit or
enhance Neutrokine-alpha mediated responses.
[0687] As a means of activating monocytes/macrophages to defend
against parasitic diseases that effect monocytes such as
Leshmania.
[0688] As pretreatment of bone marrow samples prior to transplant.
Such treatment would increase B cell representation and thus
accelerate recover.
[0689] As a means of regulating secreted cytokines that are
elicited by Neutrokine-alpha.
[0690] Neutrokine-alpha or Neutrokine-alphaSV polypeptides or
polynucleotides of the invention, or agonists may be used to
modulate IgE concentrations in vitro or in vivo.
[0691] Additionally, Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides or polynucleotides of the invention, or agonists
thereof, may be used to treat, prevent, and/or diagnose
IgE-mediated allergic reactions. Such allergic reactions include,
but are not limited to, asthma, rhinitis, and eczema.
[0692] In a specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate selective IgA deficiency.
[0693] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate ataxia-telangiectasia.
[0694] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate common variable immunodeficiency.
[0695] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate X-linked agammaglobulinemia.
[0696] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate severe combined immunodeficiency
(SCID).
[0697] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate Wiskott-Aldrich syndrome.
[0698] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate X-linked Ig deficiency with hyper
IgM.
[0699] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists or antagonists (e.g., anti-Neutrokine-alpha
antibodies) thereof, is administered to treat, prevent, and/or
diagnose chronic myelogenous leukemia, acute myelogenous leukemia,
leukemia, hystiocytic leukemia, monocytic leukemia (e.g., acute
monocytic leukemia), leukemic reticulosis, Shilling Type monocytic
leukemia, and/or other leukemias derived from monocytes and/or
monocytic cells and/or tissues.
[0700] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate monocytic leukemoid reaction, as seen,
for example, with tuberculosis.
[0701] In another specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent,
diagnose, and/or ameliorate monocytic leukocytosis, monocytic
leukopenia, monocytopenia, and/or monocytosis.
[0702] In a specific embodiment, Neutrokine-alpha, and
Neutrokine-alphaSV polynucleotides or polypeptides of the
invention, and/or anti-Neutrokine-alpha antibodies and/or agonists
or antagonists thereof, are used to treat, prevent, detect, and/or
diagnose primary B lymphocyte disorders and/or diseases, and/or
conditions associated therewith. In one embodiment, such primary B
lymphocyte disorders, diseases, and/or conditions are characterized
by a complete or partial loss of humoral immunity. Primary B
lymphocyte disorders, diseases, and/or conditions associated
therewith that are characterized by a complete or partial loss of
humoral immunity and that may be prevented, treated, detected
and/or diagnosed with compositions of the invention include, but
are not limited to, X-Linked Agammaglobulinemia (XLA), severe
combined immunodeficiency disease (SCID), and selective IgA
deficiency.
[0703] In a preferred embodiment, Neutrokine-alpha and
Neutrokine-alphaSV polynucleotides, polypeptides, and/or agonists
and/or antagonists thereof are used to treat, prevent, and/or
diagnose diseases or disorders affecting or conditions associated
with any one or more of the various mucous membranes of the body.
Such diseases or disorders include, but are not limited to, for
example, mucositis, mucoclasis, mucocolitis, mucocutaneous
leishmaniasis (such as, for example, American leishmaniasis,
leishmaniasis americana, nasopharyngeal leishmaniasis, and New
World leishmaniasis), mucocutaneous lymph node syndrome (for
example, Kawasaki disease), mucoenteritis, mucoepidermoid
carcinoma, mucoepidermoid tumor, mucoepithelial dysplasia, mucoid
adenocarcinoma, mucoid degeneration, myxoid degeneration;
myxomatous degeneration; myxomatosis, mucoid medial degeneration
(for example, cystic medial necrosis), mucolipidosis (including,
for example, mucolipidosis I, mucolipidosis II, mucolipidosis III,
and mucolipidosis IV), mucolysis disorders, mucomembranous
enteritis, mucoenteritis, mucopolysaccharidosis (such as, for
example, type I mucopolysaccharidosis (i.e., Hurler's syndrome),
type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type V
mucopolysaccharidosis), type II mucopolysaccharidosis (i.e.,
Hunter's syndrome), type III mucopolysaccharidosis (i.e.,
Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e.,
Morquio's syndrome), type VI mucopolysaccharidosis (i.e.,
Maroteaux-Lamy syndrome), type VII mucopolysaccharidosis (i.e,
mucopolysaccharidosis due to beta-glucuronidase deficiency), and
mucosulfatidosis), mucopolysacchariduria, mucopurulent
conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis), mucosal
disease (i.e., bovine virus diarrhea), mucous colitis (such as, for
example, mucocolitis and myxomembranous colitis), and
mucoviscidosis (such as, for example, cystic fibrosis, cystic
fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic
disease of the pancreas, mucoviscidosis, and viscidosis). In a
highly preferred embodiment, Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides, polypeptides, and/or agonists
and/or antagonists thereof are used to treat, prevent, and/or
diagnose mucositis, especially as associated with chemotherapy.
[0704] In a preferred embodiment, Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides, polypeptides, and/or agonists
and/or antagonists thereof are used to treat, prevent, and/or
diagnose diseases or disorders affecting or conditions associated
with sinusitis.
[0705] An additional condition, disease or symptom that can be
treated, prevented, and/or diagnosed by Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or agonists of
Neutrokine-alpha, and/or Neutrokine-alphaSV, is osteomyelitis.
[0706] An additional condition, disease or symptom that can be
treated, prevented, and/or diagnosed by Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or agonists of
Neutrokine-alpha, and/or Neutrokine-alphaSV, is endocarditis.
[0707] All of the above described applications as they may apply to
veterinary medicine.
[0708] Antagonists of Neutrokine-alpha include binding and/or
inhibitory antibodies, antisense nucleic acids, ribozymes, and
Neutrokine-alpha polypeptides of the invention. These would be
expected to reverse many of the activities of the ligand described
above as well as find clinical or practical application as:
[0709] A means of blocking various aspects of immune responses to
foreign agents or self. Examples include autoimmune disorders such
as lupus, and arthritis, as well as immunoresponsiveness to skin
allergies, inflammation, bowel disease, injury and pathogens.
Although our current data speaks directly to the potential role of
Neutrokine-alpha in B cell and monocyte related pathologies, it
remains possible that other cell types may gain expression or
responsiveness to Neutrokine-alpha. Thus, Neutrokine-alpha may,
like CD40 and its ligand, be regulated by the status of the immune
system and the microenvironment in which the cell is located.
[0710] A therapy for preventing the B cell proliferation and Ig
secretion associated with autoimmune diseases such as idiopathic
thrombocytopenic purpura, systemic lupus erythematosus and MS.
[0711] An inhibitor of graft versus host disease or transplant
rejection.
[0712] A therapy for B cell malignancies such as ALL, Hodgkins
disease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia,
plasmacytomas, multiple myeloma, Burkitt's lymphoma, and
EBV-transformed diseases.
[0713] A therapy for chronic hypergammaglobulinemeia evident in
such diseases as monoclonalgammopathy of undetermined significance
(MGUS), Waldenstrom's disease, related idiopathic
monoclonalgammopathies, and plasmacytomas.
[0714] A therapy for decreasing cellular proliferation of Large
B-cell Lymphomas.
[0715] A means of decreasing the involvement of B cells and Ig
associated with Chronic Myelogenous Leukemia.
[0716] An immunosuppressive agent(s).
[0717] Neutrokine-alpha or Neutrokine-alphaSV polypeptides or
polynucleotides of the invention, or antagonists may be used to
modulate IgE concentrations in vitro or in vivo.
[0718] In another embodiment, administration of Neutrokine-alpha or
Neutrokine-alphaSV polypeptides or polynucleotides of the
invention, or antagonists thereof, may be used to treat, prevent,
and/or diagnose IgE-mediated allergic reactions including, but not
limited to, asthma, rhinitis, and eczema.
[0719] An inhibitor of signaling pathways involving ERK1, COX2 and
Cyclin D2 which have been associated with Neutrokine-alpha induced
B cell activation.
[0720] The above-recited applications have uses in a wide variety
of hosts. Such hosts include, but are not limited to, human,
murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat,
non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig,
sheep, dog or cat. In preferred embodiments, the host is a mammal.
In most preferred embodiments, the host is a human.
[0721] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described herein.
[0722] The antagonists may be employed for instance to inhibit
Neutrokine-alpha-mediated and/or Neutrokine-alphaSV-mediated
chemotaxis and activation of macrophages and their precursors, and
of neutrophils, basophils, B lymphocytes and some T-cell subsets,
e.g., activated and CD8 cytotoxic T cells and natural killer cells,
in certain auto-immune and chronic inflammatory and infective
diseases. Examples of auto-immune diseases include multiple
sclerosis, and insulin-dependent diabetes. The antagonists may also
be employed to treat, prevent, and/or diagnose infectious diseases
including silicosis, sarcoidosis, idiopathic pulmonary fibrosis by
preventing the recruitment and activation of mononuclear
phagocytes. They may also be employed to treat, prevent, and/or
diagnose idiopathic hyper-eosinophilic syndrome by preventing
eosinophil production and migration. Endotoxic shock may also be
treated by the antagonists by preventing the migration of
macrophages and their production of the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the present invention. The
antagonists may also be employed for treating atherosclerosis, by
preventing monocyte infiltration in the artery wall. The
antagonists may also be employed to treat, prevent, and/or diagnose
histamine-mediated allergic reactions and immunological disorders
including late phase allergic reactions, chronic urticaria, and
atopic dermatitis by inhibiting chemokine-induced mast cell and
basophil degranulation and release of histamine. IgE-mediated
allergic reactions such as allergic asthma, rhinitis, and eczema
may also be treated. The antagonists may also be employed to treat,
prevent, and/or diagnose chronic and acute inflammation by
preventing the attraction of monocytes to a wound area. They may
also be employed to regulate normal pulmonary macrophage
populations, since chronic and acute inflammatory pulmonary
diseases are associated with sequestration of mononuclear
phagocytes in the lung. Antagonists may also be employed to treat,
prevent, and/or diagnose rheumatoid arthritis by preventing the
attraction of monocytes into synovial fluid in the joints of
patients. Monocyte influx and activation plays a significant role
in the pathogenesis of both degenerative and inflammatory
arthropathies. The antagonists may be employed to interfere with
the deleterious cascades attributed primarily to IL-1 and TNF,
which prevents the biosynthesis of other inflammatory cytokines. In
this way, the antagonists may be employed to prevent inflammation.
The antagonists may also be employed to inhibit
prostaglandin-independent fever induced by Neutrokine-alpha and/or
Neutrokine-alphaSV. The antagonists may also be employed to treat,
prevent, and/or diagnose cases of bone marrow failure, for example,
aplastic anemia and myelodysplastic syndrome. The antagonists may
also be employed to treat, prevent, and/or diagnose asthma and
allergy by preventing eosinophil accumulation in the lung. The
antagonists may also be employed to treat, prevent, and/or diagnose
subepithelial basement membrane fibrosis which is a prominent
feature of the asthmatic lung. The antagonists may also be employed
to treat, prevent, and/or diagnose lymphomas (e.g., one or more of
the extensive, but not limiting, list of lymphomas provided
herein).
[0723] All of the above described applications as they may apply to
veterinary medicine. Moreover, all applications described herein
may also apply to veterinary medicine.
[0724] Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides
or polypeptides of the invention and/or agonists and/or antagonists
thereof, may be used to treat, prevent, and/or diagnose various
immune system-related disorders and/or conditions associated with
these disorders, in mammals, preferably humans. Many autoimmune
disorders result from inappropriate recognition of self as foreign
material by immune cells. This inappropriate recognition results in
an immune response leading to the destruction of the host tissue.
Therefore, the administration of Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof that can inhibit an
immune response, particularly the proliferation of B cells and/or
the production of immunoglobulins, may be an effective therapy in
treating and/or preventing autoimmune disorders. Thus, in preferred
embodiments, Neutrokine-alpha and/or Neutrokine-alphaSV antagonists
of the invention (e.g., polypeptide fragments of Neutrokine-alpha
and/or Neutrokine-alphaSV and anti-Neutrokine-alpha antibodies) are
used to treat, prevent, and/or diagnose an autoimmune disorder.
[0725] Autoimmune disorders and conditions associated with these
disorders that may be treated, prevented, and/or diagnosed with the
Neutrokine-alpha polynucleotides, polypeptides, and/or antagonists
of the invention (e.g., anti-Neutrokine-alpha antibodies), include,
but are not limited to, autoimmune hemolytic anemia, autoimmune
neutropenia, autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,
myocarditis, relapsing polychondritis, rheumatic heart disease,
glomerulonephritis (e.g, IgA nephropathy), dense deposit disease,
Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary
Inflammation, Guillain-Barre Syndrome, gluten sensitive
enteropathy, insulin dependent diabetes mellitis, discoid lupus,
and autoimmune inflammatory eye disease.
[0726] Additional autoimmune disorders (that are highly probable)
that may be treated, prevented, and/or diagnosed with the
compositions of the invention include, but are not limited to,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis) (often characterized, e.g., by cell-mediated and
humoral thyroid cytotoxicity), systemic lupus erythematosus (often
characterized, e.g., by circulating and locally generated immune
complexes), Goodpasture's syndrome (often characterized, e.g., by
anti-basement membrane antibodies), Pemphigus (often characterized,
e.g., by epidermal acantholytic antibodies), Receptor
autoimmunities such as, for example, (a) Graves' Disease (often
characterized, e.g., by TSH receptor antibodies), (b) Myasthenia
Gravis (often characterized, e.g., by acetylcholine receptor
antibodies), and (c) insulin resistance (often characterized, e.g.,
by insulin receptor antibodies), autoimmune hemolytic anemia (often
characterized, e.g., by phagocytosis of antibody-sensitized RBCs),
autoimmune thrombocytopenic purpura (often characterized, e.g., by
phagocytosis of antibody-sensitized platelets.
[0727] Additional autoimmune disorders (that are probable) that may
be treated, prevented, and/or diagnosed with the compositions of
the invention include, but are not limited to, rheumatoid arthritis
(often characterized, e.g., by immune complexes in joints),
scleroderma with anti-collagen antibodies (often characterized,
e.g., by nucleolar and other nuclear antibodies), mixed connective
tissue disease (often characterized, e.g., by antibodies to
extractable nuclear antigens (e.g., ribonucleoprotein)),
polymyositis/dermatomyositis (often characterized, e.g., by
nonhistone ANA), pernicious anemia (often characterized, e.g., by
antiparietal cell, microsomes, and intrinsic factor antibodies),
idiopathic Addison's disease (often characterized, e.g., by humoral
and cell-mediated adrenal cytotoxicity, infertility (often
characterized, e.g., by antispermatozoal antibodies),
glomerulonephritis (often characterized, e.g., by glomerular
basement membrane antibodies or immune complexes) such as primary
glomerulonephritis and IgA nephropathy, bullous pemphigoid (often
characterized, e.g., by IgG and complement in basement membrane),
Sjogren's syndrome (often characterized, e.g., by multiple tissue
antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes
millitus (often characterized, e.g., by cell-mediated and humoral
islet cell antibodies), and adrenergic drug resistance (including
adrenergic drug resistance with asthma or cystic fibrosis) (often
characterized, e.g., by beta-adrenergic receptor antibodies).
[0728] Additional autoimmune disorders (that are possible) that may
be treated, prevented, and/or diagnosed with the compositions of
the invention include, but are not limited to, chronic active
hepatitis (often characterized, e.g., by smooth muscle antibodies),
primary biliary cirrhosis (often characterized, e.g., by
mitchondrial antibodies), other endocrine gland failure (often
characterized, e.g., by specific tissue antibodies in some cases),
vitiligo (often characterized, e.g., by melanocyte antibodies),
vasculitis (often characterized, e.g., by Ig and complement in
vessel walls and/or low serum complement), post-MI (often
characterized, e.g., by myocardial antibodies), cardiotomy syndrome
(often characterized, e.g., by myocardial antibodies), urticaria
(often characterized, e.g., by IgG and IgM antibodies to IgE),
atopic dermatitis (often characterized, e.g., by IgG and IgM
antibodies to IgE), asthma (often characterized, e.g., by IgG and
IgM antibodies to IgE), inflammatory myopathies, and many other
inflammatory, granulamatous, degenerative, and atrophic
disorders.
[0729] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, and/or diagnosed
using anti-Neutrokine-alpha antibodies and/or
anti-Neutrokine-alphaSV.
[0730] In a specific preferred embodiment, rheumatoid arthritis is
treated, prevented, and/or diagnosed using anti-Neutrokine-alpha
antibodies and/or anti-Neutrokine-alphaSV antibodies and/or other
antagonist of the invention.
[0731] In a specific preferred embodiment, lupus is treated,
prevented, and/or diagnosed using anti-Neutrokine-alpha antibodies
and/or anti-Neutrokine-alphaSV antibodies and/or other antagonist
of the invention.
[0732] In a specific preferred embodiment, Sjogren's Syndrome is
treated, prevented, and/or diagnosed using anti-Neutrokine-alpha
antibodies and/or anti-Neutrokine-alphaSV antibodies and/or other
antagonist of the invention.
[0733] In a specific preferred embodiment, AIDS is treated,
prevented, and/or diagnosed using anti-Neutrokine-alpha antibodies
and/or anti-Neutrokine-alphaSV antibodies and/or other antagonist
of the invention.
[0734] In a specific preferred embodiment, HIV infection is
treated, prevented, and/or diagnosed using anti-Neutrokine-alpha
antibodies and/or anti-Neutrokine-alphaSV antibodies and/or other
antagonist of the invention.
[0735] In a specific preferred embodiment, nephritis associated
with lupus is treated, prevented, and/or diagnosed using
anti-Neutrokine-alpha antibodies and/or anti-Neutrokine-alphaSV
antibodies and/or other antagonist of the invention.
[0736] In a specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or antagonists
thereof (e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alphaSV
antibodies) are used to treat or prevent systemic lupus
erythematosus and/or diseases, disorders or conditions associated
therewith. Lupus-associated diseases, disorders, or conditions that
may be treated or prevented with Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides, or antagonists
of the invention, include, but are not limited to, hematologic
disorders (e.g., hemolytic anemia, leukopenia, lymphopenia, and
thrombocytopenia), immunologic disorders (e.g., anti-DNA
antibodies, and anti-Sm antibodies), rashes, photosensitivity, oral
ulcers, arthritis, fever, fatigue, weight loss, serositis (e.g.,
pleuritus (pleuricy)), renal disorders (e.g., nephritis),
neurological disorders (e.g., seizures, peripheral neuropathy, CNS
related disorders), gastroinstestinal disorders, Raynaud
phenomenon, and pericarditis. In a preferred embodiment, the
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or
polypeptides, or antagonists thereof (e.g., anti-Neutrokine-alpha
and/or anti-Neutrokine-alphaSV antibodies) are used to treat or
prevent renal disorders associated with systemic lupus
erythematosus. In a most preferred embodiment, Neutrokine-alpha
and/or Neutrokine-alphaSV polynucleotides or polypeptides, or
antagonists thereof (e.g., anti-Neutrokine-alpha and/or
anti-Neutrokine-alphaSV antibodies) are used to treat or prevent
nephritis associated with systemic lupus erythematosus.
[0737] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention and/or agonists
and/or antagonists thereof. Moreover, these molecules can be used
to treat, prevent, and/or diagnose anaphylaxis, hypersensitivity to
an antigenic molecule, or blood group incompatibility.
[0738] Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides
or polypeptides of the invention and/or agonists and/or antagonists
thereof, may also be used to treat, prevent, and/or diagnose organ
rejection or graft-versus-host disease (GVHD) and/or conditions
associated therewith. Organ rejection occurs by host immune cell
destruction of the transplanted tissue through an immune response.
Similarly, an immune response is also involved in GVHD, but, in
this case, the foreign transplanted immune cells destroy the host
tissues. The administration of Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, that inhibits an immune
response, particularly the proliferation, differentiation, or
chemotaxis of T-cells, may be an effective therapy in preventing
organ rejection or GVHD.
[0739] Similarly, Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention and/or agonists
and/or antagonists thereof, may also be used to modulate
inflammation. For example, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, may inhibit the
proliferation and differentiation of cells involved in an
inflammatory response. These molecules can be used to treat,
prevent, and/or diagnose inflammatory conditions, both chronic and
acute conditions, including chronic prostatitis, granulomatous
prostatitis and malacoplakia, inflammation associated with
infection (e.g., septic shock, sepsis, or systemic inflammatory
response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated hyperacute rejection,
nephritis, cytokine or chemokine induced lung injury, inflammatory
bowel disease, Crohn's disease, or resulting from over production
of cytokines (e.g., TNF or IL-1.)
[0740] In a specific embodiment, anti-Neutrokine-alpha antibodies
and/or anti-Neutrokine-alphaSV antibodies of the invention are used
to treat, prevent, modulate, detect, and/or diagnose
inflammation.
[0741] In a specific embodiment, anti-Neutrokine-alpha antibodies
and/or anti-Neutrokine-alphaSV antibodies of the invention are used
to treat, prevent, modulate, detect, and/or diagnose inflamatory
disorders.
[0742] In another specific embodiment, anti-Neutrokine-alpha
antibodies and/or anti-Neutrokine-alphaSV antibodies of the
invention are used to treat, prevent, modulate, detect, and/or
diagnose allergy and/or hypersensitivity.
[0743] In another embodiment, therapeutic or pharmaceutical
compositions of the invention (e.g., Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or antagonists thereof) are administered to an animal to treat,
prevent or ameliorate ischemia and arteriosclerosis. Examples of
such disorders include, but are not limited to, reperfusion damage
(e.g., in the heart and/or brain) and cardiac hypertrophy.
[0744] Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides
or polypeptides of the invention and/or antagonists thereof, may
also be used to modulate blood clotting and to treat or prevent
blood clotting disorders, such as, for example, antibody-mediated
thrombosis (i.e., antiphospholipid antibody syndrome (APS)). For
example, Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides
or polypeptides of the invention and/or antagonists thereof, may
inhibit the proliferation and differentiation of cells involved in
producing anticardiolipin antibodies. These compositions of the
invention can be used to treat, prevent, and/or diagnose,
thrombotic related events including, but not limited to, stroke
(and recurrent stroke), heart attack, deep vein thrombosis,
pulmonary embolism, myocardial infarction, coronary artery disease
(e.g., antibody-mediated coronary artery disease), thrombosis,
graft reocclusion following cardiovascular surgery (e.g., coronary
arterial bypass grafts, recurrent fetal loss, and recurrent
cardiovascular thromboembolic events.
[0745] Antibodies against Neutrokine-alpha and/or
Neutrokine-alphaSV may be employed to bind to and inhibit
Neutrokine-alpha and/or Neutrokine-alphaSV activity to treat,
prevent, and/or diagnose ARDS, by preventing infiltration of
neutrophils into the lung after injury. The agonists and
antagonists of the instant may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as described
hereinafter.
[0746] Neutrokine-alpha and/or Neutrokine-alphaSV and/or
Neutrokine-alpha receptor polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof, are used to
treat, prevent, and/or diagnose diseases and disorders of the
pulmonary system (e.g., bronchi such as, for example, sinopulmonary
and bronchial infections and conditions associated with such
diseases and disorders and other respiratory diseases and
disorders. In specific embodiments, such diseases and disorders
include, but are not limited to, bronchial adenoma, bronchial
asthma, pneumonia (such as, e.g., bronchial pneumonia,
bronchopneumonia, and tuberculous bronchopneumonia), chronic
obstructive pulmonary disease (COPD), bronchial polyps,
bronchiectasia (such as, e.g., bronchiectasia sicca, cylindrical
bronchiectasis, and saccular bronchiectasis), bronchiolar
adenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as,
e.g., exudative bronchiolitis, bronchiolitis fibrosa obliterans,
and proliferative bronchiolitis), bronchiolo-alveolar carcinoma,
bronchitic asthma, bronchitis (such as, e.g., asthmatic bronchitis,
Castellani's bronchitis, chronic bronchitis, croupous bronchitis,
fibrinous bronchitis, hemorrhagic bronchitis, infectious avian
bronchitis, obliterative bronchitis, plastic bronchitis,
pseudomembranous bronchitis, putrid bronchitis, and verminous
bronchitis), bronchocentric granulomatosis, bronchoedema,
bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic
cyst, broncholithiasis, bronchomalacia, bronchomycosis (such as,
e.g., bronchopulmonary aspergillosis), bronchopulmonary
spirochetosis, hemorrhagic bronchitis, bronchorrhea, bronchospasm,
bronchostaxis, bronchostenosis, Biot's respiration, bronchial
respiration, Kussmaul respiration, Kussmaul-Kien respiration,
respiratory acidosis, respiratory alkalosis, respiratory distress
syndrome of the newborn, respiratory insufficiency, respiratory
scleroma, respiratory syncytial virus, and the like.
[0747] In a specific embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, are used to treat,
prevent, and/or diagnose chronic obstructive pulmonary disease
(COPD).
[0748] In another embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, are used to treat,
prevent, and/or diagnose fibroses and conditions associated with
fibroses, such as, for example, but not limited to, cystic fibrosis
(including such fibroses as cystic fibrosis of the pancreas,
Clarke-Hadfield syndrome, fibrocystic disease of the pancreas,
mucoviscidosis, and viscidosis), endomyocardial fibrosis,
idiopathic retroperitoneal fibrosis, leptomeningeal fibrosis,
mediastinal fibrosis, nodular subepidermal fibrosis, pericentral
fibrosis, perimuscular fibrosis, pipestem fibrosis, replacement
fibrosis, subadventitial fibrosis, and Symmers' clay pipestem
fibrosis.
[0749] The TNF family ligands are known to be among the most
pleiotropic cytokines, inducing a large number of cellular
responses, including cytotoxicity, anti-viral activity,
immunoregulatory activities, and the transcriptional regulation of
several genes (D. V. Goeddel et al., "Tumor Necrosis Factors: Gene
Structure and Biological Activities," Symp. Quant. Biol. 51:597-609
(1986), Cold Spring Harbor; B. Beutler and A. Cerami, Annu. Rev.
Biochem. 57:505-518 (1988); L. J. Old, Sci. Am. 258:59-75 (1988);
W. Fiers, FEBS Lett. 285:199-224 (1991)). The TNF-family ligands,
including Neutrokine-alpha and/or Neutrokine-alphaSV of the present
invention, induce such various cellular responses by binding to
TNF-family receptors. Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides are believed to elicit a potent cellular response
including any genotypic, phenotypic, and/or morphologic change to
the cell, cell line, tissue, tissue culture or patient. As
indicated, such cellular responses include not only normal
physiological responses to TNF-family ligands, but also diseases
associated with increased apoptosis or the inhibition of apoptosis.
Apoptosis-programmed cell death is a physiological mechanism
involved in the deletion of peripheral B and/or T lymphocytes of
the immune system, and its disregulation can lead to a number of
different pathogenic processes (J. C. Ameisen, AIDS 8:1197-1213
(1994); P. H. Krammer et al., Curr. Opin. Immunol. 6:279-289
(1994)).
[0750] Diseases associated with increased cell survival, or the
inhibition of apoptosis that may be diagnosed, treated, or
prevented with the Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention, and agonists and
antagonists thereof, include cancers (such as follicular lymphomas,
carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to, colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders (such as systemic lupus
erythematosus and immune-related glomerulonephritis rheumatoid
arthritis); viral infections (such as herpes viruses, pox viruses
and adenoviruses); inflammation; graft vs. host disease; acute
graft rejection and chronic graft rejection. Thus, in preferred
embodiments Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention and/or agonists or
antagonists thereof, are used to treat, prevent, and/or diagnose
autoimmune diseases and/or inhibit the growth, progression, and/or
metastasis of cancers, including, but not limited to, those cancers
disclosed herein, such as, for example, lymphocytic leukemias
(including, for example, MLL and chronic lymphocytic leukemia
(CLL)) and follicular lymphomas. In another embodiment
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or
polypeptides of the invention are used to activate, differentiate
or proliferate cancerous cells or tissue (e.g., B cell lineage
related cancers (e.g., CLL and MLL), lymphocytic leukemia, or
lymphoma) and thereby render the cells more vulnerable to cancer
therapy (e.g., chemotherapy or radiation therapy).
[0751] Moreover, in other embodiments, Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
or agonists or antagonists thereof, are used to inhibit the growth,
progression, and/or metastases of malignancies and related
disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0752] In specific embodiments Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
are used to inhibit the growth, progression, and/or metastases of
multiple myeloma. In even more specific embodiments, radiolabeled
Neutrokine-alpha polypeptides comprising, or alternatively
consisting of amino acids 134-285 of SEQ ID NO:2 (e.g.,
radiolabeled Neutrokine alpha trimers comprising three polypeptide
chains consisting of amino acids 134-285 of SEQ ID NO:2) are used
to inhibit the growth, progression, and/or metastases of multiple
myeloma. In particular embodiments, the radiolabeled
Neutrokine-alpha polypeptides are radiolabeled with an .sup.131I
radioisotope.
[0753] In specific embodiments Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
are used to inhibit the growth, progression, and/or metastases of
non-Hodgkin's lymphoma. In even more specific embodiments,
radiolabeled Neutrokine-alpha polypeptides comprising, or
alternatively consisting of amino acids 134-285 of SEQ ID NO:2
(e.g., radiolabeled Neutrokine alpha trimers comprising three
polypeptide chains consisting of amino acids 134-285 of SEQ ID
NO:2) are used to inhibit the growth, progression, and/or
metastases of non-Hodgkin's lymphoma. In particular embodiments,
the radiolabeled Neutrokine-alpha polypeptides are radiolabeled
with an .sup.131I radioisotope.
[0754] In specific embodiments Neutrokine-alpha and/or
Neutrokine-alphaSV polynucleotides or polypeptides of the invention
are used to inhibit the growth, progression, and/or metastases of
chronic lymphocytic leukemia (CLL). In even more specific
embodiments, radiolabeled Neutrokine-alpha polypeptides comprising,
or alternatively consisting of amino acids 134-285 of SEQ ID NO:2
(e.g., radiolabeled Neutrokine alpha trimers comprising three
polypeptide chains consisting of amino acids 134-285 of SEQ ID
NO:2) are used to inhibit the growth, progression, and/or
metastases of CLL. In particular embodiments, the radiolabeled
Neutrokine-alpha polypeptides are radiolabeled with an .sup.131I
radioisotope.
[0755] Diseases associated with increased apoptosis apoptosis that
may be diagnosed, treated, or prevented with the Neutrokine-alpha
and/or Neutrokine-alphaSV polynucleotides or polypeptides of the
invention, and agonists and antagonists thereof, include AIDS;
neurodegenerative disorders (such as Alzheimer's disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis
pigmentosa, Cerebellar degeneration); myelodysplastic syndromes
(such as aplastic anemia), ischemic injury (such as that caused by
myocardial infarction, stroke and reperfusion injury),
toxin-induced liver disease (such as that caused by alcohol),
septic shock, cachexia and anorexia. Thus, in preferred embodiments
Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or
polypeptides of the invention and/or agonists or antagonists
thereof, are used to treat, prevent, and/or diagnose the diseases
and disorders listed above.
[0756] In preferred embodiments, Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention and/or agonists or
antagonists thereof (e.g., anti-Neutrokine-alpha antibodies)
inhibit the growth of human histiocytic lymphoma U-937 cells in a
dose-dependent manner. In additional preferred embodiments,
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the
invention and/or agonists or antagonists thereof (e.g.,
anti-Neutrokine-alpha antibodies) inhibit the growth of PC-3 cells,
HT-29 cells, HeLa cells, MCF-7 cells, and A293 cells. In highly
preferred embodiments, Neutrokine-alpha and/or Neutrokine-alphaSV
polynucleotides or polypeptides of the invention and/or agonists or
antagonists thereof (e.g., anti-Neutrokine-alpha antibodies) are
used to inhibit growth, progression, and/or metastasis of prostate
cancer, colon cancer, cervical carcinoma, and breast carcinoma.
[0757] Thus, in additional preferred embodiments, the present
invention is directed to a method for enhancing apoptosis induced
by a TNF-family ligand, which involves administering to a cell
which expresses a Neutrokine-alpha and/or Neutrokine-alphaSV
receptor an effective amount of Neutrokine-alpha and/or
Neutrokine-alphaSV, or an agonist or antagonist thereof, capable of
increasing or decreasing Neutrokine-alpha and/or Neutrokine-alphaSV
mediated signaling. Preferably, Neutrokine-alpha and/or
Neutrokine-alphaSV mediated signaling is increased or decreased to
treat, prevent, and/or diagnose a disease wherein decreased
apoptosis or decreased cytokine and adhesion molecule expression is
exhibited. An agonist or antagonist can include soluble forms of
Neutrokine-alpha and/or Neutrokine-alphaSV and monoclonal
antibodies directed against the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide.
[0758] In a further aspect, the present invention is directed to a
method for inhibiting apoptosis induced by a TNF-family ligand,
which involves administering to a cell which expresses the
Neutrokine-alpha and/or Neutrokine-alphaSV receptor an effective
amount of an agonist or antagonist capable of increasing or
decreasing Neutrokine-alpha and/or Neutrokine-alphaSV mediated
signaling. Preferably, Neutrokine-alpha and/or Neutrokine-alphaSV
mediated signaling is increased or decreased to treat, prevent,
and/or diagnose a disease wherein increased apoptosis or NF-kappaB
expression is exhibited. An agonist or antagonist can include
soluble forms of Neutrokine-alpha and/or Neutrokine-alphaSV and
monoclonal antibodies directed against the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide.
[0759] Because Neutrokine-alpha and/or Neutrokine-alphaSV belong to
the TNF superfamily, the polypeptides should also modulate
angiogenesis. In addition, since Neutrokine-alpha and/or
Neutrokine-alphaSV inhibit immune cell functions, the polypeptides
will have a wide range of anti-inflammatory activities.
Neutrokine-alpha and/or Neutrokine-alphaSV may be employed as an
anti-neovascularizing agent to treat, prevent, and/or diagnose
solid tumors by stimulating the invasion and activation of host
defense cells, e.g., cytotoxic T cells and macrophages and by
inhibiting the angiogenesis of tumors. Those of skill in the art
will recognize other non-cancer indications where blood vessel
proliferation is not wanted. They may also be employed to enhance
host defenses against resistant chronic and acute infections, for
example, myobacterial infections via the attraction and activation
of microbicidal leukocytes. Neutrokine-alpha and/or
Neutrokine-alphaSV may also be employed to inhibit T-cell
proliferation by the inhibition of IL-2 biosynthesis for the
treatment of T-cell mediated auto-immune diseases and lymphocytic
leukemias (including, for example, chronic lymphocytic leukemia
(CLL)). Neutrokine-alpha and/or Neutrokine-alphaSV may also be
employed to stimulate wound healing, both via the recruitment of
debris clearing and connective tissue promoting inflammatory cells.
In this same manner, Neutrokine-alpha and/or Neutrokine-alphaSV may
also be employed to treat, prevent, and/or diagnose other fibrotic
disorders, including liver cirrhosis, osteoarthritis and pulmonary
fibrosis. Neutrokine-alpha and/or Neutrokine-alphaSV also increases
the presence of eosinophils that have the distinctive function of
killing the larvae of parasites that invade tissues, as in
schistosomiasis, trichinosis and ascariasis. It may also be
employed to regulate hematopoiesis, by regulating the activation
and differentiation of various hematopoietic progenitor cells, for
example, to release mature leukocytes from the bone marrow
following chemotherapy, i.e., in stem cell mobilization.
Neutrokine-alpha and/or Neutrokine-alphaSV may also be employed to
treat, prevent, and/or diagnose sepsis.
[0760] Polynucleotides and/or polypeptides of the invention and/or
agonists and/or antagonists thereof are useful in the diagnosis and
treatment or prevention of a wide range of diseases and/or
conditions. Such diseases and conditions include, but are not
limited to, cancer (e.g., immune cell related cancers, breast
cancer, prostate cancer, ovarian cancer, follicular lymphoma,
cancer associated with mutation or alteration of p53, brain tumor,
bladder cancer, uterocervical cancer, colon cancer, colorectal
cancer, non-small cell carcinoma of the lung, small cell carcinoma
of the lung, stomach cancer, etc.), lymphoproliferative disorders
(e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.)
infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirus
infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV,
HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human
papilloma virus infection, hepatitis infection (e.g., HAV, HBV,
HCV, etc.), Helicobacter pylori infection, invasive Staphylococcia,
etc.), parasitic infection, nephritis, bone disease (e.g.,
osteoporosis), atherosclerosis, pain, cardiovascular disorders
(e.g., neovascularization, hypovascularization or reduced
circulation (e.g., ischemic disease (e.g., myocardial infarction,
stroke, etc.)), AIDS, allergy, inflammation, neurodegenerative
disease (e.g., Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar
degeneration, etc.), graft rejection (acute and chronic), graft vs.
host disease, diseases due to osteomyelodysplasia (e.g., aplastic
anemia, etc.), joint tissue destruction in rheumatism, liver
disease (e.g., acute and chronic hepatitis, liver injury, and
cirrhosis), autoimmune disease (e.g., multiple sclerosis,
rheumatoid arthritis, systemic lupus erythematosus, immune complex
glomerulonephritis, autoimmune diabetes, autoimmune
thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis,
etc.), cardiomyopathy (e.g., dilated cardiomyopathy), diabetes,
diabetic complications (e.g., diabetic nephropathy, diabetic
neuropathy, diabetic retinopathy), influenza, asthma, psoriasis,
glomerulonephritis, septic shock, and ulcerative colitis.
[0761] In specific embodiments, polynucleotides and/or polypeptides
of the invention and/or agonists and/or antagonists thereof are
useful in the diagnosis and treatment or prevention of dilated
cardiomyopathy. In specific embodiments, antagonistic
anti-neutrokine-alpha antibodies are useful in the diagnosis and
treatment or prevention of dilated cardiomyopathy. In other
specific embodiments, Neutrokine-alpha polypeptides and/or
Neutrokine-alpha SV polypeptides conjugated to or otherwise
associated with a cytotoxic prodrug or toxin may be used to
treatment or prevention of dilated cardiomyopathy.
[0762] In specific embodiments, polynucleotides and/or polypeptides
of the invention and/or agonists and/or antagonists thereof are
useful in the diagnosis and treatment or prevention of diseases
involving increased hypergammaglobulinemia or elevated autoantibody
titers. In specific embodiments, antagonists of Neutrokine-alpha,
such as an anti-Neutrokine-alpha specific antibody, are useful in
the diagnosis and treatment or prevention of diseases involving
increased hypergammaglobulinemia or elevated autoantibody
titers.
[0763] In specific embodiments, polynucleotides and/or polypeptides
of the invention and/or agonists and/or antagonists thereof are
useful in the diagnosis and treatment or prevention of liver
diseases, including but not limited to, primary biliary cirrhosis,
primary sclerosing cholangitis, chronic hepatitis C infection,
autoimmune hepatitis and alcoholic liver disease. In specific
embodiments, antagonists of Neutrokine-alpha, such as an
anti-Neutrokine-alpha specific antibody, are useful in the
diagnosis and treatment or prevention of liver diseases, including
but not limited to, primary biliary cirrhosis, primary sclerosing
cholangitis, chronic hepatitis C infection, and alcoholic liver
disease.
[0764] Polynucleotides and/or polypeptides of the invention and/or
agonists and/or antagonists thereof are useful in promoting
angiogenesis, wound healing (e.g., wounds, burns, and bone
fractures). Polynucleotides and/or polypeptides of the invention
and/or agonists and/or antagonists thereof are also useful as an
adjuvant to enhance immune responsiveness to specific antigen,
anti-viral immune responses.
[0765] More generally, polynucleotides and/or polypeptides of the
invention and/or agonists and/or antagonists thereof are useful in
regulating (i.e., elevating or reducing) immune response. For
example, polynucleotides and/or polypeptides of the invention may
be useful in preparation or recovery from surgery, trauma,
radiation therapy, chemotherapy, and transplantation, or may be
used to boost immune response and/or recovery in the elderly and
immunocompromised individuals. Alternatively, polynucleotides
and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are useful as immunosuppressive agents, for
example in the treatment or prevention of autoimmune disorders. In
specific embodiments, polynucleotides and/or polypeptides of the
invention are used to treat or prevent chronic inflammatory,
allergic or autoimmune conditions, such as those described herein
or are otherwise known in the art.
[0766] Preferably, treatment using Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotides or polypeptides, and/or agonists
or antagonists of Neutrokine-alpha, and/or Neutrokine-alphaSV
(e.g., anti-Neutrokine-alpha antibody), could either be by
administering an effective amount of Neutrokine-alpha, and/or
Neutrokine-alphaSV polypeptide of the invention, or agonist or
antagonist thereof, to the patient, or by removing cells from the
patient, supplying the cells with Neutrokine-alpha, and/or
Neutrokine-alphaSV polynucleotide, and returning the engineered
cells to the patient (ex vivo therapy). Moreover, as further
discussed herein, the Neutrokine-alpha, and/or Neutrokine-alphaSV
polypeptide or polynucleotide can be used as an adjuvant in a
vaccine to raise an immune response against infectious disease.
Formulations and Administration
[0767] The Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
composition (preferably containing a polypeptide which is a soluble
form of the Neutrokine-alpha and/or Neutrokine-alphaSV
extracellular domains) will be formulated and dosed in a fashion
consistent with good medical practice, taking into account the
clinical condition of the individual patient (especially the side
effects of treatment with Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide alone), the site of delivery of the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide composition,
the method of administration, the scheduling of administration, and
other factors known to practitioners. The "effective amount" of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide for purposes
herein is thus determined by such considerations.
[0768] As a general proposition, the total pharmaceutically
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide administered parenterally per dose will be in the range
of about 1 microgram/kg/day to 10 mg/kg/day of patient body weight,
although, as noted above, this will be subject to therapeutic
discretion. More preferably, this dose is at least 0.01 mg/kg/day,
and most preferably for humans between about 0.01 and 1
mg/kg/day.
[0769] In another embodiment, the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide of the invention is administered to
a human at a dose between 0.0001 and 0.045 mg/kg/day, preferably,
at a dose between 0.0045 and 0.045 mg/kg/day, and more preferably,
at a dose of about 45 microgram/kg/day in humans; and at a dose of
about 3 mg/kg/day in mice.
[0770] If given continuously, the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide is typically administered at a dose
rate of about 1 microgram/kg/hour to about 50 micrograms/kg/hour,
either by 1-4 injections per day or by continuous subcutaneous
infusions, for example, using a mini-pump. An intravenous bag
solution may also be employed.
[0771] The length of treatment needed to observe changes and the
interval following treatment for responses to occur appears to vary
depending on the desired effect.
[0772] In a specific embodiment, the total pharmaceutically
effective amount of Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide administered parenterally per dose will be in the range
of about 0.1 microgram/kg/day to 45 micrograms/kg/day of patient
body weight, although, as noted above, this will be subject to
therapeutic discretion. More preferably, this dose is at least 0.1
microgram/kg/day, and most preferably for humans between about 0.01
and 50 micrograms/kg/day for the protein. Neutrokine-alpha and/or
Neutrokine-alphaSV may be administered as a continuous infusion,
multiple discreet injections per day (e.g., three or more times
daily, or twice daily), single injection per day, or as discreet
injections given intermitently (e.g., twice daily, once daily,
every other day, twice weekly, weekly, biweekly, monthly,
bimonthly, and quarterly). If given continuously, the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide is typically
administered at a dose rate of about 0.001 to 10 microgram/kg/hour
to about 50 micrograms/kg/hour, either by 1-4 injections per day or
by continuous subcutaneous infusions, for example, using a
mini-pump.
[0773] Effective dosages of the compositions of the present
invention to be administered may be determined through procedures
well known to those in the art which address such parameters as
biological half-life, bioavailability, and toxicity. Such
determination is well within the capability of those skilled in the
art, especially in light of the detailed disclosure provided
herein.
[0774] Bioexposure of an organism to Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide during therapy may also play an
important role in determining a therapeutically and/or
pharmacologically effective dosing regime. Variations of dosing
such as repeated administrations of a relatively low dose of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide for a
relatively long period of time may have an effect which is
therapeutically and/or pharmacologically distinguishable from that
achieved with repeated administrations of a relatively high dose of
Neutrokine-alpha and/or Neutrokine-alphaSV for a relatively short
period of time. See, for instance, the serum immunoglobulin level
experiments presented in Example 6.
[0775] Using the equivalent surface area dosage conversion factors
supplied by Freireich, E. J., et al. (Cancer Chemotherapy Reports
50(4):219-44 (1966)), one of ordinary skill in the art is able to
conveniently convert data obtained from the use of Neutrokine-alpha
and/or Neutrokine-alphaSV in a given experimental system into an
accurate estimation of a pharmaceutically effective amount of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide to be
administered per dose in another experimental system. Experimental
data obtained through the administration of Neutrokine-alpha in
mice (see, for instance, Example 6) may converted through the
conversion factors supplied by Freireich, et al., to accurate
estimates of pharmaceutically effective doses of Neutrokine-alpha
in rat, monkey, dog, and human. The following conversion table
(Table III) is a summary of the data provided by Freireich, et al.
Table III gives approximate factors for converting doses expressed
in terms of mg/kg from one species to an equivalent surface area
dose expressed as mg/kg in another species tabulated.
TABLE-US-00003 TABLE III Equivalent Surface Area Dosage Conversion
Factors. TO Mouse Rat Monkey Dog Human FROM (20 g) (150 g) (3.5 kg)
(8 kg) (60 kg) Mouse 1 1/2 1/4 1/6 1/12 Rat 2 1 1/2 1/4 1/7 Monkey
4 2 1 3/5 1/3 Dog 6 4 5/3 1 1/2 Human 12 7 3 2 1
[0776] Thus, for example, using the conversion factors provided in
Table III, a dose of 50 mg/kg in the mouse converts to an
appropriate dose of 12.5 mg/kg in the monkey because (50
mg/kg).times.(1/4)=12.5 mg/kg. As an additional example, doses of
0.02, 0.08, 0.8, 2, and 8 mg/kg in the mouse equate to effect doses
of 1.667 micrograms/kg, 6.67 micrograms/kg, 66.7 micrograms/kg,
166.7 micrograms/kg, and 0.667 mg/kg, respectively, in the
human.
[0777] In certain embodiments, administration of radiolabeled forms
of Neutrokine-alpha, Neutrokine-alphaSV, anti-Neutrokine-alpha
antibodies and/or anti-Neutrokine-alphaSV antibodies is
contemplated. The radiometric dosage to be applied can vary
substantially. The radiolabeled Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibody and/or
anti-Neutrokine-alphaSV antibody composition can be administered at
a dose of about 0.1 to about 100 mCi per 70 kg body weight. In
another embodiment, the radiolabeled Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibody and/or
anti-Neutrokine-alphaSV antibody composition can be administered at
a dose of about 0.1 to about 50 mCi per 70 kg body weight. In
another embodiment, the radiolabeled Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibody and/or
anti-Neutrokine-alphaSV antibody composition can be administered at
a dose of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60,
70, 80, 90 or 100 mCi per 70 kg body weight.
[0778] The radiolabeled Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibody and/or anti-Neutrokine-alphaSV
antibody composition can be administered at a dose of about 0.1 to
about 10 mCi/kg body weight. In another embodiment, the
radiolabeled Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibody and/or anti-Neutrokine-alphaSV
antibody composition can be administered at a dose of about 0.25 to
about 5 mCi/kg body weight. In specific embodiments, the
radiolabeled Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibody and/or anti-Neutrokine-alphaSV
antibody composition can be administered at a dose of about 0.35,
0.70, 1.35, 1.70, 2.0, 2.5 or 3.0 mCi/kg.
[0779] The radiolabeled Neutrokine-alpha, Neutrokine-alphaSV,
anti-Neutrokine-alpha antibody and/or anti-Neutrokine-alphaSV
antibody composition can be administered at a dose of about 1 to
about 50 mCi/m.sup.2. In another embodiment, the radiolabeled
Neutrokine-alpha, Neutrokine-alphaSV, anti-Neutrokine-alpha
antibody and/or anti-Neutrokine-alphaSV antibody composition can be
administered at a dose of about 10 to about 30 mCi/m.sup.2. In
specific embodiments, the radiolabeled Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibody and/or
anti-Neutrokine-alphaSV antibody composition can be administered at
a dose of about 10, 15, 20, 25, or 30 mCi/m2.
[0780] The concentration of total Neutrokine-alpha protein,
Neutrokine-alphaSV protein, anti-Neutrokine-alpha antibody and/or
anti-Neutrokine-alphaSV antibody in a radiolabelled
Neutrokine-alpha, Neutrokine-alphaSV, anti-Neutrokine-alpha
antibody and/or anti-Neutrokine-alphaSV antibody composition may
also vary, for example from about 1 microgram/kg to about 1 mg/kg.
In specific embodiments, the total concentration of
Neutrokine-alpha protein, Neutrokine-alphaSV protein,
anti-Neutrokine-alpha antibody and/or anti-Neutrokine-alphaSV
antibody antibody in a radiolabelled Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibody and/or
anti-Neutrokine-alphaSV antibody composition may be about 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95
100 micrograms/kg.
[0781] For example, lymphomas are known to be radiosensitive
tumors. For immunodiagnostic imaging, trace-labeling by the complex
may be used, typically 1-20 mg of Neutrokine-alpha protein is
labeled with about 1 to 60 mCi of radioisotope. The dose may be
somewhat dependent upon the isotope used for imaging; amounts in
the higher end of the range, preferably 40 to 60 mCi, may be used
with .sup.99mTc; amounts in the lower end of the range, preferably
1-20 mCi, may be used with .sup.111In. For imaging purposes, about
1 to about 30 mg of Neutrokine-alpha complex can be given to the
subject. For radioimmunotherapeutic purposes, the Neutrokine-alpha
complex is administered to a subject in sufficient amount so that
the whole body dose received is up to about 1100 cGy, but
preferably less than or equal to 500 cGy. The total amount of
Neutrokine-alpha protein administered to a subject, including
Neutrokine-alpha protein, Neutrokine-alpha conjugate and
Neutrokine-alpha complex, can range from 1.0 .quadrature.g/kg to
1.0 mg/kg of patient body weight. In another embodiment, total
amount of Neutrokine-alpha protein administered to a subject, can
range from 20 .quadrature.g/kg to 100 .quadrature.g/kg of patient
body weight.
[0782] An amount of radioactivity which would provide approximately
500 cGy to the whole body of a human is estimated to be about 825
mCi of .sup.131I. The amounts of radioactivity to be administered
depend, in part, upon the isotope chosen. For .sup.90Y therapy,
from about 1 to about 200 mCi amounts of radioactivity are
considered appropriate, with preferable amounts being 1 to 150 mCi,
and 1 to 100 mCi (e.g., 60 mCi) being most preferred. The preferred
means of estimating tissue doses from the amount of administered
radioactivity is to perform an imaging or other pharmacokinetic
regimen with a tracer dose, so as to obtain estimates of predicted
dosimetry. In determining the appropriate dosage of
radiopharmaceutical to administer to an individual, it is necessary
to consider the amount of radiation that individual organs will
receive compared to the maximum tolerance for such organs. Such
information is known to those skilled in the art, for example, see
Emami et al., International Journal of Radiation Oncology, Biology,
Physics 21:109-22 (1991); and Meredith, Cancer Biotherapy &
Radiopharmaceuticals 17:83-99 (2002), both of which are hereby
incorporated by reference in their entireties.
[0783] A "high-dose" protocol, for example in the range of 200 to
600 cGy (or higher) to the whole body, may require the support of a
bone-marrow replacement protocol, as the bone-marrow is the tissue
which limits the radiation dosage due to toxicity.
[0784] In specific embodiments, radiolabelled Neutrokine-alpha,
Neutrokine-alphaSV, anti-Neutrokine-alpha antibody and/or
anti-Neutrokine-alphaSV antibody compositions can be used to target
radiotherapy to cancers, such as B cells cancers (including
non-Hodgkins lymphoma and multiple myeloma) while delivering low
radiation doses to normal critical organs, such as the lung, liver,
kidney, and red marrow.
[0785] Pharmaceutical compositions containing Neutrokine-alpha
and/or Neutrokine-alphaSV polypeptides of the invention may be
administered orally, rectally, parenterally, subcutaneously,
intracisternally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray (e.g., via inhalation of a vapor or powder).
In one embodiment, "pharmaceutically acceptable carrier" means a
non-toxic solid, semisolid or liquid filler, diluent, encapsulating
material or formulation auxiliary of any type. In a specific
embodiment, "pharmaceutically acceptable" means approved by a
regulatory agency of the federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly humans. Nonlimiting
examples of suitable pharmaceutical carriers according to this
embodiment are provided in "Remington's Pharmaceutical Sciences" by
E. W. Martin, and include sterile liquids, such as water and oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can be employed as liquid
carriers, particularly for injectable solutions. The composition,
if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. These compositions can
take the form of solutions, suspensions, emulsion, tablets, pills,
capsules, powders, sustained-release formulations and the like.
[0786] The term "parenteral" as used herein refers to modes of
administration which include intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous and intraarticular
injection and infusion.
[0787] In a preferred embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV compositions of the invention (including
polypeptides, polynucleotides, and antibodies, and agonists and/or
antagonists thereof) are administered subcutaneously.
[0788] In another preferred embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV compositions of the invention (including
polypeptides, polynucleotides, and antibodies, and agonists and/or
antagonists thereof) are administered intravenously.
[0789] Neutrokine-alpha and/or Neutrokine-alphaSV compositions of
the invention are also suitably administered by sustained-release
systems. Suitable examples of sustained-release compositions
include suitable polymeric materials (such as, for example,
semi-permeable polymer matrices in the form of shaped articles,
e.g., films, or microcapsules), suitable hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, and sparingly soluble derivatives (such as, for example, a
sparingly soluble salt).
[0790] Sustained-release matrices include polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556
(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.)
or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0791] In a preferred embodiment, Neutrokine-alpha and/or
Neutrokine-alphaSV compositions of the invention are formulated in
a biodegradable, polymeric drug delivery system, for example as
described in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202;
5,324,519; 5,340,849; and 5,487,897 and in International
Publication Numbers WO01/35929, WO00/24374, and WO00/06117 which
are hereby incorporated by reference in their entirety. In specific
preferred embodiments the Neutrokine-alpha and/or
Neutrokine-alphaSV compositions of the invention are formulated
using the ATRIGEL.RTM. Biodegradable System of Atrix Laboratories,
Inc. (Fort Collins, Colo.). In other specific embodiments,
Neutrokine-alpha and/or Neutrokine-alphaSV compositions of the
invention are formulated using the ProLease.RTM. sustained release
system available from Alkermes, Inc. (Cambridge, Mass.).
[0792] Examples of biodegradable polymers which can be used in the
formulation of Neutrokine-alpha and/or Neutrokine-alphaSV
compositions, include but are not limited to, polylactides,
polyglycolides, polycaprolactones, polyanhydrides, polyamides,
polyurethanes, polyesteramides, polyorthoesters, polydioxanones,
polyacetals, polyketals, polycarbonates, polyorthocarbonates,
polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,
polyalkylene oxalates, polyalkylene succinates, poly(malic acid),
poly(amino acids), poly(methyl vinyl ether), poly(maleic
anhydride), polyvinylpyrrolidone, polyethylene glycol,
polyhydroxycellulose, chitin, chitosan, and copolymers,
terpolymers, or combinations or mixtures of the above materials.
The preferred polymers are those that have a lower degree of
crystallization and are more hydrophobic. These polymers and
copolymers are more soluble in the biocompatible solvents than the
highly crystalline polymers such as polyglycolide and chitin which
also have a high degree of hydrogen-bonding. Preferred materials
with the desired solubility parameters are the polylactides,
polycaprolactones, and copolymers of these with glycolide in which
there are more amorphous regions to enhance solubility. In specific
preferred embodiments, the biodegradable polymers which can be used
in the formulation of Neutrokine-alpha and/or Neutrokine-alphaSV
compositions are poly(lactide-co-glycolides). Polymer properties
such as molecular weight, hydrophobicity, and lactide/glycolide
ratio may be modified to obtain the desired drug Neutrokine-alpha
and/or Neutrokine-alphaSV release profile (See, e.g., Ravivarapu et
al., Journal of Pharmaceutical Sciences 89:732-741 (2000), which is
hereby incorporated by reference in its entirety).
[0793] It is also preferred that the solvent for the biodegradable
polymer be non-toxic, water miscible, and otherwise biocompatible.
Examples of such solvents include, but are not limited to,
N-methyl-2-pyrrolidone, 2-pyrrolidone, C2 to C6 alkanols, C1 to C15
alcohols, dils, triols, and tetraols such as ethanol, glycerine
propylene glycol, butanol; C3 to C15 alkyl ketones such as acetone,
diethyl ketone and methyl ethyl ketone; C3 to C15 esters such as
methyl acetate, ethyl acetate, ethyl lactate; alkyl ketones such as
methyl ethyl ketone, C1 to C15 amides such as dimethylformamide,
dimethylacetamide and caprolactam; C3 to C20 ethers such as
tetrahydrofuran, or solketal; tweens, triacetin, propylene
carbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid,
1-dodecylazacycloheptan-2-one, Other preferred solvents are benzyl
alcohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl
oleate, glycerin, glycofurol, isopropyl myristate, isopropyl
palmitate, oleic acid, polyethylene glycol, propylene carbonate,
and triethyl citrate. The most preferred solvents are
N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide,
triacetin, and propylene carbonate because of the solvating ability
and their compatibility.
[0794] Additionally, formulations comprising Neutrokine-alpha
and/or Neutrokine-alphaSV compositions and a biodegradable polymer
may also include release-rate modification agents and/or
pore-forming agents. Examples of release-rate modification agents
include, but are not limited to, fatty acids, triglycerides, other
like hydrophobic compounds, organic solvents, plasticizing
compounds and hydrophilic compounds. Suitable release rate
modification agents include, for example, esters of mono-, di-, and
tricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate,
ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl
phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate,
dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl
triethyl citrate, glycerol triacetate, di(n-butyl) sebecate, and
the like; polyhydroxy alcohols, such as propylene glycol,
polyethylene glycol, glycerin, sorbitol, and the like; fatty acids;
triesters of glycerol, such as triglycerides, epoxidized soybean
oil, and other epoxidized vegetable oils; sterols, such as
cholesterol; alcohols, such as C.sub.6-C.sub.12 alkanols,
2-ethoxyethanol, and the like. The release rate modification agent
may be used singly or in combination with other such agents.
Suitable combinations of release rate modification agents include,
but are not limited to, glycerin/propylene glycol,
sorbitol/glycerine, ethylene oxide/propylene oxide, butylene
glycol/adipic acid, and the like. Preferred release rate
modification agents include, but are not limited to, dimethyl
citrate, triethyl citrate, ethyl heptanoate, glycerin, and
hexanediol. Suitable pore-forming agents that may be used in the
polymer composition include, but are not limited to, sugars such as
sucrose and dextrose, salts such as sodium chloride and sodium
carbonate, polymers such as hydroxylpropylcellulose,
carboxymethylcellulose, polyethylene glycol, and
polyvinylpyrrolidone. Solid crystals that will provide a defined
pore size, such as salt or sugar, are preferred.
[0795] In specific preferred embodiments the Neutrokine-alpha
and/or Neutrokine-alphaSV compositions of the invention are
formulated using the BEMA.TM. BioErodible Mucoadhesive System,
MCA.TM. MucoCutaneous Absorption System, SMP.TM. Solvent
MicroParticle System, or BCP.TM. BioCompatible Polymer System of
Atrix Laboratories, Inc. (Fort Collins, Colo.).
[0796] Sustained-release compositions also include liposomally
entrapped compositions of the invention (see generally, Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)).
Liposomes containing Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide my be prepared by methods known per se: DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985);
Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP
52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.
Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324. Ordinarily, the liposomes are of the small (about 200-800
Angstroms) unilamellar type in which the lipid content is greater
than about 30 mol. percent cholesterol, the selected proportion
being adjusted for the optimal Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide therapy.
[0797] In another embodiment sustained release compositions of the
invention include crystal formulations known in the art.
[0798] In yet an additional embodiment, the compositions of the
invention are delivered by way of a pump (see Langer, supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574
(1989)).
[0799] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0800] For parenteral administration, in one embodiment, the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide is
formulated generally by mixing it at the desired degree of purity,
in a unit dosage injectable form (solution, suspension, or
emulsion), with a pharmaceutically acceptable carrier, i.e., one
that is non-toxic to recipients at the dosages and concentrations
employed and is compatible with other ingredients of the
formulation. For example, the formulation preferably does not
include oxidizing agents and other compounds that are known to be
deleterious to polypeptides.
[0801] Generally, the formulations are prepared by contacting the
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide uniformly
and intimately with liquid carriers or finely divided solid
carriers or both. Then, if necessary, the product is shaped into
the desired formulation. Preferably the carrier is a parenteral
carrier, more preferably a solution that is isotonic with the blood
of the recipient. Examples of such carrier vehicles include water,
saline, Ringer's solution, and dextrose solution. Non-aqueous
vehicles such as fixed oils and ethyl oleate are also useful
herein, as well as liposomes.
[0802] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, sucrose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; preservatives,
such as cresol, phenol, chlorobutanol, benzyl alcohol and parabens,
and/or nonionic surfactants such as polysorbates, poloxamers, or
PEG.
[0803] The Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
is typically formulated in such vehicles at a concentration of
about 0.001 mg/ml to 100 mg/ml, or 0.1 mg/ml to 100 mg/ml,
preferably 1-10 mg/ml or 1-10 mg/ml, at a pH of about 3 to 10, or 3
to 8, more preferably 5-8, most preferably 6-7. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide salts.
[0804] Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide to be
used for therapeutic administration must be sterile. Sterility is
readily accomplished by filtration through sterile filtration
membranes (e.g., 0.2 micron membranes). Therapeutic
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide compositions
generally are placed into a container having a sterile access port,
for example, an intravenous solution bag or vial having a stopper
pierceable by a hypodermic injection needle.
[0805] Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
ordinarily will be stored in unit or multi-dose containers, for
example, sealed ampoules or vials, as an aqueous solution or as a
lyophilized formulation for reconstitution. As an example of a
lyophilized formulation, 10-ml vials are filled with 5 ml of
sterile-filtered 1% (w/v) aqueous Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide solution, and the resulting mixture
is lyophilized. The infusion solution is prepared by reconstituting
the lyophilized Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide using bacteriostatic Water-for-Injection.
[0806] Alternatively, Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide is stored in single dose containers in lyophilized
form. The infusion selection is reconstituted using a sterile
carrier for injection.
[0807] In specific embodiments, a composition of the invention is a
radiolabelled form of Neutrokine-alpha. A composition of the
invention may comprise Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide that is radiolabeled, for example, with radioactive
isotopes of iodine. Compositions comprising iodinated forms of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides or
fragments or variants thereof, may also comprise radioprotectants
and plasma expanders such as sodium ascorbate, gentran-40, and
glycerol. In specific embodiments, compositions of the invention
comprising iodinated forms of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides or fragments or variants are
formulated in 10.0 mM sodium citrate, 140.0 mM sodium chloride, 8.7
mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) Genetran-40. The
above described compositions may be used as pharmaceutical
compositions.
[0808] In specific embodiments, a composition of the invention
comprises, at least 1 mg/mL of an iodinated form of amino acid
residues 134-285 of SEQ ID NO:2, 10.0 mM sodium citrate, 140.0 mM
sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium ascorbate, 3.3%
(w/v) Gentran-40. In specific embodiments, a composition of the
invention comprises, at least 2 mg/mL of an iodinated form of amino
acid residues 134-285 of SEQ ID NO:2, 10.0 mM sodium citrate, 140.0
mM sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium ascorbate, 3.3%
(w/v) Gentran-40. In specific embodiments, a composition of the
invention comprises, at least 3 mg/mL of an iodinated form of amino
acid residues 134-285 of SEQ ID NO:2, 10.0 mM sodium citrate, 140.0
mM sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium ascorbate, 3.3%
(w/v) Gentran-40. In specific embodiments, a composition of the
invention comprises, at least 4 mg/mL of an iodinated form of amino
acid residues 134-285 of SEQ ID NO:2, 10.0 mM sodium citrate, 140.0
mM sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium ascorbate, 3.3%
(w/v) Gentran-40. In particular embodiments, a composition of the
invention comprises, about 4.6 mg/mL of an iodinated form of amino
acid residues 134-285 of SEQ ID NO:2, 10.0 mM sodium citrate, 140.0
mM sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium ascorbate, 3.3%
(w/v) Gentran-40. The above described compositions may be used as
pharmaceutical compositions.
[0809] In specific embodiments, a composition of the invention
comprises, about between 0.1 mg/mL and 20 mg/mL of an iodinated
form of amino acid residues 134-285 of SEQ ID NO:2, 10.0 mM sodium
citrate, 140.0 mM sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium
ascorbate, 3.3% (w/v) Gentran-40. In specific embodiments, a
composition of the invention comprises, between 1 mg/mL and 10
mg/mL of an iodinated form of amino acid residues 134-285 of SEQ ID
NO:2, 10.0 mM sodium citrate, 140.0 mM sodium chloride, 8.7 mM
HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) Gentran-40. In
specific embodiments, a composition of the invention comprises,
between 2 mg/mL and 8 mg/mL of an iodinated form of amino acid
residues 134-285 of SEQ ID NO:2, 10.0 mM sodium citrate, 140.0 mM
sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium ascorbate, 3.3%
(w/v) Gentran-40. In specific embodiments, a composition of the
invention comprises, between 3 mg/mL and 6 mg/mL of an iodinated
form of amino acid residues 134-285 of SEQ ID NO:2, 10.0 mM sodium
citrate, 140.0 mM sodium chloride, 8.7 mM HEPES, 4% (w/v) sodium
ascorbate, 3.3% (w/v) Gentran-40. The above described compositions
may be used as pharmaceutical compositions.
[0810] In other embodiments, a composition of the invention is an
anti-neutrokine-alpha antibody. In other embodiments, a composition
of the invention is an antibody that specifically binds
Neutrokine-alpha. In other embodiments, a composition of the
invention is an antagonistic anti-neutrokine-alpha antibody. In
other embodiments, a composition of the invention is an antibody
that specifically binds Neutrokine-alpha and neutralizes
neutrokine-alpha biological activity. In other embodiments, a
composition of the invention is an anti-neutrokine-alpha antibody
that binds a recombinant Neutrokine-alpha protein purified from a
cell culture wherein said recombinant Neutrokine-alpha protein is
encoded by a polynucleotide encoding at least amino acids 134 to
285 of SEQ ID NO:2. In other embodiments, a composition of the
invention is an antibody that specifically binds Neutrokine-alpha
wherein said antibody binds a recombinant Neutrokine-alpha protein
purified from a cell culture wherein said recombinant
Neutrokine-alpha protein is encoded by a polynucleotide encoding at
least amino acids 134 to 285 of SEQ ID NO:2.
[0811] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally, associated with such container(s) is a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. In addition, the polypeptides of the
present invention may be employed in conjunction with other
therapeutic compounds.
[0812] The compositions of the invention may be administered alone
or in combination with other adjuvants. Adjuvants that may be
administered with the compositions of the invention include, but
are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE
(Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a
specific embodiment, compositions of the invention are administered
in combination with alum. In another specific embodiment,
compositions of the invention are administered in combination with
QS-21. Further adjuvants that may be administered with the
compositions of the invention include, but are not limited to,
Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,
CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.
Vaccines that may be administered with the compositions of the
invention include, but are not limited to, vaccines directed toward
protection against MMR (measles, mumps, rubella), polio, varicella,
tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae
B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,
cholera, yellow fever, Japanese encephalitis, poliomyelitis,
rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23.TM..
Combinations may be administered either concomitantly, e.g., as an
admixture, separately but simultaneously or concurrently; or
sequentially. This includes presentations in which the combined
agents are administered together as a therapeutic mixture, and also
procedures in which the combined agents are administered separately
but simultaneously, e.g., as through separate intravenous lines
into the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[0813] In a specific embodiment, compositions of the invention
(e.g., Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of
the invention, Neutrokine-alpha and/or Neutrokine-alphaSV fragments
and variants, and anti-Neutokine-alpha and/or
anti-Neutrokine-alphaSV antibodies) may be administered to patients
as vaccine adjuvants. In a further specific embodiment,
compositions of the invention may be administered as vaccine
adjuvants to patients suffering from an immune-deficiency. In a
further specific embodiment, compositions of the invention may be
administered as vaccine adjuvants to patients suffering from
HIV.
[0814] In a specific embodiment, compositions of the invention may
be used to increase or enhance antigen-specific antibody responses
to standard and experimental vaccines. In a specific embodiment,
compositions of the invention may be used to enhance seroconversion
in patients treated with standard and experimental vaccines. In
another specific embodiment, compositions of the invention may be
used to increase the number of unique epitopes recognized by
antibodies elicited by standard and experimental vaccination.
[0815] In another specific embodiment, compositions of the
invention are used in combination with PNEUMOVAX-23.TM. to treat,
prevent, and/or diagnose infection and/or any disease, disorder,
and/or condition associated therewith. In one embodiment,
compositions of the invention are used in combination with
PNEUMOVAX-23.TM. to treat, prevent, and/or diagnose any Gram
positive bacterial infection and/or any disease, disorder, and/or
condition associated therewith. In another embodiment, compositions
of the invention are used in combination with PNEUMOVAX-23.TM. to
treat, prevent, and/or diagnose infection and/or any disease,
disorder, and/or condition associated with one or more members of
the genus Enterococcus and/or the genus Streptococcus. In another
embodiment, compositions of the invention are used in any
combination with PNEUMOVAX-23.TM. to treat, prevent, and/or
diagnose infection and/or any disease, disorder, and/or condition
associated with one or more members of the Group B streptococci. In
another embodiment, compositions of the invention are used in
combination with PNEUMOVAX-23.TM. to treat, prevent, and/or
diagnose infection and/or any disease, disorder, and/or condition
associated with Streptococcus pneumoniae.
[0816] The compositions of the invention may be administered alone
or in combination with other therapeutic agents, including but not
limited to, chemotherapeutic agents, antibiotics, antivirals,
steroidal and non-steroidal anti-inflammatories, conventional
immunotherapeutic agents and cytokines. Combinations may be
administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[0817] In one embodiment, the compositions of the invention are
administered in combination with other members of the TNF family.
TNF, TNF-related or TNF-like molecules that may be administered
with the compositions of the invention include, but are not limited
to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also
known as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), AIM-II (International
Publication No. WO 97/34911), APRIL (J. Exp. Med.
188(6):1185-1190), endokine-alpha (International Publication No. WO
98/07880), TR6 (International Publication No. WO 98/30694), OPG,
and neutrokine-alpha (International Publication No. WO 98/18921,
OX40, and nerve growth factor (NGF), and soluble forms of Fas,
CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/33904), DR4
(International Publication No. WO 98/32856), TR5 (International
Publication No. WO 98/30693), TR6 (International Publication No. WO
98/30694), TR7 (International Publication No. WO 98/41629), TRANK,
TR9 (International Publication No. WO 98/56892), TR10
(International Publication No. WO 98/54202), 312C2 (International
Publication No. WO 98/06842), and TR12.
[0818] In another embodiment, the compositions of the invention are
invention are administered in combination with Neutrokine-alpha
receptors and/or Neurtokine-alpha SV receptors (e.g., TACI, BCMA
and BAFF-R) In preferred in embodiments the Neutrokine-alpha
receptors and/or Neurtokine-alpha SV receptors are soluble. In
other preferred embodiments the Neutrokine-alpha receptors and/or
Neurtokine-alpha SV receptors are fused to the FC region of an
immunoglobulin molecule (e.g, amino acid residues 1-154 of TACI
(GenBank accession number AAC51790), amino acids 1-48 of BCMA
(GenBank accession number NP.sub.--001183 or amino acids 1 to 81 of
BAFF-R (GenBank Acession Number NP.sub.--443177) fused to the Fc
region of an IgG molecule) fused to the Fc region of an IgG
molecule.
[0819] In a preferred embodiment, the compositions of the invention
are administered in combination with CD40 ligand (CD40L), a soluble
form of CD40L (e.g., AVREND.TM.), biologically active fragments,
variants, or derivatives of CD40L, anti-CD40L antibodies (e.g.,
agonistic or antagonistic antibodies), and/or anti-CD40 antibodies
(e.g, agonistic or antagonistic antibodies).
[0820] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-angiogenic agent(s). Anti-angiogenic agents that may be
administered with the compositions of the invention include, but
are not limited to, Angiostatin (Entremed, Rockville, Md.),
Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol),
Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor
of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1,
Plasminogen Activator Inhibitor-2, and various forms of the lighter
"d group" transition metals.
[0821] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[0822] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[0823] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[0824] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include, but are not limited to, platelet
factor 4; protamine sulphate; sulphated chitin derivatives
(prepared from queen crab shells), (Murata et al., Cancer Res.
51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex
(SP-PG) (the function of this compound may be enhanced by the
presence of steroids such as estrogen, and tamoxifen citrate);
Staurosporine; modulators of matrix metabolism, including for
example, proline analogs, cishydroxyproline,
d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl,
aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin
(Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin
Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et
al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
(Takeuchi et al., Agents Actions 36:312-316, 1992); and
metalloproteinase inhibitors such as BB94.
[0825] Additional anti-angiogenic factors that may also be utilized
within the context of the present invention include Thalidomide,
(Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and
J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v
beta 3 antagonist (C. Storgard et al., J. Clin. Invest. 103:47-54
(1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI)
(National Cancer Institute, Bethesda, Md.); Conbretastatin A-4
(CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin
Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap
Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London,
UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251
(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;
Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide
(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88;
Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen
(Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine);
and 5-Fluorouracil.
[0826] Anti-angiogenic agents that may be administered in
combination with the compositions of the invention may work through
a variety of mechanisms including, but not limited to, inhibiting
proteolysis of the extracellular matrix, blocking the function of
endothelial cell-extracellular matrix adhesion molecules, by
antagonizing the function of angiogenesis inducers such as growth
factors, and inhibiting integrin receptors expressed on
proliferating endothelial cells. Examples of anti-angiogenic
inhibitors that interfere with extracellular matrix proteolysis and
which may be administered in combination with the compositions of
the invention include, but are not limited to, AG-3340 (Agouron, La
Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291
(Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East
Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and
Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic
inhibitors that act by blocking the function of endothelial
cell-extracellular matrix adhesion molecules and which may be
administered in combination with the compositions of the invention
include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt,
Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune,
Gaithersburg, Md.). Examples of anti-angiogenic agents that act by
directly antagonizing or inhibiting angiogenesis inducers and which
may be administered in combination with the compositions of the
invention include, but are not limited to, Angiozyme (Ribozyme,
Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco,
Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101
(Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn,
Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic
agents act to indirectly inhibit angiogenesis. Examples of indirect
inhibitors of angiogenesis which may be administered in combination
with the compositions of the invention include, but are not limited
to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12
(Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown
University, Washington, D.C.).
[0827] In particular embodiments, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
an autoimmune disease, such as for example, an autoimmune disease
described herein.
[0828] In a particular embodiment, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
arthritis. In a more particular embodiment, the use compositions of
the invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
rheumatoid arthritis.
[0829] In particular embodiments, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
strokes.
[0830] In another embodiment, compositions of the invention are
administered in combination with an anticoagulant. Anticoagulants
that may be administered with the compositions of the invention
include, but are not limited to, heparin, warfarin, and aspirin. In
a specific embodiment, compositions of the invention are
administered in combination with heparin and/or warfarin. In
another specific embodiment, compositions of the invention are
administered in combination with warfarin. In another specific
embodiment, compositions of the invention are administered in
combination with warfarin and aspirin. In another specific
embodiment, compositions of the invention are administered in
combination with heparin. In another specific embodiment,
compositions of the invention are administered in combination with
heparin and aspirin.
[0831] In another embodiment, compositions of the invention are
administered in combination with an agent that suppresses the
production of anticardiolipin antibodies. In specific embodiments,
the polynucleotides of the invention are administered in
combination with an agent that blocks and/or reduces the ability of
anticardiolipin antibodies to bind phospholipid-binding plasma
protein beta 2-glycoprotein I (b2GPI).
[0832] In certain embodiments, compositions of the invention are
administered in combination with antiretroviral agents, nucleoside
reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors. Nucleoside
reverse transcriptase inhibitors that may be administered in
combination with the compositions of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). Non-nucleoside reverse transcriptase
inhibitors that may be administered in combination with the
compositions of the invention, include, but are not limited to,
VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM. (delavirdine), and
SUSTIVA.TM. (efavirenz). Protease inhibitors that may be
administered in combination with the compositions of the invention,
include, but are not limited to, CRIXIVAN.TM. (indinavir),
NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and VIRACEPT.TM.
(nelfinavir). In a specific embodiment, antiretroviral agents,
nucleoside reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors may be used in
any combination with compositions of the invention to treat,
prevent, and/or diagnose AIDS and/or to treat, prevent, and/or
diagnose HIV infection.
[0833] In certain embodiments, compositions of the invention are
administered in combination with antiretroviral agents,
nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs),
non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or
protease inhibitors (PIs). NRTIs that may be administered in
combination with the compositions of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). NNRTIs that may be administered in
combination with the compositions of the invention, include, but
are not limited to, VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM.
(delavirdine), and SUSTIVA.TM. (efavirenz). Protease inhibitors
that may be administered in combination with the compositions of
the invention, include, but are not limited to, CRIXIVAN.TM.
(indinavir), NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and
VIRACEPT.TM. (nelfinavir). In a specific embodiment, antiretroviral
agents, nucleoside reverse transcriptase inhibitors, non-nucleoside
reverse transcriptase inhibitors, and/or protease inhibitors may be
used in any combination with compositions of the invention to treat
AIDS and/or to prevent or treat HIV infection.
[0834] Additional NRTIs include LODENOSINE.TM. (F-ddA; an
acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL.TM.
(emtricitabine/FTC; structurally related to lamivudine (3TC) but
with 3- to 10-fold greater activity in vitro; Triangle/Abbott);
dOTC (BCH-10652, also structurally related to lamivudine but
retains activity against a substantial proportion of
lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused
approval for anti-HIV therapy by FDA; Gilead Sciences);
PREVEON.RTM. (Adefovir Dipivoxil, the active prodrug of adefovir;
its active form is PMEA-pp); TENOFOVIR.TM. (bis-POC PMPA, a PMPA
prodrug; Gilead); DAPD/DXG (active metabolite of DAPD;
Triangle/Abbott); D-D4FC (related to 3TC, with activity against
AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN.TM.
(abacavir/159U89; Glaxo Wellcome Inc.); CS-87
(3'azido-2',3'-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl
(SATE)-bearing prodrug forms of .beta.-L-FD4C and .beta.-L-FddC (WO
98/17281).
[0835] Additional NNRTIs include COACTINON.TM. (Emivirine/MKC-442,
potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE.TM.
(AG-1549/S-1153, a next generation NNRTI with activity against
viruses containing the K103N mutation; Agouron); PNU-142721 (has
20- to 50-fold greater activity than its predecessor delavirdine
and is active against K103N mutants; Pharmacia & Upjohn);
DPC-961 and DPC-963 (second-generation derivatives of efavirenz,
designed to be active against viruses with the K103N mutation;
DuPont); GW-420867X (has 25-fold greater activity than HBY097 and
is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A
(naturally occurring agent from the latex tree; active against
viruses containing either or both the Y181C and K103N mutations);
and Propolis (WO 99/49830).
[0836] Additional protease inhibitors include LOPINAVIR.TM.
(ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide;
Bristol-Myres Squibb); TIPRANAVIR.TM. (PNU-140690, a non-peptic
dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic
dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide;
Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck);
DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a
peptidomimetic with in vitro activity against protease
inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate
prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755
(Ciba); and AGENERASE.TM. (amprenavir; Glaxo Wellcome Inc.).
[0837] Additional antiretroviral agents include fusion
inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include
T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane
protein ectodomain which binds to gp41 in its resting state and
prevents transformation to the fusogenic state; Trimeris) and
T-1249 (a second-generation fusion inhibitor; Trimeris).
[0838] Additional antiretroviral agents include fusion
inhibitors/chemokine receptor antagonists. Fusion
inhibitors/chemokine receptor antagonists include CXCR4 antagonists
such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C
(a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and
the T22 analogs T134 and T140; CCR5 antagonists such as RANTES
(9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4
antagonists such as NSC 651016 (a distamycin analog). Also included
are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptor agonists
such as RANTES, SDF-1, MIP-1.alpha., MIP-1.beta., etc., may also
inhibit fusion.
[0839] Additional antiretroviral agents include integrase
inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA)
acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid);
quinalizarin (QLC) and related anthraquinones; ZINTEVIR.TM. (AR
177, an oligonucleotide that probably acts at cell surface rather
than being a true integrase inhibitor; Arondex); and naphthols such
as those disclosed in WO 98/50347.
[0840] Additional antiretroviral agents include hydroxyurea-like
compounds such as BCX-34 (a purine nucleoside phosphorylase
inhibitor; Biocryst); ribonucleotide reductase inhibitors such as
DIDOX.TM. (Molecules for Health); inosine monophosphate
dehydrogenase (IMPDH) inhibitors such a as VX-497 (Vertex); and
myvopholic acids such as CellCept (mycophenolate mofetil;
Roche).
[0841] Additional antiretroviral agents include inhibitors of viral
integrase, inhibitors of viral genome nuclear translocation such as
arylene bis(methylketone) compounds; inhibitors of HIV entry such
as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble
complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100;
nucleocapsid zinc finger inhibitors such as dithiane compounds;
targets of HIV Tat and Rev; and pharmacoenhancers such as
ABT-378.
[0842] Other antiretroviral therapies and adjunct therapies include
cytokines and lymphokines such as MIP-1.alpha., MIP-1.beta.,
SDF-1.alpha., IL-2, PROLEUKIN.TM. (aldesleukin/L2-7001; Chiron),
IL-4, IL-8, IL-10, IL-12, and IL-13; interferons such as
IFN-.alpha.2a; antagonists of TNFs, NF.kappa.B, GM-CSF, M-CSF, and
IL-10; agents that modulate immune activation such as cyclosporin
and prednisone; vaccines such as Remune.TM. (HIV Immunogen), APL
400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E)
recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2
rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched
synthetic peptide derived from discontinuous gp120 C3/C4 domain,
fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines;
gene-based therapies such as genetic suppressor elements (GSEs; WO
98/54366), and intrakines (genetically modified CC chemokines
targeted to the ER to block surface expression of newly synthesized
CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med.
3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5,
the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and
PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3
antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D,
268-D and 50.1, anti-Tat antibodies, anti-TNF-.alpha. antibodies,
and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor
agonists and antagonists such as TCDD,
3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and
.alpha.-naphthoflavone (WO 98/30213); and antioxidants such as
.gamma.-L-glutamyl-L-cysteine ethyl ester (.gamma.-GCE; WO
99/56764).
[0843] In other embodiments, compositions of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with the compositions of the invention, include, but
are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLE.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
compositions of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat, prevent, and/or
diagnose an opportunistic Pneumocystis carinii pneumonia infection.
In another specific embodiment, compositions of the invention are
used in any combination with ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., and/or ETHAMBUTOL.TM. to prophylactically treat,
prevent, and/or diagnose an opportunistic Mycobacterium avium
complex infection. In another specific embodiment, compositions of
the invention are used in any combination with RIFABUTIN.TM.,
CLARITHROMYCIN.TM., and/or AZITHROMYCIN.TM. to prophylactically
treat, prevent, and/or diagnose an opportunistic Mycobacterium
tuberculosis infection. In another specific embodiment,
compositions of the invention are used in any combination with
GANCICLOVIR.TM., FOSCARNET.TM., and/or CIDOFOVIR.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
cytomegalovirus infection. In another specific embodiment,
compositions of the invention are used in any combination with
FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
fungal infection. In another specific embodiment, compositions of
the invention are used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat, prevent, and/or diagnose
an opportunistic herpes simplex virus type I and/or type II
infection. In another specific embodiment, compositions of the
invention are used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat, prevent, and/or diagnose
an opportunistic Toxoplasma gondii infection. In another specific
embodiment, compositions of the invention are used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
bacterial infection.
[0844] In a further embodiment, the compositions of the invention
are administered in combination with an antiviral agent. Antiviral
agents that may be administered with the compositions of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, and remantidine.
[0845] In a further embodiment, the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, amoxicillin,
aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin,
ciprofloxacin, erythromycin, fluoroquinolones, macrolides,
metronidazole, penicillins, quinolones, rifampin, streptomycin,
sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamthoxazole, and vancomycin.
[0846] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the compositions of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs cyclophosphamide, cyclophosphamide IV,
methylprednisolone, prednisolone, azathioprine, FK-506,
15-deoxyspergualin, and other immunosuppressive agents that act by
suppressing the function of responding T cells. Other
immunosuppressive agents, that may be administered in combination
with the compositions of the invention include, but are not limited
to, prednisolone, methotrexate, thalidomide, methoxsalen,
rapamycin, leflunomide, mizoribine (BREDININ.TM.), brequinar,
deoxyspergualin, and azaspirane (SKF 105685).
[0847] In specific embodiments, compositions of the invention are
administered in combination with immunosuppressants.
Immunosuppressant preparations that may be administered with the
compositions of the invention include, but are not limited to,
ORTHOCLONE OKT.RTM. 3 (muromonab-CD3), SANDIMMUNE.TM., NEORAL.TM.,
SANGDYA.TM. (cyclosporine), PROGRAF.RTM. (FK506, tacrolimus),
CELLCEPT.RTM. (mycophenolate motefil, of which the active
metabolite is mycophenolic acid), IMURAN.TM. (azathioprine),
glucorticosteroids, adrenocortical steroids such as DELTASONE.TM.
(prednisone) and HYDELTRASOL.TM. (prednisolone), FOLEX.TM. and
MEXATE.TM. (methotrexate), OXSORALEN-ULTRA.TM. (methoxsalen) and
RAPAMUNE.TM. (sirolimus). In a specific embodiment,
immunosuppressants may be used to prevent rejection of organ or
bone marrow transplantation.
[0848] In a preferred embodiment, the compositions of the invention
are administered in combination with steroid therapy. Steroids that
may be administered in combination with the compositions of the
invention, include, but are not limited to, oral corticosteroids,
prednisone, and methylprednisolone (e.g., IV methylprednisolone).
In a specific embodiment, compositions of the invention are
administered in combination with prednisone. In a further specific
embodiment, the compositions of the invention are administered in
combination with prednisone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the
compositions of the invention and prednisone are those described
herein, and include, but are not limited to, azathioprine,
cylophosphamide, and cyclophosphamide IV. In a another specific
embodiment, compositions of the invention are administered in
combination with methylprednisolone. In a further specific
embodiment, the compositions of the invention are administered in
combination with methylprednisolone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the
compositions of the invention and methylprednisolone are those
described herein, and include, but are not limited to,
azathioprine, cylophosphamide, and cyclophosphamide IV.
[0849] In a preferred embodiment, the compositions of the invention
are administered in combination with an antimalarial. Antimalarials
that may be administered with the compositions of the invention
include, but are not limited to, hydroxychloroquine, chloroquine,
and/or quinacrine.
[0850] In a preferred embodiment, the compositions of the invention
are administered in combination with an NSAID.
[0851] In a nonexclusive embodiment, the compositions of the
invention are administered in combination with one, two, three,
four, five, ten, or more of the following drugs: NRD-101 (Hoechst
Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium
(Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed
disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto),
eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech
Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS
Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis),
JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong
Wha), darbufelone mesylate (Warner-Lambert), soluble TNF receptor 1
(synergen; Amgen), IPR-6001 (Institute for Pharmaceutical
Research), trocade (Hoffman-La Roche), EF-5 (Scotia
Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149
(Boehringer Ingelheim), LeukoVax (Inflammatics), MK-663 (Merck),
ST-1482 (Sigma-Tau), and butixocort propionate (WarnerLambert).
[0852] In one embodiment, the compositions of the invention are
administered in combination with one or more of the following
drugs: Infliximab (also known as Remicade.TM. Centocor, Inc.),
Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava.TM.
from Hoechst Marion Roussel), Kineret.TM. (an IL-1 Receptor
antagonist also known as Anakinra from Amgen, Inc.), SCIO-469 (p38
kinase inhibitor from Scios, Inc), Humira.RTM. (adalimumab from
Abbott Laboratories) and/or ASLERA.TM. (prasterone,
dehydroepiandrosterone, GL701) from Genelabs Technologies Inc.
[0853] In a preferred embodiment, the compositions of the invention
are administered in combination with one, two, three, four, five or
more of the following drugs: methotrexate, sulfasalazine, sodium
aurothiomalate, auranofin, cyclosporine, penicillamine,
azathioprine, an antimalarial drug (e.g., as described herein),
cyclophosphamide, chlorambucil, gold, ENBREL.TM. (Etanercept),
anti-TNF antibody, LJP 394 (La Jolla Pharmaceutical Company, San
Diego, Calif.), and prednisolone.
[0854] In a more preferred embodiment, the compositions of the
invention are administered in combination with an antimalarial,
methotrexate, anti-TNF antibody, ENBREL.TM. and/or sulfasalazine.
In one embodiment, the compositions of the invention are
administered in combination with methotrexate. In another
embodiment, the compositions of the invention are administered in
combination with anti-TNF antibody. In another embodiment, the
compositions of the invention are administered in combination with
methotrexate and anti-TNF antibody. In another embodiment, the
compositions of the invention are administered in combination with
sulfasalazine. In another specific embodiment, the compositions of
the invention are administered in combination with methotrexate,
anti-TNF antibody, and sulfasalazine. In another embodiment, the
compositions of the invention are administered in combination
ENBREL.TM.. In another embodiment, the compositions of the
invention are administered in combination with ENBREL.TM. and
methotrexate. In another embodiment, the compositions of the
invention are administered in combination with ENBREL.TM.,
methotrexate and sulfasalazine. In another embodiment, the
compositions of the invention are administered in combination with
ENBREL.TM., and sulfasalazine. In other embodiments, one or more
antimalarials is combined with one of the above-recited
combinations. In a specific embodiment, the compositions of the
invention are administered in combination with an antimalarial
(e.g., hydroxychloroquine), ENBREL.TM., methotrexate and
sulfasalazine. In another specific embodiment, the compositions of
the invention are administered in combination with an antimalarial
(e.g., hydroxychloroquine), sulfasalazine, anti-TNF antibody, and
methotrexate.
[0855] In an additional embodiment, compositions of the invention
are administered alone or in combination with one or more
intravenous immune globulin preparations. Intravenous immune
globulin preparations that may be administered with the
compositions of the invention include, but not limited to,
GAMMAR.TM., IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM., and
GAMIMUNE.TM.. In a specific embodiment, compositions of the
invention are administered in combination with intravenous immune
globulin preparations in transplantation therapy (e.g., bone marrow
transplant).
[0856] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the compositions of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0857] In specific embodiments, the compositions of the invention
are administered alone or in combination with anti-CD4 antibody. In
one embodiment, coadministration of the compositions of the
invention with anti-CD4 antibody is envisioned for treatment of
rheumatoid arthritis.
[0858] In specific embodiments, the compositions of the invention
are administered alone or in combination with anti-IL-15 antibody.
In one embodiment, coadministration of the compositions of the
invention with anti-IL-15 antibody is envisioned for treatment of
rheumatoid arthritis.
[0859] In specific embodiments, the compositions of the invention
are administered alone or in combination with CTLA4-Ig and LEA29Y.
In one embodiment, coadministration of the compositions of the
invention with CTLA4-Ig and LEA29Y is envisioned for treatment of
rheumatoid arthritis.
[0860] In specific embodiments, the compositions of the invention
are administered alone or in combination with anti-IL-6 Receptor
antibody. In one embodiment, coadministration of the compositions
of the invention with anti-IL-6 Receptor antibody is envisioned for
treatment of rheumatoid arthritis.
[0861] In specific embodiments, the compositions of the invention
are administered alone or in combination with anti-C5 (complement
component) antibody. In one embodiment, coadministration of the
compositions of the invention with anti-C5 antibody is envisioned
for treatment of rheumatoid arthritis.
[0862] In specific embodiments, the compositions of the invention
are administered alone or in combination with complement cascade
inhibitors. Complement cascade inhibitors include, but are not
limited to, anti-properdin antibodies (Gliatech); TP-10, a
recombinant soluble type I complement receptor (AVANT
Immunotheragenetics Inc.); Pexelizmab, a Complement C5 inhibitor
(Alexion Pharmaceuticals Inc.); and 5G1.1, a monoclonal antibody
that prevents cleavage of complement component C5 into its
pro-inflammatory components. In one embodiment, coadministration of
the compositions of the invention with complement cascade
inhibitors are is envisioned for treatment of Inflammation,
Rheumatoid arthritis, and/or cardiovascular disorders.
[0863] In another embodiment, compostions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[0864] In a specific embodiment, compositions of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or combination of one or
more of the components of CHOP. In one embodiment, the compositions
of the invention are administered in combination with anti-CD20
antibodies, human monoclonal anti-CD20 antibodies. In another
embodiment, the compositions of the invention are administered in
combination with anti-CD20 antibodies and CHOP, or anti-CD20
antibodies and any combination of one or more of the components of
CHOP, particularly cyclophosphamide and/or prednisone. In a
specific embodiment, compositions of the invention are administered
in combination with Rituximab. In a further embodiment,
compositions of the invention are administered with Rituximab and
CHOP, or Rituximab and any combination of one or more of the
components of CHOP, particularly cyclophosphamide and/or
prednisone. In a specific embodiment, compositions of the invention
are administered in combination with tositumomab (anti-CD20
antibody from Coulter Pharmaceuticals, San Francisco, Calif.). In a
further embodiment, compositions of the invention are administered
with tositumomab and CHOP, or tositumomab and any combination of
one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. Tositumomab may optionally be
associated with 131I. The anti-CD20 antibodies may optionally be
associated with radioisotopes, toxins or cytotoxic prodrugs.
[0865] In another specific embodiment, the compositions of the
invention are administered in combination Zevalin.TM.. In a further
embodiment, compositions of the invention are administered with
Zevalin.TM. and CHOP, or Zevalin.TM. and any combination of one or
more of the components of CHOP, particularly cyclophosphamide
and/or prednisone. Zevalin.TM. may be associated with one or more
radioisotopes. Particularly preferred isotopes are .sup.90Y and
.sup.111In.
[0866] In additional preferred embodiments, compositions of the
invention are administered in combination with Rituximab
(Rituxan.TM.) and/or Ibritumomab Tiuxetan (Zevalin.TM., e.g.,
either (In-111) Ibritumomab Tiuxetan or (Y-90) Ibritumomab
Tiuxetan). In a specific embodiment, compositions of the invention
are administered in combination with Rituximab and/or Ibritumomab
Tiuxetan for the treatment of non-Hodgkin's lymphoma.
[0867] In additional preferred embodiments, compositions of the
invention are administered in combination with imatinib mesylate
(Gleevec.RTM.:
4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyri-
midinyl]amino]-phenyl]benzamide methanesulfonate). In a specific
embodiment, compositions of the invention are administered in
combination with imatinib mesylate for the treatment of chronic
myelogenous leukemia.
[0868] In additional preferred embodiments, compositions of the
invention are administered in combination with bortezomib
(Velcade.TM.
[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl-
]amino]butyl]boronic acid). In a specific embodiment, compositions
of the invention are administered in combination with bortezomib
for the treatment of multiple myeloma.
[0869] In additional preferred embodiments, compositions of the
invention are administered in combination with Alemtuzumab
(Campath.RTM.). In a specific embodiment, compositions of the
invention are administered in combination with Alemtuzumab for the
treatment of chronic lymphocytic leukemia.
[0870] In additional preferred embodiments, compositions of the
invention are administered in combination with fludarabine
phosphate (Fludara.RTM.: 9H-Purin-6-amine,
2-fluoro-9-(5-O-phosphono-.quadrature.-D-arabinofuranosyl)(2-fluoro-ara-A-
MP)). In a specific embodiment, compositions of the invention are
administered in combination with fludarabine phosphate for the
treatment of chronic lymphocytic leukemia.
[0871] In further particular embodiments, compositions of the
present invention are used to treat, ameliorate and/or prevent
hematological cancers. Compositions of the present invention may be
used in combination with one or more surgical and/or radiological
procedures and/or therapeutic agents to treat, ameliorate and/or
prevent hematological cancers. Hematological cancers which may be
treated using compositions of the present invention include, but
are not limited to, non-Hodgkin's lymphoma (e.g., small lymphocytic
lymphoma, follicular center cell lymphoma, lymphoplasmacytoid
lymphoma, marginal zone lymphoma, mantle cell lymphoma,
immunoblastic lymphoma, burkitt's lymphoma, lymphoblastic lymphoma,
peripheral T-cell lymphoma, anaplastic large cell lymphoma and
intestinal T-cell lymphoma), leukemia, acute lymphocytic leukemia,
chronic lymphocytic leukemia and plasma cell neoplasms including
multiple myeloma.
[0872] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are used to treat, ameliorate and/or
prevent hematological cancers. Compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) may be used in combination with one
or more surgical and/or radiological procedures and/or therapeutic
agents to treat, ameliorate and/or prevent hematological cancers.
Hematological cancers which may be treated using compositions of
the present invention include, but are not limited to,
non-Hodgkin's lymphoma (e.g., small lymphocytic lymphoma,
follicular center cell lymphoma, lymphoplasmacytoid lymphoma,
marginal zone lymphoma, mantle cell lymphoma, immunoblastic
lymphoma, burkitt's lymphoma, lymphoblastic lymphoma, peripheral
T-cell lymphoma, anaplastic large cell lymphoma and intestinal
T-cell lymphoma), leukemia, acute lymphocytic leukemia, chronic
lymphocytic leukemia and plasma cell neoplasms including multiple
myeloma.
[0873] In one preferred embodiment, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are used to treat plasma cell
neoplasms. In a specific embodiment, that plasma cell neoplasm is
multiple myeloma.
[0874] In another preferred embodiment, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are used to treat non-Hodgkin's
lymphoma.
[0875] In another preferred embodiment, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are used to treat leukemia. In a
specific embodiment, that leukemia is acute lymphocytic leukemia.
In another specific embodiment, that leukemia is chronic
lymphocytic leukemia.
[0876] compositions of the present invention (e.g. Neutrokine-alpha
polypeptides in association with toxins or cytotoxic prodrugs) may
be administered in combination with one or more surgical and/or
radiological procedures useful in the treatment of hematological
cancer including, but not limited to, bone marrow transplantation,
external beam radiation and total body irradiation.
[0877] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more surgical and/or radiological procedures useful in the
treatment of hematological cancer including, but not limited to,
bone marrow transplantation, external beam radiation and total body
irradiation.
[0878] In one preferred embodiment, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) may be administered in combination
with one or more surgical and/or radiological procedures useful in
the treatment of multiple myeloma including, but not limited to,
allogeneic bone marrow transplantation and peripheral stem cell
support.
[0879] In another preferred embodiment, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) may be administered in combination
with one or more surgical and/or radiological procedures useful in
the treatment of non-Hodgkin's lymphoma including, but not limited
to, allogeneic bone marrow transplantation and peripheral stem cell
support.
[0880] In further specific embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) may be administered in combination
with one or more surgical and/or radiological procedures useful in
the treatment of leukemia including, but not limited to, allogeneic
bone marrow transplantation and peripheral stem cell support. In
one specific preferred embodiment, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) of the invention are used to treat
acute lymphocytic leukemia (ALL). In another specific preferred
embodiment, compositions of the present invention (e.g.
Neutrokine-alpha polypeptides in association with toxins or
cytotoxic prodrugs) are used to treat chronic lymphocytic leukemia
(CLL).
[0881] Compositions of the present invention may be administered in
combination with one or more therapeutic agents useful in the
treatment of multiple myeloma including, but not limited to,
Alkylating agents, Anthracyclines, Carmustine (DTI-015, BCNU,
BiCNU, Gliadel Wafer.RTM.), Cyclophosphamide (Cytoxan.RTM.,
Neosar.RTM., CTX), Dexamethasone (Decadron.RTM.), Doxorubicin
(Adriamycin.RTM., Doxil.RTM., Rubex.RTM.), Melphalan (L-PAM,
Alkeran.RTM., Phenylalanine mustard), Prednisone, Thalidomide and
Vincristine (Oncovorin.RTM., Onco TCS.RTM., VCR,
Leurocristine.RTM.).
[0882] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agents in the
treatment, amelioration and/or prevention of multiple myeloma.
[0883] Preferred combinations of therapeutic agents useful in the
treatment of multiple myeloma which may be administered in
combination with compositions of the present invention include, but
are not limited to, Cyclophosphamide+Prednisone,
Melphalan+Prednisone (MP),
Vincristine+Adriamycin.RTM.+Dexamethasone (VAD),
Vincristine+Carmustine+Melphalan+Cyclophosphamide+Prednisone
(VBMCP; the M2 protocol), and
Vincristine+Melphalan+Cyclophosphamide+Prednisone alternating with
Vincristine+Carmustine+Doxorubicin+Prednisone (VMCP/VBAP).
[0884] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agent combinations
in the treatment, amelioration and/or prevention of multiple
myeloma.
[0885] Compositions of the present invention may be administered in
combination with one or more therapeutic agents useful in the
treatment of non-Hodgkin's lymphoma including, but not limited to,
2-chlorodeoxyadenosine, Amifostine (Ethyol.RTM., Ethiofos.RTM.,
WR-272), Bexarotene (Targretin.RTM., Targretin Gel.RTM., Targretin
Oral.RTM., LGD1069), Bleomycin (Blenoxane.RTM.), Busulfan
(Busulfex.RTM., Myleran.RTM.), Carboplatin (Paraplatin.RTM.,
CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer.RTM.),
Chlorambucil (Leukeran.RTM.), Cisplatin (Platinol.RTM., CDDP),
Cladribine (2-CdA, Leustatin.RTM.), Cyclophosphamide (Cytoxan.RTM.,
Neosar.RTM., CTX), Cytarabine (Cytosar-U.RTM., ara-C, cytosine
arabinoside, DepoCyt.RTM.), Dacarbazine (DTIC), Daunorubicin
(Daunomycin, DaunoXome.RTM., Daunorubicin.RTM., Cerubidine.RTM.),
Denileukin diftitox (Ontak.RTM.), Dexamethasone (Decadron.RTM.),
Dolasetron mesylate (Anzemet.RTM.), Doxorubicin (Adriamycin.RTM.,
Doxil.RTM., Rubex.RTM.), Erythropoietin (EPO.RTM., Epogen.RTM.,
Procrit.RTM.), Etoposide phosphate (Etopophos.RTM.), Etoposide
(VP-16, Vepesid.RTM.), Fludarabine (Fludara.RTM., FAMP),
Granisetron (Kytril.RTM.), Hydrocortisone, Idarubicin
(Idamycin.RTM., DMDR, IDA), Ifosfamide (IFEX.RTM.), Interferon
alpha (Alfaferone.RTM., Alpha-IF.RTM.), Interferon alpha 2a (Intron
A.RTM.), Mechlorethamine (Nitrogen Mustard, HN.sub.2,
Mustargen.RTM.), Melphalan (L-PAM, Alkeran.RTM., Phenylalanine
mustard), Methotrexate.RTM. (MTX, Mexate.RTM., Folex.RTM.),
Methylprednisolone (Solumedrol.RTM.), Mitoxantrone
(Novantrone.RTM., DHAD), Ondansetron (Zofran.RTM.), Pentostatin
(Nipent.RTM., 2-deoxycoformycin), Perfosfamide
(4-hydroperoxycyclophosphamide, 4-HC), Prednisone, Procarbazine
(Matulane.RTM.), Rituximab.RTM. (Rituxan.RTM., anti-CD20 MAb),
Thiotepa (triethylenethiophosphaoramide, Thioplex.RTM.), Topotecan
(Hycamtin.RTM., SK&F-104864, NSC-609699, Evotopin.RTM.),
Vinblastine (Velban.RTM., VLB), Vincristine (Oncovin.RTM., Onco
TCS.RTM., VCR, Leurocristine.RTM.) and Vindesine (Eldisine.RTM.,
Fildesin.RTM.).
[0886] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agents in the
treatment, amelioration and/or prevention of non-Hodgkin's
lymphoma.
[0887] Preferred combinations of therapeutic agents useful in the
treatment of non-Hodgkin's lymphoma which may be administered in
combination with compositions of the present invention include, but
are not limited to,
Adriamycin.RTM.+Blenoxane+Vinblastine+Dacarbazine (ABVD),
Anti-idiotype therapy (BsAb)+Interferon alpha, Anti-idiotype
therapy (BsAb)+Chlorambucil, Anti-idiotype therapy
(BsAb)+Interleukin-2, BCNU (Carmustine)+Etoposide+Ara-C
(Cytarabine)+Melphalen (BEAM),
Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+Vincristine+Procarbazine+-
Prednisone (BEACOPP), Bryostatin+Vincristine, Cyclophosphamide+BCNU
(Carmustine)+VP-16 (Etoposide) (CBV),
Cyclophosphamide+Vincristine+Prednisone (CVP),
Cyclophosphamide+Adriamycin.RTM. (Hydroxyldaunomycin)+Vincristine
(Oncovorin)+Prednisone (CHOP), Cyclophosphamide+Novantrone.RTM.
(Mitoxantrone)+Vincristine (Oncovorin)+Prednisone (CNOP),
Cyclophosphamide+Doxorubicin+Teniposide+Prednisone,
Cyclophosphamide+Adriamycin.RTM. (Hydroxyldaunomycin)+Vincristine
(Oncovorin)+Prednisone+Rituximab (CHOP+Rituximab),
Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon
alpha, Cytarabine+Bleomycin+Vincristine+Methotrexate (CytaBOM),
Dexamethasone+Cytarabine+Cisplatin (DHAP),
Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE),
Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+P-
rednisone (Stanford V), Etoposide+Vinblastine+Adriamycin (EVA),
Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP),
Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC), Fludarabine,
Mitoxantrone+Dexamethasone (FMD), Fludarabine, Dexamethasone,
Cytarabine (ara-C), +Cisplatin (Platinol.RTM.) (FluDAP),
Ifosfamide+Cisplatin+Etoposide (ICE), Mechlorethamine+Oncovin.RTM.
(Vincristine)+Procarbazine+Prednisone (MOPP),
Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate with
leucovorin
rescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone
(m-BACOD),
Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide
(ProMACE), Thiotepa+Busulfan+Cyclophosphamide,
Thiotepa+Busulfan+Melphalan, Topotecan+Paclitaxel, and Vincristine
(Oncovin.RTM.)+Adriamycin.RTM.+Dexamethasone (VAD).
[0888] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agent combinations
in the treatment, amelioration and/or prevention of non-Hodgkin's
lymphoma.
[0889] Further examples of therapeutic agents useful in the
treatment of non-Hodgkin's lymphoma which may be administered in
combination with compositions of the present invention include, but
are not limited to, A007
(4-4'-dihydroxybenzophenone-2,4-dinitrophenylhydrazone), AG-2034
(AG-2024, AG-2032, GARFT [glycinamide ribonucleoside
transformylase] inhibitor), Aldesleukin (IL-2, Proleukin.RTM.),
Alemtuzumab (Campath.RTM.), Alitretinoin (Panretin.RTM., LGN-1057),
Altretamine (Hexylen.RTM., hexamethylmelamine, Hexastat.RTM.),
Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071),
Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv, anti-NHL MAb), Anti-idiotype
therapy (BsAb), Arabinosylguanine (Ara-G, GW506U78), Arsenic
trioxide (Trisenox.RTM., ATO), B43-Genistein (anti-CD19
Ab/genistein conjugate), B7 antibody conjugates, Betathine
(Beta-LT), BLyS antagonists, Bryostatin-1 (Bryostatin.RTM.,
BMY-45618, NSC-339555), CHML (Cytotropic Heterogeneous Molecular
Lipids), Clofarabine (chloro-fluoro-araA), Daclizumab
(Zenapax.RTM.), Depsipeptide (FR901228, FK228), Dolastatin-10
(DOLA-10, NSC-376128), Epirubicin (Ellence.RTM., EPI, 4'
epi-doxorubicin), Epratuzumab (Lymphocide.RTM., humanized
anti-CD22, HAT), Fly3/flk2 ligand (Mobista.RTM.), G3139
(Genasense.RTM., GentaAnticode.RTM., Bcl-2 antisense), Hu1D10
(anti-HLA-DR MAb, SMART 1D10), HumaLYM (anti-CD20 MAb), Ibritumomab
tiuxetan (Zevalin.RTM.), Interferon gamma (Gamma-interferon, Gamma
100.RTM., Gamma-IF), Irinotecan (Camptosar.RTM., CPT-11,
Topotecin.RTM., CaptoCPT-1), ISIS-2053, ISIS-3521 (PKC-alpha
antisense), Lmb-2 immunotoxin (anti-CD25 recombinant immuno toxin,
anti-Tac(Fv)-PE38), Leuvectin.RTM. (cytofectin+IL-2 gene, IL-2 gene
therapy), Lym-1 (131-I LYM-1), Lymphoma vaccine (Genitope),
Nelarabine (Compound 506, U78), Neugene compounds (Oncomyc-NG.RTM.,
Resten-NG.RTM., myc antisense), NovoMAb-G2 scFv (NovoMAb-G2 IgM),
06-benzylguanine (BG, Procept.RTM.), Oxaliplatin (Eloxatine.RTM.,
Eloxatin.RTM.), Paclitaxel (Paxene.RTM., Taxol.RTM.),
Paclitaxel-DHA (Taxoprexin.RTM.), Peldesine (BCX-34, PNP
inhibitor), Rebeccamycin and Rebeccamycin analogues, SCH-66336,
Sobuzoxane (MST-16, Perazolin.RTM.), SU5416 (Semaxanib.RTM., VEGF
inhibitor), TER-286, Thalidomide, TNP-470 (AGM-1470), Tositumomab
(Bexxar.RTM.), Valspodar (PSC 833), Vaxid (B-cell lymphoma DNA
vaccine), Vinorelbine (Navelbine.RTM.), WF10 (macrophage regulator)
and XR-9576 (XR-9351, P-glycoprotein/MDR inhibitor).
[0890] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agents in the
treatment, amelioration and/or prevention of non-Hodgkin's
lymphoma.
[0891] Compositions of the present invention may be administered in
combination with one or more therapeutic agents useful in the
treatment of acute lymphocytic leukemia including, but not limited
to, Amsacrine, Carboplatin (Paraplatin.RTM., CBDCA), Carmustine
(DTI-015, BCNU, BiCNU, Gliadel Wafer.RTM.), Cholecaliferol,
Cyclophosphamide (Cytoxan.RTM., Neosar.RTM., CTX), Cytarabine
(Cytosar-U.RTM., ara-C, cytosine arabinoside, DepoCyt.RTM.),
Daunorubicin (Daunomycin, DaunoXome.RTM., Daunorubicin.RTM.,
Cerubidine.RTM.), Dexamethasone (Decadron.RTM.), Doxorubicin
(Adriamycin.RTM., Doxil.RTM., Rubex.RTM.), Etoposide (VP-16,
Vepesid.RTM.), Filgrastam.RTM. (Neupogen.RTM., G-CSF,
Leukine.RTM.), Fludarabine (Fludara.RTM., FAMP), Idarubicin
(Idamycin.RTM., DMDR, IDA), Ifosfamide (IFEX.RTM.), Imatinib
mesylate (STI-571, Imatinib.RTM., Glivec.RTM., Gleevec.RTM., Abl
tyrosine kinase inhibitor), Interferon gamma (Gamma-interferon,
Gamma 100.RTM., Gamma-IF), L-asparaginase (Elspar.RTM.,
Crastinin.RTM., Asparaginase Medac.RTM., Kidrolase.RTM.),
Mercaptopurine (6-mercaptopurine, 6-MP), Methotrexate.RTM. (MTX,
Mexate.RTM., Folex.RTM.), Mitoxantrone (Novantrone.RTM., DHAD),
Pegaspargase.RTM. (Oncospar.RTM.), Prednisone, Retinoic acid,
Teniposide (VM-26, Vumon.RTM.), Thioguanine (6-thioguanine, 6-TG),
Topotecan (Hycamtin.RTM., SK&F-104864, NSC-609699,
Evotopin.RTM.), Tretinoin (Retin-A.RTM., Atragen.RTM., ATRA,
Vesanoid.RTM.) and Vincristine (Oncovorin.RTM., Onco TCS.RTM., VCR,
Leurocristine.RTM.).
[0892] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agents in the
treatment, amelioration and/or prevention of acute lymphocytic
leukemia.
[0893] Further examples of therapeutic agents useful in the
treatment of acute lymphocytic leukemia which may be administered
in combination with compositions of the present invention include,
but are not limited to, Aminocamptothecin (9-AC,
9-Aminocamptothecin, NSC 603071), Aminopterin, Annamycin (AR-522,
annamycin LF, Aronex.RTM.), Arabinosylguanine (Ara-G, GW506U78,
Nelzarabine.RTM.), Arsenic trioxide (Trisenox.RTM., ATO,
Atrivex.RTM.), B43-Genistein (anti-CD19 Ab/genistein conjugate),
B43-PAP (anti-CD19 Ab/pokeweed antiviral protein conjugate),
Cordycepin, CS-682, Decitabine (5-aza-2'-deoxycytidine),
Dolastatin-10 (DOLA-10, NSC-376128), G3139 (Genasense.RTM.,
GentaAnticode.RTM., Bcl-2 antisense), Irofulven (MGI-114,
Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Quinine,
TNP-470 (AGM-1470, Fumagillin), Trimetrexate (Neutrexin.RTM.),
Troxacitabine (BCH-204, BCH-4556, Troxatyl.RTM.), UCN-01
(7-hydroxystaurosporine), WHI-P131 and WT1 Vaccine.
[0894] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agents in the
treatment, amelioration and/or prevention of acute lymphocytic
leukemia.
[0895] Preferred combinations of therapeutic agents useful in the
treatment of acute lymphocytic leukemia which may be administered
in combination with compositions of the present invention include,
but are not limited to, Carboplatin+Mitoxantrone,
Carmustine+Cyclophosphamide+Etoposide, Cytarabine+Daunorubicin,
Cytarabine+Doxorubicin, Cytarabine+Idarubicin,
Cytarabine+Interferon gamma, Cytarabine+L-asparaginase,
Cytarabine+Mitoxantrone, Cytarabine+Fludarabine and Mitoxantrone,
Etoposide+Cytarabine, Etoposide+Ifosfamide, Etoposide+Mitoxantrone,
Ifosfamide+Etoposide+Mitoxantrone, Ifosfamide+Teniposide,
Methotrexate+Mercaptopurine,
Methotrexate+Mercaptopurine+Vincristine+Prednisone,
Phenylbutyrate+Cytarabine, Phenylbutyrate+Etoposide,
Phenylbutyrate+Topotecan, Phenylbutyrate+Tretinoin,
Quinine+Doxorubicin, Quinine+Mitoxantrone+Cytarabine,
Thioguanine+Cytarabine+Amsacrine, Thioguanine+Etoposide+Idarubicin,
Thioguanine+Retinoic acid+Cholecaliferol, Vincristine+Prednisone,
Vincristine+Prednisone and L-asparaginase,
Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubici-
n,
Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubi-
cin+Filgrastim,
Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubici-
n+Cyclophosphamide+Methotrexate, and
Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubici-
n+Cyclophosphamide+Methotrexate+Filgrastim.
[0896] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agent combinations
in the treatment, amelioration and/or prevention of acute
lymphocytic leukemia.
[0897] Compositions of the present invention may be administered in
combination with one or more therapeutic agents useful in the
treatment of chronic lymphocytic leukemia including, but not
limited to, Chlorambucil (Leukeran.RTM.), Cladribine (2-CdA,
Leustatin.RTM.), Cyclophosphamide (Cytoxan.RTM., Neosar.RTM., CTX),
Cytarabine (Cytosar-U.RTM., ara-C, cytosine arabinoside,
DepoCyt.RTM., cytarabine ocfosfate, ara-CMP), Doxorubicin
(Adriamycin.RTM., Doxil.RTM., Rubex.RTM.), Fludarabine
(Fludara.RTM., FAMP), Pentostatin (Nipent.RTM., 2-deoxycoformycin),
Prednisone and Vincristine (Oncovorin.RTM., Onco TCS.RTM., VCR,
Leurocristine.RTM.).
[0898] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agents in the
treatment, amelioration and/or prevention of chronic lymphocytic
leukemia.
[0899] Further examples of therapeutic agents useful in the
treatment of chronic lymphocytic leukemia which may be administered
in combination with compositions of the present invention include,
but are not limited to, Alemtuzumab (Campath.RTM.),
Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071),
Aminopterin, Annamycin (AR-522, annamycin LF, Aronex.RTM.),
Arabinosylguanine (Ara-G, GW506U78, Nelzarabine.RTM., Compound
506U78), Arsenic trioxide (Trisenox.RTM., ATO, Atrivex.RTM.),
Bryostatin-1 (Bryostatin.RTM., BMY-45618, NSC-339555), CS-682,
Dolastatin-10 (DOLA-10, NSC-376128), Filgrastim (Neupogen.RTM.,
G-CSF, Leukine), Flavopiridol (NSC-649890, HMR-1275), G3139
(Genasense.RTM., GentaAnticode.RTM., Bcl-2 antisense), Irofulven
(MGI-114, Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate,
Rituximab.RTM. (Rituxan.RTM., anti-CD20 Ab), Thalidomide,
Theophylline, TNP-470 (AGM-1470, Fumagillin), UCN-01
(7-hydroxystaurosporine) and WHI-P131.
[0900] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agents in the
treatment, amelioration and/or prevention of chronic lymphocytic
leukemia.
[0901] Preferred combinations of therapeutic agents useful in the
treatment of chronic lymphocytic leukemia which may be administered
in combination with compositions of the present invention include,
but are not limited to, Fludarabine+Prednisone, and
Cyclophosphamide+Doxorubicin+Vincristine+Prednisone (CHOP).
[0902] In preferred embodiments, compositions of the present
invention (e.g. Neutrokine-alpha polypeptides in association with
toxins or cytotoxic prodrugs) are administered in combination with
one or more of the above-described therapeutic agent combinations
in the treatment, amelioration and/or prevention of chronic
lymphocytic leukemia.
[0903] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5,
IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha,
IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In another
embodiment, compositions of the invention may be administered with
any interleukin, including, but not limited to, IL-1alpha,
IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,
IL-20, IL-21, and IL-22. In preferred embodiments, the compositions
of the invention are administered in combination with IL4 and IL10.
Both IL4 and IL10 have been observed by the inventors to enhance
Neutrokine-alpha mediated B cell proliferation.
[0904] In vitro, IFN gamma and IL-10 have each been observed by the
inventors to enhance cell surface expression of Neutrokine-alpha in
monocytes and macrophages (macrophages were obtained by culturing
primary monocytes with 20 ng/mL of M-CSF for 12-15 days), whereas
IL-4 treatment decreased cell surface expression of
Neutrokine-alpha in monocytes and macrophages. IL-4 administered
with IL-10 resulted in a complete inhibition of the IL-10 induced
cell surface expression of Neutrokine-alpha. IL-4 administered with
IFN-gamma resulted in increased cell-surface expression of
Neutrokine-alpha. Treatment of macrophages with IFN-gamma and IL-10
resulted in a 3 fold increase of soluble (active) Neutrokine-alpha
released into the culture medium compared to untreated
macrophages.
[0905] In an additional embodiment, the compositions of the
invention are administered with a chemokine. In another embodiment,
the compositions of the invention are administered with chemokine
beta-8, chemokine beta-1, and/or macrophage inflammatory protein-4.
In a preferred embodiment, the compositions of the invention are
administered with chemokine beta-8.
[0906] In an additional embodiment, the compositions of the
invention are administered in combination with an IL-4 antagonist.
IL-4 antagonists that may be administered with the compositions of
the invention include, but are not limited to: soluble IL-4
receptor polypeptides, multimeric forms of soluble IL-4 receptor
polypeptides; anti-IL-4 receptor antibodies that bind the IL-4
receptor without transducing the biological signal elicited by
IL-4, anti-IL4 antibodies that block binding of IL-4 to one or more
IL-4 receptors, and muteins of IL-4 that bind IL-4 receptors but do
not transduce the biological signal elicited by IL-4. Preferably,
the antibodies employed according to this method are monoclonal
antibodies (including antibody fragments, such as, for example,
those described herein).
[0907] In an additional embodiment, the compositions of the
invention are administered in combination with hematopoietic growth
factors. Hematopoietic growth factors that may be administered with
the compositions of the invention include, but are not limited to,
LEUKINE.TM. (SARGRAMOSTIM.TM.) and NEUPOGEN.TM.
(FILGRASTIM.TM.).
[0908] In an additional embodiment, the compositions of the
invention are administered in combination with fibroblast growth
factors. Fibroblast growth factors that may be administered with
the compositions of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0909] Additionally, the compositions of the invention may be
administered alone or in combination with other therapeutic
regimens, including but not limited to, radiation therapy. Such
combinatorial therapy may be administered sequentially and/or
concomitantly.
Agonists and Antagonists--Assays and Molecules
[0910] The invention also provides a method of screening compounds
to identify those which enhance or block the action of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide on cells,
such as its interaction with Neutrokine-alpha and/or
Neutrokine-alphaSV binding molecules such as receptor molecules. An
agonist is a compound which increases the natural biological
functions of Neutrokine-alpha and/or Neutrokine-alphaSV or which
functions in a manner similar to Neutrokine-alpha and/or
Neutrokine-alphaSV while antagonists decrease or eliminate such
functions.
[0911] In another embodiment, the invention provides a method for
identifying a receptor protein or other ligand-binding protein
which binds specifically to a Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide. For example, a cellular
compartment, such as a membrane or a preparation thereof, may be
prepared from a cell that expresses a molecule that binds
Neutrokine-alpha and/or Neutrokine-alphaSV. The preparation is
incubated with labeled Neutrokine-alpha and/or Neutrokine-alphaSV
and complexes of Neutrokine-alpha and/or Neutrokine-alphaSV bound
to the receptor or other binding protein are isolated and
characterized according to routine methods known in the art.
Alternatively, the Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide may be bound to a solid support so that binding
molecules solubilized from cells are bound to the column and then
eluted and characterized according to routine methods.
[0912] In the assay of the invention for agonists or antagonists, a
cellular compartment, such as a membrane or a preparation thereof,
may be prepared from a cell that expresses a molecule that binds
Neutrokine-alpha and/or Neutrokine-alphaSV such as a molecule of a
signaling or regulatory pathway modulated by Neutrokine-alpha
and/or Neutrokine-alphaSV. The preparation is incubated with
labeled Neutrokine-alpha and/or Neutrokine-alphaSV in the absence
or the presence of a candidate molecule which may be a
Neutrokine-alpha and/or Neutrokine-alphaSV agonist or antagonist.
The ability of the candidate molecule to bind the binding molecule
is reflected in decreased binding of the labeled ligand. Molecules
which bind gratuitously, i.e., without inducing the effects of
Neutrokine-alpha on binding the Neutrokine-alpha and/or
Neutrokine-alphaSV binding molecule, are most likely to be good
antagonists. Molecules that bind well and elicit effects that are
the same as or closely related to Neutrokine-alpha and/or
Neutrokine-alphaSV are agonists.
[0913] Neutrokine-alpha- and/or Neutrokine-alphaSV-like effects of
potential agonists and antagonists may by measured, for instance,
by determining activity of a second messenger system following
interaction of the candidate molecule with a cell or appropriate
cell preparation, and comparing the effect with that of
Neutrokine-alpha and/or Neutrokine-alphaSV or molecules that elicit
the same effects as Neutrokine-alpha and/or Neutrokine-alphaSV.
Second messenger systems that may be useful in this regard include
but are not limited to AMP guanylate cyclase, ion channel or
phosphoinositide hydrolysis second messenger systems.
[0914] Another example of an assay for Neutrokine-alpha and/or
Neutrokine-alphaSV antagonists is a competitive assay that combines
Neutrokine-alpha and/or Neutrokine-alphaSV and a potential
antagonist with membrane-bound receptor molecules or recombinant
Neutrokine-alpha and/or Neutrokine-alphaSV receptor molecules under
appropriate conditions for a competitive inhibition assay.
Neutrokine-alpha and/or Neutrokine-alphaSV can be labeled, such as
by radioactivity, such that the number of Neutrokine-alpha and/or
Neutrokine-alphaSV molecules bound to a receptor molecule can be
determined accurately to assess the effectiveness of the potential
antagonist.
[0915] Potential antagonists include small organic molecules,
peptides, polypeptides (e.g., IL-13), and antibodies that bind to a
polypeptide of the invention and thereby inhibit or extinguish its
activity. Potential antagonists also may be small organic
molecules, a peptide, a polypeptide such as a closely related
protein or antibody that binds the same sites on a binding
molecule, such as a receptor molecule, without inducing
Neutrokine-alpha and/or Neutrokine-alphaSV induced activities,
thereby preventing the action of Neutrokine-alpha and/or
Neutrokine-alphaSV by excluding Neutrokine-alpha and/or
Neutrokine-alphaSV from binding.
[0916] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56:
560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, Fla. (1988). Antisense
technology can be used to control gene expression through antisense
DNA or RNA, or through triple-helix formation. Antisense techniques
are discussed for example, in Okano, J., Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Triple helix formation is
discussed in, for instance Lee et al., Nucleic Acids Research 6:
3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et
al., Science 251: 1360 (1991). The methods are based on binding of
a polynucleotide to a complementary DNA or RNA. For example, the 5'
coding portion of a polynucleotide that encodes the extracellular
domain of the polypeptide of the present invention may be used to
design an antisense RNA oligonucleotide of from about 10 to 40 base
pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
thereby preventing transcription and the production of
Neutrokine-alpha and/or Neutrokine-alphaSV. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide. The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of
Neutrokine-alpha and/or Neutrokine-alphaSV.
[0917] In one embodiment, the Neutrokine-alpha and/or
Neutrokine-alphaSV antisense nucleic acid of the invention is
produced intracellularly by transcription from an exogenous
sequence. For example, a vector or a portion thereof, is
transcribed, producing an antisense nucleic acid (RNA) of the
invention. Such a vector would contain a sequence encoding the
Neutrokine-alpha and/or Neutrokine-alphaSV antisense nucleic acid.
Such a vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired
antisense RNA. Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid,
viral, or others know in the art, used for replication and
expression in vertebrate cells. Expression of the sequence encoding
Neutrokine-alpha and/or Neutrokine-alphaSV, or fragments thereof,
can be by any promoter known in the art to act in vertebrate,
preferably human cells. Such promoters can be inducible or
constitutive. Such promoters include, but are not limited to, the
SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310
(1981), the promoter contained in the 3' long terminal repeat of
Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the
herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci.
U.S.A. 78:1441-1445 (1981), the regulatory sequences of the
metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)),
etc.
[0918] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a Neutrokine-alpha and/or Neutrokine-alphaSV gene. However,
absolute complementarity, although preferred, is not required. A
sequence "complementary to at least a portion of an RNA," referred
to herein, means a sequence having sufficient complementarity to be
able to hybridize with the RNA, forming a stable duplex; in the
case of double stranded Neutrokine-alpha and/or Neutrokine-alphaSV
antisense nucleic acids, a single strand of the duplex DNA may thus
be tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid Generally, the larger the
hybridizing nucleic acid, the more base mismatches with a
Neutrokine-alpha and/or Neutrokine-alphaSV RNA it may contain and
still form a stable duplex (or triplex as the case may be). One
skilled in the art can ascertain a tolerable degree of mismatch by
use of standard procedures to determine the melting point of the
hybridized complex.
[0919] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R.,
1994, Nature 372:333-335. Thus, oligonucleotides complementary to
either the 5'- or 3'-non-translated, non-coding regions of
Neutrokine-alpha and Neutrokine-alphaSV shown in FIGS. 1A-B and
5A-B, respectively, could be used in an antisense approach to
inhibit translation of endogenous Neutrokine-alpha and/or
Neutrokine-alphaSV mRNA. Oligonucleotides complementary to the 5'
untranslated region of the mRNA should include the complement of
the AUG start codon. Antisense oligonucleotides complementary to
mRNA coding regions are less efficient inhibitors of translation
but could be used in accordance with the invention. Whether
designed to hybridize to the 5'-, 3'- or coding region of
Neutrokine-alpha and/or Neutrokine-alphaSV mRNA, antisense nucleic
acids should be at least six nucleotides in length, and are
preferably oligonucleotides ranging from 6 to about 50 nucleotides
in length. In specific aspects the oligonucleotide is at least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at
least 50 nucleotides.
[0920] The polynucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof,
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization,
etc. The oligonucleotide may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;
Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652 (1987); PCT
Publication No. WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents.
(See, e.g., Krol et al., BioTechniques 6:958-976 (1988)) or
intercalating agents. (See, e.g., Zon, Pharm. Res. 5:539-549
(1988)). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0921] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0922] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0923] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0924] In yet another embodiment, the antisense oligonucleotide is
an alpha-anomeric oligonucleotide. An alpha-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual beta-units, the
strands run parallel to each other (Gautier et al., Nucl. Acids
Res. 15:6625-6641 (1987)). The oligonucleotide is a
2-O-methylribonucleotide (Inoue et al., Nucl. Acids Res.
15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al.,
FEBS Lett. 215:327-330 (1997)).
[0925] Polynucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(Nucl. Acids Res. 16:3209 (1988)), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451 (1988)), etc.
[0926] While antisense nucleotides complementary to the
Neutrokine-alpha and/or Neutrokine-alphaSV coding region sequence
could be used, those complementary to the transcribed untranslated
region are most preferred.
[0927] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme (See, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al,
Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site specific recognition sequences can be used to destroy
Neutrokine-alpha and/or Neutrokine-alphaSV mRNAs, the use of
hammerhead ribozymes is preferred. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Haseloff
and Gerlach, Nature 334:585-591 (1988). There are numerous
potential hammerhead ribozyme cleavage sites within the nucleotide
sequence of Neutrokine-alpha and Neutrokine-alphaSV (FIGS. 1A-B and
5A-B, respectively). Preferably, the ribozyme is engineered so that
the cleavage recognition site is located near the 5' end of the
Neutrokine-alpha and/or Neutrokine-alphaSV mRNA; i.e., to increase
efficiency and minimize the intracellular accumulation of
non-functional mRNA transcripts.
[0928] As in the antisense approach, the ribozymes of the invention
can be composed of modified oligonucleotides (e.g. for improved
stability, targeting, etc.) and should be delivered to cells which
express Neutrokine-alpha and/or Neutrokine-alphaSV in vivo. DNA
constructs encoding the ribozyme may be introduced into the cell in
the same manner as described above for the introduction of
antisense encoding DNA. A preferred method of delivery involves
using a DNA construct "encoding" the ribozyme under the control of
a strong constitutive promoter, such as, for example, pol III or
pol II promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous Neutrokine-alpha
and/or Neutrokine-alphaSV messages and inhibit translation. Since
ribozymes unlike antisense molecules, are catalytic, a lower
intracellular concentration is required for efficiency.
[0929] Endogenous gene expression can also be reduced by
inactivating or "knocking out" the Neutrokine-alpha and/or
Neutrokine-alphaSV gene and/or its promoter using targeted
homologous recombination. (E.g., see Smithies et al., Nature
317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987);
Thompson et al., Cell 5:313-321 (1989); each of which is
incorporated by reference herein in its entirety). For example, a
mutant, non-functional polynucleotide of the invention (or a
completely unrelated DNA sequence) flanked by DNA homologous to the
endogenous polynucleotide sequence (either the coding regions or
regulatory regions of the gene) can be used, with or without a
selectable marker and/or a negative selectable marker, to transfect
cells that express polypeptides of the invention in vivo. In
another embodiment, techniques known in the art are used to
generate knockouts in cells that contain, but do not express the
gene of interest. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the targeted
gene. Such approaches are particularly suited in research and
agricultural fields where modifications to embryonic stem cells can
be used to generate animal offspring with an inactive targeted gene
(e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra).
However this approach can be routinely adapted for use in humans
provided the recombinant DNA constructs are directly administered
or targeted to the required site in vivo using appropriate viral
vectors that will be apparent to those of skill in the art. The
contents of each of the documents recited in this paragraph is
herein incorporated by reference in its entirety.
[0930] In other embodiments, antagonists according to the present
invention include soluble forms of Neutrokine-alpha and/or
Neutrokine-alphaSV (e.g., fragments of Neutrokine-alpha shown in
FIGS. 1A-B that include the ligand binding domain, TNF conserved
domain, and/or extracellular domain of Neutrokine-alpha and/or
Neutrokine-alphaSV and fragments of Neutrokine-alphaSV shown in
FIGS. 5A-B that include the ligand binding domain, TNF conserved
domain, and/or extracellular domain of Neutrokine-alpha and/or
Neutrokine-alphaSV). Such soluble forms of the Neutrokine-alpha
and/or Neutrokine-alphaSV, which may be naturally occurring or
synthetic, antagonize Neutrokine-alpha and/or Neutrokine-alphaSV
mediated signaling by competing with native Neutrokine-alpha and/or
Neutrokine-alphaSV for binding to Neutrokine-alpha and/or
Neutrokine-alphaSV receptors (e.g., DR5 (See, International
Publication No. WO 98/41629), TR10 (See, International Publication
No. WO 98/54202), 312C2 (See, International Publication No. WO
98/06842), and TR11, TR11SV1, and TR11SV2 (See, U.S. application
Ser. No. 09/176,200)), and/or by forming a multimer that may or may
not be capable of binding the receptor, but which is incapable of
inducing signal transduction. Preferably, these antagonists inhibit
Neutrokine-alpha and/or Neutrokine-alphaSV mediated stimulation of
lymphocyte (e.g., B-cell) proliferation, differentiation, and/or
activation. Antagonists of the present invention also include
antibodies specific for TNF-family ligands (e.g., CD30) and
Neutrokine-alpha-Fc and/or Neutrokine-alphaSV-Fc fusion
proteins.
[0931] By a "TNF-family ligand" is intended naturally occurring,
recombinant, and synthetic ligands that are capable of binding to a
member of the TNF receptor family and inducing and/or blocking the
ligand/receptor signaling pathway. Members of the TNF ligand family
include, but are not limited to, TNF-alpha, lymphotoxin-alpha
(LT-alpha, also known as TNF-beta), LT-beta (found in complex
heterotrimer LT-alpha2-beta), FasL, CD40L, (TNF-gamma
(International Publication No. WO 96/14328), AIM-I (International
Publication No. WO 97/33899), AIM-II (International Publication No.
WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha
(International Publication No. WO 98/07880), neutrokine-alpha
(International Publication No. WO 98/18921), CD27L, CD30L, 4-1BBL,
OX40L, CD27, CD30, 4-1BB, OX40, and nerve growth factor (NGF). In
preferred embodiments, the Neutrokine-alpha and/or
Neutrokine-alphaSV TNF-family ligands of the invention are DR5
(See, International Publication No. WO 98/41629), TR10 (See,
International Publication No. WO 98/54202), 312C2 (See,
International Publication No. WO 98/06842), and TR11, TR11SV1, and
TR11SV2 (See, U.S. application Ser. No. 09/176,200).
[0932] Antagonists of the present invention also include antibodies
specific for TNF-family receptors or the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention. Antibodies
according to the present invention may be prepared by any of a
variety of standard methods using Neutrokine-alpha and/or
Neutrokine-alphaSV immunogens of the present invention. As
indicated, such Neutrokine-alpha and/or Neutrokine-alphaSV
immunogens include the complete Neutrokine-alpha and
Neutrokine-alphaSV polypeptides depicted in FIGS. 1A-B (SEQ ID
NO:2) and FIGS. 5A-B (SEQ ID NO:19), respectively, (which may or
may not include the leader sequence) and Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide fragments comprising, for example,
the ligand binding domain, TNF-conserved domain, extracellular
domain, transmembrane domain, and/or intracellular domain, or any
combination thereof.
[0933] Polyclonal and monoclonal antibody agonists or antagonists
according to the present invention can be raised according to the
methods disclosed in Tartaglia and Goeddel, J. Biol. Chem.
267(7):4304-4307 (1992)); Tartaglia et al., Cell 73:213-216
(1993)), and PCT Application WO 94/09137 and are preferably
specific to (i.e., bind uniquely to polypeptides of the invention
having the amino acid sequence of SEQ ID NO:2. The term "antibody"
(Ab) or "monoclonal antibody" (mAb) as used herein is meant to
include intact molecules as well as fragments thereof (such as, for
example, Fab and F(ab') fragments) which are capable of binding an
antigen. Fab, Fab' and F(ab') fragments lack the Fc fragment intact
antibody, clear more rapidly from the circulation, and may have
less non-specific tissue binding of an intact antibody (Wahl et
al., J. Nucl. Med., 24:316-325 (1983)).
[0934] In a preferred method, antibodies according to the present
invention are mAbs. Such mAbs can be prepared using hybridoma
technology (Kohler and Millstein, Nature 256:495-497 (1975) and
U.S. Pat. No. 4,376,110; Harlow et al., Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1988; Monoclonal Antibodies and Hybridomas: A New Dimension
in Biological Analyses, Plenum Press, New York, N.Y., 1980;
Campbell, "Monoclonal Antibody Technology," In: Laboratory
Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon
et al., eds.), Elsevier, Amsterdam (1984)).
[0935] Proteins and other compounds which bind the Neutrokine-alpha
and/or Neutrokine-alphaSV domains are also candidate agonists and
antagonists according to the present invention. Such binding
compounds can be "captured" using the yeast two-hybrid system
(Fields and Song, Nature 340:245-246 (1989)). A modified version of
the yeast two-hybrid system has been described by Roger Brent and
his colleagues (Gyuris, Cell 75:791-803 (1993); Zervos et al., Cell
72:223-232 (1993)). Preferably, the yeast two-hybrid system is used
according to the present invention to capture compounds which bind
to the ligand binding domain, extracellular, intracellular,
transmembrane, and death domain of the Neutrokine-alpha and/or
Neutrokine-alphaSV. Such compounds are good candidate agonists and
antagonists of the present invention.
[0936] For example, using the two-hybrid assay described above, the
extracellular or intracellular domain of the Neutrokine-alpha
and/or Neutrokine-alphaSV receptor, or a portion thereof, may be
used to identify cellular proteins which interact with
Neutrokine-alpha and/or Neutrokine-alphaSV the receptor in vivo.
Such an assay may also be used to identify ligands with potential
agonistic or antagonistic activity of Neutrokine-alpha and/or
Neutrokine-alphaSV receptor function. This screening assay has
previously been used to identify protein which interact with the
cytoplasmic domain of the murine TNF-RII and led to the
identification of two receptor associated proteins. Rothe et al.,
Cell 78:681 (1994). Such proteins and amino acid sequences which
bind to the cytoplasmic domain of the Neutrokine-alpha and/or
Neutrokine-alphaSV receptors are good candidate agonist and
antagonist of the present invention.
[0937] Other screening techniques include the use of cells which
express the polypeptide of the present invention (for example,
transfected CHO cells) in a system which measures extracellular pH
changes caused by receptor activation, for example, as described in
Science, 246:181-296 (1989). In another example, potential agonists
or antagonists may be contacted with a cell which expresses the
polypeptide of the present invention and a second messenger
response, e.g., signal transduction may be measured to determine
whether the potential antagonist or agonist is effective.
[0938] Agonists according to the present invention include
naturally occurring and synthetic compounds such as, for example,
TNF family ligand peptide fragments, transforming growth factor,
neurotransmitters (such as glutamate, dopamine,
N-methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells
and antimetabolites. Preferred agonists include chemotherapeutic
drugs such as, for example, cisplatin, doxorubicin, bleomycin,
cytosine arabinoside, nitrogen mustard, methotrexate and
vincristine. Others include ethanol and -amyloid peptide. (Science
267:1457-1458 (1995)).
[0939] Preferred agonists are fragments of Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention which stimulate
lymphocyte (e.g., B cell) proliferation, differentiation and/or
activation. Further preferred agonists include polyclonal and
monoclonal antibodies raised against the Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptides of the invention, or a fragment
thereof. Such agonist antibodies raised against a TNF-family
receptor are disclosed in Tartaglia et al., Proc. Natl. Acad. Sci.
USA 88:9292-9296 (1991); and Tartaglia et al., J. Biol. Chem.
267:4304-4307 (1992). See, also, PCT Application WO 94/09137.
[0940] In an additional embodiment, immunoregulatory molecules such
as, for example, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13,
IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha, may be used as
agonists of Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides
of the invention which stimulate lymphocyte (e.g., B cell)
proliferation, differentiation and/or activation. In a specific
embodiment, IL4 and/or IL10 are used to enhance the
Neutrokine-alpha- and/or Neutrokine-alphaSV-mediated proliferation
of B cells.
[0941] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the polypeptides of the invention. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
[0942] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, e.g., genetically engineered fibroblasts
can be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each
of which is incorporated by reference herein in its entirety).
[0943] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
[0944] In yet another embodiment of the invention, the activity of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide can be
reduced using a "dominant negative." To this end, constructs which
encode defective Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptide, such as, for example, mutants lacking all or a portion
of the TNF-conserved domain, can be used in gene therapy approaches
to diminish the activity of Neutrokine-alpha and/or
Neutrokine-alphaSV on appropriate target cells. For example,
nucleotide sequences that direct host cell expression of
Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide in which all
or a portion of the TNF-conserved domain is altered or missing can
be introduced into monocytic cells or other cells or tissues
(either by in vivo or ex vivo gene therapy methods described herein
or otherwise known in the art). Alternatively, targeted homologous
recombination can be utilized to introduce such deletions or
mutations into the subject's endogenous Neutrokine-alpha and/or
Neutrokine-alphaSV gene in monocytes. The engineered cells will
express non-functional Neutrokine-alpha and/or Neutrokine-alphaSV
polypeptides (i.e., a ligand (e.g., multimer) that may be capable
of binding, but which is incapable of inducing signal
transduction).
Chromosome Assays
[0945] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0946] In certain preferred embodiments in this regard, the cDNA
and/or polynucleotides herein disclosed is used to clone genomic
DNA of a Neutrokine-alpha and/or Neutrokine-alphaSV gene. This can
be accomplished using a variety of well known techniques and
libraries, which generally are available commercially. The genomic
DNA then is used for in situ chromosome mapping using well known
techniques for this purpose.
[0947] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Fluorescence
in situ hybridization ("FISH") of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal
location in one step. This technique can be used with probes from
the cDNA as short as 50 or 60 bp. For a review of this technique,
see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,
Pergamon Press, New York (1988).
[0948] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0949] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0950] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0951] Utilizing the techniques described above, the chromosomal
location of Neutrokine-alpha and Neutrokine-alphaSV was determined
with high confidence using a combination of somatic cell hybrids
and radiation hybrids to chromosome position 13q34.
EXAMPLES
[0952] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting. Many of the following examples are set forth referring
specifically to Neutrokine-alpha polynucleotides and polypeptides
of the invention. Each example may also be practiced to generate
and/or examine Neutrokine-alphaSV polynucleotides and/or
polypeptides of the invention. One of ordinary skill in the art
would easily be able to direct the following examples to
Neutrokine-alphaSV.
Example 1a
Expression and Purification of "His-tagged" Neutrokine-Alpha in E.
coli
[0953] The bacterial expression vector pQE9 (pD10) is used for
bacterial expression in this example. (QIAGEN, Inc., supra). pQE9
encodes ampicillin antibiotic resistance ("Ampr") and contains a
bacterial origin of replication ("ori"), an IPTG inducible
promoter, a ribosome binding site ("RBS"), six codons encoding
histidine residues that allow affinity purification using
nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin sold by
QIAGEN, Inc., supra, and suitable single restriction enzyme
cleavage sites. These elements are arranged such that an inserted
DNA fragment encoding a polypeptide expresses that polypeptide with
the six His residues (i.e., a "6.times.His tag") covalently linked
to the amino terminus of that polypeptide.
[0954] The DNA sequence encoding the desired portion of the
Neutrokine-alpha protein comprising the extracellular domain
sequence is amplified from the deposited cDNA clone using PCR
oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of the protein and to sequences in
the deposited construct 3' to the cDNA coding sequence. Additional
nucleotides containing restriction sites to facilitate cloning in
the pQE9 vector are added to the 5' and 3' primer sequences,
respectively.
[0955] For cloning the extracellular domain of the protein, the 5'
primer has the sequence 5'-GTG GGA TCC AGC CTC CGG GCA GAG CTG-3'
(SEQ ID NO:10) containing the underlined Bam HI restriction site
followed by 18 nucleotides of the amino terminal coding sequence of
the extracellular domain of the sequence in FIGS. 1A and 1B. One of
ordinary skill in the art would appreciate, of course, that the
point in the protein coding sequence where the 5' primer begins may
be varied to amplify a DNA segment encoding any desired portion of
the complete Neutrokine a protein shorter or longer than the
extracellular domain of the form. The 3' primer has the sequence
5'-GTG AAG CTT TTA TTA CAG CAG TTT CAA TGC ACC-3' (SEQ ID NO:11)
containing the underlined Hind III restriction site followed by two
stop codons and 18 nucleotides complementary to the 3' end of the
coding sequence of the DNA sequence in FIGS. 1A and 1B.
[0956] The amplified DNA fragment and the vector pQE9 are digested
with Bam HI and Hind III and the digested DNAs are then ligated
together. Insertion of the DNA into the restricted pQE9 vector
places the protein coding region downstream from the IPTG-inducible
promoter and in-frame with an initiating AUG and the six histidine
codons.
[0957] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described in Sambrook
et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E.
coli strain M15/rep4, containing multiple copies of the plasmid
pREP4, which expresses the lac repressor and confers kanamycin
resistance ("Kan.sup.r"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing protein, is available commercially
from QIAGEN, Inc., supra. Transformants are identified by their
ability to grow on LB plates in the presence of ampicillin and
kanamycin. Plasmid DNA is isolated from resistant colonies and the
identity of the cloned DNA confirmed by restriction analysis, PCR
and DNA sequencing. Clones containing the desired constructs are
grown overnight ("O/N") in liquid culture in LB media supplemented
with both ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml).
The O/N culture is used to inoculate a large culture, at a dilution
of approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-beta-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0958] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the is loaded on to
a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(available from QIAGEN, Inc., supra). Proteins with a 6.times.His
tag bind to the Ni-NTA resin with high affinity and can be purified
in a simple one-step procedure (for details see: The
QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the
supernatant is loaded on to the column in 6 M guanidine-HCl, pH 8,
the column is first washed with 10 volumes of 6 M guanidine-HCl, pH
8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and
finally the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide
is eluted with 6 M guanidine-HCl, pH 5.
[0959] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM imidazole.
Imidazole is removed by a final dialyzing step against PBS or 50 mM
sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein
is stored at 4.degree. C. or frozen at -80.degree. C.
Example 1b
Expression and Purification of Neutrokine-Alpha in E. coli
[0960] The bacterial expression vector pQE60 is used for bacterial
expression in this example. (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibiotic
resistance ("Ampr") and contains a bacterial origin of replication
("ori"), an IPTG inducible promoter, a ribosome binding site
("RBS"), six codons encoding histidine residues that allow affinity
purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin sold by QIAGEN, Inc., supra, and suitable single
restriction enzyme cleavage sites. These elements are arranged such
that a DNA fragment encoding a polypeptide may be inserted in such
as way as to produce that polypeptide with the six His residues
(i.e., a "6.times.His tag") covalently linked to the carboxyl
terminus of that polypeptide. However, in this example, the
polypeptide coding sequence is inserted such that translation of
the six His codons is prevented and, therefore, the polypeptide is
produced with no 6.times.His tag.
[0961] The DNA sequence encoding the desired portion of the protein
comprising the extracellular domain sequence is amplified from the
deposited cDNA clone using PCR oligonucleotide primers which anneal
to the amino terminal sequences of the desired portion of the
protein and to sequences in the deposited construct 3' to the cDNA
coding sequence. Additional nucleotides containing restriction
sites to facilitate cloning in the pQE60 vector are added to the 5'
and 3' sequences, respectively.
[0962] For cloning the extracellular domain of the protein, the 5'
primer has the sequence 5'-GTG TCA TGA GCC TCC GGG CAG AGC TG-3'
(SEQ ID NO:12) containing the underlined Bsp HI restriction site
followed by 17 nucleotides of the amino terminal coding sequence of
the extracellular domain of the sequence in FIGS. 1A and 1B. One of
ordinary skill in the art would appreciate, of course, that the
point in the protein coding sequence where the 5' primer begins may
be varied to amplify a desired portion of the complete protein
shorter or longer than the extracellular domain of the form. The 3'
primer has the sequence 5'-GTG AAG CTT TTA TTA CAG CAG TTT CAA TGC
ACC-3' (SEQ ID NO:13) containing the underlined Hind III
restriction site followed by two stop codons and 18 nucleotides
complementary to the 3' end of the coding sequence in the DNA
sequence in FIGS. 1A and 1B.
[0963] The amplified DNA fragments and the vector pQE60 are
digested with Bsp HI and Hind III and the digested DNAs are then
ligated together. Insertion of the DNA into the restricted pQE60
vector places the protein coding region including its associated
stop codon downstream from the IPTG-inducible promoter and in-frame
with an initiating AUG. The associated stop codon prevents
translation of the six histidine codons downstream of the insertion
point.
[0964] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described in Sambrook
et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E.
coli strain M15/rep4, containing multiple copies of the plasmid
pREP4, which expresses the lac repressor and confers kanamycin
resistance ("Kan.sup.r"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing protein, is available commercially
from QIAGEN, Inc., supra. Transformants are identified by their
ability to grow on LB plates in the presence of ampicillin and
kanamycin. Plasmid DNA is isolated from resistant colonies and the
identity of the cloned DNA confirmed by restriction analysis, PCR
and DNA sequencing.
[0965] One of ordinary skill in the art recognizes that any of a
number of bacterial expression vectors may be useful in place of
pQE9 and pQE60 in the expression protocols presented in this
example. For example, the novel pHE4 series of bacterial expression
vectors, in particular, the pHE4-5 vector may be used for bacterial
expression in this example (ATCC Accession No. 209311; and
variations thereof). The plasmid DNA designated pHE4-5/MPIFD23 in
ATCC Deposit No. 209311 is vector plasmid DNA which contains an
insert which encodes another ORF. The construct was deposited with
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209, on Sep. 30, 1997. Using the Nde I and Asp
718 restriction sites flanking the irrelevant MPIF ORF insert, one
of ordinary skill in the art could easily use current molecular
biological techniques to replace the irrelevant ORF in the pHE4-5
vector with the Neutrokine-alpha ORF of the present invention.
[0966] The pHE4-5 bacterial expression vector includes a neomycin
phosphotransferase gene for selection, an E. coli origin of
replication, a T5 phage promoter sequence, two lac operator
sequences, a Shine-Delgarno sequence, and the lactose operon
repressor gene (lacIq). These elements are arranged such that an
inserted DNA fragment encoding a polypeptide expresses that
polypeptide with the six His residues (i.e., a "6.times.His tag")
covalently linked to the amino terminus of that polypeptide. The
promoter and operator sequences of the pHE4-5 vector were made
synthetically. Synthetic production of nucleic acid sequences is
well known in the art (CLONETECH 95/96 Catalog, pages 215-216,
CLONETECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303).
[0967] Clones containing the desired Neutrokine-alpha constructs
are grown overnight ("O/N") in liquid culture in LB media
supplemented with both ampicillin (100 .mu.g/ml) and kanamycin (25
.mu.g/ml). The O/N culture is used to inoculate a large culture, at
a dilution of approximately 1:25 to 1:250. The cells are grown to
an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
isopropyl-beta-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0968] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the Neutrokine a is
dialyzed against 50 mM Na-acetate buffer pH 6, supplemented with
200 mM NaCl. Alternatively, the protein can be successfully
refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM
Tris/HCl pH 7.4, containing protease inhibitors. After renaturation
the protein can be purified by ion exchange, hydrophobic
interaction and size exclusion chromatography. Alternatively, an
affinity chromatography step such as an antibody column can be used
to obtain pure protein. The purified protein is stored at 4.degree.
C. or frozen at -80.degree. C.
[0969] In certain embodiments, it is preferred to generate
expression constructs as detailed in this Example to mutate one or
more of the three cysteine residues in the Neutrokine-alpha
polypeptide sequence. The cysteine residues in the Neutrokine-alpha
polypeptide sequence are located at positions 147, 232, and 245 as
shown in SEQ ID NO:2 and at positions 213 and 226 of the
Neutrokine-alpha polypeptide sequence as shown in SEQ ID NO:19
(there is no cysteine in the Neutrokine-alphaSV polypeptide
sequence which corresponds to Cys-147 in the Neutrokine-alpha
polypeptide sequence because amino acid residues 143-160 of the
Neutrokine-alpha polypeptide sequence are not present in the
Neutrokine-alphaSV polypeptide sequence).
Example 2
Cloning, Expression, and Purification of Neutrokine-Alpha Protein
in a Baculovirus Expression System
[0970] In this illustrative example, the plasmid shuttle vector
pA2GP is used to insert the cloned DNA encoding the extracellular
domain of the protein, lacking its naturally associated
intracellular and transmembrane sequences, into a baculovirus to
express the extracellular domain of the Neutrokine-alpha protein,
using a baculovirus leader and standard methods as described in
Summers et al., A Manual of Methods for Baculovirus Vectors and
Insect Cell Culture Procedures, Texas Agricultural Experimental
Station Bulletin No. 1555 (1987). This expression vector contains
the strong polyhedrin promoter of the Autographa californica
nuclear polyhedrosis virus (AcMNPV) followed by the secretory
signal peptide (leader) of the baculovirus gp67 protein and
convenient restriction sites such as Bam HI, Xba I and Asp 718. The
polyadenylation site of the simian virus 40 ("SV40") is used for
efficient polyadenylation. For easy selection of recombinant virus,
the plasmid contains the beta-galactosidase gene from E. coli under
control of a weak Drosophila promoter in the same orientation,
followed by the polyadenylation signal of the polyhedrin gene. The
inserted genes are flanked on both sides by viral sequences for
cell-mediated homologous recombination with wild-type viral DNA to
generate viable virus that expresses the cloned polynucleotide.
[0971] Many other baculovirus vectors could be used in place of the
vector above, such as pAc373, pVL941 and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[0972] The cDNA sequence encoding an N-terminally deleted form of
the extracellular domain of the Neutrokine-alpha protein in the
deposited clone, lacking the AUG initiation codon, the naturally
associated intracellular and transmembrane domain sequences, and
amino acids Gln-73 through Leu-79 shown in FIGS. 1A and 1B (SEQ ID
NO:2), is amplified using PCR oligonucleotide primers corresponding
to the 5' and 3' sequences of the gene. The 5' primer has the
sequence 5'-GTG GGA TCC CCG GGC AGA GCT GCA GGG C-3' (SEQ ID NO:
14) containing the underlined Bam HI restriction enzyme site
followed by 18 nucleotides of the sequence of the extracellular
domain of the Neutrokine-alpha protein shown in FIGS. 1A and 1B,
beginning with the indicated N-terminus of the extracellular domain
of the protein. The 3' primer has the sequence 5'-GTG GGA TCC TTA
TTA CAG CAG TTT CAA TGC ACC-3' (SEQ ID NO:15) containing the
underlined Bam HI restriction site followed by two stop codons and
18 nucleotides complementary to the 3' coding sequence in FIGS. 1A
and 1B.
[0973] In certain other embodiments, constructs designed to express
the entire predicted extracellular domain of the Neutrokine-alpha
(i.e., amino acid residues Gln-73 through Leu-285) are preferred.
One of skill in the art would be able to use the polynucleotide and
polypeptide sequences provided as SEQ ID NO:1 and SEQ ID NO:2,
respectively, to design polynucleotide primers to generate such a
clone.
[0974] In a further preferred embodiment, a pA2GP expression
construct encodes amino acid residues Leu-112 through Leu-285 of
the Neutrokine-alpha polypeptide sequence shown as SEQ ID NO:2.
[0975] In another preferred embodiment, a pA2GP expression
construct encodes amino acid residues Ser-78 through Leu-285 of the
Neutrokine-alpha polypeptide sequence shown as SEQ ID NO:2.
[0976] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with Bam HI and again
is purified on a 1% agarose gel. This fragment is designated herein
F1.
[0977] The plasmid is digested with the restriction enzymes Bam HI
and optionally, can be dephosphorylated using calf intestinal
phosphatase, using routine procedures known in the art. The DNA is
then isolated from a 1% agarose gel using a commercially available
kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). This vector DNA
is designated herein "VI".
[0978] Fragment F1 and the dephosphorylated plasmid VI are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria are identified that contain the plasmid
with the human gene by digesting DNA from individual colonies using
Bam HI and then analyzing the digestion product by gel
electrophoresis. The sequence of the cloned fragment is confirmed
by DNA sequencing. This plasmid is designated herein
pA2GP-Neutrokine-alpha.
[0979] Five micrograms of the plasmid pA2GP-Neutrokine-alpha is
co-transfected with 1.0 microgram of a commercially available
linearized baculovirus DNA ("BaculoGold.TM. baculovirus DNA",
Pharmingen, San Diego, Calif.), using the lipofection method
described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:
7413-7417 (1987). One .mu.g of BaculoGold.TM. virus DNA and 5
micrograms of the plasmid pA2GP Neutrokine-alpha are mixed in a
sterile well of a microtiter plate containing 50 microliters of
serum-free Grace's medium (Life Technologies Inc., Gaithersburg,
Md.). Afterwards, 10 microliters Lipofectin plus 90 microliters
Grace's medium are added, mixed and incubated for 15 minutes at
room temperature. Then the transfection mixture is added drop-wise
to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue
culture plate with 1 ml Grace's medium without serum. The plate is
then incubated for 5 hours at 27.degree. C. The transfection
solution is then removed from the plate and 1 ml of Grace's insect
medium supplemented with 10% fetal calf serum is added. Cultivation
is then continued at 27.degree. C. for four days.
[0980] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Rockville,
Md.) is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Rockville,
Md., page 9-10). After appropriate incubation, blue stained plaques
are picked with the tip of a micropipettor (e.g., Eppendorf). The
agar containing the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 microliters of Grace's medium
and the suspension containing the recombinant baculovirus is used
to infect Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture dishes are harvested and then they
are stored at 4.degree. C. The recombinant virus is called
V-Neutrokine-alpha.
[0981] To verify the expression of the Neutrokine-alpha gene Sf9
cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus V-Neutrokine-alpha at a multiplicity of infection
("MOI") of about 2. If radiolabeled proteins are desired, 6 hours
later the medium is removed and is replaced with SF900 II medium
minus methionine and cysteine (available from Life Technologies
Inc., Rockville, Md.). After 42 hours, 5 microcuries of
.sup.35S-methionine and 5 microcuries .sup.35S-cysteine (available
from Amersham) are added. The cells are further incubated for 16
hours and then are harvested by centrifugation. The proteins in the
supernatant as well as the intracellular proteins are analyzed by
SDS-PAGE followed by autoradiography (if radiolabeled).
[0982] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the extracellular domain of the protein and
thus the cleavage point and length of the secretory signal
peptide.
[0983] In a specific experimental example, recombinant
Neutrokine-alpha was purified from baculovirus infected Sf9 cell
supernatants as follows. The insect cells were grown in EXCEL401
medium (JRH Scientific) with 1% (v/v) fetal bovine serum. At 92
hours post-infection, the harvested supernatant was clarified by
centrifugation at 18,000.times.g followed by 0.45 m depth
filtration. A de-lipid filtration step might be also used to remove
the lipid contaminants and in turn to improve initial capturing of
the Neutrokine-alpha protein.
[0984] The supernatant was loaded on to a set of Poros HS-50/HQ-50
in tandem mode. As alternatives, Toyopearl QAE, Toyopearl Super Q
(Tosohass), Q-Sepharose (Pharmacia) and equivalent resins might be
used. This step is used as a negative purification step to remove
strong anion binding contaminants. The HS/HQ flow through material
was adjusted to pH 7.5 with 1 M Tris-HCl pH 8, diluted with equal
volume of 50 mM Tris-HCl pH 8, and loaded onto a poros PI-20 or
PI-50 column. The PI column was washed first with 4 column volumes
of 75 mM sodium chloride in 50 mM Tris-HCl at pH 7.5, then eluted
using 3 to 5 column volumes of a stepwise gradient of 300 mM, 750
mM, 1500 mM sodium chloride in 50 mM Tris-HCl pH 7.5.
Neutrokine-alpha protein appears as a 17 KD band on reduced
SDS-PAGE and is present in the 0.75 M to 1.5M Sodium chloride
fractions.
[0985] The PI fraction was further purified through a Sephacryl
S100 HR (Pharmacia) size exclusion column equilibrated with 0.15 M
sodium chloride, 50 mM sodium acetate at pH 6. The S200 fractions
were mixed with sodium chloride to a final concentration of 3 M and
loaded onto a Toyopearl Hexyl 650C (Tosohass) column. The Hexyl
column was eluted with a linear gradient from 3 M to 0.05 M sodium
chloride in 50 mM Sodium acetate pH 6 in 5 to 15 column volumes.
The sodium chloride gradient can also be replaced by ammonium
sulfate gradient of 1M to 0 M in 50 mM sodium acetate pH 6 in the
Hexyl chromatographic step. Fractions containing purified
Neutrokine-alpha as analyzed through SDS-PAGE were combined and
dialyzed against a buffer containing 150 mM Sodium chloride, 50 mM
Sodium acetate, pH 6.
[0986] The final purified Neutrokine-alpha protein expressed in a
baculovirus system as explained herein has an N-terminus sequence
which begins with amino acid residue Ala-134 of SEQ ID NO:2.
RP-HPLC analysis shows a single peak of greater than 95% purity.
Endotoxin level was below the detection limit in LAL assay.
[0987] In another example, recombinant Neutrokine-alpha was
purified from baculovirus infected Sf9 cell supernatants containing
0.25% bovine serum as follows.
[0988] The Sf9 supernatant was harvested by centrifugation at
18,000.times.g. The supernatant was then treated with 10 mM calcium
chloride in slightly alkaline conditions for 10-15 minutes followed
by centrifugation and then 0.22 micrometer depth filtration. The
resulting Sf-9 cell supernatant was then diluted 2-fold and loaded
on to a Poros PI-50 column (available from PE Biosystems). The
column was equilibrated with 50 mM Tris (pH=7.4). The PI-50 column
was washed with 1 CV of 50 mM Tris (pH=7.4) and then eluted with
1.5 M NaCl in 50 mM NaOAc (pH=6) over 3 CV. The PI fraction was
loaded on to a Sephacryl S200 column equilibrated with 50 mM NaOAc
(pH=6), 125 mM NaCl. The S200 fraction was mixed with salts to
final concentrations of 0.7 M ammonium sulfate and 0.6 M NaCl and
loaded on to a Toyopearl Hexyl 650C column (available from Toso
Haas) that had been equilibrated in a buffer containing 0.6 M NaCl,
0.7 M ammonium sulfate in 50 mM NaOAc (pH=6). The column was then
washed with 2 CV of the same buffer. Recombinant Neutrokine-alpha
was then eluted stepwise with 3 CV of 50 mM NaOAc (pH=6) followed
by 2 CV of 20% ethanol wash. The recombinant Neutrokine-alpha
protein was then eluted at the end of the ammonium sulfate (0.3 to
0 M salt) gradient. The appropriate fractions were pooled and
dialyzed against a buffer containing 50 mM NaOAc (pH=6), and then
passed through a Poros 50 HQ column. The HQ flow-through was
diluted to 4 ms and loaded on to a Toyopearl DEAD 650M column and
then eluted with 25 mM NaCitrate, 125 mM NaCl.
[0989] In another example, recombinant Neutrokine-alpha was
expressed and purified using a baculoviral vector system in
Sf+insect cells.
[0990] First, a polynucleotide encoding amino acid residues Ser-78
through Leu-285 of the Neutrokine-alpha polypeptide sequence shown
in FIGS. 1A and 1B (which is exactly identical to amino acid
residues Ser-78 through Leu-285 of the Neutrokine-alpha polypeptide
sequence shown as SEQ ID NO:2) was subcloned into the baculovirus
transfer construct PSC to generate a baculovirus expression
plasmid. The pA2GP transfer vector, derived from pVL941, contains
the gp67 signal peptide, a modified multiple cloning site, and the
lac Z gene cloned downstream of the Drosophila heat-shock promoter
for selection of blue plaques. Using the sequence of
Neutrokine-alpha (SEQ ID NO:2) and the sequence of the pA2GP
vector, a cloning strategy was designed for seamlessly fusing the
PSC signal peptide coding sequence to the Neutrokine-alpha coding
sequence at Ala-134 (SEQ ID NO:2 and FIGS. 1A and 1B) and inserting
it into a PSC baculovirus transfer plasmid. The strategy involved
the use of a two-stage polymerase chain reaction (PCR) procedure.
First, primers were designed for amplifying the Neutrokine-alpha
sequences. The 5' primer consisted of the sequence encoding Ala-134
and following residues (5'-GGT CGC CGT TTC TAA CGC GGC CGT TCA GGG
TCC AGA AG-3'; SEQ ID NO:31), preceded by the sequence encoding the
PSC signal peptide C-terminus. The 3' primer (5'-CTG GTT CGG CCC
AAG GTA CCA AGC TTG TAC CTT AGA TCT TTT CTA GAT C-3'; SEQ ID NO:32)
consisted of the reverse complement of the pA2GP vector sequence
immediately downstream from the Neutrokine-alpha coding sequence,
preceded by a Kpn I restriction endonuclease site and a spacer
sequence (for increased cutting efficiency by Kpn I). PCR was
performed with the pA2GP containing Neutrokine-alpha plasmid
template and primers O-1887 and O-1888, and the resulting PCR
product was purified using standard techniques.
[0991] An additional PCR reaction was performed using the PSC
baculovirus transfer plasmid pMGS12 as a template. The pMGS12
plasmid consists of the AcNPV EcoRI "I" fragment inserted into
pUC8, with the polyhedrin coding sequences after the ATG start
codon replaced with the PSC signal peptide and a polylinker site.
The PCR reaction used pMGS12 as a template, a 5' primer (5'-CTG GTA
GTT CTT CGG AGT GTG-3'; SEQ ID NO:33) which annealed in AcNPV
ORF603 upstream of the unique NgoM IV and EcoR V sites, and a 3'
primer (5'-CGC GTT AGA AAC GGC GAC C-3'; SEQ ID NO:34) which
annealed to the 3' end of the sequence encoding the PSC signal
peptide.
[0992] To generate a PCR product in which the PSC signal peptide
was seamlessly fused to the Ala-134 of the Neutrokine-alpha coding
sequence, the PCR product was combined with the PSC signal
peptide-polyhedrin upstream region PCR product and subjected to an
additional round of PCR. Because the 3' end of the PSC signal
peptide PCR product (pMGS12/O-959/O-1044) overlapped the 5' end of
the Neutrokine-alpha PCR product prepared with primers
O-1887/O-1888, the two PCR products were combined and
overlap-extended by PCR using primers O-959 and O-1888.
[0993] The resulting overlap-extended PCR product containing the
PSC signal peptide fused to the Neutrokine-alpha sequence
subsequently was inserted into baculovirus transfer plasmid pMGS12.
The PCR product was digested with NgoM IV and Kpn I, and the
fragment was purified and ligated into NgoM IV-Kpn I-cut pMGS12.
After transformation of competent E. coli DH5alpha cells with the
ligation mix, colonies were picked and plasmid DNA mini-preps were
prepared. Several positive clones from each ligation were
identified by restriction digestion analysis of the plasmid DNA,
and three clones (pAcC9669, pAcC9671, and pAcC9672) were selected
for large scale plasmid purification. The resulting plasmid DNA was
subjected to DNA sequence analysis to confirm and sequence the
Neutrokine-alpha insert.
[0994] The following steps describe the recovery and purification
process of recombinant Neutrokine-alpha from Sf+insect cells.
Unless stated otherwise, the process is conducted at 2-8.degree.
C.
Recovery
Step 1. CaCl.sub.2 Treatment
[0995] Sf+cell supernatant was harvested by centrifugation at
8,000.times.g. Recovery buffer-1 (1M CaCl.sub.2) was added to the
supernatant so that the final concentration of CaCl.sub.2 was 10
mM. (In a further preferred embodiment, 1M ZnCl.sub.2 is used in
place of 1M CaCl.sub.2.) The pH of the solution was adjusted to
7.7+ with Recovery buffer-2 (1M Tris pH 8 (+0.2)). The solution was
incubated for 15 minutes and then centrifuged at 8,000.times.g.
Purification
Step 1. Chromatography on Poros PI-50 Column
[0996] Sf+cell supernatant was loaded on to a Poros PI-50 column
(PE Biosystem). The column was equilibrated in PI-1 buffer (50 mM
Tris, 50 mM NaCl, pH 7.4 (.+-.0.2)). The PI-50 column was washed
with 1-2 CV of PI-1 buffer and then eluted with PI-2 buffer (50 mM
Na Citrate pH 6 (.+-.0.2)) over 3 CV linear gradient. The elution
was monitored by ultraviolet (UV) absorbance at 280 nm. Fractions
were collected across the eluate peak and analyzed by SDS page.
Appropriate fractions were pooled.
Step 2. Chromatography on Toyopearl Hexyl 650C Column
[0997] The PI pool was mixed with salts to final concentrations of
0.7M (NH.sub.4).sub.2SO.sub.4 and loaded on to a Toyopearl Hexyl
650C (Toso Haas) column equilibrated in HIC-1 buffer (50 mM NaOAc,
0.6M NaCl, 0.7M (NH.sub.4).sub.2SO.sub.4 pH 6 (.+-.0.2)). The
column was then washed with 2 CV of HIC-1 buffer. Subsequently,
recombinant Neutrokine-alpha was then eluted stepwise with 3-5 CV
of HIC-2 buffer (50 mM NaOAc pH 6.0 (+0.2)) followed by a 2 CV 20%
ethanol wash. The elution was monitored by UV absorbance at 280 nm
and conductivity. Fractions were collected across the eluate peak
and analyzed by SDS-PAGE. The appropriate fractions were then
pooled.
Step 3. Chromatography on SP Sepharose FF
[0998] The Hexyl fraction was dialyzed and adjusted to pH 4.5 with
SP-1 buffer (50 mM sodium acetate pH 4.5 (.+-.0.2)), diluted to 4
ms and loaded through a SP sepharose (cation exchanger, Pharmacia)
column equilibrated with SP-1 buffer (50 mM sodium acetate pH 4.5
(.+-.0.2)). Recombinant Neutrokine-alpha protein was then eluted
from the SP column with SP-2 buffer (50 mM sodium acetate pH 5.5
(.+-.0.2)) at pH 5.5. The elution was then monitored by ultraviolet
(UV) absorbance at 280 nm. Fractions were collected across the
eluate peak and analyzed by SDS page. Appropriate fractions were
pooled.
Step 4. Dialysis of Recombinant Neutrokine-alpha
[0999] The SP fractions were placed into a 6-8 kd cutoff membrane
device and then dialyzed or diafiltered into Dialysis Buffer (10 mM
sodium citrate, 140 mM sodium chloride pH 6 (.+-.0.2))
overnight.
Step 5. Filtration and Fill
[1000] The protein concentration of the recombinant
Neutrokine-alpha solution from Step 6 was determined by
bicinchoninic acid (BCA) protein assay. Recombinant
Neutrokine-alpha formulation was adjusted to the final protein
concentration with the appropriate buffer and filtered under
controlled conditions. The filtrate (bulk substance) was stored in
suitable sterilized containers below -20.degree. C.
[1001] In a specific embodiment, Neutrokine-alpha protein of the
invention produced as described infra was adjusted to a final
protein concentration of 1 to 5 mg/ml and buffered in 10 mM sodium
citrate, 140 mM sodium chloride, pH=6.0.+-.(0.4) and stored at or
below -20.degree. C. in Type 1 glass vials.
[1002] During chromatography runs, the processes are monitored by
UV absorbance at 280 nm. When applicable, in-process chromatography
intermediates are tested for conductivity, pH, and monitored by SDS
and/or RP-HPLC.
[1003] Columns and purification equipment are cleaned and sanitized
with 0.2 or 0.5 M NaOH followed by deionized water and then 0.1 or
0.5 M acetic acid. The column and purification equipment are rinsed
with deionized water and, if necessary, stored in the appropriate
storage solution. Prior to use, the equipment is equilibrated with
appropriate buffers (as described herein or as is well known in the
art).
[1004] In a further preferred embodiment, 1M ZnCl.sub.2 is used in
place of 1M CaCl.sub.2 in Step 1 of the Recovery section described
above. Also, in this embodiment, a combination of ZnCl.sub.2 and
CaCl.sub.2 may be used. Many combinations of 0.1 M ZnCl.sub.2 and
0.9 M CaCl.sub.2, may be used in the Recovery process of
recombinant Neutrokine-alpha protein such as, for example, but not
limited to, a combination of 0.1 M ZnCl.sub.2 and 0.9 M CaCl.sub.2,
0.2 M ZnCl.sub.2 and 0.8 M CaCl.sub.2, 0.3 M ZnCl.sub.2 and 0.7 M
CaCl.sub.2, 0.4 M ZnCl.sub.2 and 0.6 M CaCl.sub.2, 0.5 M ZnCl.sub.2
and 0.5 M CaCl.sub.2, 0.6 M ZnCl.sub.2 and 0.4 M CaCl.sub.2, 0.7 M
ZnCl.sub.2 and 0.3 M CaCl.sub.2, 0.8 M ZnCl.sub.2 and 0.2 M
CaCl.sub.2, 0.9 M ZnCl.sub.2 and 0.1 M CaCl.sub.2, and others.
However, the presence of EDTA will inhibit the recovery process.
Moreover, the presence of ZnCl.sub.2 and/or CaCl.sub.2 in Recovery
Buffer-1 will induce the formation of larger amounts of higher
molecular weight (or molecular mass) Neutrokine-alpha
multimers.
Example 3
Cloning and Expression of Neutrokine-Alpha in Mammalian Cells
[1005] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and
pBC12MI (ATCC 67109). Mammalian host cells that could be used
include, human HeLa, 293, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells,
Chinese hamster ovary (CHO) cells CHO-K1, NSO and HEK 293
cells.
[1006] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells.
[1007] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
proteins.
[1008] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites Bam
HI, Xba I and Asp 718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 3(a)
Cloning and Expression in COS Cells
[1009] The expression plasmid, pNeutrokine-alpha-HA, is made by
cloning a portion of the deposited cDNA encoding the extracellular
domain of the protein into the expression vector pcDNAI/Amp or
pcDNAIII (which can be obtained from Invitrogen, Inc.). To produce
a soluble, secreted form of the polypeptide, the extracellular
domain is fused to the secretory leader sequence of the human IL-6
gene.
[1010] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a cDNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the
target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pcDNAIII contains, in addition, the selectable neomycin
marker.
[1011] A DNA fragment encoding the extracellular domain of the
Neutrokine-alpha polypeptide is cloned into the polylinker region
of the vector so that recombinant protein expression is directed by
the CMV promoter. The plasmid construction strategy is as follows.
The Neutrokine-alpha cDNA of the deposited clone is amplified using
primers that contain convenient restriction sites, much as
described above for construction of vectors for expression of
Neutrokine-alpha in E. coli. Suitable primers include the
following, which are used in this example. The 5' primer,
containing the underlined Bam HI site, a Kozak sequence, an AUG
start codon, a sequence encoding the secretory leader peptide from
the human IL-6 gene, and 18 nucleotides of the 5' coding region of
the extracellular domain of Neutrokine-alpha protein, has the
following sequence: 5'-GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA
AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT
GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3' (SEQ
ID NO:16). The 3' primer, containing the underlined Bam HI
restriction site and 18 of nucleotides complementary to the 3'
coding sequence immediately before the stop codon, has the
following sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC-3'
(SEQ ID NO:17).
[1012] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with Bam HI and then ligated. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037), and the transformed culture is plated on ampicillin media
plates which then are incubated to allow growth of ampicillin
resistant colonies. Plasmid DNA is isolated from resistant colonies
and examined by restriction analysis or other means for the
presence of the fragment encoding the Neutrokine-alpha
extracellular domain.
[1013] For expression of recombinant Neutrokine-alpha, COS cells
are transfected with an expression vector, as described above,
using DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under
conditions for expression of Neutrokine-alpha by the vector.
[1014] Expression of the Neutrokine-alpha-HA fusion protein is
detected by radiolabeling and immunoprecipitation, using methods
described in, for example Harlow et al., Antibodies: A Laboratory
Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1988). To this end, two days after transfection, the
cells are labeled by incubation in media containing
.sup.35S-cysteine for 8 hours. The cells and the media are
collected, and the cells are washed and the lysed with
detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS,
1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et
al. cited above. Proteins are precipitated from the cell lysate and
from the culture media using an HA-specific monoclonal antibody.
The precipitated proteins then are analyzed by SDS-PAGE and
autoradiography. An expression product of the expected size is seen
in the cell lysate, which is not seen in negative controls.
Example 3(b)
Cloning and Expression in CHO Cells
[1015] The vector pC4 is used for the expression of
Neutrokine-alpha protein. Plasmid pC4 is a derivative of the
plasmid pSV2-dhfr (ATCC Accession No. 37146). To produce a soluble,
secreted form of the Neutrokine-alpha polypeptide, the portion of
the deposited cDNA encoding the extracellular domain is fused to
the secretory leader sequence of the human IL-6 gene. The vector
plasmid contains the mouse DHFR gene under control of the SV40
early promoter. Chinese hamster ovary- or other cells lacking
dihydrofolate activity that are transfected with these plasmids can
be selected by growing the cells in a selective medium (alpha minus
MEM, Life Technologies) supplemented with the chemotherapeutic
agent methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented (see,
e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R.
T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C.
1990, Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and
Sydenham, M. A. 1991, Biotechnology 9:64-68). Cells grown in
increasing concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach may be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained which contain
the amplified gene integrated into one or more chromosome(s) of the
host cell.
[1016] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rouse
Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology,
March 1985:438-447) plus a fragment isolated from the enhancer of
the immediate early gene of human cytomegalovirus (CMV) (Boshart et
al., Cell 41:521-530 (1985)). Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: BamHI, Xba I, and Asp718. Behind these
cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for the expression, e.g., the
human beta-actin promoter, the SV40 early or late promoters or the
long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
Clontech's Tet-Off and Tet-On gene expression systems and similar
systems can be used to express the Neutrokine-alpha in a regulated
way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc.
Natl. Acad. Sci. USA 89: 5547-5551). For the polyadenylation of the
mRNA other signals, e.g., from the human growth hormone or globin
genes can be used as well. Stable cell lines carrying a gene of
interest integrated into the chromosomes can also be selected upon
co-transfection with a selectable marker such as gpt, G418 or
hygromycin. It is advantageous to use more than one selectable
marker in the beginning, e.g., G418 plus methotrexate.
[1017] The plasmid pC4 is digested with the restriction enzymes Bam
HI and then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[1018] The DNA sequence encoding the extracellular domain of the
Neutrokine-alpha protein is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene. The
5' primer, containing the underlined Bam HI site, a Kozak sequence,
an AUG start codon, a sequence encoding the secretory leader
peptide from the human IL-6 gene, and 18 nucleotides of the 5'
coding region of the extracellular domain of Neutrokine-alpha
protein, has the following sequence: 5'-GCG GGA TCC GCC ACC ATG AAC
TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC
CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG
GCC AGC-3' (SEQ ID NO:16). The 3' primer, containing the underlined
Bam HI and 18 of nucleotides complementary to the 3' coding
sequence immediately before the stop codon, has the following
sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC-3' (SEQ ID
NO:17).
[1019] The amplified fragment is digested with the endonuclease Bam
HI and then purified again on a 1% agarose gel. The isolated
fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed
and bacteria are identified that contain the fragment inserted into
plasmid pC4 using, for instance, restriction enzyme analysis.
[1020] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using lipofectin
(Felgner et al., supra). The plasmid pSV2-neo contains a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that
confers resistance to a group of antibiotics including G418. The
cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
After 2 days, the cells are trypsinized and seeded in hybridoma
cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After
about 10-14 days single clones are trypsinized and then seeded in
6-well petri dishes or 10 ml flasks using different concentrations
of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10
.mu.M, 20 .mu.M). The same procedure is repeated until clones are
obtained which grow at a concentration of 100-200 .mu.M. Expression
of the desired gene product is analyzed, for instance, by SDS-PAGE
and Western blot or by reversed phase HPLC analysis.
[1021] At least six Neutrokine-alpha expression constructs have
been generated by the inventors herein to facilitate the production
of Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of
several sizes and in several systems. The expression constructs are
as follows: (1) pNa.A71-L285 (expresses amino acid residues Ala-71
through Leu-285), (2) pNa.A81-L285 (expresses amino acid residues
Ala-81 through Leu-285), (3) pNa.L112-L285 (expresses amino acid
residues Leu-112 through Leu-285), (4) pNa.A134-L285 (expresses
amino acid residues Ala-134 through Leu-285), (5) pNa.L147-L285
(expresses amino acid residues Leu-147 through Leu-285), and (6)
pNa.G161-L285 (expresses amino acid residues Gly-161 through
Leu-285).
[1022] In preferred embodiments, the expression constructs are used
to express various Neutrokine-alpha muteins from bacterial,
baculoviral, and mammalian systems.
[1023] In certain additional preferred embodiments, the constructs
express a Neutrokine-alpha polypeptide fragment fused at the N-
and/or C-terminus to a heterologous polypeptide, e.g., the signal
peptide from human IL-6, the signal peptide from CK-beta8 (amino
acids -21 to -1 of the CK-beta8 sequence disclosed in published PCT
application PCT/US95/09058), or the human IgG Fc region. Other
sequences could be used which are known to those of skill in the
art.
Example 4
Tissue Distribution of Neutrokine-Alpha mRNA Expression
[1024] Northern blot analysis is carried out to examine
Neutrokine-alpha gene expression in human tissues, using methods
described by, among others, Sambrook et al., cited above. A cDNA
probe containing the entire nucleotide sequence of the
Neutrokine-alpha protein (SEQ ID NO:1) is labeled with .sup.32P
using the Rediprime.TM. DNA labeling system (Amersham Life
Science), according to manufacturer's instructions. After labeling,
the probe is purified using a CHROMA SPIN-100.TM. column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number
PT1200-1. The purified labeled probe is then used to examine
various human tissues for Neutrokine-alpha and/or Neutrokine-alpha
mRNA.
[1025] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) are obtained
from Clontech and are examined with the labeled probe using
ExpressHyb.TM. hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots are mounted and exposed to film at
-70.degree. C. overnight, and films developed according to standard
procedures.
[1026] To determine the pattern of Neutrokine-alpha and/or
Neutrokine-alpha expression a panel of multiple tissue Northern
blots were probed. This revealed predominant expression of single
2.6 kb mRNA in peripheral blood leukocytes, spleen, lymph node and
bone marrow, and detectable expression in placenta, heart, lung,
fetal liver, thymus and pancreas. Analysis of a panel of cell lines
demonstrated high expression of Neutrokine-alpha and/or
Neutrokine-alpha in HL60 cells, detectable expression in K562, but
no expression in Raji, HeLa, or MOLT-4 cells. Overall it appears
that Neutrokine-alpha and/or Neutrokine-alpha mRNA expression is
enriched in the immune system.
Example 5
Gene Therapy Using Endogenous Neutrokine-Alpha Gene
[1027] Another method of gene therapy according to the present
invention involves operably associating the endogenous
Neutrokine-alpha sequence with a promoter via homologous
recombination as described, for example, in U.S. Pat. No.
5,641,670, issued Jun. 24, 1997; International Publication No. WO
96/29411, published Sep. 26, 1996; International Publication No. WO
94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad.
Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989). This method involves the activation of a gene
which is present in the target cells, but which is not expressed in
the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and
targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous Neutrokine-alpha, flanking the promoter. The
targeting sequence will be sufficiently near the 5' end of
Neutrokine-alpha so the promoter will be operably linked to the
endogenous sequence upon homologous recombination. The promoter and
the targeting sequences can be amplified using PCR. Preferably, the
amplified promoter contains distinct restriction enzyme sites on
the 5' and 3' ends. Preferably, the 3' end of the first targeting
sequence contains the same restriction enzyme site as the 5' end of
the amplified promoter and the 5' end of the second targeting
sequence contains the same restriction site as the 3' end of the
amplified promoter.
[1028] The amplified promoter and the amplified targeting sequences
are digested with the appropriate restriction enzymes and
subsequently treated with calf intestinal phosphatase. The digested
promoter and digested targeting sequences are added together in the
presence of T4 DNA ligase. The resulting mixture is maintained
under conditions appropriate for ligation of the two fragments. The
construct is size fractionated on an agarose gel then purified by
phenol extraction and ethanol precipitation.
[1029] In this Example, the polynucleotide constructs are
administered as naked polynucleotides via electroporation. However,
the polynucleotide constructs may also be administered with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, precipitating agents, etc. Such methods
of delivery are known in the art.
[1030] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous Neutrokine-alpha sequence. This results in the
expression of Neutrokine-alpha in the cell. Expression may be
detected by immunological staining, or any other method known in
the art.
[1031] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in DMEM+10% fetal calf serum.
Exponentially growing or early stationary phase fibroblasts are
trypsinized and rinsed from the plastic surface with nutrient
medium. An aliquot of the cell suspension is removed for counting,
and the remaining cells are subjected to centrifugation. The
supernatant is aspirated and the pellet is resuspended in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl,
0.7 mM Na2 HPO4, 6 mM dextrose). The cells are recentrifuged, the
supernatant aspirated, and the cells resuspended in electroporation
buffer containing 1 mg/ml acetylated bovine serum albumin. The
final cell suspension contains approximately 3.times.10.sup.6
cells/ml. Electroporation should be performed immediately following
resuspension.
[1032] Plasmid DNA is prepared according to standard techniques.
For example, to construct a plasmid for targeting to the
Neutrokine-alpha locus, plasmid pUC18 (MBI Fermentas, Amherst,
N.Y.) is digested with HindIII. The CMV promoter is amplified by
PCR with an XbaI site on the 5' end and a BamHI site on the 3' end.
Two Neutrokine-alpha non-coding sequences are amplified via PCR:
one Neutrokine-alpha non-coding sequence (Neutrokine-alpha fragment
1) is amplified with a HindIII site at the 5' end and an Xba site
at the 3' end; the other Neutrokine-alpha non-coding sequence
(Neutrokine-alpha fragment 2) is amplified with a BamHI site at the
5' end and a HindIII site at the 3' end. The CMV promoter and
Neutrokine-alpha fragments are digested with the appropriate
enzymes (CMV promoter--XbaI and BamHI; Neutrokine-alpha fragment
1--XbaI; Neutrokine-alpha fragment 2--BamHI) and ligated together.
The resulting ligation product is digested with HindIII, and
ligated with the HindIII-digested pUC18 plasmid.
[1033] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1.5.times.106 cells) is then added to the cuvette,
and the cell suspension and DNA solutions are gently mixed.
Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 mSec should
be observed.
[1034] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37.degree. C. The following
day, the media is aspirated and replaced with 10 ml of fresh media
and incubated for a further 16-24 hours.
[1035] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product. The fibroblasts can then be introduced into a patient as
described above.
Example 6
Neutrokine-Alpha, a Novel Member of the Tumor Necrosis Factor
Ligand Family that Functions as a B Lymphocyte Stimulator
[1036] A 285 amino acid protein was identified in a human
neutrophil/monocyte-derived cDNA library that shared significant
homology within its predicted extracellular receptor-ligand binding
domain to APRIL (28.7%) (Hahne, M., et al., J. Exp. Med. 188,
1185-90 (1998)), TNF-alpha (16.2%) (Pennica, D., et al., Nature
312, 724-729 (1984)) and LT-alpha (14.1%) (Gray, Nature 312,
721-724 (1984)) (FIGS. 7A-1 and 7A-2). We have designated this
cytokine Neutrokine-alpha (we have also designated this molecule as
B Lymphocyte Stimulator (BLyS) based on its biological activity).
Hydrophobicity analyses of the Neutrokine-alpha protein sequence
have revealed a potential transmembrane spanning domain between
amino acid residues 47 and 73 which is preceded by non-hydrophobic
amino acids suggesting that Neutrokine-alpha, like other members of
the TNF ligand family, is a type II membrane bound protein (Cosman,
D. Stem. Cells. 12:440-55 (1994)). Expression of this cDNA in
mammalian cells (HEK 293 and Chinese Hamster Ovary) and Sf9 insect
cells identified a 152 amino acid soluble form with an N-terminal
sequence beginning with the alanine residue at amino acid 134
(arrow in FIGS. 7A-1 and 7A-2). Reconstruction of the mass to
charge ratio defined a mass for Neutrokine-alpha of 17,038 Daltons,
a value in consistent with that predicted for this 152 amino acid
protein with a single disulfide bond (17037.5 Daltons).
[1037] Using human/hamster somatic cell hybrids and a
radiation-hybrid mapping panel, the gene encoding Neutrokine-alpha
was found linked to marker SHGC-36171 which maps to human
chromosome 13q34, a region not previously associated with any other
member of the TNF superfamily of genes (Cosman, D. Stem. Cells.
12:440-55 (1994)).
[1038] The expression profile of Neutrokine-alpha was assessed by
Northern blot (FIG. 7B) and flow cytometric analyses (Table V and
FIGS. 8A, 8B and 8C). Neutrokine-alpha is encoded by a single 2.6
kb mRNA found at high levels in peripheral blood leukocytes,
spleen, lymph node and bone marrow. Lower expression levels were
detected in placenta, heart, lung, fetal liver, thymus and
pancreas. Among a panel of cell lines, Neutrokine-alpha mRNA was
detected in HL-60 and K562, but not in Raji, HeLa, or MOLT-4 cells.
These results were confirmed by flow cytometric analyses using the
Neutrokine-alpha-specific mAb 2E5. As shown in Table V,
Neutrokine-alpha expression is not detected on T or B lineage cells
but rather restricted to cells within the myeloid origin. Further
analyses of normal blood cell types demonstrated significant
expression on resting monocytes that was upregulated approximately
4-fold following exposure of cells to IFN-gamma (100 U/mL) for
three days (FIGS. 8A and 8B). A concomitant increase in
Neutrokine-alpha-specific mRNA was also detected (FIG. 8C). By
contrast, Neutrokine-alpha was not expressed on freshly isolated
peripheral blood granulocytes, T cells, B cells, or NK cells.
[1039] Purified recombinant Neutrokine-alpha ("rNeutrokine-alpha")
was assessed for its ability to induce activation, proliferation,
differentiation or death in numerous cell based assays involving B
cells, T cells, monocytes, NK cells, hematopoietic progenitors, and
a variety of cell types of endothelial and epithelial origin. Among
these assays, Neutrokine-alpha was specifically found to increase B
cell proliferation in a standard co-stimulatory assay in which
purified tonsillar B cells are cultured in the presence of either
formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized
anti-human IgM as priming agents (Sieckmann, D. G., et al., J. Exp.
Med. 147:814-29 (1978); Ringden, O., et al., Scand. J. Immunol.
6:1159-69 (1977)). As shown in FIG. 9A, recombinant
Neutrokine-alpha induced a dose-dependent proliferation of
tonsillar B cells. This response was similar to that of rIL2 over
the dose range from 0.1 to 10,000 ng/mL. Neutrokine-alpha also
induces B cell proliferation when cultured with cells co-stimulated
with immobilized anti-IgM (FIG. 9B). A dose-dependent response is
readily observed as the amount of crosslinking agent increases in
the presence of a fixed concentration of either IL2 or
rNeutrokine-alpha.
[1040] In an attempt to correlate the specific biological activity
on B cells with receptor expression, purified Neutrokine-alpha was
biotinylated. The resultant biotin-Neutrokine-alpha protein
retained biological function in the standard B cell proliferation
assays. Lineage-specific analyses of whole human peripheral blood
cells indicated that binding of biotinylated Neutrokine-alpha was
undetectable on T cells, monocytes, NK cells and granulocytes as
assessed by CD3, CD14, CD56, and CD66b respectively (FIGS. 10A,
10B, 10C, 10D and 10E). In contrast, biotinylated Neutrokine-alpha
bound peripheral CD20.sup.+ B cells. Receptor expression was also
detected on the B cell tumor lines REH, ARH-77, Raji, Namalwa, RPMI
8226, and IM-9 but not any of the myeloid-derived lines tested
including THP-1, HL-60, K-562, and U-937. Representative flow
cytometric profiles for the myeloma cell line IM-9 and the
histiocytic line U-937 are shown in FIGS. 10F and 10G. Similar
results were also obtained using a biologically active FLAG-tagged
Neutrokine-alpha protein instead of the chemically modified
biotin-Neutrokine-alpha. Taken together, these results confirm that
Neutrokine-alpha displays a clear B cell tropism in both its
receptor distribution and biological activity. It remains to be
shown whether cellular activation may induce expression of
Neutrokine-alpha receptors on peripheral blood cells, other normal
cell types or established cell lines.
[1041] To examine the species specificity of Neutrokine-alpha,
mouse splenic B cells were cultured in the presence of human
Neutrokine-alpha and SAC. Results demonstrate that
rNeutrokine-alpha induced in vitro proliferation of murine splenic
B cells and bound to a cell surface receptor on these cells.
Interestingly, immature surface Ig negative B cell precursors
isolated from mouse bone marrow did not proliferate in response to
Neutrokine-alpha nor did they bind the ligand.
[1042] To assess the in vivo activity of rNeutrokine-alpha, BALB/c
mice (3/group) were injected (i.p.) twice per day with buffer only,
or 0.08 mg/kg, 0.8 mg/kg, 2 mg/kg or 8 mg/kg of rNeutrokine-alpha.
Mice received this treatment for 4 consecutive days at which time
they were sacrificed and various tissues and serum collected for
analyses. In an alternative embodiment, BALB/c mice may be injected
(i.p.) twice per day with any amount of rNeutrokine-alpha in a
range of 0.01 to 10 mg/kg. In a preferred embodiment, BALB/c mice
are injected (i.p.) twice per day with any amount of
rNeutrokine-alpha in a range of 0.01 to 3 mg/kg (specific preferred
exemplary dosages in this embodiment include, but are not limited
to, 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg,
0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2
mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8
mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4
mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9
mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5
mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, and 3.0 mg/kg).
In an additional preferred embodiment, BALB/c mice are injected
(i.p.) twice per day with any amount of rNeutrokine-alpha in a
range of 0.02 to 2 mg/kg (specific preferred exemplary dosages in
this embodiment include, but are not limited to, 0.02 mg/kg, 0.03
mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg,
0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg,
0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg,
1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.6 mg/kg,
1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, and 2.0 mg/kg).
[1043] Microscopically, the effects of Neutrokine-alpha
administration were clearly evident in sections of spleen stained
with routine hematoxylin and eosin (H&E) and
immunohistochemically with a mAb specific for CD45R(B220) (FIG.
11A). Normal splenic architecture was altered by a dramatic
expansion of the white pulp marginal zone and a distinct increase
in cellularity of the red pulp (FIG. 11A). Marginal zone expansion
appeared to be the result of increased numbers of lymphocytes
expressing the B cell marker CD45R(B220). In addition, the T cell
dense periarteriolar lymphoid sheath (PALS) areas were also
infiltrated by moderate numbers of CD45R(B220) positive cells. This
suggests the white pulp changes were due to increased numbers of B
cells. The densely packed cell population that frequently filled
red pulps spaces did not stain with CD45R(B220). Additional
experiments will be required to characterize all the cell types
involved and further define the mechanism by which Neutrokine-alpha
alters splenic architecture.
[1044] Flow cytometric analyses of the spleens from mice treated
with 2 mg/kg Neutrokine-alpha-treated indicated that
Neutrokine-alpha increased the proportion of mature
(CD45R(B220).sup.dull, ThB.sup.bright) B cells approximately
10-fold over that observed in control mice (FIGS. 11B and 11C).
Further analyses performed in which mice were treated with buffer,
0.08 mg/kg, 0.8 mg/kg, 2 mg/kg, or 8 mg/kg Neutrokine-alpha
indicated that 0.08 mg/kg, 0.8 mg/kg, and 2 mg/kg each increased
the proportion of mature (CD45R(B220).sup.dull, ThB.sup.bright) B
cells approximately 10-fold over that observed in control mice,
whereas buffer and 8 mg/kg produced approximately equal proportions
of mature B cells. See, Table IV.
TABLE-US-00004 TABLE IV FACS Analysis of Mouse Spleen B cell
Population. Neutrokine-alpha % Mature % CD45R- (mg/kg) B Cells (R2)
positive (R1) Control (buffer) 1.26 52.17 0.08 mg/kg 16.15 56.53
0.8 mg/kg 18.54 57.56 2 mg/kg 16.54 57.55 8 mg/kg 1.24 61.42
[1045] A potential consequence of increased mature B cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgA, IgG and IgM levels were compared between
buffer and Neutrokine-alpha-treated mice (FIGS. 11D, 11E, and 11F).
Neutrokine-alpha administration resulted in a 2- and 5-fold
increase in IgA and IgM serum levels respectively. Interestingly,
circulating levels of IgG did not increase.
[1046] Moreover, a dose-dependent response was observed in serum
IgA titers in mice treated with various amounts of Neutrokine-alpha
over a period of four days, whereas no apparent dose-dependency was
observed by administration of the same amounts of Neutrokine-alpha
over a period of two days. In the case of administration over four
days, administration of 8, 2, 0.8, 0.08, and 0 mg/kg
Neutrokine-alpha resulted in serum IgA titers of approximately 800
micrograms/ml, 700 micrograms/ml, 400 micrograms/ml, 200
micrograms/ml and 200 micrograms/ml. That is, administration of 8,
2, 0.8, and 0.08 mg/kg Neutrokine-alpha over four days resulted in
approximately 4-fold, 3.75-fold, 2-fold, and minimal-fold,
respectively, increases in IgA serum levels over background or
basal levels observed by administration of buffer only. In an
alternative embodiment, these experiments may be performed with any
amount of rNeutrokine-alpha in a range of 0.01 to 10 mg/kg. In a
preferred embodiment, Neutrokine-alpha is administered in a range
of 0.01 to 3 mg/kg (specific preferred exemplary dosages in this
embodiment include, but are not limited to, 0.01 mg/kg, 0.02 mg/kg,
0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08
mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5
mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1
mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.6
mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2
mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8
mg/kg, 2.9 mg/kg, and 3.0 mg/kg). In an additional preferred
embodiment, Neutrokine-alpha is administered in a range of 0.02 to
2 mg/kg (specific preferred exemplary dosages in this embodiment
include, but are not limited to, 0.02 mg/kg, 0.03 mg/kg, 0.04
mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg,
0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg,
0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg,
1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.6 mg/kg, 1.7 mg/kg,
1.8 mg/kg, 1.9 mg/kg, and 2.0 mg/kg).
[1047] The data presented herein define Neutrokine-alpha, as a
novel member of the TNF-ligand superfamily that induces both in
vivo and in vitro B cell proliferation and differentiation.
Neutrokine-alpha is distinguished from other B cell growth and
differentiation factors such as IL2 (Metzger, D. W., et al., Res.
Immunol. 146:499-505 (1995)), IL4 (Armitage, R. J., et al., Adv.
Exp. Med. Biol. 292:121-30 (1991); Yokota, T., et al., Proc. Natl.
Acad. Sci. U.S.A. 83:5894-98 (1986)), IL5 (Takatsu, K., et al.,
Proc. Natl. Acad. Sci. U.S.A. 84:4234-38 (1987); Bertolini, J. N.,
et al., Eur. J. Immunol. 23:398-402 (1993)), IL6 (Poupart, P., et
al., EMBO J. 6:1219-24 (1987); Hirano, T., et al., Nature 324:73-76
(1986)) IL7 (Goodwin, R. G., et al., Proc. Natl. Acad. Sci. U.S.A.
86:302-06 (1989); Namen, A. E., et al., Nature 333:571-73 (1988)),
IL13 (Punnonen, J., et al., Allergy. 49:576-86 (1994)), IL15
(Armitage, R. J., et al., J. Immunol. 154:483-90 (1995)), CD40L
(Armitage, R. J., et al., Nature 357:80-82 (1992); Van Kooten, C.
and Banchereau, J. Int. Arch. Allergy. Immunol. 113:393-99 (1997))
or CD27L (CD70) (Oshima, H., et al., Int. Immunol. 10:517-26
(1998); Lens, S. M., et al., Semin. Immunol. 10:491-99 (1998)) by
its monocyte-specific gene/protein expression pattern and its
specific receptor distribution and biological activity on B
lymphocytes. Taken together these data suggest that
Neutrokine-alpha is likely involved in the exchange of signals
between B cells and monocytes or their differentiated progeny.
Although all B cells may utilize this mode of signaling, the
restricted expression patterns and Ig secretion suggest a role for
Neutrokine-alpha in the activation of CD5.sup.+ or "unconventional"
B cell responses. These B cells provide a critical component to the
innate immune system and provide protection from environmental
pathogens through their secretion of polyreactive IgM and IgA
antibodies (Pennell, C. A., et al., Eur. J. Immunol. 19:1289-95
(1989); Hayakawa, K., et al., Proc. Natl. Acad. Sci. U.S.A.
81:2494-98 (1984)). Alternatively, Neutrokine-alpha may function as
a regulator of T cell independent responses in a manner analogous
to that of CD40 and CD40L in T cell dependent antigen activation
(van den Eertwegh, A. J., et al., J. Exp. Med. 178:1555-65 (1993);
Grabstein, K. H., et al., J. Immunol. 150:3141-47 (1993)). As such,
Neutrokine-alpha, its receptor or related antagonists have utility
in the treatment of B cell disorders associated with autoimmunity,
neoplasia and/or immunodeficient syndromes.
Methods
[1048] Mice. BALB/cAnNCR (6-8 weeks) were purchased from Charles
River Laboratories, Inc. and maintained according to recommended
standards (National Research Council, Guide for the care and use of
laboratory animals (1999)) in microisolator cages with recycled
paper bedding (Harlan Sprague Dawley, Inc., Indianapolis, Ind.) and
provided with pelleted rodent diet (Harlan Sprague Dawley, Inc) and
bottled drinking water on an ad libitum basis. The animal protocols
used in this study were reviewed and approved by the HGS
Institutional Animal Care and Use Committee.
[1049] Isolation of full length Neutrokine-alpha cDNA. The BLAST
algorithm was used to search the Human Genome Sciences Inc.
expressed sequence tag (EST) database for sequences with homology
to the receptor-binding domain of the TNF family. A full length
Neutrokine-alpha clone was identified, sequenced and submitted to
GenBank (Accession number AF132600). The Neutrokine-alpha open
reading frame was PCR amplified utilizing a 5' primer (5'-CAG ACT
GGA TCC GCC ACC ATG GAT GAC TCC ACA GAA AG-3') annealing at the
predicted start codon and a 3' primer (5'-CAG ACT GGT ACC GTC CTG
CGT GCA CTA CAT GGC-3') designed to anneal at the predicted
downstream stop codon. The resulting amplicon was tailed with Bam
HI and Asp 718 restriction sites and subcloned into a mammalian
expression vector. Neutrokine-alpha was also expressed in p-CMV-1
(Sigma Chemicals).
[1050] Purification of recombinant human Neutrokine-alpha. The full
length cDNA encoding Neutrokine-alpha was subcloned into the
baculovirus expression vector pA2 and transfected into Sf9 insect
cells (Patel, V. P., et al., J. Exp. Med. 185:1163-72 (1997)).
Recombinant Neutrokine-alpha was purified from cell supernatants at
92 h post-infection using a combination of anion-exchange, size
exclusion, and hydrophobic interaction columns. The purified
protein was formulated in a buffer containing 0.15 M NaCl, 50 mM
NaOAc at pH 6, sterile filtered and stored at 4.degree. C. until
needed. Both SDS-PAGE and RP-HPLC analyses indicate that
rNeutrokine-alpha is greater than 95% pure. Endotoxin levels were
below the detection limit in the LAL assay (Associates of Cape Cod,
Falmouth, Mass.). The final purified Neutrokine-alpha protein has
an N-terminus sequence of Ala-Val-Gln-Gly-Pro. This corresponds
identically to the sequence of soluble Neutrokine-alpha derived
from CHO cell lines stably transfected with the full length
Neutrokine-alpha gene.
[1051] Monoclonal antibody generation. BALB/cAnNCR mice were
immunized with 50 micrograms of HisTag-Neutrokine-alpha suspended
in complete Freund's adjuvant followed by 2 challenges in
incomplete Freund's adjuvant. Hybridomas and monoclonal antibodies
were prepared as described (Gefter, M. L., et al., Somatic. Cell
Genet. 3:231-36 (1977); Akerstrom, B., et al., J. Immunol.
135:2589-92 (1985)).
[1052] Cell lines. All human cell lines were purchased from ATCC
(American Type Culture Collection, Manassas, Va.).
[1053] FACS analysis. Neutrokine-alpha expression was assessed on
human cell lines, freshly isolated normal peripheral blood
nucleated cells, and in vitro cultured monocytes, a mouse
anti-human Neutrokine-alpha mAb 2E5 (IgG1) followed by
PE-conjugated F(ab')2 goat antibody to mouse IgG (CALTAG
Laboratories, Burlingame, Calif.). Cells were analyzed using a
FACScan (Becton Dickinson Immunocytometry Systems, San Jose,
Calif.) with propidium iodide to exclude dead cells.
Neutrokine-alpha binding was assessed using rNeutrokine-alpha
biotinylated with a N-hydroxysuccinimidobiotin reagent (Pierce,
Rockford, Ill.) followed by PE-conjugated streptavidin (Dako Corp,
Glostrup, Denmark).
[1054] Chromosomal mapping. To determine the chromosomal location
of the Neutrokine-alpha gene, a panel of monochromosomal somatic
cell hybrids (Quantum Biotechnology, Canada) retaining individual
chromosomes was screened by PCR using Neutrokine-alpha specific
primers (5' primer: 5'-TGG TGT CTT TCT ACC AGG TGG-3' and 3'
primer: 5'-TTT CTT CTG GAC CCT GAA CGG-3'). The predicted 233 bp
PCR product was only detected in human chromosome 13 hybrids. Using
a panel of 83 radiation hybrids (Research Genetics, St. Louis, Mo.)
and the Stanford Human Genome Center Database,
(http://www.shgc.stanford.edu.RH/rhserver). Neutrokine-alpha was
found linked to the SHGC-36171 marker on chromosome 13.
Superposition of this map with the cytogenetic map of human
chromosome 13 allowed the assignment of human Neutrokine-alpha to
chromosomal band 13q34.
[1055] B lymphocyte proliferation assay. Human tonsillar B cells
were purified by magnetic bead (MACS) depletion of CD3-positive
cells. The resulting cell population was routinely greater than 95%
B cells as assessed by expression of CD19 and CD20. Various
dilutions of human rNeutrokine-alpha or the control protein
recombinant human IL2 were placed into individual wells of a
96-well plate to which was added 10.sup.5 B cells suspended in
culture medium (RPMI 1640 containing 10% FBS, 5.times.10.sup.-5M
2ME, 100 U/ml penicillin, 100 microgram/ml streptomycin, and
10.sup.-5 dilution of Pansorbin (SAC) or anti-IgM) in a total
volume of 150 microliters. Proliferation was quantitated by a 20 h
pulse (1 microCi/well) of .sup.3H-thymidine (6.7 Ci/mM) beginning
72 h post factor addition.
[1056] Histological analyses. Spleens were fixed in 10% neutral
buffered formalin, embedded in paraffin, sectioned at 5
micrometers, mounted on glass slides and stained with hematoxylin
and eosin or by enzyme-labeled indirect method immunohistochemistry
for CD45R(B220) (Hilbert, D. M., et al., Eur. J. Immunol.
23:2412-18 (1993)).
TABLE-US-00005 TABLE V Neutrokine-alpha cell surface expression
Neutrokine- Cell line alpha cell expression Cellular Morphology
surface Monocytic lineage U-937 Lymphoma, histiocytic/macrophage +
BL-60 Leukemia, acutepromyelocytic + K-562 Leukemia,
chronlcmyelogenous + THP-1 Leukemia, acutemonocytic + T-lineage
Jurkat Leukemia, T lymphocytic - SUP-T13 Leukemia, T lymphoblastic
- MOLT-4 Leukemia, T lymphoblastic - B-lineage Daudi Burkitt's,
lymphoblastic - Namalwa Burkitt's, lymphocyte - Raji Burkitt's,
lymphocyte - Reh Leukemia, lymphocytic - ARH-77 Leukemia, plasma
cell - IM9 Myeloma - RPMI 8226 Myeloma -
Example 7
Assays to Detect Stimulation or Inhibition of B Cell Proliferation
and Differentiation
[1057] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL5, IL6, IL-7, IL10, IL-13, IL14 and IL15.
Interestingly, these signals are by themselves weak effectors but
can, in combination with various co-stimulatory proteins, induce
activation, proliferation, differentiation, homing, tolerance and
death among B cell populations. One of the best studied classes of
B-cell co-stimulatory proteins is the TNF-superfamily. Within this
family CD40, CD27, and CD30 along with their respective ligands
CD154, CD70, and CD153 have been found to regulate a variety of
immune responses. Assays which allow for the detection and/or
observation of the proliferation and differentiation of these
B-cell populations and their precursors are valuable tools in
determining the effects various proteins may have on these B-cell
populations in terms of proliferation and differentiation. Listed
below are two assays designed to allow for the detection of the
differentiation, proliferation, or inhibition of B-cell populations
and their precursors.
[1058] In Vitro assay-Purified Neutrokine-alpha and/or
Neutrokine-alphaSV protein, or truncated forms thereof, is assessed
for its ability to induce activation, proliferation,
differentiation or inhibition and/or death in B-cell populations
and their precursors. The activity of Neutrokine-alpha and/or
Neutrokine-alphaSV protein on purified human tonsillar B cells,
measured qualitatively over the dose range from 0.1 to 10,000
ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay
in which purified tonsillar B cells are cultured in the presence of
either formalin-fixed Staphylococcus aureus Cowan I (SAC) or
immobilized anti-human IgM antibody as the priming agent. Second
signals such as IL-2 and IL-15 synergize with SAC and IgM
crosslinking to elicit B cell proliferation as measured by
tritiated-thymidine incorporation. Novel synergizing agents can be
readily identified using this assay. The assay involves isolating
human tonsillar B cells by magnetic bead (MACS) depletion of
CD3-positive cells. The resulting cell population is greater than
95% B cells as assessed by expression of CD45R(B220). Various
dilutions of each sample are placed into individual wells of a
96-well plate to which are added 10.sup.5 B-cells suspended in
culture medium (RPMI 1640 containing 10% FBS, 5.times.10.sup.-5M
2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10.sup.-5
dilution of SAC) in a total volume of 150 ul. Proliferation or
inhibition is quantitated by a 20 h pulse (1 uCi/well) with
.sup.3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition.
The positive and negative controls are IL2 and medium
respectively.
[1059] Agonists (including Neutrokine-alpha and/or
Neutrokine-alphaSV polypeptide fragments) demonstrate an increased
B cell proliferation when compared to that observed when the same
number of B cells is contacted with the same concentration of
priming agent. Antagonists according to the invention exhibit a
decreased B cell proliferation when compared to controls containing
the same number of B cells, the same concentration of priming
agent, and the same concentration of a soluble form of
Neutrokine-alpha that elicits an increase in B cell proliferative
activity (e.g., 71-285, 81-285, 112-285 or 134-285 of the
Neutrokine-alpha polypeptide shown in SEQ ID NO:2) in the absence
the antagonist.
[1060] In Vivo assay-BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of Neutrokine-alpha and/or
Neutrokine-alphaSV protein, or truncated forms thereof. Mice
receive this treatment for 4 consecutive days, at which time they
are sacrificed and various tissues and serum collected for
analyses. Comparison of H&E sections from normal and
Neutrokine-alpha and/or Neutrokine-alphaSV protein-treated spleens
identify the results of the activity of Neutrokine-alpha and/or
Neutrokine-alphaSV protein on spleen cells, such as the diffusion
of peri-arterial lymphatic sheaths, and/or significant increases in
the nucleated cellularity of the red pulp regions, which may
indicate the activation of the differentiation and proliferation of
B-cell populations. Immunohistochemical studies using a B cell
marker, anti-CD45R(B220), are used to determine whether any
physiological changes to splenic cells, such as splenic
disorganization, are due to increased B-cell representation within
loosely defined B-cell zones that infiltrate established T-cell
regions.
[1061] Flow cytometric analyses of the spleens from
Neutrokine-alpha and/or Neutrokine-alphaSV protein-treated mice is
used to indicate whether Neutrokine-alpha and/or Neutrokine-alphaSV
protein specifically increases the proportion of ThB+,
CD45R(B220)dull B cells over that which is observed in control
mice.
[1062] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgM and IgA levels are compared between buffer
and Neutrokine-alpha and/or Neutrokine-alphaSV protein-treated
mice.
Example 8
Effect of Neutrokine-Alpha and its Agonists in Treating
Graft-Versus-Host Disease Associated Lymphoid Atrophy and
Hypoplasia in Mice
[1063] An analysis of the use of Neutrokine-alpha to treat,
prevent, and/or diagnose graft-versus-host disease
(GVHD)-associated lymphoid hypoplasia/atrophy is performed through
the use of a C57BL/6 parent into (BALB/c X C57BL/6) F1 (CBF1) mouse
model. This parent into F1 mouse model is a well-characterized and
reproducible animal model of GVHD in bone marrow transplant
patients, which is well know to one of ordinary skill in the art
(see, Gleichemann, et al., Immunol. Today 5:324, 1984). Soluble
Neutrokine-alpha is expected to induced the proliferation and
differentiation of B lymphocyte, and correct the lymphoid
hypoplasia and atrophy observed in this animal model of GVHD
(Piguet, et al., J. Exp. Med. 166:1280 (1987); Hattori, et al.,
Blood 90:542 (1997)).
[1064] Initiation of the GVHD condition is induced by the
intravenous injection of approximately 1-5.times.10.sup.8 spleen
cells from C57BL/6 mice into (BALB/c X C57BL/6) F1 mice (both are
available from Jackson Lab, Bar Harbor, Me.). Groups of 6 to 8 mice
receive daily either 0.1 to 5.0 mg/kg of Neutrokine-alpha or buffer
control intraperitoneally, intramuscularly or intradermally
starting from the days when lymphoid hypoplasia and atrophy are
mild (.about.day 5), moderate (.about.day 12) or severe (.about.day
20) following the parental cell injection. The effect of
Neutrokine-alpha on lymphoid hypoplasia and atrophy of spleen is
analyzed by FACS and histopathology at multiple time points (3-4)
between day 10-30. Briefly, splenocytes are prepared from normal
CBF1, GVHD or Neutrokine-alpha-treated mice, and stained with
fluorescein phycoerythrin-conjugated anti-H-2 Kb, biotin-conjugated
anti-H-2 Kd, and FITC-conjugated anti-CD4, anti-CD8, or anti-B220,
followed by a CyChrome-conjugated avidin. All of these conjugated
antibodies can be purchased from PharMingen (San Diego, Calif.).
Cells are then analysis on a FACScan (Becton Dickinson, San Jose,
Calif.). Recipient and donor lymphocytes are identified as H-2
Kb+Kd+ and H-2 Kb+Kd-cells, respectively. Cell numbers of CD4+T,
CD8+T and B220+B cells of recipient or donor origin are calculated
from the total numbers of splenocytes recovered and the percentages
of each subpopulation are determined by the three color analysis.
Histological evaluation of the relative degree of tissue damage in
other GVHD-associated organs (liver, skin and intestine) may be
conducted after sacrificing the animals.
[1065] Finally, Neutrokine-alpha and buffer-treated animals undergo
a clinical evaluation every other day to assess cachexia, body
weight and lethality.
[1066] Neutrokine-alpha agonists and antagonists may also be
examined in this acute GVHD murine model.
Example 9
Isolation of Antibody Fragments Directed Against Neutrokine-Alpha
Polypeptides from a Library of scFvs
[1067] Naturally occurring V-genes isolated from human PBLs are
constructed into a large library of antibody fragments which
contain reactivities against Neutrokine-alpha and/or
Neutrokine-alphaSV to which the donor may or may not have been
exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein in
its entirety by reference).
[1068] Rescue of the Library.
[1069] A library of scFvs is constructed from the RNA of human PBLs
as described in WO92/01047 (which is hereby incorporated by
reference in its entirety). To rescue phage displaying antibody
fragments, approximately 10.sup.9 E. coli harboring the phagemid
are used to inoculate 50 ml of 2.times.TY containing 1% glucose and
100 micrograms/ml of ampicillin (2.times.TY-AMP-GLU) and grown to
an O.D. of 0.8 with shaking. Five ml of this culture is used to
inoculate 50 ml of 2.times.TY-AMP-GLU, 2.times.10.sup.8 TU of delta
gene 3 helper (M13 delta gene III, see WO92/01047) are added and
the culture incubated at 37.degree. C. for 45 minutes without
shaking and then at 37.degree. C. for 45 minutes with shaking. The
culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100 micrograms/ml
ampicillin and 50 micrograms/ml kanamycin and grown overnight.
Phage are prepared as described in WO92/01047.
[1070] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells were spun down (IEC-Centra 8, 4000 revs/min for 10
min), resuspended in 300 ml 2.times.TY broth containing 100
micrograms ampicillin/ml and 25 micrograms kanamycin/ml
(2.times.TY-AMP-KAN) and grown overnight, shaking at 37.degree. C.
Phage particles are purified and concentrated from the culture
medium by two PEG-precipitations (Sambrook et al., 1990),
resuspended in 2 ml PBS and passed through a 0.45 micrometer filter
(Minisart NML; Sartorius) to give a final concentration of
approximately 1013 transducing units/ml (ampicillin-resistant
clones).
[1071] Panning the Library.
[1072] Immunotubes (Nunc) are coated overnight in PBS with 4 ml of
either 100 micrograms/ml or 10 micrograms/ml of a polypeptide of
the present invention. Tubes are blocked with 2% Marvel-PBS for 2
hours at 37.degree. C. and then washed 3 times in PBS.
Approximately 1013 TU of phage is applied to the tube and incubated
for 30 minutes at room temperature tumbling on an over and under
turntable and then left to stand for another 1.5 hours. Tubes are
washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage
are eluted by adding 1 ml of 100 mM triethylamine and rotating 15
minutes on an under and over turntable after which the solution is
immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage
are then used to infect 10 ml of mid-log E. coli TG1 by incubating
eluted phage with bacteria for 30 minutes at 37.degree. C. The E.
coli are then plated on TYE plates containing 1% glucose and 100
micrograms/ml ampicillin. The resulting bacterial library is then
rescued with delta gene 3 helper phage as described above to
prepare phage for a subsequent round of selection. This process is
then repeated for a total of 4 rounds of affinity purification with
tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20
times with PBS for rounds 3 and 4.
[1073] Characterization of Binders.
[1074] Eluted phage from the third and fourth rounds of selection
are used to infect E. coli HB 2151 and soluble scFv is produced
(Marks, et al., 1991) from single colonies for assay. ELISAs are
performed with microtiter plates coated with either 10 picograms/ml
of the polypeptide of the present invention in 50 mM bicarbonate pH
9.6. Clones positive in ELISA are further characterized by PCR
fingerprinting (see e.g., WO92/01047) and then by sequencing.
Example 10
Neutralization of Neutrokine-Alpha/Neutrokine-Alpha Receptor
Interaction with an Anti-Neutrokine-Alpha Monoclonal Antibody
[1075] Monoclonal antibodies were generated against
Neutrokine-alpha protein according to the following method.
Briefly, mice were given a subcutaneous injection (front part of
the dorsum) of 50 micrograms of His-tagged Neutrokine-alpha protein
produced by the method of Example 2 in 100 microliters of PBS
emulsified in 100 microliters of complete Freunds adjuvant. Three
additional subcutaneous injections of 25 micrograms of
Neutrokine-alpha in incomplete Freunds adjuvant were given at
2-week intervals. The animals were rested for a month before they
received the final intraperitoneal boost of 25 micrograms of
Neutrokine-alpha in PBS. Four days later mice were sacrificed and
splenocytes taken for fusion.
[1076] The process of "Fusion" was accomplished by fusing
splenocytes from one spleen were with 2x10E7 P3X63Ag8.653
plasmacytoma cells using PEG 1500 (Boehringer Mannheim), according
to the manufacturer's modifications of an earlier described method.
(See, Gefter, M. L., et al. Somatic Cell Genet. 3:231-36 (1977);
Boehringer Mannheim, PEG 1500 (Cat. No. 783641), product
description.)
[1077] After fusion, the cells were resuspended in 400 ml of HAT
medium supplemented with 20% FBS and 4% Hybridoma Supplement
(Boehringer Mannheim) and distributed to 96 well plates at a
density of 200 microliters per well. At day 7 post-fusion, 100
microliters of medium was aspirated and replaced with 100
microliters of fresh medium. At day 14 post-fusion, the hybridomas
were screened for antibody production.
[1078] Hybridoma supernatants were screened by ELISA for binding to
Neutrokine-alpha protein immobilized on plates. Plates were coated
with Neutrokine-alpha by overnight incubation of 100 microliters
per well of Neutrokine-alpha in PBS at a concentration of 2
micrograms per ml. Hybridoma supernatants were diluted 1:10 with
PBS were placed in individual wells of Neutrokine-alpha-coated
plates and incubated overnight at 4.degree. C. On the following
day, the plates were washed 3 times with PBS containing 0.1%
Tween-20 and developed using the anti-mouse IgG ABC system (Vector
Laboratories). The color development reaction was stopped with the
addition of 25 ml/well of 2M H.sub.2SO.sub.4. The plates were then
read at 450 nm.
[1079] Hybridoma supernatants were checked for Ig isotype using
Isostrips. Cloning was done by the method of limiting dilutions on
HT medium. About 3x10E6 cells in 0.9 ml of HBSS were injected in
pristane-primed mice. After 7-9 days, ascitic fluid was collected
using a 19 g needle. All antibodies were purified by protein G
affinity chromatography using the Acta FPLC system (Pharmacia).
[1080] After primary and two consecutive subcutaneous injections,
all three mice developed a strong immune response; the serum titer
was 10E-7 as assessed by ELISA on Neutrokine-alpha-coated
plates.
[1081] In one experiment, using the splenocytes from the positive
mouse more than 1000 primary hybridomas were generated. 917 of them
were screened for producing anti-Neutrokine-alpha antibody.
Screening was performed using 1:1 diluted supernatants in order to
detect all positive clones. Of 917 hybridomas screened, 76 were
found to be positive and 17 of those were found to be IgG
producers. After affinity testing and cloning, 9 of them were
chosen for further expansion and purification.
[1082] All purified monoclonal antibodies were able to bind
different forms of Neutrokine-alpha (including His-tagged and
protein produced from a baculoviral system (see Example 2)) in both
Western blot analysis and ELISA. Six of nine clones were also able
to bind Neutrokine-alpha on the surface of THP-1 cells. However,
none of the antibodies tested were able to capture Neutrokine-alpha
from solution.
[1083] High affinity anti-Neutrokine-alpha monoclonal antibodies
were generated that recognize Neutrokine-alpha expressed on the
cell surface but not in solution can be used for neutralization
studies in vivo and in monocyte and B cell assays in vitro. These
antibodies are also useful for sensitive detection of
Neutrokine-alpha on Western blots.
[1084] In an independent experiment, using the splenocytes from the
positive mouse, more than 1000 primary hybridomas were generated.
729 of the primary hybridomas were then screened for the production
of an anti-Neutrokine-alpha antibody. Screening was performed under
stringent conditions using 1:10 diluted supernatants in order to
pick up only clones of higher affinity. Of 729 hybridomas screened,
23 were positive, including 16 IgM and 7 IgG producers (among the
latter, 4 gave a strong IgM background). In this experiment, the
isotype distribution of IgG antibodies was biased towards the IgG2
subclasses. Three of seven IgG hybridomas produced antibodies of
IgG2a subclass and two produced an antibody of IgG2b subclass,
while the remaining two were IgG1 producers.
[1085] Supernatants from all positive hybridomas generated in the
second experiment were tested for the ability to inhibit
Neutrokine-alpha-mediated proliferation of B cells. In the first
screening experiment, two hybridomas producing IgG-neutralizing
antibodies were detected (these are antibodies 16C9 and 12C5). In
additional experiments, the IgG-neutralizing activity of the
hybridomas (i.e., 16C9 and 12C5) were confirmed and two additional
strongly neutralizing supernatants from hybridomas 15C10 and 4A6
were identified.
[1086] Three clones were subsequently expanded in vivo (a single
clone, i.e., 15C10, was also expanded in a hollow fiber system),
and the antibody purified by affinity chromatography. All three of
the clones were able to bind Neutrokine-alpha on the surface of
THP-1 cells and were also able to bind (i.e., "capture")
Neutrokine-alpha from solution.
[1087] Specifically, experiments were performed using the
anti-Neutrokine-alpha monoclonal antibodies described in the second
experiment above to determine whether the antibodies neutralize
Neutrokine-alpha/Neutrokine-alpha Receptor binding. Briefly,
Neutrokine-alpha protein was biotinylated using the EZ-link T
NHS-biotin reagent (Pierce, Rockford, Ill.). Biotinylated
Neutrokine-alpha was then used to identify cell surface proteins
that bind Neutrokine-alpha. Preliminary experiments demonstrated
that Neutrokine-alpha binds to a receptor on B lymphoid cells.
[1088] The inclusion of anti-Neutrokine-alpha antibodies generated
in the second experiment described above neutralized binding of
Neutrokine-alpha to a Neutrokine-alpha receptor. In a specific
embodiment, anti-Neutrokine-alpha antibody 15C10 neutralizes
binding of Neutrokine-alpha to a Neutrokine-alpha Receptor.
[1089] Thus, the anti-Neutrokine-alpha monoclonal antibodies
generated in the second experiment described above (in particular,
antibody 15C10) recognize and bind to both membrane-bound and
soluble Neutrokine-alpha protein and neutralize
Neutrokine-alpha/Neutrokine-alpha Receptor binding in vitro.
Example 11
Neutrokine-Alpha Induced Signalling in B Cells
[1090] Total RNA was prepared from tonsillar B cells unstimulated
or stimulated with SAC or SAC plus soluble Neutrokine-alpha (amino
acids 134-285 of SEQ ID NO:2, 100 ng/mL) for 12 hours. Messenger
RNA levels of ERK-1 and PLK was determined by real time
quantitative PCR using ABI 7700 Taqman sequence detector.
Amplification primers and probes were designed to span the region
from nucleotides 252-332 of the human PLK sequence and nucleotides
373 to 446 of the human ERK-1 mRNA (Genbank accession numbers
X75932 and X60188, respectively). For quantitation of RNA, the
comparative delta CT method was used (Perkin-Elmer user Bulletin #2
and #4, 1997) using an 18S ribosomal RNA probes endogenous
reference. Expression levels were characterized relative to
observed levels in unstimulated B-cells.
Example 12
Rapid and Specific Targeting of Radiolabeled Neutrokine-Alpha to
Lymphoid Tissues
[1091] Here, biodistribution studies of radiolabeled
Neutrokine-alpha are reported that demonstrate high in vivo
targeting specificity of Neutrokine-alpha for lymphoid tissues.
Neutrokine-alpha was radiolabeled with .sup.125I and injected
intravenously into BALB/c mice. Three doses and 4 timepoints over a
24-hr period were studied. Biodistribution was measured by direct
counting of the radioactivity in dissected whole organs or tissues
and by whole body quantitative autoradiography (QAR).
[1092] Spleen and lymph nodes showed the highest concentration of
radioactivity among the dissected organs and tissues. Three hr
after injection of 0.01 mg/kg Neutrokine-alpha, 63% and 23%
injected dose (ID)/g were measured in spleen and lymph node,
respectively, compared to .about.5% for both kidney and liver. As
the dose was increased, the % ID/g in spleen and lymph node
decreased but was unchanged in liver and kidney, suggesting that
targeting to spleen and lymph nodes is mediated by saturable
binding. With increasing time, the ratio of the concentration in
spleen and lymph node to the concentration in either kidney or
liver increased. QAR confirmed the high uptake of radiolabeled
Neutrokine-alpha in spleen and lymph nodes at 3 hr, and revealed
high uptake in bone marrow, gut-associated lymphoid tissue (GALT)
and intestinal contents as well. At 24 hr, spleen, lymph nodes and
GALT were still strongly positive for radiolabeled Neutrokine-alpha
by QAR whereas liver and kidney no longer had observable levels. A
cytotoxic radionuclide coupled to Neutrokine-alpha could irradiate
neoplastic B-cells trafficking through or residing in lymphoid
tissues. Thus, the rapid and highly specific targeting of
radiolabeled Neutrokine-alpha to lymphoid tissues provides a
rationale for its application in the treatment of B-cell
malignancies.
Example 13
Pharmacological Effects of .sup.131I-labeled Neutrokine-alpha in
BCL1 Tumor-Bearing Mice and J558 Tumor Bearing Mice
.sup.131I-Neutrokine-Alpha Administration to BCL1 Tumor-Bearing
Mice
[1093] The BCL1 tumor cell line was derived from a spontaneous
murine B cell tumor. Intraperitoneal inoculation of the BCL1 cell
line in BALB/c mice results in splenomegaly, and subsequent death.
The BCL1 tumor cell phenotype is IgM positive, complement receptor
negative, Fc receptor positive and has marginal IgD expression
(Knapp et al., J. Immunol. 123:992-999 (1979) and Vitetta et al
Blood 89:4425-36. (1997)). Based on FACS analysis using
biotinylated Neutrokine-alpha, BCL1 cells freshly isolated from the
spleens of BALB/c mice express Neutrokine-alpha receptors on their
cell surface. The BCL1 tumor model is a relevant mouse model for
human B cell lymphoma, providing a means to test the ability of
.sup.131I-labeled Neutrokine-alpha to kill leukemic B cells and
consequently prolong survival of tumor-bearing mice. Three lots of
.sup.131I-labeled Neutrokine-alpha (Lots TX1, TX2 and TX3) were
prepared by MDS Nordion (Ontario, Canada) and used in 3 different
experiments to evaluate the effects of .sup.131I-labeled
Neutrokine-alpha in this murine model.
[1094] Female BALB/c mice were injected intraperitoneally (ip) on
Day 0 with 1.times.10.sup.5 viable BCL1 cells that had been
propagated in vivo. Treatment groups for the 3 experiments are
described in Table VI. Ten days after injection of tumor cells, the
animals were administered .sup.131I-labeled Neutrokine-alpha iv in
110 .mu.L. The doses administered were 11.9 or 15.3 mCi/kg (TX1),
17.5 mCi/kg (TX2), or 37.7 mCi/kg (TX3) for the 3 experiments. To
identify potentially toxic effects of the administered
.sup.131I-labeled Neutrokine-alpha, age-matched control BALB/c mice
without BCL1 tumors were injected with identical doses of the
.sup.131I-labeled protein. An additional group of BALB/c mice,
bearing BCL1 tumors and receiving an iv injection of the vehicle,
served as the normal tumor control group. Survival was then
monitored for 48, 44, or 40 days for the TX1, TX2, and TX3
experiments, respectively.
TABLE-US-00006 TABLE VI Treatment groups for TX1, TX2 and TX3
experiments .sup.131I-Neutrokine- BCL1 Tumor alpha Dose Inoculated
ip Exp. Group (mCi/kg) n (No. of cells) 1 1 Vehicle 0 15 1 .times.
10.sup.5 (TX1) 2 .sup.131I-Neutrokine- 11.9 10 1 .times. 10.sup.5
alpha 3 .sup.131I-Neutrokine- 15.3 10 1 .times. 10.sup.5 alpha 4
.sup.131I-Neutrokine- 11.9 10 0 alpha 5 .sup.131I-Neutrokine- 15.3
10 0 alpha 2 1 Vehicle 0 12 1 .times. 10.sup.5 (TX2) 2
.sup.131I-Neutrokine- 17.5 12 1 .times. 10.sup.5 alpha 3
.sup.131I-Neutrokine- 17.5 8 0 alpha 3 1 Vehicle 0 14 1 .times.
10.sup.5 (TX3) 2 .sup.131I-Neutrokine- 37.7 14 1 .times. 10.sup.5
alpha 3 .sup.131I-Neutrokine- 37.7 8 0 alpha
[1095] The endpoint monitored in the 3 experiments was survival
(days) following ip inoculation of BCL1 tumor cells. All animals
were examined daily. The day post-inoculation that mice were either
found dead or in moribund condition (the latter being immediately
euthanized for humane reasons) was recorded.
[1096] A single iv administration of either 11.9 or 15.3 mCi/kg
(TX1), 17.5 mCi/kg (TX2), or 37.7 mCi/kg (TX3) of .sup.131I-labeled
Neutrokine-alpha injected 10 days after intraperitoneal inoculation
of BCL1 cells in BALB/c mice significantly improved survival
compared with mice inoculated with tumor and treated with the
.sup.131I-labeled Neutrokine-alpha vehicle (FIGS. 12-14; in FIGS.
12-14, .sup.131I-labeled Neutrokine-alpha is indicated as LR131).
The median survival time for the vehicle-treated, tumor-bearing
mice was 18, 21, and 19 days post-tumor cell injection for the TX1,
TX2, and TX3 experiments, respectively. In the TX1 experiment,
.sup.131I-labeled Neutrokine-alpha administration at dose levels of
11.9 and 15.3 mCi/kg doubled the median survival time of
tumor-bearing mice to 35.5 (11.9 mCi/kg) and 34 (15.3 mCi/kg) days
post-treatment, respectively. In the TX2 and TX3 experiments,
.sup.131I-labeled Neutrokine-alpha administration at a dose of 17.5
or 37.7 mCi/kg increased the median survival time of tumor-bearing
mice to 30 and 22 days post-treatment, respectively. Tumor-bearing
mice treated with all doses of .sup.131I-labeled Neutrokine-alpha
in the 3 experiments had a significantly lower risk of dying than
tumor-bearing mice treated with vehicle (Table VII).
TABLE-US-00007 TABLE VII Incidence of mortality for TX1-TX3
experiments Median Survival Time Experiment Treatment Group (Days)
TX1 1, BCL1 + .sup.131I-labeled Neutrokine-alpha (11.9 mCi/kg) 35.5
2, BCL1 + .sup.131I-labeled Neutrokine-alpha (15.3 mCi/kg) 34 3,
BCL1 Tumor Only 18 4, No Tumor + .sup.131I-labeled Neutrokine-alpha
>48 (11.9 mCi/kg) 5, No Tumor + .sup.131I-labeled
Neutrokine-alpha >48 (15.3 mCi/kg) TX2 1, BCL1 +
.sup.131I-labeled Neutrokine-alpha (17.5 mCi/kg) 30 2, BCL1 Tumor
Only + vehicle 21 3, No Tumor + .sup.131I-labeled Neutrokine-alpha
>44 (17.5 mCi/kg) TX3 1, BCL1 + vehicle 19 2, BCL1 +
.sup.131I-labeled Neutrokine-alpha (37.7 mCi/kg) 22 3, No tumor +
.sup.131I-labeled Neutrokine-alpha >40 (37.7 mCi/kg)
[1097] In the TX1-TX3 series of experiments, the effect that
increasing the dose of .sup.131I-labeled Neutrokine-alpha had on
the survival of the BCL1 tumor-bearing animals was investigated. A
maximal survival benefit was achieved with the low doses of
.sup.131I-labeled Neutrokine-alpha (11.9 and 15.3 mCi/kg). The much
reduced effectiveness of .sup.131I-labeled Neutrokine-alpha in TX3
may be due to toxicity associated with the high dose of the
material used.
[1098] In conclusion, a single iv administration of
.sup.131I-labeled Neutrokine-alpha administered to mice bearing
BCL1 leukemia cell splenic tumors significantly improved survival
compared with tumor-bearing mice treated with vehicle.
[1099] 131I-Neutrokine-Alpha administration to J558 Tumor-Bearing
Mice
[1100] In a similar experiment as that described above, BALB/c mice
were injected subcutaneously with J558 plasmacytoma cells (ATCC #
TIB-6) and treated with a single intravenous treatment of 25mCi/kg
of .sup.131I-labeled Neutrokine-alpha. 24 BALB/c mice (NCI, 4 weeks
old, average weight 18 g) were divided into 2 groups (12 mice per
group) and injected sc with 2.5.times.10.sup.5 J558 cells in 100 mL
of PBS. At Day 9 after injection, mice in Group 1 were injected
intravenously with 100 mL of formulation buffer, and mice in Group
2 were injected iv with a dose of 25 mCi/kg of
.sup.131I-Neutrokine-alpha in 100 mL of formulation buffer. The
average body weight at the time of .sup.131I-Neutrokine-alpha
injection was 19.5 g.
[1101] Two parameters were evaluated during this study the tumor
size and the time to tumor response. To evaluate tumor size the
short and long axes of the tumor were measured using an electronic
digital caliper. Tumor size was calculated by multiplication of the
lengths of the short and long axes and expressed in mm.sup.2. The
time to tumor response was characterized by the day after cell
inoculation when a visible tumor (>2 mm) was detected on a
mouse. In addition, mice were monitored for survival and signs of
radiation induced toxicity (general appearance, activity, breathing
frequency, stool consistence).
[1102] One mouse in the .sup.131I-Neutrokine-alpha-treated group
died on Day 25 (16 days after .sup.131I-Neutrokine-alpha treatment)
with no obvious signs of radiation related toxicity. A second mouse
died in the same group on Day 30, when all animals in the control
group were terminated because of large tumor size.
[1103] The first tumors of measurable size were detected at Day 14
in the buffer control group, where 4 out of 12 animals developed
tumors. In the .sup.131I-Neutrokine-alpha treated animals, tumor
formation was delayed by 6 days. Only one mouse out of 12 developed
a tumor at Day 20. At Day 22, there was only one tumor-bearing
mouse in the .sup.131I-Neutrokine-alpha treated group out of 12
animals, whereas in the buffer control group, 11 out of 12 mice
developed tumors of different sizes. At Day 27, the mean tumor size
in the buffer control group was 489 mm.sup.2 (all tumor positive
mice in this group were terminated at this time point). In the
.sup.131I-Neutrokine-alpha treated group, the mean tumor size was
32.7 mm.sup.2, 15 times smaller than in the buffer control group.
Taken together, these data suggest a strong inhibition of J558
tumor development in mice treated with .sup.131I-Neutrokine-alpha
at a dose of 25 mCi/kg and tumor load of 2.5.times.10.sup.5
cells/mouse.
[1104] In conclusion, a single intravenous administration of
.sup.131I-Neutrokine-alpha into BALB/c mice at a dose of 25 mCi/kg
significantly inhibits subcutaneous growth of J558 plasma cell
tumors. At the initial tumor load of 2.5.times.105 cells/mouse, a 6
day delay in tumor formation and a 15-fold reduction in tumor size
was observed in .sup.131I-Neutrokine-alpha treated animals.
[1105] The anti-neoplastic effects of .sup.131I-Neutrokine-alpha
were accompanied by the expected B lymphocyte hypoplasia and a
transient (<20 days) depletion of cKit.sup.+ bone marrow
precursors and peripheral platelets. Peripheral neutrophil, red
blood cell, and monocyte counts were unaffected by
.sup.131I-Neutrokine-alpha treatment. Taken together, the results
demonstrate that .sup.131I-Neutrokine-alpha inhibits in vivo tumor
growth in two models of B cell neoplasia. Moreover,
.sup.131I-Neutrokine-alpha efficacy was not accompanied by
significant bone marrow toxicities or peripheral
myelosuppression.
Example 14
Improved Method for Producing Neutrokine-Alpha Using a Stringent
Promoter and Low Expression Level
[1106] Neutrokine-alpha has been produced in Escherichia coli K-12
from the periplasmic fraction of the cell lysate. Using this
system, soluble, properly folded, active Neutrokine-alpha is not
obtainable from simple shake flask experiments. Yields of soluble
Neutrokine-alpha from complex media fermentations in small and
large-scale bioreactors are on the order of 1-5 mg/L. Greater
yields (25-38 mg/L) of soluble, properly folded, active
Neutrokine-alpha can be accomplished in bioreactors at low to
medium cell density under defined medium conditions. Moreover, this
low quantity of protein is difficult to purify via conventional
methods.
[1107] This example describes a method for the production of high
yields of soluble, properly folded, active Neutrokine-alpha in the
periplasm of Escherichia coli, which permits the use of
conventional methods for Neutrokine-alpha purification, such as
those described below or in Example 2 (paragraphs [0994]-[1004],
with modifications for E. coli, as would be apparent to one of
ordinary skill in the art). Additionally, Neutrokine-alpha protein
may be purified using affinity columns comprising Neutrokine-alpha
binding peptides such as those described in WO 02/02641, which is
herein incorporated by reference in its entirety. Purified
Neutrokine-alpha may be quantified using RP-HPLC.
[1108] This method relies on the expression of Neutrokine-alpha
protein from the bacterial phoA promoter. The phoA promoter is a
very tightly regulated system that exhibits a very low level of
transcription in the presence of excess phosphate. As the phosphate
level in the medium decreases below a threshold of .about.4
micromolar (Wanner, B. L., J Cell Biochem 51:47 (1993)),
transcription is induced about 1000-fold. The phoA promoter yields
a gradual build-up of recombinant protein, instead of a sharp
increase of induction that occurs with other systems. This gradual
or steady increase in recombinant protein minimizes the chance of
overwhelming the components of the bacterial expression system and
may also minimize the formation of inclusion bodies. Furthermore,
this gradual build up permits the expression of proteins that might
have been toxic to the cell if they were induced to high levels
over a short period of time.
Expression Vector pML124
[1109] The expression vector, pML124, was created using pBR322 as
the starting backbone. First, the endogenous NdeI site of pBR322
was eliminated by digesting it with NdeI, filling in the
overhanging ends with the Klenow enzyme, then re-ligating the two
blunt-ends back together (this created pML123). Next, pML123 was
digested with EcoRI and BamHI restriction enzymes and the linear
plasmid (loss of .about.375 bp of DNA) was agarose gel purified
(Qiagen).
[1110] The phoA promoter region was PCR-amplified from the E. coli
K-12 chromosome (W3110; ATCC Catalogue No. 27325) with EcoRI (5')
and BamHI (3') engineered sites. NdeI and KpnI sites were also
engineered downstream of the phoA promoter to facilitate cloning of
recombinant genes. Finally, the Shine-Dalgarno (SD) box was
optimized for protein expression. The wild-type SD box and its
adjacent sequence is as follows (the putative SD boxes are
underlined and in bold):
5'-TTTGTACATGGAGAAAATAAA (SEQ ID NO:56):-[ATG, start of coding
sequence]-3' Optimized SD box and adjacent sequence is as follows:
5'-CACGTAAAGGAAGTATCTCAT (SEQ ID NO:57)-[ATG, start of coding
sequence]-3'
[1111] The digested (EcoRI and BamHI) and purified phoA promoter
PCR product was ligated into the agarose gel purified pML123
(described above). The ligation mixture was transformed into highly
competent E. coli cells using standard techniques. Positive clones
were identified via restriction analysis and DNA sequencing.
[1112] pML124 contains a gene for ampicillin resistance, a ColE1
replicon (pBR322-based), Rop, phoA promoter, the optimized
Shine-Dalgamo (SD) box (above) and a multiple cloning site. FIG. 15
is a plasmid map of pML124 and SEQ ID NO:52 is the nucleotide
sequence of pML124. Additionally, plasmid pML124 was deposited at
the American Type Culture Collection (ATCC) on Oct. 8, 2001 and
given ATCC Deposit No. PTA-3778. ATCC Deposit Nos. PTA-3778 was
made pursuant to the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure. The ATCC (American Type Culture
Collection) is located at 10801 University Boulevard, Manassas, Va.
20110-2209.
Neutrokine-Alpha Expression Vector pML124-MBPssBLyS
[1113] A fusion construct of the maltose binding protein signal
sequence (MBPss) and Neutrokine-alpha was placed behind the phoA
promoter in pML124 as follows. A 549 bp NdeI/KpnI
MBPss-Neutrokine-alpha containing DNA insert was ligated into
NdeI/KpnI digested and gel purified pML124 to form
pML124-MBPss-BLyS. (FIG. 16, SEQ ID NO:53 ATCC Deposit No.
PTA-3867, deposited Nov. 16, 2001). ATCC Deposit No. PTA-3867 was
made pursuant to the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure. The ATCC (American Type Culture
Collection) is located at 10801 University Boulevard, Manassas, Va.
20110-2209.
[1114] The pML124 plasmid (FIG. 15, SEQ ID NO:52) is described in
above and in U.S. Provisional Applications 60/329,508 filed Oct.
17, 2001, 60/329,747 filed Oct. 18, 2001 and 60/331,478 filed Nov.
16, 2001 which are herein incorporated by reference in their
entireties. The phoA promoter region is located at nucleotides
111-410 SEQ ID NOs:52 and 53. The MBP signal sequence is encoded by
nucleotides 423-500 of SEQ ID NO:53 and nucleotides 501-959 of SEQ
ID NO:53 encode amino acids 134-285 of Neutrokine-alpha (SEQ ID
NO:2). The amino acid sequence of the MBP signal sequence is shown
in SEQ ID NO:54 and the amino acid sequence of the full length MBP
signal sequence-neutrokine-alpha protein encoded by the
pML124-MBPss-BLyS vector is shown in SEQ ID NO:55
Neutrokine-Alpha Expression in E. coli
[1115] Plasmid pML124-MBPss-BLyS was transformed into E. coli
cells, e.g. K-12 based strains, by standard methods. Ampicillin
resistant transformants were screened for the proper DNA insert by
restriction enzyme analysis and DNA sequence. For example,
digestion of pML124-MBPss-BLyS.TM. with NdeI and KpnI results in
two nucleotide fragments: 549 and 4,431 base pairs in length.
Positive clones were subsequently grown in City Broth-Low Phosphate
media (see recipe below). Neutrokine-alpha expression levels were
examined via SDS-PAGE and subsequent Coomassie staining. Using
simple shake flask experiments, more than 260 mg/L of
Neutrokine-alpha was obtained.
[1116] Next, positive clones were grown to high cell density in
complex media in small scale bioreactors, similar to the method
described by Joly et al., PNAS 95:2773-2777 (1998), which is hereby
incorporated by reference in its entirety. Specifically, the
initial fermentation medium for the 5L bioreactor was composed of
55.7 mM ammonium sulfate, 13.9 mM sodium monobasic phosphate, 21.9
mM potassium dibasic phosphate, 5 mM sodium citrate, 29.6 mM
potassium chloride, 14.7 mM magnesium sulfate, 1.11% NZ-amine AS,
1.11% yeast extract, 5 g/L glucose, 0.002% ferric chloride, 25
.quadrature.g/ml kanamycin. A trace element solution (2.5 ml/3.4 L)
was added containing 100 mM ferric chloride plus 30 mM of the
following components: zinc sulfate, cobalt chloride, sodium
molybdate, copper sulfate, boric acid and manganese sulfate. The
fermentor was operated at 30.degree. C., 650 rpm agitation, 10
standard liter/minute aeration. When the initial glucose was
depleted, a concentrated glucose solution (50%) was added until the
dissolved oxygen (DO) concentration reached 20% of air saturation
as measured by an on-line oxygen electrode. When the optical
density (600 nm) reached 400D600, a solution of 20% NZ amine AS,
20% yeast extract was fed at 0.2 ml/min for the rest of the
fermentation. Neutrokine-alpha production was on the order of
260-570 mg/L.
Low Phosphate Containing Media:
City Broth-Low Phosphate:
[1117] 30 mM (NH.sub.4).sub.2SO.sub.4; 2.25 mM NaCitrate-2H.sub.2O;
12 mM MgSO.sub.4; 15 mM KCl; 5% Yeast extract; 2% Casamino acids;
110 mM MOPS; 33 mM Glucose; pH 7.3
Vegan City Broth-Low Phosphate:
[1118] 30 mM (NH.sub.4).sub.2SO.sub.4; 2.25 mM NaCitrate-2H.sub.2O;
12 mM MgSO.sub.4; 15 mM KCl; 5% Phytone; 2% Casamino acids; 110 mM
MOPS; 33 mM Glucose; pH 7.3
[1119] The only difference between the two media is that Phytone is
substituted for Yeast extract in the Vegan recipe.
Purification of Neutrokine-Alpha
[1120] 10 grams of E. coli cell paste are suspended in 50
milliliters of 5 mM sodium citrate, pH 6.0 and placed at 4.degree.
C. for 1 hour with gentle shaking. Cells are then disrupted by
passing them through an M-Y110 Microfluidizer.RTM. Processor
(Microfluidics, Inc., Newton, Mass.) set at 7500 psi four times.
The suspension is then centrifuged at 22,000.times.g for twenty
minutes at 4.degree. C. using a Sorvall SLA-1500 rotor. The
supernatant is then collected and filtered through a 0.45 micron
bottle top filter (Nalgene).
[1121] Filtered supernatant is then loaded at 9 centimeters/hour on
a Fast Flow Sepharose DEAE column (Amersham Bisociences,
Piscataway, N.J.) previously equilibrated with 5 mM sodium citrate,
pH6.0 (equilibration buffer). After loading, the column is washed
with 5 to 10 column volumes of equilibration buffer. The
Neutrokine-alpha protein is eluted with a 200 mM NaCl step in
equilibration buffer. Buffers used with the Fast Flow Sepharose
DEAE chromatography column are pre-filtered using a 0.22 micron CA
bottle top filter (Nalgene) and pre-chilled to 4.degree. C. The
Fast Flow Sepharose DEAE column is used at 4.degree. C. Prior to
use, columns are cleaned with 0.5 M NaOH.
[1122] Relevant fractions, as determined by the ratio of
contaminating proteins to Neutrokine-alpha protein seen in
Coomassie stained SDS-PAGE gels, are pooled and diluted 1:1 with 10
mM sodium citrate, pH 6.0, 2M (NH.sub.4)SO.sub.4. Pooled fractions
are loaded at 17 centimeters/hour onto a Polypropylene Glycol
Hydrophobic Interaction chromatography column (Tosoh Biosep,
Montgomeryville, Pa.) previously equilibrated with 10 mM sodium
citrate, pH 6.0, 1M (NH.sub.4)SO.sub.4 (loading buffer). After
loading, the column is washed with 5-10 volumes of loading buffer.
The Neutrokine-alpha protein is eluted with a 5 column volume
gradient from loading buffer to elution buffer (10 mM sodium
citrate, pH 6.0). Neutrokine-alpha elutes in the second peak toward
the end of the gradient absorbance at 280 nm. Buffers used with the
Polypropylene Glycol Hydrophobic Interaction chromatography column
are pre-filtered using a 0.22 micron CA bottle top filter (Nalgene)
and used at room temperature. The Polypropylene Glycol Hydrophobic
Interaction chromatography column is also used at room temperature.
Prior to use, columns are cleaned with 0.5 M NaOH.
[1123] Relevant fractions, determined by the ratio of contaminating
proteins to Neutrokine-alpha protein as monitored by Coomassie
stained SDS-PAGE gels, are pooled and are dialyzed overnight (12
hours) into 50 mM Tris, pH 7.4, 50 mM NaCl at 4.degree. C. The
dialyzed pool is then loaded onto a POROS PI-50 anion exchange
chromatography column (Applied Biosystems, Foster City, Calif.),
previously equilibrated with 50 mM Tris, pH 7.4, 50 mM NaCl, at 17
centimeters/hour. After loading, column is washed with 5-10 volumes
of loading buffer. Neutrokine-alpha is eluted using a pH step from
50 mM Tris, pH 7.4, 50 mM NaCl buffer to 50 mM sodium citrate, pH
6.0. Relevant fractions, as determined by the ratio of
contaminating proteins to Neutrokine-alpha protein seen in
Coomassie stained SDS-PAGE gels, are pooled and stored at 4.degree.
C. Buffers used with the POROS PI-50 anion exchange chromatography
column are pre-filtered using a 0.22 micron CA bottle top filter
(Nalgene) and pre-chilled to 4.degree. C. The POROS PI-50 anion
exchange chromatography column is used at 4.degree. C. Prior to
use, columns are cleaned with 0.5 M NaOH.
[1124] This purification protocol yields 0.5-1 milligram per gram
of starting cell paste based on BCA protein assay (Pierce
Biotechnology, Rockford, Ill.) and absorbance at 280 nanometers.
The protein is 96% pure as determined by reverse phase-high
performance liquid chromatography (RP-HPLC). Native-PAGE and size
exclusion chromatography-HPLC (SEC-HPLC) analysis indicates the
protein is predominantly in trimeric form.
[1125] The production of MBPss-Neutrokine-alpha under control of
the phoA promoter allowed more stringent, slower expression, and
resulted in increased yields. In summary, the production of
Neutrokine-alpha from the phoA system is scaleable and achieves 10
to 20-fold more soluble, properly folded, active material than the
current system.
Example 15
Competitive Binding Studies Between Antibody 15C10 and 3D4
[1126] To determine if antibodies 15C10 and 3D4 bind similar or
distinct epitopes, competitive binding studies were performed.
[1127] Soluble Neutrokine-alpha (amino acids 134-284 of SEQ ID
NO:2) was preincubated with 15C10 or 3D4 antibodies. Hereinafter in
this example, the antibody with which Neutrokine-alpha was
preincubated will be referred to as the "competing antibody". After
preincubation, soluble Neutrokine-alpha-competing antibody
complexes were captured on an ELISA plate coated with either 3D4 or
15C10. Hereinafter in this example, the antibody coated on the
ELISA plate will be referred to as the "capture antibody". After
binding, and wash steps, soluble Neutrokine-alpha-competing
antibody complexes captured on the 3D4 or 15C10-coated ELISA plates
was detected using a biotinylated polyclonal anti-Neutrokine-alpha
antibody followed by a streptavidin-coupled detection agent such as
horse radish peroxidase or alkaline phosphatase.
[1128] If there is no competition between the competing antibody
and the capture antibody on the ELISA plate (i.e., if the two
antibodies bind non-overlapping epitopes), soluble Neutrokine-alpha
will be not prevented from binding to the capture antibody on the
ELISA plate and the ELISA will give a positive signal. On the other
hand, if there is competition between the competing antibody and
the capture antibody on the ELISA plate (i.e., if the two
antibodies bind overlapping or identical epitopes), a decreased (or
no) amount of soluble Neutrokine-alpha will be bound to the ELISA
plate and the ELISA will give a decreased signal, compared to the
signal given in the absence of competition between the two
antibodies.
[1129] When an assay similar to that described above was performed
using monoclonal antibodies 15C10 and 3D4, it was found that the
two antibodies competed with each other, irrespective of which
antibody was the competing antibody and which antibody was the
capture antibody. These results indicate that 15C10 and 3D4 at
least have overlapping epitopes. Isotype matched controls of
irrelevant specificity (non-Neutrokine-alpha binding) were not able
to compete for binding.
Example 16
Optimization of BLyS Production and Purification from Escherichia
coli
[1130] In this example numbers in square brackets ([#]) refer to
the list of references found at the end of this example.
[1131] In order to generate large quantities of sBLyS, Escherichia
coli was chosen as the host for the production of recombinant
soluble BLyS (rsBLyS). The initial E. coli expression system
utilized a high copy number plasmid, pHE4 (see, e.g., U.S. Pat. No.
6,194,168 B1), which contains a strong,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) inducible promoter.
From defined media fermentations, this system generated 25-38 mg of
rsBLyS per liter of culture. Due to this low level of expression, a
BLyS-affinity resin was required to completely capture this
quantity of protein. Despite these rsBLyS yields, this process
successfully produced sufficient quantities of rsBLyS for use in
preclinical studies and early clinical trials.
[1132] However, in order to develop a more robust manufacturing
process, rsBLyS yields needed to be increased significantly. After
testing dozens of host strain and vector combinations, and using
multiple growth and induction conditions, high yields of properly
folded, biologically active, trimeric rsBLyS were achieved using a
phoA promoter system. This promoter has been used for the high
yield production of numerous other recombinant proteins [13-18].
Briefly, protein expression is induced by a transmembrane signal
transduction system in which the PhoR and PhoB proteins are
critical for transcriptional activation [19-22]. Signaling begins
when extracellular inorganic phosphate (P.sub.i) drops below 4
.mu.M causing membrane-bound PhoR to undergo a conformational
change and autophosphorylate [20-22]. Next, PhoR transfers the
phosphate moiety to the PhoB protein, which in turn acts as a
transcriptional activator for the phoA promoter [20,22]. Activated
PhoB also acts as a self-regulating transcriptional activator and
as an inducer of PhoR production [19, 22-24]. This regulation
allows the machinery for the induction of the phoA promoter to
gradually build up once P.sub.i levels have dropped below the 4
.mu.M threshold. The result is that a gradual accumulation of
heterologous protein occurs, in contrast to the acute induction
that is characteristic of promoter systems that rely on exogenous
inducers.
[1133] This Example details the achievement of small-scale
production yields of approximately 435 mg/L using the phoA promoter
system. This constitutes at least an 11-fold increase over earlier
manufacturing production yields. With this increased level of
production, a conventional purification scheme that eliminates the
need for the BLyS-affinity resin has also been developed.
Materials and Methods
Media and Reagents
[1134] Luria-Bertani broth (LB) was prepared as previously
described [25] with the ingredients supplied by Difco. When
required, liquid and solid media were supplemented with 25 .mu.g/ml
kanamycin (Sigma). All enzymes, including modification enzymes and
restriction enzymes, were obtained from New England Biolabs. Taq
DNA polymerase was purchased from Roche. Platinum Pfx was obtained
from Invitrogen. Ligations were performed using a Fast-Link.TM. DNA
Ligation Kit (Epicentre). Oligonucleotides were purchased from
Sigma-Genosys or synthesized in-house. MultiMark.RTM. Multi-Colored
Standard and Mark12.TM. unstained standard were purchased from
Invitrogen. Low Range Rainbow Molecular Weight Marker was purchased
from Amersham Biosciences, Corp. All other chemicals were of
analytical grade.
BLyS Plasmid Construction and Bacterial Strains
[1135] To construct the phoA inducible rsBLyS production vector,
pML123 was used as the backbone. pML123 is identical to pBR322 [26]
except that its unique NdeI restriction site has been eliminated.
This was accomplished by digesting pBR322 with NdeI, filling in the
overhangs with T4 DNA polymerase, and ligating the blunt ends
together. Next, a DNA segment containing the phoA promoter region,
an optimized Shine-Dalgamo (SD) box, flanking EcoRI and BamHI
restriction sites, and a multiple cloning site that includes NdeI
and KpnI restriction sites was amplified by polymerase chain
reaction (PCR) from the E. coli genome. The resulting PCR product
was purified and digested with EcoRI and BamHI and ligated into a
similarly digested pML123 vector. This intermediate phoA promoter
plasmid was named pML124. Next, the codon-optimized rsBLyS gene
with a maltose binding protein signal sequence was isolated and gel
purified from a previous BLyS production vector by digesting it
with NdeI and KpnI. The MBPss-rsBLyS insert was ligated into
pML124, generating pML126. Finally, the ampicillin resistance gene
of pML126 was replaced with a gene conferring kanamycin resistance.
This was accomplished by PCR amplification of the kanamycin
resistance gene of pACYC177 [27] using oligonucleotides that
created flanking EcoRI and PstI sites. The PCR product was digested
with PstI and the overhanging end was filled in using the T4 DNA
polymerase. This fragment was then digested with EcoRI and ligated
into pML126 that had been digested with EcoRI and ScaI. The
resulting plasmid was designated p2615 (FIG. 2). All constructs
were confirmed by DNA sequencing and restriction endonuclease
analyses (data not shown).
[1136] For the production of rsBLyS, the phoA production vector,
p2615, was transformed into the E. coli K-12 host W3110 (F-,
.lamda.-, mcrA, mcrB, IN(rrnD-rrnE)1, rph1, ilvG) [28]. This
strain, designated 8D1, was propagated in LB supplemented with
kanamycin (25 .mu.g/ml).
Shake Flask Production of rsBLyS
[1137] Strain 8D1 was tested for rsBLyS expression in shake flasks
using a complex, low phosphate medium known as C.R.A.P. [18]. A
MOPS solution is used to buffer this medium since it contains
minimal quantities of phosphate or other buffering agents.
Typically, 800 mL of C.R.A.P. was aseptically added to a 4-L
baffled shake flask along with 25 mg/L of kanamycin for plasmid
retention. Shake flasks were inoculated to a starting OD.sub.600 of
0.1 from an overnight culture grown in LB supplemented with
kanamycin (25 mg/L). Cultures were grown for 18 hr at 30.degree. C.
and 220 rpm in an Innova 4300 shaker/incubator (New Brunswick
Scientific, Inc.). Cells were harvested by centrifugation at 5,663
rcf for 15 min and yielded about 9 grams of cell paste per liter of
culture.
SDS-PAGE and Western Blot Analysis
[1138] BLyS protein production and purification was examined via
SDS-PAGE [29,30] on 18% Novex.RTM. Tris-glycine gels (Invitrogen).
For Western blot analyses, proteins were transferred to
nitrocellulose membranes (0.22 .quadrature.m; Invitrogen) after
SDS-PAGE using a BIO-RAD Trans-Blot Semi-Dry electrophoretic cell
[31]. Western blot analyses were carried out using a BLyS-specific
mouse monoclonal antibody (internal), followed by a
phosphatase-labeled goat .quadrature.-mouse IgG obtained from
Kirkegaard & Perry Laboratories (KPL). Western Blue Stabilized
Substrate (Promega) for alkaline phosphatase was used for
detection.
Fermentation Set-Up and Operation
[1139] A starter culture of the appropriate strain was grown in 1 L
of MLB medium (12 g/L yeast extract (Difco), 7 g/L NaCl, 2.5 g/L
phytone (Becton Dickinson), and 0.2 g/L L-methionine) and kanamycin
(25 mg/L) in a 4-L baffled shake flask. Shake flask cultures were
inoculated with one seed vial (1.5 ml) and grown at 220 rpm and
30.degree. C. until the culture reached an OD.sub.600 of 2-3. The
culture was used to inoculate fermentors to a starting OD.sub.600
of 0.2.
[1140] Medium-cell density production of rsBLyS was performed using
5-L Bioflo 3000 fermentors from New Brunswick Scientific, Inc.
Fermentations were performed in semi-defined media in a manner
similar to that described previously [18] using the following
parameters: 30.degree. C., pH 7, airflow 5 Lpm (1 vvm), agitation
650 rpm, and dissolved oxygen (DO) 30%. The OD.sub.600 of the
culture was tested frequently using a Genesys 8 spectrophotometer
(Spectronic Unicam). Additional samples were tested for glucose
concentration using the YSI 2700 Select Biochemistry Analyzer
equipped with the YSI 2365 Glucose Membrane (YSI Inc.). The culture
was allowed to grow for a total of 48 hours and then harvested by
centrifugation at 4369 rcf for 25 min.
Affinity Purification of rsBLyS
[1141] E. coli cell paste (.about.10 g) expressing rsBLyS was
resuspended in 80 milliliters of 100 mM Tris pH 7 at 4.degree. C.
for 1 hr with stirring. Cells were disrupted with four passes
through an M-Y110 Microfluidizer (Microfluidics, Inc.) set to 7,500
psi, and then centrifuged at 22,000 rcf for 20 min at 4.degree. C.
The supernatant was filtered through a 0.2 .mu.m cellulose acetate
(CA) membrane (Corning, Inc.) and loaded at 100 cm/hr on a 1.6 cm
(ID).times.4 cm (h) Octyl Sepharose (Amersham Biosciences) column
in tandem with a 1.6 cm (ID).times.3.5 cm (h) BLyS-affinity column;
both were equilibrated with 100 mM Tris pH 7. The Octyl Sepharose
column was used as a negative purification step to remove host cell
proteins, DNA and lipids. Host proteins were removed with a 1 M
NaCl salt wash in the same buffer. rsBLyS was eluted with a pH step
from the load buffer to 100 mM NaAcetate pH 4.5, 50% Ethylene
Glycol. Relevant fractions containing rsBLyS as judged by the ratio
of rsBLyS to host cell proteins were pooled and dialyzed into 10 mM
NaCitrate pH 6, 140 mM NaCl and stored at 4.degree. C. Samples were
analyzed via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
Coomassie staining. RP-HPLC analyses were performed on a Waters
Alliance 2690 Separations Module with 996 Photodiode Array Detector
(Waters, Inc.) using a 5 .quadrature.m, 50 mm by 2 mm BetaBasic
CYANO column (Keystone Scientific, Inc.). To quantify BLyS protein
concentrations via HPLC, a 50 ml blank of final buffer was injected
at the beginning of each sample set and then subtracted from all
standards and samples to eliminate any potential background peaks
caused by the elution buffer. A standard curve was run with each
sample set consisting of 0.5 mg, 5 mg, and 10 mg by injecting 2.5
ml, 25 ml, and 50 ml respectively of a 0.2 mg/ml BLyS standard. The
amount of BLyS in each sample was then quantified based on the
sample peak area compared to the standard curve.
Conventional Purification of rsBLyS
[1142] E. coli cell paste (.about.7 g) was resuspended in 40
milliliters of 5 mM NaCitrate pH 6 at 4.degree. C. for 1 hr with
stirring. The cells were disrupted with four passes through an
M-Y110 Microfluidizer set to 7,500 psi. The suspension was
centrifuged at 22,000 rcf for 20 min at 4.degree. C. The
supernatant was collected and filtered through a 0.2 .mu.m CA
membrane. The filtrate was loaded at 60 cm/hr onto a 2.6 cm
(ID).times.12 cm (h) Fast Flow Sepharose DEAE (Amersham
Biosciences) column that had been equilibrated with 5 mM NaCitrate,
pH 6 (Equilibration Buffer 1). After loading, the column was washed
with 10 column volumes (CV) of Equilibration Buffer 1. Soluble,
trimeric rsBLyS was eluted with a 200 mM NaCl step in the same
buffer.
[1143] Relevant fractions were determined by the ratio of
contaminating proteins to rsBLyS as observed via SDS-PAGE followed
by Coomassie staining. Fractions were pooled (30 milliliters) and
diluted 1:1 with 10 mM NaCitrate, pH 6, 2 M
(NH.sub.4).sub.2SO.sub.4 and loaded at 150 cm/hr onto a 1.6 cm
(ID).times.11 cm (h) Polypropylene Glycol (PPG) HIC (Tosoh Biosep)
column that had been equilibrated with 10 mM NaCitrate pH 6, 1 M
(NH.sub.4).sub.2SO.sub.4 (Equilibration Buffer 2). After loading,
the column was washed with 10 CV of Equilibration Buffer 2. Elution
of rsBLyS was accomplished with a 5 CV gradient from Equilibration
Buffer 2 to 10 mM NaCitrate pH 6.
[1144] Fractions containing rsBLyS were pooled (50 milliliters),
concentrated to 15 milliliters and dialyzed in 1 liter of 50 mM
Tris pH 7.4, 50 mM NaCl for 12 hr at 4.degree. C. with stirring.
The dialyzed pool (.about.15 milliliters) was loaded at 150 cm/hr
onto a 1.6 cm (ID).times.10.4 cm (h) POROS PI-50 (Applied
Biosystems) column equilibrated with 50 mM Tris pH 7.4, 50 mM NaCl
(Equilibration Buffer 3). This column was then washed with 5 CV of
Equilibration Buffer 3 and purified rsBLyS was eluted with a pH
step from the Equilibration Buffer 3 to 50 mM NaCitrate pH 6. All
purification steps were performed at 4.degree. C. and all buffers
were filtered prior to use with a 0.2 .mu.m CA bottle top filter.
Aliquots of rsBLyS were stored at -80.degree. C. RP-HPLC analysis
was performed as described above.
Preparation of Bacterial Lysates and rsBLyS Analysis
[1145] Cells from 8D1 shake flask and fermentor cultures were
harvested at regular intervals, normalized to the same OD600 (0.5)
per ml and pelleted at 16,100 rcf. Pellets were stored at
-20.degree. C. Whole cell lysates were prepared by boiling the
normalized cell pellets in 100 .mu.L SDS-PAGE sample buffer
(Invitrogen) containing 5% .beta.-mercaptoethanol for 5 min. Then,
20 .mu.L of each sample was analyzed via SDS-PAGE or on 18%
Novex.RTM. glycine gels (Invitrogen) followed by Coomassie staining
and Western blotting.
[1146] In order to analyze the soluble and insoluble protein
fractions, cell paste was resuspended in PBS so that resuspensions
being compared were of equivalent OD.sub.600. These resuspensions
were passed through an M-Y110 Microfluidizer four times at 7,500
psi to ensure that nearly all cells had been ruptured. Aliquots of
1 ml were then taken from the cell lysates and microcentrifuged at
16,100 rcf to pellet the insoluble material. Supernatants were
transferred to a separate tube and the insoluble pellet was
resuspended in 1 ml of PBS. The resulting soluble and insoluble
fractions were each mixed in a 1:1 ratio with SDS-PAGE sample
buffer containing 5% .beta.-mercaptoethanol and boiled for 5 min.
Equal volumes were analyzed via SDS-PAGE on 18% Novex.RTM.
Tris-glycine gels in followed by Western Blotting.
B Cell Proliferation Assay
[1147] The biological activity of purified rsBLyS was tested in a
murine B cell proliferation assay as described previously [32].
Unpurified murine splenocytes were plated at 10.sup.5/well, in the
presence of rsBLyS and Staphylococcus aureus cells (SAC), using a
final dilution of 1:100,000. Cells were incubated at 37.degree. C.
for 72 hr, after which they were pulsed with 0.5 mCi/well of
.sup.3H-thymidine for 20 to 24 hr, and then harvested.
Incorporation of thymidine was used as a measure of cellular
proliferation. The BLyS positive control was from a previously
defined reference standard purified from Baculovirus [33].
Results
[1148] Expression of rsBLyS in Shake Flask Cultures
[1149] The codon-optimized gene encoding rsBLyS was cloned behind
the phoA promoter in an attempt to increase yields. This production
plasmid, p2615, was transformed into W3110, host 8D1, and rsBLyS
expression was monitored in C.R.A.P. medium [18] as described in
the Materials and methods. Yields were determined by purifying
rsBLyS using the BLyS-affinity resin and quantifying the protein
via HPLC. Initial screening of rsBLyS production from 8D1 in shake
flask cultures typically yielded 1 mg of rsBLyS per gram of cell
paste. Shake flask yields from the previous production vector (data
not shown) and host combination, production strain 3C3, averaged
only 0.04 mg/g. The use of p2615 increased shake flask yields
25-fold based on milligrams of rsBLyS per g of cell paste, or
75-fold when compared as mg of rsBLyS per L of culture (the g/L of
cell paste is increased three-fold for C.R.A.P. compared to LB).
Based on Western blot data, the overall increase in rsBLyS
production stems mainly from a sizable increase in cellular
production. Examination of soluble and insoluble fractions of the
induced cell paste showed that 8D1 actually contains a higher
percentage of insoluble monomeric BLyS. Whole cell lysates revealed
an overall increase in total cellular production of monomeric
BLyS.
rsBLyS Production in Bioreactors
[1150] Next, strain 8D1 was grown in 5-L fermentors to examine
whether the increased rsBLyS yields could be maintained, or even
amplified, at a larger scale. The 5-L fermentation strategy allowed
the culture to grow to a final OD.sub.600 of 130-140 and gave a wet
cell weight of 125-145 g/L. Typically, the batch glucose would be
exhausted at an elapsed fermentation time (EFT) of 8-9 hours at
which point the 50% glucose feed would be activated to maintain the
DO at 30%. The glucose feed reached a maximum rate around an EFT of
15 hours and then dropped off significantly over the next 8 hours.
After this dynamic flux in the glucose feed rate, a small increase
in the feed rate was observed followed by a reduction over the
remainder of the fermentation. This pattern of glucose uptake was
common to all similarly run fermentations using the 8D1 host. The
growth of the culture was characterized by an initial logarithmic
growth phase followed by a prolonged linear phase. The logarithmic
phase lasted for the first 10 hours of the fermentation while the
cells consumed the batch glucose. Once the batch glucose was
exhausted and the glucose feed was started, the culture shifted
into a steady linear growth phase that continued to the end of the
run. At an EFT of 15 hours, induction of the phoA promoter and
consummate production of rsBLyS was initiated. This induction point
also coincides with fluctuations in the glucose feed rate describe
above. rsBLyS gradually builds up per OD.sub.600 until the end of
the fermentation. Similar to the shake flask experiments, a large
portion of the BLyS produced by 8D1 in fermentors was insoluble but
overall cellular production was increased when compared to a
previous bioreactor process. The increased yields may also be
attributed in part to the increased biomass of the culture. Final
volumetric yields of periplasmic, properly folded, biologically
active, trimeric rsBLyS from 5-L fermentations reached levels above
435 mg/L (equivalent to about 3 mg of rsBLyS per g of cell
paste).
Conventional Purification and Characterization of rsBLyS
[1151] Host and vector modifications, as well as fermentation
optimization, dramatically increased rsBLyS production from E.
coli. The rsBLyS production process described in this work permits
the protein to be purified using commercially available,
conventional resins. For example, trimeric rsBLyS was purified from
E. coli cell paste using resins and buffers that were cost
efficient and amenable to a larger manufacturing scale. The first
step consisted of a Fast Flow Sepharose DEAE column (Table VIII)
and used a low concentration of sodium citrate to enhance rsBLyS
capture. A PPG HIC column was used next, followed by a POROS PI-50
to eliminate residual contaminants (Table VIII). Endotoxin levels
were measured by LAL and it was determined that the POROS PI-50
column was also effective in reducing the level of endotoxin
contamination (Table VIII). Purification yields were determined by
OD.sub.280 and RP-HPLC analyses and were found to be about 0.76 mg
of rsBLyS per gram of cell paste (.about.110 mg/L).
TABLE-US-00008 TABLE VIII Conventional Purification Scheme for
rsBLyS.sup.a Total Endotoxin Protein Percent Total Approximate
Cumulative Level Step Principle (mg) protein.sup.b mg rsBLyS Yield
(%).sup.c (EU/ml) 1 Cell lysis 900 2-3 21 100 N/D 2 Sepaharose FF
116 12 14 67 25,900 DEAE 3 Polypropylene 51 20 10 48 1,950 glycol
HIC 4 POROS PI-50 5.5 >97 5.3 25 0.245 .sup.aBased on the
purification from 7 g of fermentation cell paste. .sup.bPercentage
of rsBLyS compared to total protein. Numbers based on SDS-PAGE
analysis except for Step 4, which is based on RP-HPLC.
.sup.cPercentage of cumulative rsBLyS recovered based on SDS-PAGE
analysis. N/D = not determined
[1152] Methods to characterize the final purified product were
employed to determine the level of purity and biological activity.
The final product was judged to be 97% pure by means of RP-HPLC
(data not shown) and appeared to be identical to the material used
previously for clinical trials. Moreover, rsBLyS produced by this
process and purified via conventional resins was examined in B cell
proliferation assays. The results showed that rsBLyS produced by
this conventional scheme exhibited similar or slightly increased
biological activity.
Discussion
[1153] In this work, an expression plasmid and host combination was
generated that yielded a high concentration of rsBLyS from E. coli.
This system utilized the phoA promoter to maximize periplasmic
expression of the heterologous protein. Initial shake flask yields
were increased to 1 mg of rsBLyS per gram of cell paste over the
0.04 mg/g produced by a previous expression system. Additionally, a
5-L fermentation process was developed for this expression system
that yields 435 mg of purified rsBLyS per L of culture (3 mg of
rsBLyS per g of cell paste). This is an 11-fold increase over
earlier manufacturing production yields. The increased yields
appear to stem from a large increase in overall cellular production
and have facilitated the development of a conventional purification
scheme that eliminates the need for the BLyS-affinity resin.
[1154] The inventors have found that when optimizing IPTG-induced,
rsBLyS production in E. coli, periplasmic expression of the protein
was necessary to obtain any appreciable amounts of properly folded,
trimeric rsBLyS. Moreover, it was discovered that the MBP signal
sequence in combination with the codon-optimized BLyS gene
exhibited the best ratio of mature to precursor protein. The
expression vector was further refined to include the use of the
phoA promoter. The use of this promoter system led to the largest
increase of rsBLyS yields. Furthermore, the copy number of the
production plasmid was also reduced, however, copy number seems to
be of only minor significance since lower copy number
IPTG-inducible vectors were tested without success.
[1155] Previous work has shown the phoA promoter to be superior to
other induction systems for the production of periplasmically
targeted recombinant proteins [13-18]. This may be due to the fact
that the phoA promoter allows for the gradual build up of
recombinant protein versus the acute induction characteristic of
other promoter systems. A slower build up of recombinant protein
may not only permit or enhance proper folding but it also allows
the cells to adjust to potentially toxic proteins when compared to
the rapid accumulation indicative of other promoter systems
[34,35]. In addition, slower recombinant protein production is less
likely to overload protein synthesis components or the secretion
apparatus [35]. These factors can be highly beneficial to the
bacterial production workhorse and may allow the cell to maintain
its regular cellular functions during the induction phase.
[1156] The implementation of a phosphate depleted small-scale
fermentation was used successfully for the production of rsBLyS. In
contrast to the current production system, the exogenous inducer,
IPTG, is not required for protein expression. This is a point of
interest because it not only avoids the addition of another reagent
to the fermentor, but also eliminates a large cost component of the
fermentation process. In addition, the glucose feed has been
simplified and there is no need for other additions (i.e. salt
feeds). The only drawback to the process is that the total run time
is nearly twice that of the current manufacturing production
process. If the time necessary for protein recovery and turn over
of the fermentors is taken into consideration, as well as the
difference in rsBLyS yields between these two production systems,
this extended fermentation time is of little significance.
[1157] The increased rsBLyS yields described in this example
allowed for the development of a purification scheme that utilizes
conventional resins. This is of utmost importance since the
affinity resin currently being used to purify rsBLyS is specially
manufactured strictly for that task. This fact makes it a large
cost component of the recovery process. Therefore, the use of
conventional resins will result in a large reduction in the costs
associated with the purification of rsBLyS. Furthermore, the resins
chosen for the conventional purification of rsBLyS are completely
scaleable which is crucial to any future manufacturing plans
involving rsBLyS.
LIST OF REFERENCES FOR EXAMPLE 16
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immunoglobulins in Escherichia coli: rapid and efficient production
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Regulation of the phosphate regulon of Escherichia coli: properties
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Smith, V. Albert, W. Stohl, K. P. Baker, S. Ullrich, B. Nardelli,
D. M. Hilbert, T. S. Migone, BLyS and APRIL form biologically
active heterotrimers that are expressed in patients with systemic
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production of recombinant proteins in Escherichia coli, Appl.
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Russell, Expression of secreted foreign Proteins using the alkaline
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[1193] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[1194] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
[1195] Further, the Sequence Listing submitted herewith in both
computer and paper forms are hereby incorporated by reference in
their entireties. Additionally, the entire disclosure (including
the specification, sequence listing, and drawings) of each of the
following U.S. Provisional and Non-Provisional Patent Applications
and International Patent Applications are herein incorporated by
reference in their entireties: U.S. Provisional Application Nos.
60/543,261 filed Feb. 11, 2004, 60/580,387 filed Jun. 18, 2004,
60/617,191 filed Oct. 12, 2004, 60/368,548 filed Apr. 1, 2002,
60/336,726 filed Dec. 7, 2001, 60/331,478 filed Nov. 16, 2001,
60/330,835 filed Oct. 31, 2001, 60/329,747 filed Oct. 18, 2001, and
60/329,508 filed Oct. 17, 2001, 60/225,628 filed Aug. 15, 2000,
60/227,008 filed Aug. 23, 2000, 60/234,338 filed Sep. 22, 2000,
60/240,806 filed Oct. 17, 2000, 60/250,020 filed Nov. 30, 2000,
60/276,248 filed Mar. 6, 2001, 60/293,499 filed May 25, 2001,
60/296,122 filed Jun. 7, 2001, 60/304,809 filed Jul., 13,
2001,60/122,388 filed Mar. 2, 1999, 60/124,097 filed Mar. 12, 1999,
60/126,599 filed Mar. 26, 2000, 60/127,598 filed Apr. 2, 1999,
60/130,412 filed Apr. 16, 1999, 60/130,696 filed Apr. 23, 1999,
60/131,278 filed Apr. 27, 1999, 60/131,673 filed Apr. 29, 1999,
60/136,784 filed May 28, 1999, 60/142,659 filed Jul. 6, 1999,
60/145,824 filed Jul. 27, 1999, 60/167,239 filed Nov. 24, 1999,
60/168,624 filed Dec. 3, 1999, 60/171,108 filed Dec. 16, 1999,
60/171,626 filed Dec. 23, 1999, 60/176,015 filed Jan. 14, 2000, and
60/036,100 filed Jan. 14, 1997; U.S. Nonprovisional application
Ser. Nos.: 10,739,042 filed Dec. 19, 2003, 10/735,865 filed Dec.
16, 2003, 10/270,487 filed Oct. 16, 2002, 09/929,493, filed Aug.
14, 2001, 09/588,947 filed Jun. 8, 2000, 09/589,285 filed Jun. 8,
2000, 09/589,286 filed Jun. 8, 2000, 09/589,287 filed Jun. 8, 2000,
09/589,288 filed Jun. 8, 2000, 09/507,968 filed Feb. 22, 2000,
09/255,794 filed Feb. 23, 1999, and 09/005,874 filed Jan. 12, 1998;
and International Patent Application Serial Nos. PCT/US01/25549
filed Aug. 15, 2001, PCT/US00/04336, filed Feb. 22, 2000, and
PCT/US96/17957, filed Oct. 25, 1996.
Sequence CWU 1
1
5911100DNAHomo sapiensCDS(147)..(1001) 1aaattcagga taactctcct
gaggggtgag ccaagccctg ccatgtagtg cacgcaggac 60atcaacaaac acagataaca
ggaaatgatc cattccctgt ggtcacttat tctaaaggcc 120ccaaccttca
aagttcaagt agtgat atg gat gac tcc aca gaa agg gag cag 173 Met Asp
Asp Ser Thr Glu Arg Glu Gln 1 5tca cgc ctt act tct tgc ctt aag aaa
aga gaa gaa atg aaa ctg aag 221Ser Arg Leu Thr Ser Cys Leu Lys Lys
Arg Glu Glu Met Lys Leu Lys10 15 20 25gag tgt gtt tcc atc ctc cca
cgg aag gaa agc ccc tct gtc cga tcc 269Glu Cys Val Ser Ile Leu Pro
Arg Lys Glu Ser Pro Ser Val Arg Ser 30 35 40tcc aaa gac gga aag ctg
ctg gct gca acc ttg ctg ctg gca ctg ctg 317Ser Lys Asp Gly Lys Leu
Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu 45 50 55tct tgc tgc ctc acg
gtg gtg tct ttc tac cag gtg gcc gcc ctg caa 365Ser Cys Cys Leu Thr
Val Val Ser Phe Tyr Gln Val Ala Ala Leu Gln 60 65 70ggg gac ctg gcc
agc ctc cgg gca gag ctg cag ggc cac cac gcg gag 413Gly Asp Leu Ala
Ser Leu Arg Ala Glu Leu Gln Gly His His Ala Glu 75 80 85aag ctg cca
gca gga gca gga gcc ccc aag gcc ggc ctg gag gaa gct 461Lys Leu Pro
Ala Gly Ala Gly Ala Pro Lys Ala Gly Leu Glu Glu Ala90 95 100 105cca
gct gtc acc gcg gga ctg aaa atc ttt gaa cca cca gct cca gga 509Pro
Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala Pro Gly 110 115
120gaa ggc aac tcc agt cag aac agc aga aat aag cgt gcc gtt cag ggt
557Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn Lys Arg Ala Val Gln Gly
125 130 135cca gaa gaa aca gtc act caa gac tgc ttg caa ctg att gca
gac agt 605Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala
Asp Ser 140 145 150gaa aca cca act ata caa aaa gga tct tac aca ttt
gtt cca tgg ctt 653Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe
Val Pro Trp Leu 155 160 165ctc agc ttt aaa agg gga agt gcc cta gaa
gaa aaa gag aat aaa ata 701Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu
Glu Lys Glu Asn Lys Ile170 175 180 185ttg gtc aaa gaa act ggt tac
ttt ttt ata tat ggt cag gtt tta tat 749Leu Val Lys Glu Thr Gly Tyr
Phe Phe Ile Tyr Gly Gln Val Leu Tyr 190 195 200act gat aag acc tac
gcc atg gga cat cta att cag agg aag aag gtc 797Thr Asp Lys Thr Tyr
Ala Met Gly His Leu Ile Gln Arg Lys Lys Val 205 210 215cat gtc ttt
ggg gat gaa ttg agt ctg gtg act ttg ttt cga tgt att 845His Val Phe
Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile 220 225 230caa
aat atg cct gaa aca cta ccc aat aat tcc tgc tat tca gct ggc 893Gln
Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly 235 240
245att gca aaa ctg gaa gaa gga gat gaa ctc caa ctt gca ata cca aga
941Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro
Arg250 255 260 265gaa aat gca caa ata tca ctg gat gga gat gtc aca
ttt ttt ggt gca 989Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr
Phe Phe Gly Ala 270 275 280ttg aaa ctg ctg tgacctactt acaccatgtc
tgtagctatt ttcctccctt 1041Leu Lys Leu Leu 285tctctgtacc tctaagaaga
aagaatctaa ctgaaaatac caaaaaaaaa aaaaaaaaa 11002285PRTHomo sapiens
2Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu1 5
10 15Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu
Pro 20 25 30Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys
Leu Leu 35 40 45Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu
Thr Val Val 50 55 60Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu
Ala Ser Leu Arg65 70 75 80Ala Glu Leu Gln Gly His His Ala Glu Lys
Leu Pro Ala Gly Ala Gly 85 90 95Ala Pro Lys Ala Gly Leu Glu Glu Ala
Pro Ala Val Thr Ala Gly Leu 100 105 110Lys Ile Phe Glu Pro Pro Ala
Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125Ser Arg Asn Lys Arg
Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135 140Asp Cys Leu
Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys145 150 155
160Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser
165 170 175Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr
Gly Tyr 180 185 190Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys
Thr Tyr Ala Met 195 200 205Gly His Leu Ile Gln Arg Lys Lys Val His
Val Phe Gly Asp Glu Leu 210 215 220Ser Leu Val Thr Leu Phe Arg Cys
Ile Gln Asn Met Pro Glu Thr Leu225 230 235 240Pro Asn Asn Ser Cys
Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255Asp Glu Leu
Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270Asp
Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280
2853233PRTHomo sapiens 3Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu
Leu Ala Glu Glu Ala1 5 10 15Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly
Ser Arg Arg Cys Leu Phe 20 25 30Leu Ser Leu Phe Ser Phe Leu Ile Val
Ala Gly Ala Thr Thr Leu Phe 35 40 45Cys Leu Leu His Phe Gly Val Ile
Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60Arg Asp Leu Ser Leu Ile Ser
Pro Leu Ala Gln Ala Val Arg Ser Ser65 70 75 80Ser Arg Thr Pro Ser
Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95Gln Ala Glu Gly
Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110Leu Ala
Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120
125Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg
Ile Ala145 150 155 160Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser
Ala Ile Lys Ser Pro 165 170 175Cys Gln Arg Glu Thr Pro Glu Gly Ala
Glu Ala Lys Pro Trp Tyr Glu 180 185 190Pro Ile Tyr Leu Gly Gly Val
Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205Ser Ala Glu Ile Asn
Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220Gln Val Tyr
Phe Gly Ile Ile Ala Leu225 2304205PRTHomo sapiens 4Met Thr Pro Pro
Glu Arg Leu Phe Leu Pro Arg Val Arg Gly Thr Thr1 5 10 15Leu His Leu
Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala 20 25 30Gln Gly
Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala 35 40 45Arg
Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala 50 55
60Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg65
70 75 80Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser
Asn 85 90 95Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr
Ser Gln 100 105 110Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala
Thr Ser Ser Pro 115 120 125Leu Tyr Leu Ala His Glu Val Gln Leu Phe
Ser Ser Gln Tyr Pro Phe 130 135 140His Val Pro Leu Leu Ser Ser Gln
Lys Met Val Tyr Pro Gly Leu Gln145 150 155 160Glu Pro Trp Leu His
Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr 165 170 175Gln Gly Asp
Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val 180 185 190Leu
Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu 195 200
2055244PRTHomo sapiens 5Met Gly Ala Leu Gly Leu Glu Gly Arg Gly Gly
Arg Leu Gln Gly Arg1 5 10 15Gly Ser Leu Leu Leu Ala Val Ala Gly Ala
Thr Ser Leu Val Thr Leu 20 25 30Leu Leu Ala Val Pro Ile Thr Val Leu
Ala Val Leu Ala Leu Val Pro 35 40 45Gln Asp Gln Gly Gly Leu Val Thr
Glu Thr Ala Asp Pro Gly Ala Gln 50 55 60Ala Gln Gln Gly Leu Gly Phe
Gln Lys Leu Pro Glu Glu Glu Pro Glu65 70 75 80Thr Asp Leu Ser Pro
Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro 85 90 95Leu Lys Gly Gln
Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe 100 105 110Leu Thr
Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro 115 120
125Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg
130 135 140Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr
Leu Arg145 150 155 160Ser Ser Leu Tyr Arg Ala Gly Gly Ala Tyr Gly
Pro Gly Thr Pro Glu 165 170 175Leu Leu Leu Glu Gly Ala Glu Thr Val
Thr Pro Val Leu Asp Pro Ala 180 185 190Arg Arg Gln Gly Tyr Gly Pro
Leu Trp Tyr Thr Ser Val Gly Phe Gly 195 200 205Gly Leu Val Gln Leu
Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser 210 215 220His Pro Asp
Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly Ala225 230 235
240Val Met Val Gly6281PRTHomo sapiens 6Met Gln Gln Pro Phe Asn Tyr
Pro Tyr Pro Gln Ile Tyr Trp Val Asp1 5 10 15Ser Ser Ala Ser Ser Pro
Trp Ala Pro Pro Gly Thr Val Leu Pro Cys 20 25 30Pro Thr Ser Val Pro
Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45Pro Pro Pro Pro
Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro 50 55 60Pro Leu Pro
Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly65 70 75 80Leu
Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly 85 90
95Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser
Leu Glu 115 120 125Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys
Lys Glu Leu Arg 130 135 140Lys Val Ala His Leu Thr Gly Lys Ser Asn
Ser Arg Ser Met Pro Leu145 150 155 160Glu Trp Glu Asp Thr Tyr Gly
Ile Val Leu Leu Ser Gly Val Lys Tyr 165 170 175Lys Lys Gly Gly Leu
Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr 180 185 190Ser Lys Val
Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser 195 200 205His
Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met 210 215
220Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp
Ala225 230 235 240Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr
Ser Ala Asp His 245 250 255Leu Tyr Val Asn Val Ser Glu Leu Ser Leu
Val Asn Phe Glu Glu Ser 260 265 270Gln Thr Phe Phe Gly Leu Tyr Lys
Leu 275 2807338DNAHomo sapiensDescription of Combined DNA/RNA
Molecule n equals a, t, g, or c 7aggntaactc tcctgagggg tgagccaagc
cctgccatgt agtgcacgca ggacatcanc 60aaacacannn nncaggaaat aatccattcc
ctgtggtcac ttattctaaa ggccccaacc 120ttcaaagttc aagtagtgat
atggatgact ccacagaaag ggagcagtca cgccttactt 180cttgccttaa
gaaaagagaa gaaatgaaac tgnaaggagt gtgtttccat cctcccacgg
240aaggaaagcc cctctntccg atcctccaaa gacggaaagc tgctggctgc
aaccttgntg 300ntggcattgt gttcttgctg nctcaaggtg gtgttntt
3388509DNAHomo sapiensDescription of Combined DNA/RNA Molecule n
equals a, t, g, or c 8aattcggcan agnaaactgg ttactttttt atatatggtc
aggttttata tactgataag 60acctacgcca tgggacatct agttcagagg aagaaggtcc
atgtctttgg ggatgaattg 120agtctggtga ctttgtttcg atgtattcaa
aatatgcctg aaacactacc caataattcc 180tgctattcag ctggcattgc
aaaactggna ggaaggagat gaactccaac ttgcaatacc 240aggggaaaat
gcacaattat cactgggatg gagatgttca cattttttgg gtgccattga
300aactgctgtg acctncttac ancangtgct gttngctatt ttncctncct
nttctntggt 360aacctcttag gaaggaagga ttcttaactg ggaaataacc
caaaaaaann ttaaangggt 420angngnnana ngnggggnng ttnncnngnn
gnnttttngg nntatnttnt nntngggnnn 480ngtaaaaatg gggccnangg gggnttttt
5099497DNAHomo sapiensmisc_feature(168)n equals a, t, g, or c
9aattcggcac gagcaaggcc ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa
60tctttgaacc accagctcca ggagaaggca actccagtca gaacagcaga aataagcgtg
120ccgttcaggg tccagaagaa acagtcactc aagactgctt gcaactgntt
gcagacagtg 180aaacaccaac tatacaaaaa ggctcccttc tgntgccaca
tttgggccaa ggaatggaga 240gatttcttcg tctggaaaca ttttgccaaa
ctcttcagat actctttnct ctctgggaat 300caaaggaaaa tctctactta
gattnacaca tttgttccca tgggtntctt aagttttaaa 360aggggagtgc
ccttaggagg aaaaggggat aaatattggc caaggnactg gttantttnt
420aaatatggtc aggtttntat anctggtagg cctcgccatg ggcattnatt
canggngagg 480ncnntctttt gggntga 4971027DNAHomo
sapiensNeutrokine-alpha forward primer containing BamHI restriction
site 10gtgggatcca gcctccgggc agagctg 271133DNAHomo
sapiensNeutrokine-alpha reverse primer containing HindIII
restriction site and sequence complementary to two stop codons
11gtgaagcttt tattacagca gtttcaatgc acc 331226DNAHomo
sapiensNeutrokine-alpha forward primer containing BspHI restriction
site 12gtgtcatgag cctccgggca gagctg 261333DNAHomo
sapiensNeutrokine-alpha reverse primer containing HindIII
restriction site and sequence complementary to two stop codons
13gtgaagcttt tattacagca gtttcaatgc acc 331428DNAHomo
sapiensNeutrokine-alpha forward primer containing BamHI restriction
site 14gtgggatccc cgggcagagc tgcagggc 281533DNAHomo
sapiensNeutrokine-alpha reverse primer containing BamHI restriction
site and sequence complementary to two stop codons 15gtgggatcct
tattacagca gtttcaatgc acc 3316129DNAHomo sapiensNeutrokine-alpha
forward primer containing a Bam HI restriction site, Kozak
sequence, AUG start codon, and sequence encoding the secretory
leader peptide of human IL-6 gene 16gcgggatccg ccaccatgaa
ctccttctcc acaagcgcct tcggtccagt tgccttctcc 60ctggggctgc tcctggtgtt
gcctgctgcc ttccctgccc cagttgtgag acaaggggac 120ctggccagc
1291730DNAHomo sapiensNeutrokine-alpha reverse primerconating BamHI
restriction site 17gtgggatcct tacagcagtt tcaatgcacc 3018903DNAHomo
sapiensCDS(1)..(798) 18atg gat gac tcc aca gaa agg gag cag tca cgc
ctt act tct tgc ctt 48Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg
Leu Thr Ser Cys Leu1 5 10 15aag aaa aga gaa gaa atg aaa ctg aag gag
tgt gtt tcc atc ctc cca 96Lys Lys Arg Glu Glu Met Lys Leu Lys Glu
Cys Val Ser Ile Leu Pro 20 25 30cgg aag gaa agc ccc tct gtc cga tcc
tcc aaa gac gga aag ctg ctg 144Arg Lys Glu Ser Pro Ser Val Arg Ser
Ser Lys Asp Gly Lys Leu Leu 35 40 45gct gca acc ttg ctg ctg gca ctg
ctg tct tgc tgc ctc acg gtg gtg 192Ala Ala Thr Leu Leu Leu Ala Leu
Leu Ser Cys Cys Leu Thr Val Val 50 55 60tct ttc tac cag gtg gcc gcc
ctg caa ggg gac ctg gcc agc ctc cgg 240Ser Phe Tyr Gln Val Ala Ala
Leu Gln Gly Asp Leu Ala Ser Leu Arg65 70 75 80gca gag ctg cag ggc
cac cac gcg gag aag ctg cca gca gga gca gga 288Ala Glu Leu Gln Gly
His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95gcc ccc aag gcc
ggc ctg gag gaa gct cca gct gtc acc gcg gga ctg 336Ala Pro Lys Ala
Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105
110aaa atc ttt gaa cca cca gct cca gga gaa ggc aac tcc agt cag aac
384Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn
115 120 125agc aga aat aag cgt gcc gtt cag ggt cca gaa gaa aca gga
tct tac 432Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly
Ser Tyr 130 135 140aca ttt gtt cca tgg ctt ctc agc ttt aaa agg gga
agt gcc cta gaa 480Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly
Ser Ala Leu Glu145 150 155 160gaa aaa gag aat aaa ata ttg gtc aaa
gaa act ggt tac ttt ttt ata 528Glu Lys Glu Asn Lys Ile Leu Val Lys
Glu Thr Gly Tyr Phe Phe Ile 165 170 175tat ggt cag gtt tta tat act
gat aag acc tac gcc atg gga cat cta 576Tyr Gly Gln Val Leu Tyr Thr
Asp Lys Thr Tyr Ala Met Gly His Leu 180 185 190att cag agg aag aag
gtc cat gtc ttt ggg gat gaa ttg agt ctg gtg 624Ile Gln Arg Lys Lys
Val His Val Phe Gly Asp Glu Leu Ser Leu Val 195 200 205act ttg ttt
cga tgt att caa aat atg cct gaa aca cta ccc aat aat 672Thr Leu Phe
Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn 210 215 220tcc
tgc tat tca gct ggc att gca aaa ctg gaa gaa gga gat gaa ctc 720Ser
Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu225 230
235 240caa ctt gca ata cca aga gaa aat gca caa ata tca ctg gat gga
gat 768Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly
Asp245 250 255gtc aca ttt ttt ggt gca ttg aaa ctg ctg tgacctactt
acaccatgtc 818Val Thr Phe Phe Gly Ala Leu Lys Leu Leu260
265tgtagctatt ttcctccctt tctctgtacc tctaagaaga aagaatctaa
ctgaaaatac 878caaaaaaaaa aaaaaaaaaa aaaaa 90319266PRTHomo sapiens
19Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu1
5 10 15Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu
Pro 20 25 30Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys
Leu Leu 35 40 45Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu
Thr Val Val 50 55 60Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu
Ala Ser Leu Arg65 70 75 80Ala Glu Leu Gln Gly His His Ala Glu Lys
Leu Pro Ala Gly Ala Gly 85 90 95Ala Pro Lys Ala Gly Leu Glu Glu Ala
Pro Ala Val Thr Ala Gly Leu 100 105 110Lys Ile Phe Glu Pro Pro Ala
Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125Ser Arg Asn Lys Arg
Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr 130 135 140Thr Phe Val
Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu145 150 155
160Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile
165 170 175Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly
His Leu 180 185 190Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu
Leu Ser Leu Val 195 200 205Thr Leu Phe Arg Cys Ile Gln Asn Met Pro
Glu Thr Leu Pro Asn Asn 210 215 220Ser Cys Tyr Ser Ala Gly Ile Ala
Lys Leu Glu Glu Gly Asp Glu Leu225 230 235 240Gln Leu Ala Ile Pro
Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp 245 250 255Val Thr Phe
Phe Gly Ala Leu Lys Leu Leu 260 26520136PRTHomo sapiens 20His Ser
Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp1 5 10 15Ser
Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg 20 25
30Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val
35 40 45Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr
Met 50 55 60Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr
Leu Phe65 70 75 80Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg
Ala Tyr Asn Ser 85 90 95Cys Tyr Ser Ala Gly Val Phe His Leu His Gln
Gly Asp Ile Leu Ser 100 105 110Val Ile Ile Pro Arg Ala Arg Ala Lys
Leu Asn Leu Ser Pro His Gly 115 120 125Thr Phe Leu Gly Phe Val Lys
Leu 130 13521462DNAHomo sapiens 21atggctgttc agggtccgga agaaaccgtt
actcaggact gccttcagct gatcgcagac 60tctgaaactc cgaccatcca gaaaggttct
tacacctttg ttccttggct gctttctttc 120aaacgtggtt ctgccctgga
agagaaagaa aacaaaatcc tggttaaaga aactggttac 180ttctttatct
acggtcaggt tctttacact gataagacct acgccatggg tcacctgatt
240cagcgtaaga aagttcacgt tttcggtgac gagctgtctc tggttactct
gtttcgctgc 300attcagaaca tgccggaaac tcttcctaac aactcctgct
actctgctgg catcgcaaaa 360ctggaagagg gtgatgaact gcagctggca
attcctcgtg aaaacgcaca aatttctctg 420gacggtgatg taaccttctt
tggtgcactg aaacttctgt aa 462221040DNAMus musculusCDS(1)..(468)
22cgc gtg gta gac ctc tca gct cct cct gca cca tgc ctg cct gga tgc
48Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys1
5 10 15cgc cat tct caa cat gat gat aat gga atg aac ctc aga aac aga
act 96Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg
Thr 20 25 30tac aca ttt gtt cca tgg ctt ctc agc ttt aaa aga gga aat
gcc ttg 144Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn
Ala Leu 35 40 45gag gag aaa gag aac aaa ata gtg gtg agg caa aca ggc
tat ttc ttc 192Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly
Tyr Phe Phe 50 55 60atc tac agc cag gtt cta tac acg gac ccc atc ttt
gct atg ggt cat 240Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe
Ala Met Gly His65 70 75 80gtc atc cag agg aag aaa gta cac gtc ttt
ggg gac gag ctg agc ctg 288Val Ile Gln Arg Lys Lys Val His Val Phe
Gly Asp Glu Leu Ser Leu 85 90 95gtg acc ctg ttc cga tgt att cag aat
atg ccc aaa aca ctg ccc aac 336Val Thr Leu Phe Arg Cys Ile Gln Asn
Met Pro Lys Thr Leu Pro Asn 100 105 110aat tcc tgc tac tcg gct ggc
atc gcg agg ctg gaa gaa gga gat gag 384Asn Ser Cys Tyr Ser Ala Gly
Ile Ala Arg Leu Glu Glu Gly Asp Glu 115 120 125att cag ctt gca att
cct cgg gag aat gca cag att tca cgc aac gga 432Ile Gln Leu Ala Ile
Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly 130 135 140gac gac acc
ttc ttt ggt gcc cta aaa ctg ctg taa ctcacttgct 478Asp Asp Thr Phe
Phe Gly Ala Leu Lys Leu Leu145 150 155ggagtgcgtg atccccttcc
ctcgtcttct ctgtacctcc gagggagaaa cagacgactg 538gaaaaactaa
aagatgggga aagccgtcag cgaaagtttt ctcgtgaccc gttgaatctg
598atccaaacca ggaaatataa cagacagcca caaccgaagt gtgccatgtg
agttatgaga 658aacggagccc gcgctcagaa agaccggatg aggaagaccg
ttttctccag tcctttgcca 718acacgcaccg caaccttgct ttttgccttg
ggtgacacat gttcagaatg cagggagatt 778tccttgtttt gcgatttgcc
atgagaagag ggcccacaac tgcaggtcac tgaagcattc 838acgctaagtc
tcaggattta ctctcccttc tcatgctaag tacacacacg ctcttttcca
898ggtaatacta tgggatacta tggaaaggtt gtttgttttt aaatctagaa
gtcttgaact 958ggcaatagac aaaaatcctt ataaattcaa gtgtaaaata
aacttaatta aaaaggttta 1018agtgtgaaaa aaaaaaaaaa aa 104023155PRTMus
musculus 23Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro
Gly Cys1 5 10 15Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg
Asn Arg Thr 20 25 30Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg
Gly Asn Ala Leu 35 40 45Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln
Thr Gly Tyr Phe Phe 50 55 60Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro
Ile Phe Ala Met Gly His65 70 75 80Val Ile Gln Arg Lys Lys Val His
Val Phe Gly Asp Glu Leu Ser Leu 85 90 95Val Thr Leu Phe Arg Cys Ile
Gln Asn Met Pro Lys Thr Leu Pro Asn 100 105 110Asn Ser Cys Tyr Ser
Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu 115 120 125Ile Gln Leu
Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly 130 135 140Asp
Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu145 150 1552426DNAHomo
sapiens 24ccaccagctc caggagaagg caactc 262519DNAHomo sapiens
25accgcgggac tgaaaatct 192623DNAHomo sapiens 26cacgcttatt
tctgctgttc tga 2327657DNAMacaca irus 27taccaggtgg cggccgtgca
aggggacctg gccagcctcc gggcagagct gcagggccac 60cacgcggaga agctgccagc
aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120gctgtcaccg
caggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt
180cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat
tcaagactgc 240ttgcaactga ttgcagacag tgaaacacca actatacaaa
aaggatctta cacatttgtt 300ccatggcttc tcagctttaa aaggggaagt
gccctagaag aaaaagagaa taaaatattg 360gtcaaagaaa ctggttactt
ttttatatat ggtcaggttt tatacactga taagacctat 420gccatgggac
atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg
480gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa
ttcctgctat 540tcagctggca ttgcaaaact ggaagaagga gatgaacttc
aacttgcaat accacgagaa 600aatgcacaaa tatcactgga tggagatgtc
acattttttg gtgccctcaa actgctg 65728219PRTMacaca irus 28Tyr Gln Val
Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu1 5 10 15Leu Gln
Gly His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro 20 25 30Lys
Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile 35 40
45Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg
50 55 60Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp
Cys65 70 75 80Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln
Lys Gly Ser 85 90 95Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg
Gly Ser Ala Leu 100 105 110Glu Glu Lys Glu Asn Lys Ile Leu Val Lys
Glu Thr Gly Tyr Phe Phe 115 120 125Ile Tyr Gly Gln Val Leu Tyr Thr
Asp Lys Thr Tyr Ala Met Gly His 130 135 140Leu Ile Gln Arg Lys Lys
Val His Val Phe Gly Asp Glu Leu Ser Leu145 150 155 160Val Thr Leu
Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn 165 170 175Asn
Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu 180 185
190Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly
195 200 205Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 210
21529657DNAMacaca mulatta (Rhesus Monkey) 29taccaggtgg cggccgtgca
aggggacctg gccagcctcc gggcagagct gcagagccac 60cacgcggaga agctgccagc
aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120gctgtcaccg
cgggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt
180cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat
tcaagactgc 240ttgcaactga ttgcagacag tgaaacacca actatacaaa
aaggatctta cacatttgtt 300ccatggcttc tcagctttaa aaggggaagt
gccctagaag aaaaagagaa taaaatattg 360gtcaaagaaa ctggttactt
ttttatatat ggtcaggttt tatacactga taagacctat 420gccatgggac
atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg
480gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa
ttcctgctat 540tcagctggca ttgcaaaact ggaagaaggg gatgaacttc
aacttgcaat accacgagaa 600aatgcacaaa tatcactgga tggagatgtc
acattttttg gtgccctcaa actgctg 65730219PRTMacaca mulatta (Rhesus
Monkey) 30Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg
Ala Glu1 5 10 15Leu Gln Ser His His Ala Glu Lys Leu Pro Ala Arg Ala
Arg Ala Pro 20 25 30Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala
Gly Leu Lys Ile 35 40 45Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser
Ser Gln Ser Ser Arg 50 55 60Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu
Thr Val Ile Gln Asp Cys65 70 75 80Leu Gln Leu Ile Ala Asp Ser Glu
Thr Pro Thr Ile Gln Lys Gly Ser 85 90 95Tyr Thr Phe Val Pro Trp Leu
Leu Ser Phe Lys Arg Gly Ser Ala Leu 100 105 110Glu Glu Lys Glu Asn
Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe 115 120 125Ile Tyr Gly
Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His 130 135 140Leu
Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu145 150
155 160Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro
Asn 165 170 175Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu
Gly Asp Glu 180 185 190Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln
Ile Ser Leu Asp Gly 195 200 205Asp Val Thr Phe Phe Gly Ala Leu Lys
Leu Leu 210 2153138DNAHomo sapiensNeutrokine-alpha forward primer
containing sequence encoding PSC signal peptide C-terminus
31ggtcgccgtt tctaacgcgg ccgttcaggg tccagaag 383249DNAHomo
sapiensReverse primer for amplifying Neutrokine-alpha containing
reverse complement sequence of the pA2GP vectorand Kpn I
restriction site 32ctggttcggc ccaaggtacc aagcttgtac cttagatctt
ttctagatc 493321DNAHomo sapiensforward primer that anneals to PSC
baculovirus transfer plasmid pMGS12 33ctggtagttc ttcggagtgt g
213419DNAHomo sapiensreverse primer that anneals to PSC baculovirus
transfer plasmid pMGS12 34cgcgttagaa acggcgacc 193522DNAHomo
sapiensmisc_feature(7)n equals deoxyinosine or dideoxyinosine
35taccagntgg cngccntgca ag 223622DNAHomo sapiensmisc_feature(3)n
equals deoxyinosine or dideoxyinosine 36gtnacagcag tttnanngca cc
2237867DNAMus musculus 37atggatgagt ctgcaaagac cctgccacca
ccgtgcctct gtttttgctc cgagaaagga 60gaagatatga aagtgggata tgatcccatc
actccgcaga aggaggaggg tgcctggttt 120gggatctgca gggatggaag
gctgctggct gctaccctcc tgctggccct gttgtccagc 180agtttcacag
cgatgtcctt gtaccagttg gctgccttgc aagcagacct gatgaacctg
240cgcatggagc tgcagagcta ccgaggttca gcaacaccag ccgccgcggg
tgctccagag 300ttgaccgctg gagtcaaact cctgacaccg gcagctcctc
gaccccacaa ctccagccgc 360ggccacagga acagacgcgc cttccaggga
ccagaggaaa cagaacaaga tgtagacctc 420tcagctcctc ctgcaccatg
cctgcctgga tgccgccatt ctcaacatga tgataatgga 480atgaacctca
gaaacatcat tcaagactgt ctgcagctga ttgcagacag cgacacgccg
540gccttggagg agaaagagaa caaaatagtg gtgaggcaaa caggctattt
cttcatctac 600agccaggttc tatacacgga ccccatcttt gctatgggtc
atgtcatcca gaggaagaaa 660gtacacgtct ttggggacga gctgagcctg
gtgaccctgt tccgatgtat tcagaatatg 720cccaaaacac tgcccaacaa
ttcctgctac tcggctggca tcgcgaggct ggaagaagga 780gatgagattc
agcttgcaat tcctcgggag aatgcacaga tttcacgcaa cggagacgac
840accttctttg gtgccctaaa actgctg 86738289PRTMus musculus 38Met Asp
Glu Ser Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys1 5 10 15Ser
Glu Lys Gly Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25
30Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu
35 40 45Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr
Ala 50 55 60Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu Met
Asn Leu65 70 75 80Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala Thr
Pro Ala Ala Ala 85 90 95Gly Ala Pro Glu Leu Thr Ala Gly Val Lys Leu
Leu Thr Pro Ala Ala 100 105 110Pro Arg Pro His Asn Ser Ser Arg Gly
His Arg Asn Arg Arg Ala Phe 115 120 125Gln Gly Pro Glu Glu Thr Glu
Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140Ala Pro Cys Leu Pro
Gly Cys Arg His Ser Gln His Asp Asp Asn Gly145 150 155 160Met Asn
Leu Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp 165 170
175Ser Asp Thr Pro Ala Leu Glu Glu Lys Glu Asn Lys Ile Val Val Arg
180 185
190Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro
195 200 205Ile Phe Ala Met Gly His Val Ile Gln Arg Lys Lys Val His
Val Phe 210 215 220Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys
Ile Gln Asn Met225 230 235 240Pro Lys Thr Leu Pro Asn Asn Ser Cys
Tyr Ser Ala Gly Ile Ala Arg 245 250 255Leu Glu Glu Gly Asp Glu Ile
Gln Leu Ala Ile Pro Arg Glu Asn Ala 260 265 270Gln Ile Ser Arg Asn
Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu 275 280 285Leu
39309PRTMus musculus 39Met Asp Glu Ser Ala Lys Thr Leu Pro Pro Pro
Cys Leu Cys Phe Cys1 5 10 15Ser Glu Lys Gly Glu Asp Met Lys Val Gly
Tyr Asp Pro Ile Thr Pro 20 25 30Gln Lys Glu Glu Gly Ala Trp Phe Gly
Ile Cys Arg Asp Gly Arg Leu 35 40 45Leu Ala Ala Thr Leu Leu Leu Ala
Leu Leu Ser Ser Ser Phe Thr Ala 50 55 60Met Ser Leu Tyr Gln Leu Ala
Ala Leu Gln Ala Asp Leu Met Asn Leu65 70 75 80Arg Met Glu Leu Gln
Ser Tyr Arg Gly Ser Ala Thr Pro Ala Ala Ala 85 90 95Gly Ala Pro Glu
Leu Thr Ala Gly Val Lys Leu Leu Thr Pro Ala Ala 100 105 110Pro Arg
Pro His Asn Ser Ser Arg Gly His Arg Asn Arg Arg Ala Phe 115 120
125Gln Gly Pro Glu Glu Thr Glu Gln Asp Val Asp Leu Ser Ala Pro Pro
130 135 140Ala Pro Cys Leu Pro Gly Cys Arg His Ser Gln His Asp Asp
Asn Gly145 150 155 160Met Asn Leu Arg Asn Ile Ile Gln Asp Cys Leu
Gln Leu Ile Ala Asp 165 170 175Ser Asp Thr Pro Thr Ile Arg Lys Gly
Thr Tyr Thr Phe Val Pro Trp 180 185 190Leu Leu Ser Phe Lys Arg Gly
Asn Ala Leu Glu Glu Lys Glu Asn Lys 195 200 205Ile Val Val Arg Gln
Thr Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu 210 215 220Tyr Thr Asp
Pro Ile Phe Ala Met Gly His Val Ile Gln Arg Lys Lys225 230 235
240Val His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys
245 250 255Ile Gln Asn Met Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr
Ser Ala 260 265 270Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu Ile Gln
Leu Ala Ile Pro 275 280 285Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly
Asp Asp Thr Phe Phe Gly 290 295 300Ala Leu Lys Leu
Leu30540290PRTMus musculus 40Met Asp Glu Ser Ala Lys Thr Leu Pro
Pro Pro Cys Leu Cys Phe Cys1 5 10 15Ser Glu Lys Gly Glu Asp Met Lys
Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30Gln Lys Glu Glu Gly Ala Trp
Phe Gly Ile Cys Arg Asp Gly Arg Leu 35 40 45Leu Ala Ala Thr Leu Leu
Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala 50 55 60Met Ser Leu Tyr Gln
Leu Ala Ala Leu Gln Ala Asp Leu Met Asn Leu65 70 75 80Arg Met Glu
Leu Gln Ser Tyr Arg Gly Ser Ala Thr Pro Ala Ala Ala 85 90 95Gly Ala
Pro Glu Leu Thr Ala Gly Val Lys Leu Leu Thr Pro Ala Ala 100 105
110Pro Arg Pro His Asn Ser Ser Arg Gly His Arg Asn Arg Arg Ala Phe
115 120 125Gln Gly Pro Glu Glu Thr Glu Gln Asp Val Asp Leu Ser Ala
Pro Pro 130 135 140Ala Pro Cys Leu Pro Gly Cys Arg His Ser Gln His
Asp Asp Asn Gly145 150 155 160Met Asn Leu Arg Asn Arg Thr Tyr Thr
Phe Val Pro Trp Leu Leu Ser 165 170 175Phe Lys Arg Gly Asn Ala Leu
Glu Glu Lys Glu Asn Lys Ile Val Val 180 185 190Arg Gln Thr Gly Tyr
Phe Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp 195 200 205Pro Ile Phe
Ala Met Gly His Val Ile Gln Arg Lys Lys Val His Val 210 215 220Phe
Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn225 230
235 240Met Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile
Ala 245 250 255Arg Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro
Arg Glu Asn 260 265 270Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe
Phe Gly Ala Leu Lys 275 280 285Leu Leu 29041152PRTRattus sp 41Ala
Phe Gln Gly Pro Glu Glu Thr Val Ile Gln Asp Cys Leu Gln Leu1 5 10
15Ile Ala Asp Ser Asn Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe
20 25 30Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu Glu
Lys 35 40 45Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe Ile
Tyr Ser 50 55 60Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His
Val Ile Gln65 70 75 80Arg Lys Lys Ile His Val Phe Gly Asp Glu Leu
Ser Leu Val Thr Leu 85 90 95Phe Arg Cys Ile Gln Asn Met Pro Lys Thr
Leu Pro Asn Asn Ser Cys 100 105 110Tyr Ser Ala Gly Ile Ala Lys Leu
Glu Glu Gly Asp Glu Val Gln Leu 115 120 125Ala Ile Pro Arg Glu Asn
Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr 130 135 140Phe Phe Gly Ala
Leu Lys Leu Leu145 15042165PRTRattus sp 42Ala Phe Gln Gly Pro Glu
Glu Thr Glu Gln Asp Val Asp Leu Ser Ala1 5 10 15Thr Pro Val Pro Ser
Leu Pro Gly Asn Cys His Ala Ser His His Asp 20 25 30Glu Asn Gly Leu
Asn Leu Arg Thr Arg Thr Tyr Thr Phe Val Pro Trp 35 40 45Leu Leu Ser
Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys 50 55 60Ile Val
Val Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu65 70 75
80Tyr Thr Asp Pro Ile Phe Ala Met Gly His Val Ile Gln Arg Lys Lys
85 90 95Ile His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg
Cys 100 105 110Ile Gln Asn Met Pro Lys Thr Leu Pro Asn Asn Ser Cys
Tyr Ser Ala 115 120 125Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Ile
Gln Leu Ala Ile Pro 130 135 140Arg Glu Asn Ala Gln Ile Ser Arg Asn
Gly Asp Asp Thr Phe Phe Gly145 150 155 160Ala Leu Lys Leu Leu
16543184PRTRattus sp 43Ala Phe Gln Gly Pro Glu Glu Thr Glu Gln Asp
Val Asp Leu Ser Ala1 5 10 15Thr Pro Ala Pro Ser Leu Pro Gly Asn Cys
His Ala Ser His His Asp 20 25 30Glu Asn Gly Leu Asn Leu Arg Thr Ile
Ile Gln Asp Cys Leu Gln Leu 35 40 45Ile Ala Asp Ser Asn Thr Pro Thr
Ile Arg Lys Gly Thr Tyr Thr Phe 50 55 60Val Pro Trp Leu Leu Ser Phe
Lys Arg Gly Asn Ala Leu Glu Glu Lys65 70 75 80Glu Asn Lys Ile Val
Val Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser 85 90 95Gln Val Leu Tyr
Thr Asp Pro Ile Phe Ala Met Gly His Val Ile Gln 100 105 110Arg Lys
Lys Ile His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu 115 120
125Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn Asn Ser Cys
130 135 140Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Ile
Gln Leu145 150 155 160Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg
Asn Gly Asp Asp Thr 165 170 175Phe Phe Gly Ala Leu Lys Leu Leu
18044133PRTRattus sp 44Ala Phe Gln Gly Pro Glu Glu Thr Gly Thr Tyr
Thr Phe Val Pro Trp1 5 10 15Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu
Glu Glu Lys Glu Asn Lys 20 25 30Ile Val Val Arg Gln Thr Gly Tyr Phe
Phe Ile Tyr Ser Gln Val Leu 35 40 45Tyr Thr Asp Pro Ile Phe Ala Met
Gly His Val Ile Gln Arg Lys Lys 50 55 60Ile His Val Phe Gly Asp Glu
Leu Ser Leu Val Thr Leu Phe Arg Cys65 70 75 80Ile Gln Asn Met Pro
Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala 85 90 95Gly Ile Ala Lys
Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro 100 105 110Arg Glu
Asn Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly 115 120
125Ala Leu Lys Leu Leu 1304517PRTHomo sapiens 45Met Leu Gln Asn Ser
Ala Val Leu Leu Leu Leu Val Ile Ser Ala Ser1 5 10
15Ala4622PRTArtificial SequenceSIGNAL(1)..(22)consensus signal
sequence 46Met Pro Thr Trp Ala Trp Trp Leu Phe Leu Val Leu Leu Leu
Ala Leu1 5 10 15Trp Ala Pro Ala Arg Gly 2047250PRTHomo sapiens
47Met Pro Ala Ser Ser Pro Phe Leu Leu Ala Pro Lys Gly Pro Pro Gly1
5 10 15Asn Met Gly Gly Pro Val Arg Glu Pro Ala Leu Ser Val Ala Leu
Trp 20 25 30Leu Ser Trp Gly Ala Ala Leu Gly Ala Val Ala Cys Ala Met
Ala Leu 35 40 45Leu Thr Gln Gln Thr Glu Leu Gln Ser Leu Arg Arg Glu
Val Ser Arg 50 55 60Leu Gln Gly Thr Gly Gly Pro Ser Gln Asn Gly Glu
Gly Tyr Pro Trp65 70 75 80Gln Ser Leu Pro Glu Gln Ser Ser Asp Ala
Leu Glu Ala Trp Glu Asn 85 90 95Gly Glu Arg Ser Arg Lys Arg Arg Ala
Val Leu Thr Gln Lys Gln Lys 100 105 110Lys Gln His Ser Val Leu His
Leu Val Pro Ile Asn Ala Thr Ser Lys 115 120 125Asp Asp Ser Asp Val
Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg 130 135 140Gly Arg Gly
Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala145 150 155
160Gly Val Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe
165 170 175Thr Met Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln
Glu Thr 180 185 190Leu Phe Arg Cys Ile Arg Ser Met Pro Ser His Pro
Asp Arg Ala Tyr 195 200 205Asn Ser Cys Tyr Ser Ala Gly Val Phe His
Leu His Gln Gly Asp Ile 210 215 220Leu Ser Val Ile Ile Pro Arg Ala
Arg Ala Lys Leu Asn Leu Ser Pro225 230 235 240His Gly Thr Phe Leu
Gly Phe Val Lys Leu 245 2504838DNAArtificial
SequenceOligonucleotide primer 48cagactggat ccgccaccat ggatgactcc
acagaaag 384933DNAArtificial SequenceOligonucleotide Primer
49cagactggta ccgtcctgcg tgcactacat ggc 335021DNAArtificial
SequenceOligonucleotide Primer 50tggtgtcttt ctaccaggtg g
215121DNAArtificial SequenceOligonucleotide primer 51tttcttctgg
accctgaacg g 21524458DNAArtificialpML124 52gaattcaact tctccatact
ttggataagg aaatacagac atgaaaaatc tcattgctga 60gttgttattt aagcttgccc
aaaaagaaga agagtcgaat gaactgtgtg cgcaggtaga 120agctttggag
attatcgtca ctgcaatgct tcgcaatatg gcgcaaaatg accaacagcg
180gttgattgat caggtagagg gggcgctgta cgaggtaaag cccgatgcca
gcattcctga 240cgacgatacg gagctgctgc gcgattacgt aaagaagtta
ttgaagcatc ctcgtcagta 300aaaagttaat cttttcaaca gctgtcataa
agttgtcacg gccgagactt atagtcgctt 360tgtttttatt ttttaatgta
tttgtaacta gtacgcaagt tcacgtaaag gaagtatctc 420atatgcagat
cttcgtgtaa accggtaccg gcaaaaccat cactctagaa gtggatcctc
480tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
tggcgcctat 540atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
tcgggctcat gagcgcttgt 600ttcggcgtgg gtatggtggc aggccccgtg
gccgggggac tgttgggcgc catctccttg 660catgcaccat tccttgcggc
ggcggtgctc aacggcctca acctactact gggctgcttc 720ctaatgcagg
agtcgcataa gggagagcgt cgaccgatgc ccttgagagc cttcaaccca
780gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
gactgtcttc 840tttatcatgc aactcgtagg acaggtgccg gcagcgctct
gggtcatttt cggcgaggac 900cgctttcgct ggagcgcgac gatgatcggc
ctgtcgcttg cggtattcgg aatcttgcac 960gccctcgctc aagccttcgt
cactggtccc gccaccaaac gtttcggcga gaagcaggcc 1020attatcgccg
gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt cgcgacgcga
1080ggctggatgg ccttccccat tatgattctt ctcgcttccg gcggcatcgg
gatgcccgcg 1140ttgcaggcca tgctgtccag gcaggtagat gacgaccatc
agggacagct tcaaggatcg 1200ctcgcggctc ttaccagcct aacttcgatc
actggaccgc tgatcgtcac ggcgatttat 1260gccgcctcgg cgagcacatg
gaacgggttg gcatggattg taggcgccgc cctatacctt 1320gtctgcctcc
ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa
1380gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat
caattcttgc 1440ggagaactgt gaatgcgcaa accaaccctt ggcagaacat
atccatcgcg tccgccatct 1500ccagcagccg cacgcggcgc atctcgggca
gcgttgggtc ctggccacgg gtgcgcatga 1560tcgtgctcct gtcgttgagg
acccggctag gctggcgggg ttgccttact ggttagcaga 1620atgaatcacc
gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
1680gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
acgcggaagt 1740cagcgccctg caccattatg ttccggatct gcatcgcagg
atgctgctgg ctaccctgtg 1800gaacacctac atctgtatta acgaagcgct
ggcattgacc ctgagtgatt tttctctggt 1860cccgccgcat ccataccgcc
agttgtttac cctcacaacg ttccagtaac cgggcatgtt 1920catcatcagt
aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
1980tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
accgccctta 2040acatggcccg ctttatcaga agccagacat taacgcttct
ggagaaactc aacgagctgg 2100acgcggatga acaggcagac atctgtgaat
cgcttcacga ccacgctgat gagctttacc 2160gcagctgcct cgcgcgtttc
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 2220agacggtcac
agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
2280cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
gatagcggag 2340tgtatactgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc accatatatg 2400cggtgtgaaa taccgcacag atgcgtaagg
agaaaatacc gcatcaggcg ctcttccgct 2460tcctcgctca ctgactcgct
gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 2520tcaaaggcgg
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
2580gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
gtttttccat 2640aggctccgcc cccctgacga gcatcacaaa aatcgacgct
caagtcagag gtggcgaaac 2700ccgacaggac tataaagata ccaggcgttt
ccccctggaa gctccctcgt gcgctctcct 2760gttccgaccc tgccgcttac
cggatacctg tccgcctttc tcccttcggg aagcgtggcg 2820ctttctcata
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
2880ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
taactatcgt 2940cttgagtcca acccggtaag acacgactta tcgccactgg
cagcagccac tggtaacagg 3000attagcagag cgaggtatgt aggcggtgct
acagagttct tgaagtggtg gcctaactac 3060ggctacacta gaaggacagt
atttggtatc tgcgctctgc tgaagccagt taccttcgga 3120aaaagagttg
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
3180gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
tttgatcttt 3240tctacggggt ctgacgctca gtggaacgaa aactcacgtt
aagggatttt ggtcatgaga 3300ttatcaaaaa ggatcttcac ctagatcctt
ttaaattaaa aatgaagttt taaatcaatc 3360taaagtatat atgagtaaac
ttggtctgac agttaccaat gcttaatcag tgaggcacct 3420atctcagcga
tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
3480actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
gcgagaccca 3540cgctcaccgg ctccagattt atcagcaata aaccagccag
ccggaagggc cgagcgcaga 3600agtggtcctg caactttatc cgcctccatc
cagtctatta attgttgccg ggaagctaga 3660gtaagtagtt cgccagttaa
tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg 3720gtgtcacgct
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
3780gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
tccgatcgtt 3840gtcagaagta agttggccgc agtgttatca ctcatggtta
tggcagcact gcataattct 3900cttactgtca tgccatccgt aagatgcttt
tctgtgactg gtgagtactc aaccaagtca 3960ttctgagaat agtgtatgcg
gcgaccgagt tgctcttgcc cggcgtcaac acgggataat 4020accgcgccac
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
4080aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
tcgtgcaccc 4140aactgatctt cagcatcttt tactttcacc agcgtttctg
ggtgagcaaa aacaggaagg 4200caaaatgccg caaaaaaggg aataagggcg
acacggaaat gttgaatact catactcttc 4260ctttttcaat attattgaag
catttatcag ggttattgtc tcatgagcgg atacatattt 4320gaatgtattt
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
4380cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
gcgtatcacg 4440aggccctttc gtcttcaa 4458534980DNAArtificial
SequencepML124MBPss-BLyS 53gaattcaact tctccatact ttggataagg
aaatacagac atgaaaaatc tcattgctga
60gttgttattt aagcttgccc aaaaagaaga agagtcgaat gaactgtgtg cgcaggtaga
120agctttggag attatcgtca ctgcaatgct tcgcaatatg gcgcaaaatg
accaacagcg 180gttgattgat caggtagagg gggcgctgta cgaggtaaag
cccgatgcca gcattcctga 240cgacgatacg gagctgctgc gcgattacgt
aaagaagtta ttgaagcatc ctcgtcagta 300aaaagttaat cttttcaaca
gctgtcataa agttgtcacg gccgagactt atagtcgctt 360tgtttttatt
ttttaatgta tttgtaacta gtacgcaagt tcacgtaaag gaagtatctc
420atatgaaaat aaaaacaggt gcacgcatcc tcgcattatc cgcattaacg
acgatgatgt 480tttccgcctc ggctctcgcc gctgttcagg gtccggaaga
aaccgttact caggactgcc 540ttcagctgat cgcagactct gaaactccga
ccatccagaa aggttcttac acctttgttc 600cttggctgct ttctttcaaa
cgtggttctg ccctggaaga gaaagaaaac aaaatcctgg 660ttaaagaaac
tggttacttc tttatctacg gtcaggttct ttacactgat aagacctacg
720ccatgggtca cctgattcag cgtaagaaag ttcacgtttt cggtgacgag
ctgtctctgg 780ttactctgtt tcgctgcatt cagaacatgc cggaaactct
tcctaacaac tcctgctact 840ctgctggcat cgcaaaactg gaagagggtg
atgaactgca gctggcaatt cctcgtgaaa 900acgcacaaat ttctctggac
ggtgatgtaa ccttctttgg tgcactgaaa cttctgtaat 960aataaggtac
cggcaaaacc atcactctag aagtggatcc tctacgccgg acgcatcgtg
1020gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
catcaccgat 1080ggggaagatc gggctcgcca cttcgggctc atgagcgctt
gtttcggcgt gggtatggtg 1140gcaggccccg tggccggggg actgttgggc
gccatctcct tgcatgcacc attccttgcg 1200gcggcggtgc tcaacggcct
caacctacta ctgggctgct tcctaatgca ggagtcgcat 1260aagggagagc
gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
1320gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
gcaactcgta 1380ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
accgctttcg ctggagcgcg 1440acgatgatcg gcctgtcgct tgcggtattc
ggaatcttgc acgccctcgc tcaagccttc 1500gtcactggtc ccgccaccaa
acgtttcggc gagaagcagg ccattatcgc cggcatggcg 1560gccgacgcgc
tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc
1620attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc
catgctgtcc 1680aggcaggtag atgacgacca tcagggacag cttcaaggat
cgctcgcggc tcttaccagc 1740ctaacttcga tcactggacc gctgatcgtc
acggcgattt atgccgcctc ggcgagcaca 1800tggaacgggt tggcatggat
tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg 1860cgtcgcggtg
catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta
1920acggattcac cactccaaga attggagcca atcaattctt gcggagaact
gtgaatgcgc 1980aaaccaaccc ttggcagaac atatccatcg cgtccgccat
ctccagcagc cgcacgcggc 2040gcatctcggg cagcgttggg tcctggccac
gggtgcgcat gatcgtgctc ctgtcgttga 2100ggacccggct aggctggcgg
ggttgcctta ctggttagca gaatgaatca ccgatacgcg 2160agcgaacgtg
aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
2220tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
tgcaccatta 2280tgttccggat ctgcatcgca ggatgctgct ggctaccctg
tggaacacct acatctgtat 2340taacgaagcg ctggcattga ccctgagtga
tttttctctg gtcccgccgc atccataccg 2400ccagttgttt accctcacaa
cgttccagta accgggcatg ttcatcatca gtaacccgta 2460tcgtgagcat
cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
2520acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
cgctttatca 2580gaagccagac attaacgctt ctggagaaac tcaacgagct
ggacgcggat gaacaggcag 2640acatctgtga atcgcttcac gaccacgctg
atgagcttta ccgcagctgc ctcgcgcgtt 2700tcggtgatga cggtgaaaac
ctctgacaca tgcagctccc ggagacggtc acagcttgtc 2760tgtaagcgga
tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
2820gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
ggcttaacta 2880tgcggcatca gagcagattg tactgagagt gcaccatata
tgcggtgtga aataccgcac 2940agatgcgtaa ggagaaaata ccgcatcagg
cgctcttccg cttcctcgct cactgactcg 3000ctgcgctcgg tcgttcggct
gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 3060ttatccacag
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
3120gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
cccccctgac 3180gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
acccgacagg actataaaga 3240taccaggcgt ttccccctgg aagctccctc
gtgcgctctc ctgttccgac cctgccgctt 3300accggatacc tgtccgcctt
tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 3360tgtaggtatc
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
3420cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
caacccggta 3480agacacgact tatcgccact ggcagcagcc actggtaaca
ggattagcag agcgaggtat 3540gtaggcggtg ctacagagtt cttgaagtgg
tggcctaact acggctacac tagaaggaca 3600gtatttggta tctgcgctct
gctgaagcca gttaccttcg gaaaaagagt tggtagctct 3660tgatccggca
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
3720acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
gtctgacgct 3780cagtggaacg aaaactcacg ttaagggatt ttggtcatga
gattatcaaa aaggatcttc 3840acctagatcc ttttaaatta aaaatgaagt
tttaaatcaa tctaaagtat atatgagtaa 3900acttggtctg acagttacca
atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3960tttcgttcat
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
4020ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
ggctccagat 4080ttatcagcaa taaaccagcc agccggaagg gccgagcgca
gaagtggtcc tgcaacttta 4140tccgcctcca tccagtctat taattgttgc
cgggaagcta gagtaagtag ttcgccagtt 4200aatagtttgc gcaacgttgt
tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt 4260ggtatggctt
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
4320ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
taagttggcc 4380gcagtgttat cactcatggt tatggcagca ctgcataatt
ctcttactgt catgccatcc 4440gtaagatgct tttctgtgac tggtgagtac
tcaaccaagt cattctgaga atagtgtatg 4500cggcgaccga gttgctcttg
cccggcgtca acacgggata ataccgcgcc acatagcaga 4560actttaaaag
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
4620ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
ttcagcatct 4680tttactttca ccagcgtttc tgggtgagca aaaacaggaa
ggcaaaatgc cgcaaaaaag 4740ggaataaggg cgacacggaa atgttgaata
ctcatactct tcctttttca atattattga 4800agcatttatc agggttattg
tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4860aaacaaatag
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
4920attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
tcgtcttcaa 49805426PRTArtificial SequenceMBP signal sequence 54Met
Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr1 5 10
15Thr Met Met Phe Ser Ala Ser Ala Leu Ala 20 2555178PRTArtificial
SequenceMBPss-Neutrokine-alpha 55Met Lys Ile Lys Thr Gly Ala Arg
Ile Leu Ala Leu Ser Ala Leu Thr1 5 10 15Thr Met Met Phe Ser Ala Ser
Ala Leu Ala Ala Val Gln Gly Pro Glu 20 25 30Glu Thr Val Thr Gln Asp
Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr 35 40 45Pro Thr Ile Gln Lys
Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser 50 55 60Phe Lys Arg Gly
Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val65 70 75 80Lys Glu
Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp 85 90 95Lys
Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val His Val 100 105
110Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn
115 120 125Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly
Ile Ala 130 135 140Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile
Pro Arg Glu Asn145 150 155 160Ala Gln Ile Ser Leu Asp Gly Asp Val
Thr Phe Phe Gly Ala Leu Lys 165 170 175Leu Leu5621DNAArtificial
Sequencewild-type Shine Dalgarno box and its adjacent sequence
56tttgtacatg gagaaaataa a 215721DNAArtificial Sequenceoptimized
Shine Dalgarno box and its adjacent sequence 57cacgtaaagg
aagtatctca t 2158120PRTMus musculus 58Gln Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys
Arg Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30Thr Met His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Ile
Pro Arg Asn Thr Tyr Thr Thr Phe Asn Gln Lys Phe 50 55 60Lys Asn Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr65 70 75 80Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95Ala Arg His Tyr Gly Gly Gly Tyr Trp Phe Phe Asp Val Trp Gly Ala
100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 12059113PRTMus
musculus 59Glu Leu Val Met Thr Gln Thr Pro Ala Ser Leu Ala Val Ser
Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Gly Ser Glu Ser Val
Asp Ser Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu
Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp
Phe Thr Leu Thr Ile Tyr65 70 75 80Pro Val Glu Ala Asp Asp Val Ala
Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Asp Asp Pro Met Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg
* * * * *
References