U.S. patent application number 09/932613 was filed with the patent office on 2003-05-15 for binding polypeptides and methods based thereon.
Invention is credited to Beltzer, James P., Fleming, Tony J., Potter, M. Daniel, Potter, Marilou, Rosen, Craig A..
Application Number | 20030091565 09/932613 |
Document ID | / |
Family ID | 22850040 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030091565 |
Kind Code |
A1 |
Beltzer, James P. ; et
al. |
May 15, 2003 |
Binding polypeptides and methods based thereon
Abstract
Binding polypeptides that specifically bind BLyS protein or
BLyS-like polypeptides can be used in methods of the invention for
detecting, diagnosing, or prognosing a disease or disorder
associated with aberrant BLyS or BLyS receptor expression or
inappropriate function of BLyS or BLyS receptor, comprising BLyS
binding polypeptides or fragments or variants thereof, that
specifically bind to BLyS. The present invention further relates to
methods and compositions for preventing, treating or ameliorating a
disease or disorder associated with aberrant BLyS or BLyS receptor
expression or inappropriate BLyS function or BLyS receptor
function, comprising administering to an animal, preferably a
human, an effective amount of one or more BLyS binding polypeptides
or fragments or variants thereof, that specifically bind to
BLyS.
Inventors: |
Beltzer, James P.;
(Carlisle, MA) ; Potter, M. Daniel; (US) ;
Potter, Marilou; (Acton, MA) ; Fleming, Tony J.;
(Waltham, MA) ; Rosen, Craig A.; (Laytonsville,
MD) |
Correspondence
Address: |
Leon R. Yankwich, Esq.
Yankwich & Associates
130 Bishop Allen Drive
Cambridge
MA
02139
US
|
Family ID: |
22850040 |
Appl. No.: |
09/932613 |
Filed: |
August 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60226700 |
Aug 18, 2000 |
|
|
|
Current U.S.
Class: |
424/144.1 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 7/08 20130101; A61K 38/00 20130101; A61P 37/00 20180101; C07K
14/7151 20130101; C07K 2319/00 20130101; C07K 14/70578
20130101 |
Class at
Publication: |
424/144.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of treating, preventing or ameliorating a disease or
disorder associated with aberrant B Lymphocyte Stimulator (BLyS) or
BLyS receptor expression or activity, comprising administering to
an animal in which such treatment, prevention or amelioration is
desired, a BLyS binding polypeptide in an amount effective to
treat, prevent or ameliorate the disease or disorder.
2. The method of claim 1, wherein the disease or disorder is an
immune system disease or disorder.
3. The method of claim 2, wherein the immune system disease or
disorder is an autoimmune disease or disorder.
4. The method of claim 2, wherein the immune system disease or
disorder is an immunodeficiency.
5. The method of claim 3, wherein the autoimmune disease or
disorder is lupus.
6. The method of claim 1, wherein the disease or disorder is
glomerular nephritis.
7. The method of claim 2, wherein the immune system disease or
disorder is rheumatoid arthritis, multiple sclerosis,
hypogammaglobulinemia or hypergammaglobulinemia.
8. The method of claim 2 wherein the immune system disease or
disorder is graft vs. host disease.
9. The method of claim 2, wherein the immune system disease or
disorder is a proliferative disease or disorder.
10. The method of claim 9, wherein the proliferative disorder is
cancer.
11. The method of claim 1, wherein the disease or disorder is an
infectious disease or disorder.
12. A method of treating, preventing, or ameliorating an immune
system disease or disorder, comprising administering to an animal
in which such treatment, prevention, or amelioration is desired, a
BLyS binding polypeptide in an amount effective to treat, prevent,
or ameliorate the immune system disease or disorder.
13. The method of claim 12, wherein the immune system disease or
disorder is an autoimmune disease or disorder.
14. The method of claim 12, wherein the immune system disease or
disorder is an immunodeficiency.
15. The method of claim 13, wherein the autoimmune disease or
disorder is lupus.
16. The method of claim 12, wherein the immune system disease or
disorder is glomerular nephritis, rheumatoid arthritis, multiple
sclerosis, hypogammaglobulinemia, hypergammaglobulinemia, or graft
vs. host disease.
17. A method of treating, preventing or ameliorating a disease or
disorder of cells of hematopoietic origin, comprising administering
to an animal in which such treatment, prevention, or amelioration
is desired, a BLyS binding polypeptide in an amount effective to
treat, prevent or ameliorate the disease or disorder.
18. The method of claim 17, wherein the cells of hematopoietic
origin are selected from the group consisting of: lymphocytes,
monocytes, macrophages, or dendritic cells.
19. The method of claim 18, wherein the lymphocytes are B
cells.
20. The method of claim 18, wherein the lymphocytes are T
cells.
21. A method of inhibiting or reducing immunoglobulin production,
comprising contacting an effective amount of BLyS binding
polypeptide with BLyS, wherein the effective amount of BLyS binding
polypeptide inhibits or reduces BLyS mediated immunoglobulin
production.
22. The method of claim 21, wherein IgG production is inhibited or
reduced.
23. The method of claim 21, wherein IgM production is inhibited or
reduced.
24. The method of claim 21, wherein IgA production is inhibited or
reduced.
25. A method of inhibiting or reducing immunoglobulin production,
comprising administering to an animal in which such inhibition or
reduction is desired, a BLyS binding polypeptide in an amount
effective to inhibit or reduce immunoglobulin production.
26. The method of claim 25, wherein IgG production is inhibited or
reduced.
27. The method of claim 25, wherein IgM production is inhibited or
reduced.
28. The method of claim 25, wherein IgA production is inhibited or
reduced.
29. A method of inhibiting or reducing B cell proliferation,
comprising contacting an effective amount of BLyS binding
polypeptide with BLyS, wherein the effective amount of BLyS binding
polypeptide inhibits or reduces BLyS mediated B cell
proliferation.
30. A method of inhibiting or reducing B cell proliferation
comprising administering to an animal in which such inhibition or
reduction is desired, a BLyS binding polypeptide in an amount
effective to inhibit or reduce B cell proliferation.
31. A method of inhibiting or reducing activation of B cells,
comprising contacting an effective amount of BLyS binding
polypeptide with BLyS, wherein the effective amount of BLyS binding
polypeptide inhibits or reduces BLyS mediated B cell
activation.
32. A method of inhibiting or reducing activation of B cells,
comprising administering to an animal in which such inhibition or
reduction is desired, a BLyS binding polypeptide in an amount
effective to inhibit or reduce B cell activation.
33. A method of decreasing lifespan of B cells, comprising
contacting an effective amount of BLyS binding polypeptide with
BLyS, wherein the effective amount of BLyS binding polypeptide
inhibits or reduces BLyS regulated lifespan of B cells.
34. A method of decreasing B cell lifespan, comprising
administering to an animal in which such decrease is desired, a
BLyS binding polypeptide in an amount effective to decrease B cell
lifespan.
35. A method of inhibiting or reducing graft rejection, comprising
administering to an animal in which such inhibition or reduction is
desired, a BLyS binding polypeptide in an amount effective to
inhibit or reduce graft rejection.
36. A method of killing cells of hematopoietic origin, comprising
contacting BLyS binding polypeptides with BLyS to form a complex;
and contacting the complex with cells of hematopoietic origin.
37. The method of claim 36, wherein the cells of hematopoietic
origin are selected from the group consisting of: lymphocytes,
monocytes, macrophages, or dendritic cells.
38. The method of claim 37, wherein the lymphocytes are B
cells.
39. The method of claim 37, wherein the lymphocytes are T
cells.
40. A method of killing cells of hematopoietic origin, comprising
administering to an animal in which such killing is desired, a BLyS
binding polypeptide in an amount effective to kill cells of
hematopoietic origin.
41. The method of claim 40, wherein the cells of hematopoietic
origin are selected from the group consisting of: lymphocytes,
monocytes, macrophages, or dendritic cells.
42. The method of claim 41, wherein the lymphocytes are B
cells.
43. The method of claim 41, wherein the lymphocytes are T
cells.
44. A method of treating a proliferative disease or disorder,
comprising administering to an animal in which such treatment is
desired, a BLyS binding polypeptide in an amount effective to treat
the proliferative disease or disorder.
45. The method of claim 44, wherein the proliferative disease or
disorder is selected from the group consisting of: premalignant
conditions, benign tumors, hyperproliferative disorders, and benign
proliferative disorders.
46. The method of claim 44, wherein the proliferative disease or
disorder is a proliferative disease or disorder of a cell of
hematopoietic origin.
47. The method of claim 46, wherein the proliferative disease or
disorder is a B cell proliferative disease or disorder.
48. The method of claim 47, wherein the B cell proliferative
disease or disorder is a leukemia.
49. The method of claim 47, wherein the B cell proliferative
disease or disorder is a lymphoma.
50. The method of claim 47, wherein the B cell proliferative
disease or disorder is chronic lymphocytic leukemia, multiple
myeloma, non-Hodgkin's lymphoma, or Hodgkins disease.
51. The method of claim 46, wherein the proliferative disease or
disorder is a T cell proliferative disease or disorder.
52. The method of claim 46, wherein the proliferative disease or
disorder is a monocytic proliferative disease or disorder.
53. The method of claim 52, wherein the monocytic proliferative
disease or disorder is leukemia, lymphoma, or acute myelogenous
leukemia.
54. The method of claim 46, wherein the proliferative disease or
disorder is a macrophage proliferative disease or disorder.
55. A method of stimulating immunoglobulin production, comprising
contacting an effective amount of BLyS binding polypeptide with
BLyS, wherein the effective amount of the BLyS binding polypeptide
stimulates BLyS mediated immunoglobulin production.
56. The method of claim 55, wherein IgG production is
stimulated.
57. The method of claim 55, wherein IgM production is
stimulated.
58. The method of claim 55, wherein IgA production is
stimulated.
59. A method of stimulating immunoglobulin production comprising
administering to an animal in which such stimulation is desired, a
BLyS binding polypeptide in an amount effective to stimulate
immunoglobulin production.
60. The method of claim 59, wherein IgG production is
stimulated.
61. The method of claim 59, wherein IgM production is
stimulated.
62. The method of claim 59, wherein IgA production is inhibited or
stimulated.
63. A method of stimulating B cell proliferation, comprising
contacting an effective amount of BLyS binding polypeptide with
BLyS, wherein the effective amount of BLyS binding polypeptide
stimulates BLyS mediated B cell proliferation.
64. A method of stimulating B cell proliferation, comprising
administering to an animal in which such stimulation is desired, a
BLyS binding polypeptide in an amount effective to stimulate B cell
proliferation.
65. A method of increasing activation of B cells, comprising
contacting an effective amount of BLyS binding polypeptide with
BLyS, wherein the effective amount of BLyS binding polypeptide
increases BLyS mediated activation of B cells.
66. A method of increasing activation of B cells, comprising
administering to an animal in which such increase is desired, a
BLyS binding polypeptide in an amount effective to increase B cell
activation.
67. A method of increasing lifespan of B cells, comprising
contacting an effective amount of BLyS binding polypeptide with
BLyS, wherein the effective amount of BLyS binding polypeptide
increases BLyS mediated lifespan of B cells.
68. A method increasing lifespan of B cells, comprising
administering to an animal in which such increase is desired, a
BLyS binding polypeptide in an amount effective to increase
lifespan of B cells.
69. The method according to any one of claims 1, 12, 17, 21, 25,
29, 30, 31, 32, 33, 34, 35, 36, 40, 44, 55, 59, 63, 64, 65, 66, 67,
or 68, wherein the BLyS binding polypeptide comprises an amino acid
sequence selected from the group consisting of: (1) Asp-Xaa-Leu-Thr
(SEQ ID NO: 446), where Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys,
or Ala (preferably Pro or Ser); (2)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-Phe-X.sub.7-Trp-Glu--
Cys-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO: 1), wherein X.sub.1 is
Ala, Asn, Lys, or Ser; X.sub.2 is Ala, Glu, Met, Ser, or Val;
X.sub.3 is Ala, Asn, Lys, or Pro (preferably Lys); X.sub.5 is Phe,
Trp, or Tyr (preferably Tyr); X.sub.7 is Pro or Tyr (preferably
Pro); X.sub.11 is Ala, Gln, His, Phe, or Val; X.sub.12 is Asn, Gln,
Gly, His, Ser, or Val; and X.sub.13 is Ala, Asn, Gly, Ile, Pro, or
Ser; (3)
X.sub.1-X.sub.2-X3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-C-
ys-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO: 2), wherein X.sub.1 is
Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, Val, or is absent; X.sub.2 is Ala, Asn, Asp, Gln
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;
X.sub.3 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Trp, Tyr, or Val (preferably Asp); X.sub.5 is
Asp, Ile, Leu, or Tyr (preferably Asp or Leu); X.sub.6 is Arg, Asp,
Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val (preferably Glu or
Leu); X.sub.7 is His, Leu, Lys, or Phe (preferably His or Leu);
X.sub.8 is Leu, Pro, or Thr (preferably Thr or Pro); X.sub.9 is
Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys);
X.sub.10 is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Tip,
Tyr, or Val; X.sub.12 is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe,
Ser, Trp, Tyr, or Val; X.sub.13 is Ala, Arg, Asn, Asp, Gln, Glu,
Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and
X.sub.14 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys,
Phe, Pro, Trp, Tyr, Val, or is absent; (4)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-Cys-X.sub.13-X.sub.14-X.sub.15 (SEQ ID NO: 3),
wherein X.sub.1 is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or
Thr; X.sub.2 is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp;
X.sub.3 is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr
(preferably Ala); X.sub.5 is Asn, Asp, Pro, Ser, or Thr (preferably
Asp); X.sub.6 is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably
Ile); X.sub.7 is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or
Leu); X.sub.8 is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably
Thr); X.sub.9 is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr
(preferably Leu); X.sub.10 is Arg, Asn, Asp, Gln, Glu, Gly, Ile,
Lys, Met, Pro, Ser, or Trp; X.sub.11 is Arg, Glu, Gly, Lys, Phe,
Ser, Trp, or Tyr (preferably Ser); X.sub.13 is Gln, Glu, Ile, Leu,
Phe, Pro, Ser, Tyr, or Val (preferably Val); X.sub.14 is Asn, Gly,
Ile, Phe, Pro, THr, Trp, or Tyr; and X.sub.15 is Asn, Asp, Glu,
Leu, Lys, Met, Pro, or Thr (preferably Glu or Pro); (5)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.su-
b.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-Cys-X.sub.14-X.sub.-
15-X.sub.16 (SEQ ID NO: 4), wherein X.sub.1 is Asn, Asp, His, Leu,
Phe, Pro, Ser, Tyr, or is absent (preferably Ser); X.sub.2 is Arg,
Asn, Asp, His, Phe, Ser, or Trp (preferably Arg); X.sub.3 is Asn,
Asp, Leu, Pro, Ser, or Val (preferably Asn or Asp); X.sub.5 is Asp,
Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X.sub.6 is His, Ile,
Leu, Met, Phe, Pro, Trp, or Tyr; X.sub.7 is Asp, His, Leu, or Ser
(preferably Asp); X.sub.8 is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or
Thr (preferably Glu or Pro); X.sub.9 is Ala, Arg, Asn, or Leu
(preferably Leu); X.sub.10 is Ile, Leu, Met, Pro, Ser, or Thr
(preferably Thr); X.sub.11 is Ala, Arg, Asn, Gly, His, Lys, Ser, or
Tyr; X.sub.12 is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or
Val; X.sub.14 is Asp, Gly, Leu, Phe, Tyr, or Val (preferably Leu);
X.sub.15 is Asn, His, Leu, Pro, or Tyr (preferably His, Leu or
Pro); and X.sub.16 is Asn, Asp, His, Phe, Ser, or Tyr, (preferably
Asp or Ser); (6)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub-
.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14Cys-X.sub.16X.sub.17-X.sub.18
(SEQ ID NO: 5), wherein X.sub.1 is Arg, Asp, Gly, His, Leu, Phe,
Pro, Ser, Trp, Tyr, or is absent (preferably Arg); X.sub.2 is Ala,
Arg, Asn, Asp, Gly, Pro, Ser, or is absent (preferably Asn, Asp,
Gly, or Pro); X.sub.3 is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro,
Trp or Val (preferably Gly or Met); X.sub.5 is Arg, Asn, Gln, Glu,
His, Leu, Phe, Pro, Trp, Tyr, or Val preferably Trp, Tyr, or Val);
X.sub.6 is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr
(preferably Asp); X.sub.7 is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or
Tyr (preferably Asp); X.sub.8 is Asp, Gln, Glu, Leu, Met, Phe, Pro,
Ser, or Tyr (preferably Leu); X.sub.9 is Asp, Leu, Pro, Thr, or Val
(preferably Leu or Thr); X.sub.10 is Arg, Gln, His, Ile, Leu, Lys,
Met, Phe, THr, Trp or Tyr (preferably Lys or Thr); X.sub.11 is Ala,
Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferably Arg or
Leu); X.sub.12 is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp,
or Tyr (preferably Thr or Trp); X.sub.13 is Ala, Arg, Gln, His,
Lys, Met, Phe, Pro, Thr, Trp, or Tyr (preferably Met or Phe);
X.sub.14 is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr,
Tyr, or Val (preferably Val); X.sub.16 is Arg, Asp, Gly, His, Lys,
Met, Phe, Pro, Ser, or Trp (preferably Met); X.sub.17 is Arg, Asn,
Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr, (preferably Arg, His, or
Tyr); and X.sub.18 is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp
(preferably His or Asn); (7)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12 (SEQ ID NO: 6), wherein X.sub.1 is Ala,
Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val (preferably
Gly, Tyr, or Val); X.sub.2 is Ala, Arg, Gln, His, Ile, Leu, Phe,
Thr, Trp, or Tyr (preferably His or Tyr); X.sub.3 is Ala, Asp, Lys,
Phe, Thr, Trp or Tyr (preferably Asp or Tyr); X.sub.4 is Arg, Asp,
Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val (preferably Asp or Gln);
X.sub.5 is Asp, Leu, Lys, Phe, Pro, Ser, or Val (preferably Leu or
Ser); X.sub.6 is His, Ile, Leu, Pro, Ser, or Thr (preferably Leu or
Thr); X.sub.7 is Arg, Gly, His, Leu, Lys, Met, or Thr (preferably
Lys or Thr); X.sub.8 is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr
(preferably Leu or Lys); X.sub.9 is Ala, Asn, Arg, Asp, Glu, Gly,
His, Leu, Met, Ser, Trp, Tyr, or Val (preferably Met or Ser);
X.sub.10 is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val (preferably
Thr or Leu); X.sub.11 is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro,
Ser, Thr, Trp, Tyr, or Val (preferably Pro or Thr); and X.sub.12 is
Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val
(preferably Arg or Pro); (8)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-.sub.13 (SEQ ID NO: 7), wherein X.sub.1
is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp (preferably Glu or
Lys); X.sub.2 is Arg, Gln, His, Ile, Leu, or Pro (preferably His or
Pro); X.sub.3 is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably
Tyr); X.sub.4 is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr
(preferably Asp or Gln); X.sub.5 is Asn, Asp, His, Ile, Leu, Met,
Pro, Thr or Val (preferably Asn or Thr); X.sub.6 is Asp, Glu, His,
Leu, Lys, Pro, or Val (preferably Asp or Pro); X.sub.7 is Arg, Asn,
Gln, His, Ile, Leu, Met, Pro, or Thr (preferably Ile or Pro);
X.sub.8 is Gln, Gly, His, Leu, Met, Ser, or Thr (preferably Leu or
Thr); X.sub.9 is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr
(preferably Lys); X.sub.10 is Ala, Gly, Ile, Leu, Lys, Met, or Phe
(preferably Gly or Met); X.sub.11 is Ala, Glu, His, Ile, Leu, Met,
Ser, Thr, Trp, Tyr, or Val (preferably Ala or Thr); X.sub.12 is
Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val (preferably Arg or
His); and X.sub.13 is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or
Val (preferably His); (9) Cys-X.sub.2-Phe-X.sub.4-Trp-Glu-Cys (SEQ
ID NO: 8), wherein X.sub.2 is Phe, Trp, or Tyr (preferably Tyr);
and X.sub.4 is Pro or Tyr (preferably Pro); or (10)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-Cys (SEQ ID NO:
9), wherein X.sub.2 is Asp, Ile, Leu, or Tyr (preferably Asp or
Leu); X.sub.3 is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr,
or Val (preferably Glu or Leu); X.sub.4 is His, Leu, Lys, or Phe
(preferably His or Leu); X.sub.5 is Leu, Pro, or Thr (preferably
Thr or Pro); X.sub.6 is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp
(preferably Lys); and X.sub.7 is Ala, Asn, Gln, Glu, Gly, His, Ile,
Leu, Met, Phe, Ser, Trp, Tyr, or Val; or (11)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-- X.sub.8-Cys
(SEQ ID NO: 10), wherein X.sub.2 is Asn, Asp, Pro, Ser, or Thr
(preferably Asp); X.sub.3 is Arg, Asp, Ile, Leu, Met, Pro, or Val
(preferably Ile); X.sub.4 is Ala, Ile, Leu, Pro, Thr, or Val
(preferably Val or Leu); X.sub.5 is Asn, His, Ile, Leu, Lys, Phe,
or Thr (preferably Thr); X.sub.6 is Asn, Glu, Gly, His, Leu, Lys,
Met, Pro, or Thr (preferably Leu); X.sub.7 is Arg, Asn, Asp, Gln,
Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp; X.sub.8 is Arg, Glu,
Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser); or (12)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.s-
ub.7-X.sub.8-X.sub.9-Cys (SEQ ID NO: 11), wherein X.sub.2 is Asp,
Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X.sub.3 is His, Ile,
Leu, Met, Phe, Pro, Trp, or Tyr; X.sub.4 is Asp, His, Leu, or Ser
(preferably Asp); X.sub.5 is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or
Thr (preferably Glu or Pro); X.sub.6 is Ala, Arg, Asn, or Leu
(preferably Leu); X.sub.7 is Ile, Leu, Met, Pro, Ser, or Thr
(preferably Thr); X.sub.8 is Ala, Arg, Asn, Gly, His, Lys, Ser, or
Tyr; X.sub.9 is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or
Val; or (13) Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.s-
ub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-Cys (SEQ ID NO: 12), wherein
X.sub.2 is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val
(preferably Trp, Tyr, or Val); X.sub.3 is Arg, Asp, Gln, Gly, Ile,
Lys, Phe, Thr, Trp or Tyr (preferably Asp); X.sub.4 is Ala, Arg,
Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp); X.sub.5 is Asp,
Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu); X.sub.6
is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr); X.sub.7 is
Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr (preferably
Lys or Thr); X.sub.8 is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys,
Met, or Thr (preferably Arg or Leu); X.sub.9 is Ala, Asn, Gln, Gly,
Leu, Lys, Phe, Pro, Thr, Trp, or Tyr (preferably Thr or Trp);
X.sub.10 is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or
Tyr (preferably Met or Phe); X.sub.11 is Arg, Gln, Glu, Gly, His,
Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val (preferably Val); (14)
Ala-X.sub.2-X.sub.3-X.sub.4-Asp-X.sub.6-Leu-Thr-X.sub.9-Leu-X.sub.11-
-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO: 447), wherein X.sub.2 is
Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys, Leu, Met, Thr,
Val, Glu, Ala, Gly, Cys, or Trp (i.e., any amino acid except Arg;
preferably Asn); X.sub.3 is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly,
or Ser (preferably Trp); X.sub.4 is Tyr, Phe, Glu, Cys, Asn
(preferably Tyr); X.sub.6 is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or
Ala (preferably Pro or Ser); X.sub.9 is Lys, Asn, Gln, Gly, or Arg
(preferably Lys); X.sub.11 is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys
(preferably Trp); X.sub.12 is Leu, Phe, Val, Ile, or His
(preferably Leu); X.sub.13 is Pro, Leu, His, Ser, Arg, Asn, Gln,
Thr, Val, Ala, Cys, Ile, Phe, or Tyr (i.e., not Asp, Glu, Gly, Lys,
Met, or Trp; preferably Pro); and X.sub.14 is Asp, Glu, Asn, Val,
His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys, Ile, Thr or Leu (i.e.,
not Phe, Met, Pro, or Trp; preferably Asp); and (15)
X.sub.1-X.sub.2-Asp-X.su- b.4-Leu-Thr-X.sub.7-Leu-X.sub.9-X.sub.10
(SEQ ID NO: 448), wherein X.sub.1 is Trp, Glu, Lys, Cys, Leu, Ala,
Arg, Gly, or Ser (preferably Trp); X.sub.2 is Tyr, Phe, Glu, Cys,
Asn (preferably Tyr); X.sub.4 is Pro, Ser, Thr, Phe, Leu, Tyr, Cys,
or Ala preferably Pro or Ser); X.sub.7 is Lys, Asn, Gln, Gly, or
Arg (preferably Lys); X.sub.9 is Trp, Ser, Thr, Arg, Cys, Tyr, or
Lys (preferably Trp); and X.sub.10 is Leu, Phe, Val, Ile, or His
(preferably Leu).
70. The method according to claim 69, wherein the BLyS binding
polypeptide comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs: 20-168 and 186-435, as depicted in
Tables 1-8 and 13.
71. The method according to claim 69, wherein the BLyS binding
polypeptide comprises an amino acid sequence selected from the
group consisting of:
Ala-Gly-Lys-Glu-Pro-Cys-Tyr-Phe-Tyr-Trp-Glu-Cys-Ala-Val-Ser-Gly
(SEQ ID NO: 450);
Ala-Gly-Val-Pro-Phe-Cys-Asp-Leu-Leu-Thr-Lys-His-Cys-Phe-Glu-Ala-
-Gly (SEQ ID NO: 45 1);
Gly-Ser-Ser-Arg-Leu-Cys-His-Met-Asp-Glu-Leu-Thr-Hi-
s-Val-Cys-Val-His-Phe-Ala-Pro (SEQ ID NO: 452);
Gly-Asp-Gly-Gly-Asn-Cys-Ty-
r-Thr-Asp-Ser-Leu-Thr-Lys-Leu-His-Phe-Cys-Met-Gly-Asp-Glu (SEQ ID
NO: 453); Gly-Tyr-Asp-Val-Leu-Thr-Lys-Leu-Tyr-Phe-Val-Pro-Gly-Gly
(SEQ ID NO: 454);
Trp-Thr-Asp-Ser-Leu-Thr-Gly-Leu-Trp-Phe-Pro-Asp-Gly-Gly (SEQ ID NO:
455); Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp (SEQ
ID NO: 186); Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp (SEQ
ID NO: 456); Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO:
457); Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Val (SEQ
ID NO: 189);
Ala-Asn-Trp-Phe-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp (SEQ ID NO:
309); Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Ser-Leu-Pro-Asp (SEQ
ID NO: 458);
Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Phe-Pro-Asp (SEQ ID NO:
353); Ala-Asn-Trp-Tyr-Asp-Ser-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp (SEQ
ID NO: 327).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to therapeutic and diagnostic
uses for molecules that bind to B lymphocyte stimulator protein
(BLyS). In particular, the present invention also relates to
methods and compositions for detecting, diagnosing, or prognosing a
disease or disorder associated with aberrant BLyS or BLyS receptor
expression or inappropriate function of BLyS or BLyS receptor,
comprising BLyS binding polypeptides or fragments or variants
thereof, that specifically bind to BLyS. The present invention
further relates to methods and compositions for preventing,
treating or ameliorating a disease or disorder associated with
aberrant BLyS or BLyS receptor expression or inappropriate BLyS
function or BLyS receptor function, comprising administering to an
animal, preferably a human, an effective amount of one or more BLyS
binding polypeptides or fragments or variants thereof, that
specifically bind to BLyS.
BACKGROUND OF THE INVENTION
[0002] B lymphocyte stimulator (BLyS) is a member of the tumor
necrosis factor ("TNF") superfamily that induces both in vivo and
in vitro B cell proliferation and differentiation (Moore et al.,
Science, 285: 260-263 (1999)). BLyS is distinguishable from other B
cell growth and differentiation factors such as IL-2, IL-4, IL-5,
IL-6, IL-7, IL-1 3, IL-1 5, CD40L, or CD27 (CD70) by its
monocyte-specific gene and protein expression pattern and its
specific receptor distribution and biological activity on B
lymphocytes. BLyS expression is not detected on natural killer
("NK") cells, T cells or B cells, but is restricted to cells of
myeloid origin. BLyS expression on resting monocytes is upregulated
by interferon-gamma (IFN-gamma). The gene encoding BLyS has been
mapped to chromosome 13q34.
[0003] BLyS is expressed as a 285 amino acid type II membrane-bound
polypeptide and a soluble 152 amino acid polypeptide (Moore et al.,
1999, supra). The membrane-bound form of BLyS has a predicted
transmembrane spanning domain between amino acid residues 47 and
73. The NH.sub.2-terminus of the soluble form of BLyS begins at
Ala.sup.134 of the membrane-bound form of BLyS. Both the soluble
and membrane-bound forms of the protein form homotrimers. Soluble
recombinant BLyS has been shown to induce in vitro proliferation of
murine splenic B cells and to bind to a cell-surface receptor on
these cells (Moore et al., 1999, supra). Soluble BLyS
administration to mice has been shown to result in an increase in
the proportion of CD45R.sup.dull, Ly6D.sup.bright (also known as
ThB) B cells and an increase in serum IgM and IgA levels (Moore et
al., 1999, supra). Thus, BLyS displays a B cell tropism in both its
receptor distribution and biological activity.
[0004] Based on its expression pattern and biological activity,
BLyS has been suggested to be involved in the exchange of signals
between B cells and monocytes or their differentiated progeny. The
restricted expression patterns of BLyS receptor and ligand suggest
that BLyS 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.
[0005] Accordingly, molecules that specifically bind BLyS would
find a variety of uses in the study of the BLyS cytokine, in the
manufacture and purification of BLyS in commercial and medically
pure quantities, and in the development new therapeutic or
diagnostic reagents. BLyS binding polypeptides may also find
medical utility in, for example, the treatment of B cell and/or
monocyte disorders associated with autoimmunity, neoplasia, or
immunodeficiency syndromes.
SUMMARY OF THE INVENTION
[0006] New polypeptides that specifically bind to B lymphocyte
stimulator protein (BLyS) and/or BLyS-like polypeptides have been
discovered, and the therapeutic and diagnostic applications for
such polypeptides are disclosed herein. Particular polypeptides
useful in the methods of this invention specifically bind to a
polypeptide or polypeptide fragment of human BLyS (SEQ ID NOs: 173
and/or 174) or BLyS expressed on human monocytes; murine BLyS (SEQ
ID NOs: 175 and/or 176) or BLyS expressed on murine monocytes; rat
BLyS (either the soluble forms as given in SEQ ID NOs: 177, 178,
179 and/or 180 or in a membrane associated form, e.g., on the
surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS
polypeptides of SEQ ID NOS: 181 and/or 182, the soluble form of
monkey BLyS, or BLyS expressed on monkey monocytes), preferably
human BLyS.
[0007] In preferred methods of the invention, BLyS binding
polypeptides comprising, or alternatively consisting of, an amino
acid sequence selected from the group consisting of SEQ ID NOs:
1-12, 20-172, and 186-444, Preferably SEQ ID NOs: 163-172 and
436-444 as referred to herein and in Tables 1-8, 13 and 14, and
fragments and variants thereof, will be used.
[0008] In specific preferred embodiments, the BLyS binding
polypeptides bind BLyS and/or BLyS-like polypeptides with high
affinity. In other embodiments, the BLyS binding polypeptides
reversibly bind BLyS and/or BLyS-like polypeptides. In still other
embodiments, the BLyS binding polypeptides irreversibly bind BLyS
and/or BLyS-like polypeptides.
[0009] The cysteine residues in certain polypeptides useful in the
methods of the invention are believed to form a disulfide bond,
which would cause the polypeptide containing these cysteine
residues to form a stable loop structure under non-reducing
conditions. Especially preferred BLyS binding polypeptides useful
in the methods of the invention are polypeptide molecules that
comprise amino acid sequences that form stable loop structures or
other stable structures that bind BLyS or BLyS-like
polypeptides.
[0010] Analysis of the sequences of the BLyS binding polypeptides
described herein shows a strong selection for polypeptides
containing the tetrapeptide Asp-Xaa-Leu-Thr (SEQ ID NO: 446), and
therefore in its broadest aspects, the present invention relates to
methods for using polypeptides capable of binding to BLyS
comprising the polypeptide Asp-Xaa-Leu-Thr (SEQ ID NO: 446), where
Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or
Ser).
[0011] In addition, seven consensus sequences (SEQ ID NOs: 1-7) are
disclosed for peptides useful in the methods of the invention,
based on the specific BLyS binding polypeptides shown in Tables
1-8. In preferred methods according to the invention, BLyS binding
polypeptides comprising one or more of these sequences are used.
Such preferred methods utilize BLyS binding polypeptides including
polypeptides with the potential to form a cyclic or loop structure
between invariant Cys residues comprising, or alternatively
consisting of, an amino acid sequence selected from A-E (SEQ ID
NOs: 1-5):
[0012] (A)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-Phe-X.sub.7-Trp-Glu-Cys-X.s-
ub.11-X.sub.12-X.sub.13 (SEQ ID NO: 1),
[0013] wherein
[0014] X.sub.1 is Ala, Asn, Lys, or Ser;
[0015] X.sub.2 is Ala, Glu, Met, Ser, or Val;
[0016] X.sub.3 is Ala, Asn, Lys, or Pro (preferably Lys);
[0017] X.sub.5 is Phe, Trp, or Tyr (preferably Tyr);
[0018] X.sub.7 is Pro or Tyr (preferably Pro);
[0019] X.sub.11 is Ala, Gln, His, Phe, or Val;
[0020] X.sub.12 is Asn, Gln, Gly, His, Ser, or Val; and
[0021] X.sub.13 is Ala, Asn, Gly, Ile, Pro, or Ser,
[0022] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0023] (B)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-Cys-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO: 2),
[0024] wherein
[0025] X.sub.1 is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, Val, or is absent;
[0026] X.sub.2 is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X.sub.3 is Ala, Arg, Asn,
Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp,
Tyr, or Val (preferably Asp);
[0027] X.sub.5 is Asp, Ile, Leu, or Tyr (preferably Asp or
Leu);
[0028] X.sub.6 is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr,
or Val (preferably Glu or Leu);
[0029] X.sub.7 is His, Leu, Lys, or Phe (preferably His or
Leu);
[0030] X.sub.8 is Leu, Pro, or Thr (preferably Thr or Pro);
[0031] X.sub.9 is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp
(preferably Lys);
[0032] X.sub.10 is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,
Ser, Trp, Tyr, or Val;
[0033] X.sub.12 is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser,
Trp, Tyr, or Val;
[0034] X.sub.13 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and
[0035] X.sub.14 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile,
Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent,
[0036] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0037] (C)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-Cys-X.sub.13-X.sub.14-X.sub.15 (SEQ ID NO:
3),
[0038] wherein
[0039] X.sub.1 is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or
Thr;
[0040] X.sub.2 is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or
Trp;
[0041] X.sub.3 is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr
(preferably Ala);
[0042] X.sub.5 is Asn, Asp, Pro, Ser, or Thr (preferably Asp);
[0043] X.sub.6 is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably
Ile);
[0044] X.sub.7 is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val
or Leu);
[0045] X.sub.8 is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably
Thr);
[0046] X.sub.9 is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr
(preferably Leu);
[0047] X.sub.10 is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met,
Pro, Ser, or Trp;
[0048] X.sub.11 is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr
(preferably Ser);
[0049] X.sub.13 is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val
(preferably Val);
[0050] X.sub.14 is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr;
and
[0051] X.sub.15 is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr
(preferably Glu or Pro),
[0052] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0053] (D)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-X.sub.12-Cys-X.sub.14-X.sub.15-X.sub.16 (SEQ
ID NO: 4),
[0054] wherein
[0055] X.sub.1 is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is
absent (preferably Ser);
[0056] X.sub.2 is Arg, Asn, Asp, His, Phe, Ser, or Trp (preferably
Arg);
[0057] X.sub.3 is Asn, Asp, Leu, Pro, Ser, or Val (preferably Asn
or Asp);
[0058] X.sub.5 is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or
Thr;
[0059] X.sub.6 is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;
[0060] X.sub.7 is Asp, His, Leu, or Ser (preferably Asp);
[0061] X.sub.8 is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr
(preferably Glu or Pro);
[0062] X.sub.9 is Ala, Arg, Asn, or Leu (preferably Leu);
[0063] X.sub.10 is Ile, Leu, Met, Pro, Ser, or Thr (preferably
Thr);
[0064] X.sub.11 is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;
[0065] X.sub.12 is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or
Val;
[0066] X.sub.14 is Asp, Gly, Leu, Phe, Tyr, or Val (preferably
Leu);
[0067] X.sub.15 is Asn, His, Leu, Pro, or Tyr (preferably His, Leu
or Pro); and
[0068] X.sub.16 is Asn, Asp, His, Phe, Ser, or Tyr, (preferably Asp
or Ser),
[0069] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0070] (E)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-Cys-X.sub.16-X.sub.17-X.-
sub.18 (SEQ ID NO: 5),
[0071] wherein
[0072] X.sub.1 is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr,
or is absent (preferably Arg);
[0073] X.sub.2 is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent
(preferably Asn, Asp, Gly, or Pro);
[0074] X.sub.3 is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or
Val (preferably Gly or Met);
[0075] X.sub.5 is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr,
or Val (preferably Trp, Tyr, or Val);
[0076] X.sub.6 is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or
Tyr (preferably Asp);
[0077] X.sub.7 is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr
(preferably Asp);
[0078] X.sub.8 is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr
(preferably Leu);
[0079] X.sub.9 is Asp, Leu, Pro, Thr, or Val (preferably Leu or
Thr);
[0080] X.sub.10 is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp
or Tyr (preferably Lys or Thr);
[0081] X.sub.11 is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or
Thr (preferably Arg or Leu);
[0082] X.sub.12 is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr,
Trp, or Tyr (preferably Thr or Trp);
[0083] X.sub.13 is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr,
Trp, or Tyr (preferably Met or Phe);
[0084] X.sub.14 is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro,
Ser, Thr, Tyr, or Val (preferably Val);
[0085] X.sub.16 is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or
Trp (preferably Met);
[0086] X.sub.17 is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or
Tyr, (preferably Arg, His, or Tyr); and
[0087] X.sub.18 is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp
(preferably His or Asn),
[0088] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides.
[0089] Additional preferred embodiments include methods utilizing
linear BLyS binding polypeptides comprising, or alternatively
consisting of, an amino acid sequence selected from F and G (SEQ ID
NOs: 6 and 7):
[0090] (F)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-
-X.sub.9-X.sub.10-X.sub.11-X.sub.12 (SEQ ID NO: 6),
[0091] wherein
[0092] X.sub.1 is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr,
or Val (preferably Gly, Tyr, or Val);
[0093] X.sub.2 is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or
Tyr (preferably His or Tyr);
[0094] X.sub.3 is Ala, Asp, Lys, Phe, Thr, Trp or Tyr (preferably
Asp or Tyr);
[0095] X.sub.4 is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or
Val (preferably Asp or Gln);
[0096] X.sub.5 is Asp, Leu, Lys, Phe, Pro, Ser, or Val (preferably
Leu or Ser);
[0097] X.sub.6 is His, Ile, Leu, Pro, Ser, or Thr (preferably Leu
or Thr);
[0098] X.sub.7 is Arg, Gly, His, Leu, Lys, Met, or Thr (preferably
Lys or Thr);
[0099] X.sub.8 is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr
(preferably Leu or Lys);
[0100] X.sub.9 is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser,
Trp, Tyr, or Val (preferably Met or Ser);
[0101] X.sub.10 is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val
(preferably Thr or Leu);
[0102] X.sub.11 is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser,
Thr, Trp, Tyr, or Val (preferably Pro or Thr); and
[0103] X.sub.12 is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser,
Trp, Tyr, or Val (preferably Arg or Pro),
[0104] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0105] (G)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-.sub.X.sub.6-X.sub.7-X.-
sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO:
7),
[0106] wherein
[0107] X.sub.1 is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp
(preferably Glu, Lys);
[0108] X.sub.2 is Arg, Gln, His, Ile, Leu, or Pro (preferably His
or Pro);
[0109] X.sub.3 is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably
Tyr);
[0110] X.sub.4 is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr
(preferably Asp or Gln);
[0111] X.sub.5 is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val
(preferably Asn or Thr);
[0112] X.sub.6 is Asp, Glu, His, Leu, Lys, Pro, or Val (preferably
Asp or Pro);
[0113] X.sub.7 is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr
(preferably Ile or Pro);
[0114] X.sub.8 is Gln, Gly, His, Leu, Met, Ser, or Thr (preferably
Leu or Thr);
[0115] X.sub.9 is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr
(preferably Lys);
[0116] X.sub.10 is Ala, Gly, Ile, Leu, Lys, Met, or Phe (preferably
Gly or Met);
[0117] X.sub.11 is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp,
Tyr, or Val (preferably Ala or Thr);
[0118] X.sub.12 is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val
(preferably Arg or His); and
[0119] X.sub.13 is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val
(preferably His),
[0120] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides.
[0121] Additional polypeptides useful in the methods of the
invention include polypeptides comprising, or alternatively
consisting of, an amino acid sequence selected from H-L (SEQ ID
NOs: 8-12):
[0122] (H) Cys-X.sub.2-Phe-X.sub.4-Trp-Glu-Cys (SEQ ID NO: 8),
[0123] wherein
[0124] X.sub.2 is Phe, Trp, or Tyr (preferably Tyr); and
[0125] X.sub.4 is Pro or Tyr (preferably Pro); or
[0126] (I) Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-Cys
(SEQ ID NO: 9),
[0127] wherein
[0128] X.sub.2 is Asp, Ile, Leu, or Tyr (preferably Asp or
Leu);
[0129] X.sub.3 is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr,
or Val (preferably Glu or Leu);
[0130] X.sub.4 is His, Leu, Lys, or Phe (preferably His or
Leu);
[0131] X.sub.5 is Leu, Pro, or Thr (preferably Thr or Pro);
[0132] X.sub.6 is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp
(preferably Lys); and
[0133] X.sub.7 is Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,
Ser, Trp, Tyr, or Val; or
[0134] (J)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-Cys
(SEQ ID NO: 10),
[0135] wherein
[0136] X.sub.2 is Asn, Asp, Pro, Ser, or Thr (preferably Asp);
[0137] X.sub.3 is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably
Ile);
[0138] X.sub.4 is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val
or Leu);
[0139] X.sub.5 is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably
Thr);
[0140] X.sub.6 is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr
(preferably Leu);
[0141] X.sub.7 is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro,
Ser, or Trp;
[0142] X.sub.8 is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr
(referably Ser); or
[0143] (K)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7X.sub.8-X.su-
b.9-Cys (SEQ ID NO: 11),
[0144] wherein
[0145] X.sub.2 is Asp, Gln His, Ile, Leu, Lys, Met, Phe, or
Thr;
[0146] X.sub.3 is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;
[0147] X.sub.4 is Asp, His, Leu, or Ser (preferably Asp);
[0148] X.sub.5 is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr
(preferably Glu or Pro);
[0149] X.sub.6 is Ala, Arg, Asn, or Leu (preferably Leu);
[0150] X.sub.7 is Ile, Leu, Met, Pro, Ser, or Thr (preferably
Thr);
[0151] X.sub.8 is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;
[0152] X.sub.9 is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or
Val; or
[0153] (L)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-Cys (SEQ ID NO: 12),
[0154] wherein
[0155] X.sub.2 is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr,
or Val (preferably Trp, Tyr, or Val);
[0156] X.sub.3 is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or
Tyr (preferably Asp);
[0157] X.sub.4 is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr
(preferably Asp);
[0158] X.sub.5 is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr
(preferably Leu);
[0159] X.sub.6 is Asp, Leu, Pro, Thr, or Val (preferably Leu or
Thr);
[0160] X.sub.7 is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp
or Tyr (preferably Lys or Thr);
[0161] X.sub.8 is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or
Thr (preferably Arg or Leu);
[0162] X.sub.9 is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp,
or Tyr (preferably Thr or Trp);
[0163] X.sub.10 is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr,
Tip, or Tyr (preferably Met or Phe);
[0164] X.sub.11 is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro,
Ser, Thr, Tyr, or Val (preferably Val);
[0165] wherein said polypeptides bind BLyS and/or BLyS-like
polypeptides.
[0166] In preferred embodiments of the present invention, BLyS
binding polypeptides are used which comprise the following amino
acid sequence M (SEQ ID NO: 447):
[0167] (M)
Ala-X.sub.2-X.sub.3-X.sub.4-Asp-X.sub.6-Leu-Thr-X.sub.9-Leu-X.s-
ub.11-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO: 447),
[0168] wherein
[0169] X.sub.2 is Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys,
Leu, Met, Thr, Val, Glu, Ala, Gly, Cys, or
[0170] Trp (i.e., any amino acid except Arg; preferably Asn);
[0171] X.sub.3 is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser
(preferably Trp);
[0172] X.sub.4 is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);
[0173] X.sub.6 is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala
(preferably Pro or Ser);
[0174] X.sub.9 is Lys, Asn, Gln, Gly, or Arg (preferably Lys);
[0175] X.sub.11 is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably
Trp);
[0176] X.sub.12 is Leu, Phe, Val, Ile, or His (preferably Leu);
[0177] X.sub.13 is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val,
Ala, Cys, Ile, Phe, or Tyr (i.e., not Asp, Glu, Gly, Lys, Met, or
Trp; preferably Pro); and
[0178] X.sub.14 is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser,
Tyr, Ala, Cys, Lys, Ile, Thr or Leu (i.e., not Phe, Met, Pro, or
Trp; preferably Asp, Val or Glu).
[0179] Preferred methods will utilize polypeptides comprising a
core sequence of the formula N:
[0180] (N)
X.sub.1-X.sub.2-Asp-X.sub.4-Leu-Thr-X.sub.7-Leu-X.sub.9-X.sub.1- 0
(SEQ ID NO: 448),
[0181] wherein
[0182] X.sub.1 is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser
(preferably Trp);
[0183] X.sub.2 is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);
[0184] X.sub.4 is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala
(preferably Pro or Ser);
[0185] X.sub.7 is Lys, Asn, Gln, Gly, or Arg (preferably Lys);
[0186] X.sub.9 is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably
Trp); and
[0187] X.sub.10 is Leu, Phe, Val, Ile, or His (preferably Leu).
[0188] Especially preferred methods according to the invention will
utilize BLyS binding polypeptides which comprise the core peptide
Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 436).
[0189] BLyS binding polypeptides used in the methods of the present
invention may also have an amino terminal (N-terminal) capping or
functional group, such as an acetyl group, which, for example,
blocks the amino terminal amino group from undesirable reactions or
is useful in linking the BLyS binding polypeptide to another
molecule, matrix, resin, or solid support. BLyS binding
polypeptides may also have a carboxy terminal (C-terminal) capping
or functional group, such as an amide group, which, for example,
blocks the C-terminal carboxyl group from undesirable reactions or
provides a functional group useful in conjugating the binding
polypeptide to other molecules, matrices, resins, or solid
supports. Preferably, the N- and/or C-terminal capping groups are
polypeptide linker molecules. An especially preferred C-terminal
linker molecule that is useful for immobilizing a BLyS binding
polypeptide to a solid support or chromatographic matrix material
comprises the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys
(SEQ ID NO: 13). Another useful C-terminal linker, e.g., for
fluoresceinating peptides, is Gly-Gly-Lys (see Table 14).
[0190] In the methods of the present invention, it may be
advantageous to use BLyS binding polypeptides that have been
modified, for example, to increase or decrease the stability of the
molecule, while retaining the ability to bind BLyS and/or BLyS-like
polypeptides. An example of a modified BLyS binding polypeptide is
a polypeptide in which one of two cysteine residues is substituted
with a non-naturally occurring amino acid that is capable of
condensing with the remaining cysteine side chain to form a stable
thioether bridge, thereby generating a cyclic BLyS binding
polypeptide. Such cyclic thioether molecules of synthetic peptides
may be routinely generated using techniques known in the art, e.g.,
as described in PCT publication WO 97/46251, incorporated herein by
reference.
[0191] Some of the methods provided herein utilize BLyS binding
polypeptides that have been attached, coupled, linked or adhered to
a matrix or resin or solid support. Techniques for attaching,
linking or adhering polypeptides to matrices, resins and solid
supports are well known in the art. Suitable matrices, resins or
solid supports for these materials may be any composition known in
the art to which a BLyS binding polypeptide could be attached,
coupled, linked, or adhered, including but not limited to, a
chromatographic resin or matrix, such as SEPHAROSE-4 FF agarose
beads, the wall or floor of a well in a plastic microtiter dish,
such as used in an enzyme-liked immunosorbent assay (ELISA), or a
silica based biochip. Materials useful as solid supports on which
to immobilize binding polypeptides for use in the methods include,
but are not limited to, polyacrylamide, agarose, silica,
nitrocellulose, paper, plastic, nylon, metal, and combinations
thereof. A BLyS binding polypeptide may be immobilized on a matrix,
resin or solid support material by a non-covalent association or by
covalent bonding, using techniques known in the art.
[0192] In certain embodiments of the present invention, it is
preferred to utilize BLyS binding polypeptides or phage displaying
such binding polypeptides that irreversibly bind the BLyS protein
in its native, soluble trimeric form.
[0193] In certain embodiments of the present, it is preferred to
utilize BLyS binding polypeptides of the present invention or phage
displaying such binding polypeptides that reversibly bind the BLyS
protein in its native, soluble trimeric form.
[0194] In further embodiments of the present invention, a method
may call for the use of a composition of matter comprising isolated
nucleic acids, preferably DNA, encoding a BLyS binding polypeptide.
In specific embodiments, nucleic acid molecules encode a BLyS
binding polypeptide comprising the amino acid sequence of SEQ ID
NOs: 1-12, 20-172, or 186-444. In additional embodiments, the
nucleic acid molecules encode a polypeptide variant or fragment of
a polypeptide comprising an amino acid sequence of SEQ ID NOs:
1-12, 20-172, or 186-444. In a further additional embodiment, such
nucleic acid molecules encode a BLyS binding polypeptide, the
complementary strand of which nucleic acid hybridizes to a
polynucleotide sequence encoding a polypeptide described in Tables
1-8 and 13 and in Examples 2, 5 and 6 (SEQ ID NOs: 1-12, 20-172 and
186-444), under stringent conditions, e.g., hybridization to
filter-bound DNA in 6.times. chloride/sodium citrate (SSC) at about
45.degree. C. followed by one or more washes in 0.2.times.SSC/0.1%
SDS at about 50-65.degree. C., under highly stringent conditions,
e.g., hybridization to filter-bound nucleic acid in 6.times.SSC at
about 45.degree. C. followed by one or more washes in
0.1.times.SSC/0.2% SDS at about 68.degree. C., or under other
stringent hybridization conditions which are known to those of
skill in the art (see, for example, Ausubel, F. M. et al., eds.,
1989, Current Protocols in Molecular Biology, Vol. 1, Green
Publishing Associates, Inc. and John Wiley & Sons, Inc., New
York at pages 6.3.1-6.3.6 and 2.10.3).
[0195] In further embodiments of the invention, recombinant
bacteriophage are utilized which display BLyS binding polypeptides
on their surfaces. Such phage may be routinely generated using
techniques known in the art and are useful, for example, as
screening reagents and reagents for detecting BLyS.
[0196] In other methods according to the invention, a BLyS binding
polypeptide is used to detect or isolate BLyS or BLyS-like
polypeptides in a solution. Such solutions include, but are not
limited to, BLyS or BLyS-like polypeptides suspended or dissolved
in water or a buffer solution as well as any fluid and/or cell
obtained from an individual, biological fluid, body tissue, body
cell, cell line, tissue culture, or other source which may contain
BLyS or BLyS-like polypeptides, such as, cell culture medium, cell
extracts, and tissue homogenates. Biological fluids include, but
are not limited to, sera, plasma, lymph, blood, blood fractions,
urine, synovial fluid, spinal fluid, saliva, and mucous.
[0197] Methods according to the present invention may
advantageously utilize panels of BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants) wherein the panel
members correspond to one, two, three, four, five, ten, fifteen,
twenty, or more different BLyS binding polypeptides. Methods
according to the present invention may alternatively use mixtures
of BLyS binding polypeptides, wherein the mixture corresponds to
one, two, three, four, five, ten, fifteen, twenty, or more
different BLyS binding polypeptides. The present invention also
provides methods of using compositions comprising, or alternatively
consisting of, one, two, three, four, five, ten, fifteen, twenty,
or more BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof). Alternatively, a method according to the
invention may utilize a composition comprising, or alternatively
consisting of, nucleic acid molecules encoding one or more BLyS
binding polypeptides.
[0198] The methods of the present invention also provides for the
use of fusion proteins comprising a BLyS binding polypeptide
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof), and a
heterologous polypeptide. A composition useful in methods of the
present invention may comprise, or alternatively consist of, one,
two, three, four, five, ten, fifteen, twenty or more fusion
proteins capable of binding to BLyS. Alternatively, a composition
useful in methods of the invention may comprise, or alternatively
consist of, nucleic acid molecules encoding one, two, three, four,
five, ten, fifteen, twenty or more such fusion proteins.
[0199] The present invention encompasses methods and compositions
for detecting, diagnosing, prognosing, and/or monitoring diseases
or disorders associated with aberrant BLyS or BLyS receptor
expression or inappropriate BLyS or BLyS receptor function in an
animal, preferably a mammal, and most preferably a human,
comprising, or alternatively consisting of, use of BLyS binding
polypeptides (including molecules which comprise, or alternatively
consist of, BLyS binding polypeptide fragments or variants thereof)
that specifically bind to BLyS. Diseases and disorders which can be
detected, diagnosed, prognosed and/or monitored with the BLyS
binding polypeptides include, but are not limited to, immune system
diseases or disorders (e.g., autoimmune diseases or disorders,
immunodeficiencies, lupus, glomerular nephritis, rheumatoid
arthritis, multiple sclerosis, graft vs. host disease, myasthenia
gravis, Hashimoto's disease, and immunodeficiency syndrome),
proliferative diseases or disorders (e.g., cancer, premalignant
conditions, benign tumors, hyperproliferative disorders, benign
proliferative disorders, leukemia, lymphoma, chronic lymphocytic
leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease,
T cell proliferative diseases and disorders, B cell proliferative
diseases and disorders, monocytic proliferative diseases or
disorders, acute myelogenous leukemia, macrophage proliferative
diseases and disorders, and carcinoma), infectious diseases (e.g.,
AIDS), and inflammatory disorders (e.g., asthma, allergic
disorders, and rheumatoid arthritis).
[0200] In specific embodiments, the present invention encompasses
methods and compositions for detecting, diagnosing, prognosing
and/or monitoring diseases or disorders associated with
hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some
immunodeficiencies). In other specific embodiments, the present
invention encompasses methods and compositions for detecting,
diagnosing, prognosing and/or monitoring diseases or disorders
associated with hypogammaglobulinemia (e.g., an
immunodeficiency).
[0201] The present invention further encompasses methods and
compositions for preventing, treating and/or ameliorating diseases
or disorders associated with aberrant BLyS or BLyS receptor
expression or inappropriate BLyS or BLyS receptor 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 BLyS binding polypeptides (including molecules
which comprise, or alternatively consist of, BLyS binding
polypeptide fragments or variants thereof) in an amount effective
to treat, prevent or ameliorate the disease or disorder. Diseases
and disorders which can be prevented, treated, and/or ameliorated
with the BLyS binding polypeptides include, but are not limited to,
immune system diseases or disorders (e.g., autoimmune diseases or
disorders, immunodeficiencies, lupus, glomerular nephritis,
rheumatoid arthritis, multiple sclerosis, graft vs. host disease,
myasthenia gravis, Hashimoto's disease, immunodeficiency syndrome,
hypogammaglobulinemia, and hypergammaglobulinemia), proliferative
diseases or disorders (e.g., cancer, premalignant conditions,
benign tumors, hyperproliferative disorders, benign proliferative
disorders, leukemia, lymphoma, chronic lymphocytic leukemia,
multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, T cell
proliferative diseases and disorders, B cell proliferative diseases
and disorders, monocytic proliferative diseases or disorders, acute
myelogenous leukemia, macrophage proliferative diseases and
disorders, and carcinoma), infectious diseases (e.g., AIDS), and
inflammatory disorders (e.g., asthma, allergic disorders, and
rheumatoid arthritis).
[0202] In specific embodiments, the present invention encompasses
methods and compositions (e.g., BLyS binding polypeptides that
antagonize BLyS activity) for preventing, treating and/or
ameliorating diseases or disorders associated with
hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some
immunodeficiency syndromes). In other specific embodiments, the
present invention encompasses methods and compositions (e.g., BLyS
binding polypeptides that enhance BLyS activity) for preventing,
treating or ameliorating diseases or disorders associated with
hypogammaglobulinemia (e.g., an immunodeficiency syndrome).
[0203] In specific embodiments, the present invention encompasses
methods and compositions (e.g., BLyS binding polypeptides that
antagonize BLyS activity) for preventing, treating and/or
ameliorating immune system diseases or disorders, comprising, or
alternatively consisting of, administering to an animal in which
such treatment, prevention, and/or amelioration is desired, a BLyS
binding polypeptide in an amount effective to treat, prevent and/or
ameliorate the disease or disorder.
[0204] In specific embodiments, the present invention encompasses
methods and compositions (e.g., BLyS binding polypeptides that
antagonize BLyS activity) for preventing, treating and/or
ameliorating diseases or disorders of cells of hematopoietic
origin, comprising, or alternatively consisting of, administering
to an animal in which such treatment, prevention, and/or
amelioration is desired, a BLyS binding polypeptide in an amount
effective to treat, prevent and/or ameliorate the disease or
disorder.
[0205] Autoimmune disorders, diseases, or conditions that may be
detected, diagnosed, prognosed, monitored, treated, prevented,
and/or ameliorated using the BLyS binding polypeptides include, but
are not limited to, autoimmune hemolytic anemia, autoimmune
neonatal thrombocytopenia, idiopathic thrombocytopenia purpura,
autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, gluten-sensitive
enteropathy, 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, myocarditis, IgA glomerulonephritis, dense deposit
disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin
dependent diabetes mellitis, 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, schleroderma 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 mellitus, 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, granulomatous,
degenerative, and atrophic disorders).
[0206] Immunodeficiencies that may be detected, diagnosed,
prognosed, monitored, treated, prevented, and/or ameliorated using
the BLyS binding polypeptides 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.
[0207] The present invention further encompasses methods and
compositions for inhibiting or reducing immunoglobulin production,
comprising, or alternatively consisting of, contacting an effective
amount of BLyS binding polypeptide with BLyS, wherein the effective
amount of BLyS binding polypeptide inhibits or reduces BLyS
mediated immunoglobulin production.
[0208] 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 BLyS
binding polypeptide in an amount effective to inhibit or reduce
immunoglobulin production.
[0209] The present invention further encompasses methods and
compositions for inhibiting or reducing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of BLyS binding polypeptide with BLyS, wherein the effective
amount of BLyS binding polypeptide inhibits or reduces BLyS
mediated B cell proliferation.
[0210] The present invention further encompasses methods and
compositions for inhibiting or reducing B cell proliferation
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a BLyS
binding polypeptide in an amount effective to inhibit or reduce B
cell proliferation.
[0211] The present invention further encompasses methods and
compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of BLyS binding polypeptide with BLyS, wherein the effective
amount of BLyS binding polypeptide inhibits or reduces BLyS
mediated B cell activation.
[0212] The present invention further encompasses methods and
compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a BLyS
binding polypeptide in an amount effective to inhibit or reduce B
cell activation.
[0213] The present invention further encompasses methods and
compositions for decreasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide inhibits or reduces BLyS regulated lifespan of
B cells.
[0214] The present invention further encompasses methods and
compositions for decreasing lifespan of B cells, comprising, or
alternatively consisting of, administering to an animal in which
such decrease is desired, a BLyS binding polypeptide in an amount
effective to decrease B cell lifespan.
[0215] The present invention further encompasses methods and
compositions for inhibiting or reducing graft rejection,
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a BLyS
binding polypeptide in an amount effective to inhibit or reduce
graft rejection.
[0216] The present invention further encompasses methods and
compositions for killing cells of hematopoietic origin, comprising,
or alternatively consisting of, contacting BLyS binding
polypeptides with BLyS to form a complex; and contacting the
complex with cells of hematopoietic origin.
[0217] 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 BLyS binding polypeptide in an amount
effective to kill cells of hematopoietic origin.
[0218] The present invention further encompasses methods and
compositions for stimulating immunoglobulin production, comprising,
or alternatively consisting of, contacting an effective amount of
BLyS binding polypeptide with BLyS, wherein the effective amount of
the BLyS binding polypeptide stimulates BLyS mediated
immunoglobulin production.
[0219] 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 BLyS binding polypeptide in an
amount effective to stimulate immunoglobulin production.
[0220] The present invention further encompasses methods and
compositions for stimulating B cell proliferation, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide stimulates BLyS mediated B cell
proliferation.
[0221] The present invention further encompasses methods and
compositions for stimulating B cell proliferation, comprising, or
alternatively consisting of, administering to an animal in which
such stimulation is desired, a BLyS binding polypeptide in an
amount effective to stimulate B cell proliferation.
[0222] The present invention further encompasses methods and
compositions for increasing activation of B cells, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide increases BLyS mediated activation of B
cells.
[0223] The present invention further encompasses methods and
compositions for increasing activation of B cells, comprising, or
alternatively consisting of, administering to an animal in which
such increase is desired, a BLyS binding polypeptide in an amount
effective to increase B cell activation.
[0224] The present invention further encompasses methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide increases BLyS regulated lifespan of B
cells.
[0225] The present invention further encompasses methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, administering to an animal in which
such increase is desired, a BLyS binding polypeptide in an amount
effective to increase lifespan of B cells.
[0226] Definitions
[0227] In order that the invention may be clearly understood, the
following terms are defined:
[0228] The term "recombinant" is used to describe non-naturally
altered or manipulated nucleic acids, host cells transfected with
exogenous nucleic acids, or polypeptide molecules that are
expressed non-naturally, through manipulation of isolated nucleic
acid (typically, DNA) and transformation or transfection of host
cells. "Recombinant" is a term that specifically encompasses
nucleic acid molecules that have been constructed in vitro using
genetic engineering techniques, and use of the term "recombinant"
as an adjective to describe a molecule, construct, vector, cell,
polypeptide or polynucleotide specifically excludes naturally
occurring such molecules, constructs, vectors, cells, polypeptides
or polynucleotides.
[0229] The term "bacteriophage" is defined as a bacterial virus
containing a nucleic acid core and a protective shell built up by
the aggregation of a number of different protein molecules. The
terms "bacteriophage" and "phage" are synonymous and are used
herein interchangeably.
[0230] The term "affinity ligand" is sometimes used herein and is
synonymous with BLyS binding polypeptides.
[0231] The term "BLyS protein" as used herein encompasses both the
membrane (e.g., SEQ ID NOs: 173 and 174) and soluble forms (e.g.,
amino acids 134-285 of SEQ ID NO: 173) of BLyS. BLyS protein may be
monomeric, dimeric, or trimeric or multivalent. Preferably, BLyS
proteins are homotrimeric.
[0232] The term "BLyS-like polypeptide" as used herein encompasses
natural BLyS or full-length recombinant BLyS as well as fragments
and variants thereof, such as, a modified or truncated form of
natural BLyS or full-length recombinant BLyS, which BLyS and
BLyS-like polypeptide retain a BLyS functional activity. BLyS or
BLyS fragments that may be specifically bound by the compositions
useful according to the invention include, but are not limited to,
human BLyS (SEQ ID NOs: 173 and/or 174) or BLyS expressed on human
monocytes; murine BLyS (SEQ ID NOs: 175 and/or 176) or BLyS
expressed on murine monocytes; rat BLyS (either the soluble forms
as given in SEQ ID NOs: 177, 178, 179 and/or 180 or in a membrane
associated form, e.g., on the surface of rat monocytes); or monkey
BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NOS: 181 and/or
182, the soluble form of monkey BLyS, or BLyS expressed on monkey
monocytes) or fragments thereof. Preferably compositions useful
according to the invention bind human BLyS (SEQ ID NOs: 173 and/or
174) or fragments thereof. BLyS and BLyS-like polypeptides retain
at least one functional activity of the natural or full-length
BLyS, including but not limited to the following activities:
binding to BLyS receptor (e.g., TACI (GenBank accession number
AAC51790), and BCMA (GenBank accession number NP.sub.13 001183)),
stimulating B cell proliferation, stimulating immunoglobulin
secretion by B cells, stimulating the BLyS receptor signaling
cascade and/or being bound by an anti-BLyS antibody or other BLyS
binding polypeptide. Assays that can be used to determine the
functional activities of BLyS or BLyS like polypeptides can readily
be determined by one skilled in the art (e.g., see assays disclosed
in Moore et al., 1999, supra) "BLyS-like polypeptides" also include
fusion polypeptides in which all or a portion of BLyS is fused or
conjugated to another polypeptide. BLyS-like polypeptides that are
fusion polypeptides retain at least one functional activity of
BLyS, preferably the ability to stimulate B lymphocytes (see, for
example, Moore et al., Science, 285: 260-263 (1999)), to bind the
BLyS receptors (e.g., TACI or BCMA), and/or to be bound by an
anti-BLyS antibody or other BLyS binding polypeptide. BLyS fusion
polypeptides may be made by recombinant DNA techniques in which a
gene or other polynucleotide coding sequence for BLyS or a fragment
thereof is ligated in-frame (recombined) with the coding sequence
of another protein or polypeptide. The resulting recombinant DNA
molecule is then inserted into any of a variety of plasmid or phage
expression vectors, which enable expression of the fusion protein
molecule in an appropriate eukaryotic or prokaryotic host cell.
BLyS fusion polypeptides may be generated by synthetic or
semi-synthetic procedures as well.
[0233] The terms "BLyS target" or "BLyS target protein" are
sometimes used herein and encompass BLyS and/or BLyS-like
polypeptides. Thus, the BLyS binding polypeptides used according to
the methods of the invention bind "BLyS target proteins" and can be
used to bind, detect, remove, and/or purify "BLyS target
proteins."
[0234] The term "binding polypeptide" is used herein to refer to
any polypeptide capable of forming a binding complex with another
molecule, polypeptide, peptidomimetic or transformant.
[0235] A "BLyS binding polypeptide" is a molecule that can bind
BLyS target protein. Non-limiting examples of BLyS binding
polypeptides useful in the methods of the invention are the
polypeptide molecules having an amino acid sequence described
herein (see SEQ ID NOs: 1-12, 20-172, and 186-444). The term BLyS
binding polypeptide also encompasses BLyS binding fragments and
variants (including derivatives) of polypeptides having the
specific amino acid sequences described herein (SEQ ID NOs: 1-12,
20-172, and 186-444). By "variant" of an amino acid sequence as
described herein is meant a polypeptide that binds BLyS, but does
not necessarily comprise an identical or similar amino acid
sequence of a BLyS binding polypeptide specified herein. BLyS
binding polypeptides useful according to the invention which are
variants of a BLyS binding polypeptide specified herein satisfy at
least one of the following: (a) a polypeptide comprising, or
alternatively consisting of, an amino acid sequence that is at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95% least
99%, or 100% identical to the amino acid sequence of a BLyS binding
polypeptide sequence disclosed herein (SEQ ID NOs: 1-12, 20-172,
and 186-444), (b) a polypeptide encoded by a nucleotide sequence,
the complementary sequence of which hybridizes under stringent
conditions to a nucleotide sequence encoding a BLyS binding
polypeptide disclosed herein (e.g., a nucleic acid sequence
encoding the amino acid sequence of SEQ ID NOs: 1-12, 20-172, and
186-444), and/or a fragment of a BLyS binding polypeptide disclosed
herein, of at least 5 amino acid residues, at least 10 amino acid
residues, at least 15 amino acid residues, or at least 20 amino
acid residues. BLyS binding polypeptides useful according to the
invention also encompass polypeptide sequences that have been
modified for various applications provided that such modifications
do not eliminate the ability to bind a BLyS target. Specific,
non-limiting examples of modifications contemplated include
C-terminal or N-terminal amino acid substitutions or peptide chain
elongations for the purpose of linking the BLyS binder to a
chromatographic material or other solid support. Other
substitutions contemplated herein include substitution of one or
both of a pair of cysteine residues that normally form disulfide
links, for example with non-naturally occurring amino acid residues
having reactive side chains, for the purpose of forming a more
stable bond between those amino acid positions than the former
disulfide bond. All such modified binding polypeptides are also
considered BLyS binding polypeptides so long as the modified
polypeptides retain the ability to bind BLyS and/or BLyS-like
polypeptides, and therefore, may be used in one or more of the
various methods described herein, such as, to detect, purify, or
isolate BLyS or BLyS-like polypeptides in or from a solution. BLyS
binding polypeptides also include variants of the specific BLyS
binding polypeptide sequences disclosed herein (e.g., SEQ ID NOs:
1-12, 20-172, and 186-444) which have an amino acid sequence
corresponding to one of these polypeptide sequences, but in which
the polypeptide sequence is altered by substitutions, additions or
deletions that provide for molecules that bind BLyS. Thus, the BLyS
binding polypeptides include polypeptides containing, as a primary
amino acid sequence, all or part of the particular BLyS binding
polypeptide sequence including altered sequences in which
functionally equivalent amino acid residues are substituted for
residues within the sequence, resulting in a peptide which is
functionally active. For example, one or more amino acid residues
within the sequence can be substituted by another amino acid of a
similar polarity which acts as a functional equivalent, resulting
in a silent alteration. Conservative substitutions for an amino
acid within the sequence may be selected from other members of the
class to which the amino acid belongs. For example, the nonpolar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine, and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid. Such BLyS binding polypeptides can be made
either by chemical peptide synthesis or by recombinant production
from a nucleic acid encoding the BLyS binding polypeptide which
nucleic acid has been mutated. Any technique for mutagenesis known
in the art can be used, including but not limited to, chemical
mutagenesis, in vitro site-directed mutagenesis (Hutchinson et al.,
J. Biol. Chem., 253:6551 (1978)), use of TAB.RTM. linkers
(Pharmacia), etc.
[0236] As used and understood herein, percent homology or percent
identity of two amino acid sequences or of two nucleic acid
sequences is determined using the algorithm of Karlin and Atschul
(Proc. Natl. Acad. Sci. USA, 87: 2264-2268 (1990)), modified as in
Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873-5877
(1993)). Such an algorithm is incorporated into the NBLAST and
XBLAST programs of Altschul et al. (J. Mol. Biol., 215: 403-410
(1990)). BLAST nucleotide searches are performed with the NBLAST
program to obtain nucleotide sequences homologous to a nucleic acid
molecule described herein. BLAST protein searches are performed
with the XBLAST program to obtain amino acid sequences homologous
to a reference polypeptide. To obtain gapped alignments for
comparison purposes, Gapped BLAST is utilized as described in
Altschul et al. (Nucleic Acids Res., 25: 3389-3402 (1997)). When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) are used. See,
http://www.ncbi.nlm.nih.gov. Alternatively, the percent identity of
two amino acid sequences or of two nucleic acid sequences can be
determined once the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the sequence of a first
amino acid or nucleic acid sequence for optimal alignment with a
second amino acid or nucleic acid sequence). The amino acid
residues or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide at the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., %
identity=number of identical overlapping positions/total number of
positions.times.100%). In one embodiment, the two sequences are the
same length.
[0237] The term "polypeptide", as used herein, refers to a linear,
branched, or cyclic (e.g., all containing a loop structure) polymer
of two or more amino acid residues linked with a peptide bond. The
term "polypeptide" is not restricted to any particular upper limit
of amino acid residues. Thus, the BLyS affinity ligands that
comprise an amino acid sequence described herein are properly
referred to as "BLyS binding polypeptides" because such binding
polypeptides contain at least two amino acid residues held together
by a peptide bond, even though such molecules may also contain one
or more additional moieties or groups that are not amino acids,
such as N-terminal and/or C-terminal capping or functional groups,
and that may or may not be involved in a peptide bond. The
polypeptides may be monovalent, divalent, trivalent, or multivalent
and may comprise one or more of the BLyS binding polypeptides
having the amino acid sequence of SEQ ID NOs: 1-12, 20-172, and
186-444 and/or fragments or variants thereof. The term "peptide" is
used herein to have the same meaning as "polypeptide."
[0238] 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. As such, the
term antibody encompasses not only whole antibody molecules, but
also antibody fragments as well as variants (including derivatives)
of antibodies and antibody fragments. Examples of molecules which
are described by the term "antibody" in this application include,
but are not limited to: single chain Fvs (scFvs), Fab fragments,
Fab' fragments, F(ab').sub.2, disulfide linked Fvs (sdFvs), Fvs,
and fragments comprising or alternatively consisting of, either a
VL or a VH domain. The term "single chain Fv" or "scFv" as used
herein refers to a polypeptide comprising a VL domain of antibody
linked to a VH domain of an antibody.
[0239] "Feed stream": BLyS and BLyS-like polypeptides that are
bound by a BLyS binding polypeptide of this invention may be
produced by any method known in the art, including, but not limited
to, chemical synthesis; production in transformed host cells;
secretion into culture medium by naturally occurring cells or
recombinantly transformed bacteria, yeasts, fungi, insect cells,
plant cells, and mammalian cells; production in genetically
engineered organisms (for example, transgenic mammals); and
production in non-genetically engineered organisms. The solution,
sample, or mixture that contains a BLyS or BLyS-like polypeptide as
it is produced or is found present in a production solution will
sometimes be referred to as the "feed stream".
[0240] The term "binding" refers to the determination by standard
techniques that a binding polypeptide recognizes and binds to a
given target. Such standard techniques include, but are not limited
to, affinity chromatography, equilibrium dialysis, gel filtration,
enzyme linked immunosorbent assay (ELISA), FACS analysis, and the
monitoring of spectroscopic changes that result from binding, e.g.,
using fluorescence anisotropy, either by direct binding
measurements or competition assays with another binder.
[0241] The term "specificity" refers to a binding polypeptide
useful according to the invention that has a higher binding
affinity for one target over another. Thus, the term "BLyS target
protein specificity" refers to a molecule having a higher affinity
for BLyS target protein as compared with another molecule that is
not a BLyS target protein.
[0242] The term "epitopes" as used herein refers to portions of
BLyS having antigenic or immunogenic activity in an animal,
preferably a mammal. An epitope having immunogenic activity is a
portion of BLyS that elicits an antibody response in an animal. An
epitope having antigenic activity is a portion of BLyS to which an
antibody or BLyS binding polypeptide specifically binds as
determined by any method known in the art, for example, by the
immunoassays described herein. Antigenic epitopes need not
necessarily be immunogenic.
[0243] The term "fragment" as used herein refers to a polypeptide
comprising an amino acid sequence of at least 5 amino acid
residues, at least 6 amino acid residues, at least 7 amino acid
residues, at least 8 amino acid residues, at least 9 amino acid
residues, at least 10 amino acid residues, at least 11 amino acid
residues, at least 12 amino acid residues, at least 13 amino acid
residues, at least 14 amino acid residues, at least 15 amino acid
residues, at least 16 amino acid residues, at least 17 amino acid
residues, at least 18 amino acid residues, at least 19 amino acid
residues, at least 20 amino acid residues, at least 21 amino acid
residues, at least 22 amino acid residues, at least 23 amino acid
residues, at least 24 amino acid residues, or at least 25 amino
acid residues of the amino acid sequence of BLyS, or a BLyS binding
polypeptide (including molecules that comprise, or alternatively
consist of, BLyS binding polypeptide fragments or variants
thereof).
[0244] The term "fusion protein" as used herein refers to a
polypeptide that comprises, or alternatively consists of, an amino
acid sequence of a BLyS binding polypeptide and an amino acid
sequence of a heterologous polypeptide (i.e., a polypeptide
unrelated to the BLyS binding polypeptide).
[0245] The term "host cell" as used herein refers to the particular
subject cell transfected with a nucleic acid molecule and the
progeny or potential progeny of such a cell. Progeny may not be
identical to the parent cell transfected with the nucleic acid
molecule due to mutations or environmental influences that may
occur in succeeding generations or integration of the nucleic acid
molecule into the host cell genome.
[0246] Other terms are defined as necessary in the text below.
DETAILED DESCRIPTION OF THE INVENTION
[0247] The present invention provides methods and compositions for
detecting, diagnosing, prognosing, and/or monitoring diseases or
disorders associated with aberrant BLyS or BLyS receptor expression
or inappropriate BLyS or BLyS receptor function in an animal,
preferably a mammal, and most preferably a human, comprising, or
alternatively consisting of, use of BLyS binding polypeptides
(including molecules which comprise, or alternatively consist of,
BLyS binding polypeptide fragments or variants thereof) that
specifically bind to BLyS. Diseases and disorders which can be
detected, diagnosed, prognosed and/or monitored with the BLyS
binding polypeptides include, but are not limited to, immune system
diseases or disorders (e.g., autoimmune diseases or disorders,
immunodeficiencies, lupus, glomerular nephritis, rheumatoid
arthritis, multiple sclerosis, graft vs. host disease, myasthenia
gravis, Hashimoto's disease, and immunodeficiency syndrome),
proliferative diseases or disorders (e.g., cancer, premalignant
conditions, benign tumors, hyperproliferative disorders, benign
proliferative disorders, leukemia, lymphoma, chronic lymphocytic
leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease,
T cell proliferative diseases and disorders, B cell proliferative
diseases and disorders, monocytic proliferative diseases or
disorders, acute myelogenous leukemia, macrophage proliferative
diseases and disorders, and carcinoma), infectious diseases (e.g.,
AIDS), and inflammatory disorders (e.g., asthma, allergic
disorders, and rheumatoid arthritis).
[0248] The present invention further encompasses methods and
compositions for preventing, treating and/or ameliorating diseases
or disorders associated with aberrant BLyS or BLyS receptor
expression or inappropriate BLyS or BLyS receptor 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 BLyS binding polypeptides (including molecules
which comprise, or alternatively consist of, BLyS binding
polypeptide fragments or variants thereof) in an amount effective
to treat, prevent or ameliorate the disease or disorder. Diseases
and disorders which can be prevented, treated, and/or ameliorated
with the BLyS binding polypeptides include, but are not limited to,
immune system diseases or disorders (e.g., autoimmune diseases or
disorders, immunodeficiencies, lupus, glomerular nephritis,
rheumatoid arthritis, multiple sclerosis, graft vs. host disease,
myasthenia gravis, Hashimoto's disease, immunodeficiency syndrome,
hypogammaglobulinemia, and hypergammaglobulinemia), proliferative
diseases or disorders (e.g., cancer, premalignant conditions,
benign tumors, hyperproliferative disorders, benign proliferative
disorders, leukemia, lymphoma, chronic lymphocytic leukemia,
multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, T cell
proliferative diseases and disorders, B cell proliferative diseases
and disorders, monocytic proliferative diseases or disorders, acute
myelogenous leukemia, macrophage proliferative diseases and
disorders, and carcinoma), infectious diseases (e.g., AIDS), and
inflammatory disorders (e.g., asthma, allergic disorders, and
rheumatoid arthritis).
[0249] BLyS Binding Polypeptides
[0250] The methods of the present invention may be performed
utilizing new polypeptides and families of polypeptides that
specifically bind to B lymphocyte stimulator protein (BLyS) and/or
BLyS-like polypeptides. In particular, the invention encompasses
diagnostic and therapeutic uses for polypeptides that specifically
bind to a polypeptide or polypeptide fragment of human BLyS (SEQ ID
NOs: 173 and/or 174) or BLyS expressed on human monocytes; murine
BLyS (SEQ ID NOs: 175 and/or 176) or BLyS expressed on murine
monocytes; rat BLyS (either the soluble forms as given in SEQ ID
NOs: 177, 178, 179 and/or 180 or in a membrane associated form,
e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the
monkey BLyS polypeptides of SEQ ID NOS: 181 and/or 182, the soluble
form of monkey BLyS, or BLyS expressed on monkey monocytes);
preferably human BLyS.
[0251] In preferred embodiments, the BLyS binding polypeptides used
according to the present invention (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof), specifically bind to BLyS and do not
cross-react with any other antigens. In more preferred embodiments,
the BLyS binding polypeptides specifically bind to BLyS and do not
cross-react with TRAIL (Hahne et al., J. Exp. Med., 188(6): 1185-90
(1998)), APRIL (Wilet et al., Immunity, 3(6):673-82 (1995)),
Endokine-alpha (Kwon et al., J. Biol. Chem., 274(10):6056-61
(1999)), TNF-alpha, TNF-beta (Nedwin et al., J. Immunol.,
135(4):2492-7 (1985)), Fas-L (Suda et al., Cell, 75(6):1169-78
(1993)), or LIGHT (Mauri et al., Immunity, 8(1):21-30 (1998)).
[0252] Many BLyS binding polypeptides have been discovered which
may be used in the methods of the present invention. Specific BLyS
binding polypeptides for use in the present invention comprise, or
alternatively consist of, an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-12, 20-172, and 186-444,
Preferably SEQ ID NOs: 163-172 or 436-444 as referred to above and
in Tables 1-8, 13 and 14. In its broadest aspects, the methods of
the present invention may be carried out using a polypeptide
capable of binding to BLyS and comprising the polypeptide
Asp-Xaa-Leu-Thr (SEQ ID NO: 446), where Xaa is Pro, Ser, Thr, Phe,
Leu, Tyr, Cys, or Ala preferably Pro or Ser).
[0253] Additional polypeptides for use in the methods described
herein include polypeptides with the potential to form a cyclic or
loop structure between invariant Cys residues comprising, or
alternatively consisting of, an amino acid sequence selected from
A-E (SEQ ID NOs: 1-5):
[0254] (A)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-Phe-X.sub.7-Trp-Glu-Cys-X.s-
ub.11-X.sub.12-X.sub.13 (SEQ ID NO: 1),
[0255] wherein
[0256] X.sub.1 is Ala, Asn, Lys, or Ser;
[0257] X.sub.2 is Ala, Glu, Met, Ser, or Val;
[0258] X.sub.3 is Ala, Asn, Lys, or Pro (preferably Lys);
[0259] X.sub.5 is Phe, Trp, or Tyr (preferably Tyr);
[0260] X.sub.7 is Pro or Tyr (preferably Pro);
[0261] X.sub.11 is Ala, Gln, His, Phe, or Val;
[0262] X.sub.12 is Asn, Gln, Gly, His, Ser, or Val; and
[0263] X.sub.13 is Ala, Asn, Gly, Ile, Pro, or Ser,
[0264] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0265] (B)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-Cys-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO: 2),
[0266] wherein
[0267] X.sub.1 is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, Val, or is absent;
[0268] X.sub.2 is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val;
[0269] X.sub.3 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val (preferably Asp);
[0270] X.sub.5 is Asp, Ile, Leu, or Tyr (preferably Asp or
Leu);
[0271] X.sub.6 is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr,
or Val (preferably Glu or Leu);
[0272] X.sub.7 is His, Leu, Lys, or Phe (preferably His or
Leu);
[0273] X.sub.8 is Leu, Pro, or Thr (preferably Thr or Pro);
[0274] X.sub.9 is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp
(preferably Lys);
[0275] X.sub.10 is Ala, Gln Glu, Gly, His, lie, Leu, Met, Phe, Ser,
Trp, Tyr, or Val;
[0276] X.sub.12 is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser,
Trp, Tyr, or Val;
[0277] X.sub.13 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and
[0278] X.sub.14 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile,
Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent,
[0279] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0280] (C)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-Cys-X.sub.13-X.sub.14-X.sub.15 (SEQ ID NO:
3),
[0281] wherein
[0282] X.sub.1 is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or
Thr;
[0283] X.sub.2 is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or
Trp;
[0284] X.sub.3 is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr
(preferably Ala);
[0285] X.sub.5 is Asn, Asp, Pro, Ser, or Thr (preferably Asp);
[0286] X.sub.6 is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably
Ile);
[0287] X.sub.7 is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val
or Leu);
[0288] X.sub.8 is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably
Thr);
[0289] X.sub.9 is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr
(preferably Leu);
[0290] X.sub.10 is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met,
Pro, Ser, or Trp;
[0291] X.sub.11 is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr
(preferably Ser);
[0292] X.sub.13 is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val
(preferably Val);
[0293] X.sub.14 is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr;
and
[0294] X.sub.15 is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr
(preferably Glu or Pro),
[0295] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0296] (D)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-X.sub.12-Cys-X.sub.14-X.sub.15-X.sub.16 (SEQ
ID NO: 4),
[0297] wherein
[0298] X.sub.1 is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is
absent (preferably Ser);
[0299] X.sub.2 is Arg, Asn, Asp, His, Phe, Ser, or Trp (preferably
Arg);
[0300] X.sub.3 is Asn, Asp, Leu, Pro, Ser, or Val (preferably Asn
or Asp);
[0301] X.sub.5 is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or
Thr;
[0302] X.sub.6 is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;
[0303] X.sub.7 is Asp, His, Leu, or Ser (preferably Asp);
[0304] X.sub.8 is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr
(preferably Glu or Pro);
[0305] X.sub.9 is Ala, Arg, Asn, or Leu (preferably Leu);
[0306] X.sub.10 is Ile, Leu, Met, Pro, Ser, or Thr (preferably
Thr);
[0307] X.sub.11 is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;
[0308] X.sub.12 is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or
Val;
[0309] X.sub.14 is Asp, Gly, Leu, Phe, Tyr, or Val (preferably
Leu);
[0310] X.sub.15 is Asn, His, Leu, Pro, or Tyr (preferably His, Leu
or Pro); and
[0311] X.sub.16 is Asn, Asp, His, Phe, Ser, or Tyr, (preferably Asp
or Ser),
[0312] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0313] (E)
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-Cys-X.sub.16-X.sub.17-X.-
sub.18 (SEQ ID NO: 5),
[0314] wherein
[0315] X.sub.1 is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr,
or is absent (preferably Arg);
[0316] X.sub.2 is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent
(preferably Asn, Asp, Gly, or Pro);
[0317] X.sub.3 is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or
Val (preferably Gly or Met);
[0318] X.sub.5 is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr,
or Val (preferably Trp, Tyr, or Val);
[0319] X.sub.6 is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or
Tyr (preferably Asp);
[0320] X.sub.7 is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr
(preferably Asp);
[0321] X.sub.8 is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr
(preferably Leu);
[0322] X.sub.9 is Asp, Leu, Pro, Thr, or Val (preferably Leu or
Thr);
[0323] X.sub.10 is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp
or Tyr (preferably Lys or Thr);
[0324] X.sub.11 is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or
Thr (preferably Arg or Leu);
[0325] X.sub.12 is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr,
Trp, or Tyr (preferably Thr or Trp);
[0326] X.sub.13 is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr,
Trp, or Tyr (preferably Met or Phe);
[0327] X.sub.14 is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro,
Ser, Thr, Tyr, or Val (preferably Val);
[0328] X.sub.16 is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or
Trp (preferably Met);
[0329] X.sub.17 is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or
Tyr, (preferably Arg, His, or Tyr); and
[0330] X.sub.18 is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp
(preferably His or Asn),
[0331] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides.
[0332] Additional BLyS binding polypeptides that may be used in the
methods of the present invention include linear polypeptides
comprising, or alternatively consisting of, an amino acid sequence
selected from F and G (SEQ ID NOs: 6 and 7):
[0333] (F)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-
-X.sub.9-X.sub.10-X.sub.11-X.sub.12 (SEQ ID NO: 6),
[0334] wherein
[0335] X.sub.1 is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr,
or Val (preferably Gly, Tyr, or Val);
[0336] X.sub.2 is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or
Tyr (preferably His or Tyr);
[0337] X.sub.3 is Ala, Asp, Lys, Phe, Thr, Trp or Tyr (preferably
Asp or Tyr);
[0338] X.sub.4 is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or
Val (preferably Asp or Gln);
[0339] X.sub.5 is Asp, Leu, Lys, Phe, Pro, Ser, or Val (preferably
Leu or Ser);
[0340] X.sub.6 is His, Ile, Leu, Pro, Ser, or Thr (preferably Leu
or Thr);
[0341] X.sub.7 is Arg, Gly, His, Leu, Lys, Met, or Thr (preferably
Lys or Thr);
[0342] X.sub.8 is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr
(preferably Leu or Lys);
[0343] X.sub.9 is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser,
Trp, Tyr, or Val (preferably Met or Ser);
[0344] X.sub.10 is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val
(preferably Thr or Leu);
[0345] X.sub.1 is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr,
Trp, Tyr, or Val (preferably Pro or Thr); and
[0346] X.sub.12 is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser,
Trp, Tyr, or Val (preferably Arg or Pro),
[0347] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides; or
[0348] (G)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-
-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO: 7),
[0349] wherein
[0350] X.sub.1 is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp
(preferably Glu, Lys);
[0351] X.sub.2 is Arg, Gln, His, Ile, Leu, or Pro (preferably His
or Pro);
[0352] X.sub.3 is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably
Tyr);
[0353] X.sub.4 is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr
(preferably Asp or Gln);
[0354] X.sub.5 is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val
(preferably Asn or Thr);
[0355] X.sub.6 is Asp, Glu, His, Leu, Lys, Pro, or Val (referably
Asp or Pro);
[0356] X.sub.7 is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr
(preferably Ile or Pro);
[0357] X.sub.8 is Gln, Gly, His, Leu, Met, Ser, or Thr (preferably
Leu or Thr);
[0358] X.sub.9 is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr
(preferably Lys);
[0359] X.sub.10 is Ala, Gly, Ile, Leu, Lys, Met, or Phe (preferably
Gly or Met);
[0360] X.sub.11 is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp,
Tyr, or Val (preferably Ala or Thr);
[0361] X.sub.12 is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val
preferably Arg or His); and
[0362] X.sub.13 is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val
(preferably His),
[0363] wherein said polypeptide binds BLyS and/or BLyS-like
polypeptides.
[0364] Additional BLyS binding polypeptides that may be used in the
methods of the present invention include BLyS binding polypeptides
comprising, or alternatively consisting of, an amino acid sequence
selected from H-L (SEQ ID NOs: 8-12):
[0365] (H) Cys-X.sub.2-Phe-X.sub.4-Trp-Glu-Cys (SEQ ID NO: 8),
[0366] wherein
[0367] X.sub.2 is Phe, Trp, or Tyr (preferably Tyr); and
[0368] X.sub.4 is Pro or Tyr (preferably Pro); or
[0369] (I) Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-Cys
(SEQ ID NO: 9),
[0370] wherein
[0371] X.sub.2 is Asp, Ile, Leu, or Tyr (preferably Asp or
Leu);
[0372] X.sub.3 is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr,
or Val (preferably Glu or Leu);
[0373] X.sub.4 is His, Leu, Lys, or Phe (preferably His or
Leu);
[0374] X.sub.5 is Leu, Pro, or Thr (preferably Thr or Pro);
[0375] X.sub.6 is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp
(preferably Lys); and
[0376] X.sub.7 is Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,
Ser, Trp, Tyr, or Val; or
[0377] (J)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-Cys
(SEQ ID NO: 10),
[0378] wherein
[0379] X.sub.2 is Asn, Asp, Pro, Ser, or Thr (preferably Asp);
[0380] X.sub.3 is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably
Ile);
[0381] X.sub.4 is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val
or Leu);
[0382] X.sub.5 is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably
Thr);
[0383] X.sub.6 is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr
(preferably Leu);
[0384] X.sub.7 is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro,
Ser, or Trp;
[0385] X.sub.8 is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr
(preferably Ser); or
[0386] (K)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-Cys (SEQ ID NO: 11),
[0387] wherein
[0388] X.sub.2 is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or
Thr;
[0389] X.sub.3 is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;
[0390] X.sub.4 is Asp, His, Leu, or Ser (preferably Asp);
[0391] X.sub.5 is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr
(preferably Glu or Pro);
[0392] X.sub.6 is Ala, Arg, Asn, or Leu (preferably Leu);
[0393] X.sub.7 is Ile, Leu, Met, Pro, Ser, or Thr (preferably
Thr);
[0394] X.sub.8 is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;
[0395] X.sub.9 is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or
Val; or
[0396] (L)
Cys-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-Cys (SEQ ID NO: 12),
[0397] wherein
[0398] X.sub.2 is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr,
or Val (preferably Trp, Tyr, or Val);
[0399] X.sub.3 is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or
Tyr (preferably Asp);
[0400] X.sub.4 is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr
(preferably Asp);
[0401] X.sub.5 is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr
(preferably Leu);
[0402] X.sub.6 is Asp, Leu, Pro, Thr, or Val (preferably Leu or
Thr);
[0403] X.sub.7 is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp
or Tyr (preferably Lys or Thr);
[0404] X.sub.8 is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or
Thr (preferably Arg or Leu);
[0405] X.sub.9 is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp,
or Tyr (preferably Thr or Trp);
[0406] X.sub.10 is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr,
Trp, or Tyr (preferably Met or Phe);
[0407] X.sub.11 is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro,
Ser, Thr, Tyr, or Val (preferably Val);
[0408] wherein said polypeptides bind BLyS and/or BLyS-like
polypeptides.
[0409] Additional BLyS binding polypeptides that may be used in the
methods of the present invention include linear polypeptides
comprise the following amino acid sequence M (SEQ ID NO: 447):
[0410] (M)
Ala-X.sub.2-X.sub.3-X.sub.4-Asp-X.sub.6Leu-Thr-X.sub.9Leu-X.sub-
.11-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO: 447),
[0411] wherein
[0412] X.sub.2 is Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys,
Leu, Met, Thr, Val, Glu, Ala, Gly, Cys, or Trp (i.e., any amino
acid except Arg; preferably Asn);
[0413] X.sub.3 is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser
(preferably Trp);
[0414] X.sub.4 is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);
[0415] X.sub.6 is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala
(preferably Pro or Ser);
[0416] X.sub.9 is Lys, Asn, Gln, Gly, or Arg (preferably Lys);
[0417] X.sub.11 is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably
Trp);
[0418] X.sub.12 is Leu, Phe, Val, Ile, or His (preferably Leu);
[0419] X.sub.13 is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val,
Ala, Cys, Ile, Phe, or Tyr (i.e., not Asp, Glu, Gly, Lys, Met, or
Trp; preferably Pro); and
[0420] X.sub.14 is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser,
Tyr, Ala, Cys, Lys, Ile, Thr or Leu (i.e., not Phe, Met, Pro, or
Trp; preferably Asp, Val or Glu).
[0421] Preferred BLyS binding polypeptides that may be used in the
methods of the present invention include linear polypeptides
comprising a core sequence of the formula N:
[0422] (N)
X.sub.1-X.sub.2-Asp-X.sub.4-Leu-Thr-X.sub.7-Leu-X.sub.9-X.sub.1- 0
(SEQ ID NO: 448),
[0423] wherein
[0424] X.sub.1 is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser
(preferably Trp);
[0425] X.sub.2 is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);
[0426] X.sub.4 is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala
preferably Pro or Ser);
[0427] X.sub.7 is Lys, Asn, Gln, Gly, or Arg (preferably Lys);
[0428] X.sub.9 is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably
Tip); and
[0429] X.sub.10 is Leu, Phe, Val, Ile, or His (preferably Leu).
[0430] Especially preferred BLyS binding polypeptides that may be
used in the methods of the present invention include linear
polypeptides comprising the core peptide
Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 436).
[0431] In performing certain methods according to the present
invention, it is preferred that the BLyS binding polypeptides, or
phage displaying such binding polypeptides, irreversibly bind the
BLyS protein in its native, soluble trimeric form.
[0432] In performing certain methods according to the present
invention, it is preferred that the BLyS binding polypeptides of
the present invention, or phage displaying such binding
polypeptides, reversibly bind the BLyS protein in its native,
soluble trimeric form.
[0433] In performing certain methods according to the invention, it
may be advantageous for a BLyS binding polypeptide to bind BLys
target protein with high affinity. In specific embodiments, BLyS
binding polypeptides used in this invention will bind BLyS target
proteins with a dissociation constant of 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, BLyS binding polypeptides used in
the invention will bind BLyS targer proteins with a dissociaton
constant of 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.-8 M,
or 10.sup.-8 M. Even more preferably, BLyS binding polypeptides
used in the methods of the invention bind BLyS target proteins 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.-11 M, 5.times.10.sup.-12 M, or
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, or
10.sup.-15 M.
[0434] In certain preferred embodiments, BLyS binding polypeptides
reversibly bind BLyS and/or BLyS-like polypeptides and release
bound BLyS protein in an active form, preferably in the native
soluble trimeric form, under specific release conditions. In
specific embodiments, BLyS binding polypeptides bind BLyS target
proteins with off-rates or k.sub.off greater than or equal to
10.sup.-10 s.sup.-1, 5.times.10.sup.-9 s.sup.-1, 10.sup.-9
s.sup.-1, 5.times.10.sup.-8 s.sup.-1, 10.sup.-8 s.sup.-1,
5.times.10.sup.-7 s.sup.-1, 10.sup.-7 s.sup.-1, 5.times.10.sup.-6
s.sup.-1, 10.sup.-6 s.sup.-1, 5.times.10.sup.-5 s.sup.-1, 10.sup.-5
s.sup.-1, 5.times.10.sup.-4 s.sup.-1, 10.sup.-4 s.sup.-1,
5.times.10.sup.-3 s.sup.-1, 10.sup.-3 s.sup.-1, 5.times.10.sup.-2
s.sup.-1, 10.sup.-2 s.sup.-1, 5.times.10.sup.-1 s.sup.-1, or
10.sup.-1 s.sup.-1.
[0435] Binding experiments to determine K.sub.D and off-rates can
be performed in a number of conditions including, but not limited
to, [pH 6.0, 0.01% Tween 20], [pH 6.0, 0.1% gelatin], [pH5.0, 0.01%
Tween 20], [pH9.0, 0.1% Tween 20], [pH6.0, 15% ethylene glycol,
0.01% Tween20], [pH5.0, 15% ethylene glycol, 0.01% Tween 20], and
[pH9.0, 15% ethylene glycol, 0.01% Tween 20] The buffers in which
to make these solutions can readily be determined by one of skill
in the art, and depend largely on the desired pH of the final
solution. Low pH solutions (<pH 5.5) can be made, for example,
in citrate buffer, glycine-HCl buffer, or in succinic acid buffer.
High pH solutions can be made, for example, in Tris-HCl, phosphate
buffers, or sodium bicarbonate buffers. A number of conditions may
be used to determine K.sub.D and off-rates for the purpose of
determining, for example, optimal pH and/or salt
concentrations.
[0436] In certain embodiments, BLyS binding polypeptides reversibly
bind BLyS and/or BLyS-like polypeptides, preferably in the native
soluble, trimeric form.
[0437] In preferred embodiments, BLyS binding polypeptides
reversibly bind only the native soluble, trimeric form of BLyS.
[0438] In certain embodiments, BLyS binding polypeptides
irreversibly bind BLyS and/or BLyS-like polypeptides, preferably in
the native soluble, trimeric form.
[0439] In preferred embodiments, BLyS binding polypeptides
irreversibly bind only the native soluble, trimeric form of
BLyS.
[0440] In some screening or assay procedures, it is possible and
more convenient to use recombinant bacteriophage that display a
particular BLyS binding polypeptide instead of using isolated BLyS
binding polypeptide. Such procedures include phage-based ELISA
protocols and immobilization of phage displaying a binding
polypeptide to chromatographic materials. Such screening assays and
procedures are routine in the art and may be readily adapted for
procedures using recombinant bacteriophage such as disclosed
herein.
[0441] Specific methods of the present invention contemplate the
use of BLyS binding polypeptides that competitively inhibit the
binding of a BLyS binding molecule. Competitive inhibition can be
determined by any suitable method known in the art, for example,
using the competitive binding assays described herein. In preferred
embodiments, the polypeptide competitively inhibits the binding of
a BLyS binding molecule to BLyS 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%. In a more preferred embodiment, the BLyS binding
polypeptide competitively inhibits the binding of a BLyS binding
molecule to the native soluble trimeric form of BLyS, 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%.
[0442] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) useful in the practice of the methods of the
present invention may have one or more of the same biological
characteristics as one or more of the BLyS binding polypeptides
specifically described herein. By "biological characteristics" is
meant, the in vitro or in vivo activities or properties of the BLyS
binding polypeptides, such as, for example, the ability to bind to
BLyS (e.g., the soluble form of BLyS, the membrane-bound form of
BLyS, the soluble form and membrane-bound form of BLyS), and/or an
antigenic and/or epitope region of BLyS), the ability to
substantially block BLyS/BLyS receptor (e.g., TACI and BCMA)
binding, the ability to substantially increase BLyS/BLyS receptor
(e.g., TACI and BCMA) binding, the ability to block BLyS mediated
biological activity (e.g., stimulation of B cell proliferation and
immunoglobulin production), or, the ability to enhance or stimulate
BLyS mediated biological activity (e.g., stimulation of B cell
proliferation and immunoglobulin production). Optionally, the BLyS
binding polypeptides useful according to the invention will bind to
the same epitope as at least one of the BLyS binding polypeptides
specifically referred to herein. Such epitope binding can be
routinely determined using assays known in the art.
[0443] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) useful in the practice of the methods of the
present invention may be polypeptides that neutralize BLyS or a
fragment thereof. By a BLyS binding polypeptide that "neutralizes
BLyS or a fragment thereof" is meant a BLyS binding polypeptide
that inhibits (i.e., is effective to reduce or abolish) or
abolishes the ability of BLyS: to bind to its receptor (e.g., TACI
and BCMA), to stimulate B cell activation, to stimulate B cell
proliferation, to stimulate immunoglobulin secretion by B cells, to
increase B cell lifespan, and/or to stimulate the BLyS receptor
signalling cascade.
[0444] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) useful in the practice of the methods of the
present invention may also be effective to inhibit or abolish
BLyS-mediated B cell proliferation as determined by any method
known in the art such as, for example, the assays described in the
Examples, infra, said BLyS binding polypeptides comprising, or
alternatively consisting of, a polypeptide having an amino acid
sequence of any one of SEQ ID NOs: -1-2, 20-172, and 186-444,
preferably of SEQ ID NOs: 163-172 and 436-444, or a fragment or
variant thereof.
[0445] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) useful in the practice of the methods of the
present invention may also be effective to enhance the activity of
BLyS or a fragment thereof, said BLyS binding polypeptides
comprising, or alternatively consisting of, a polypeptide having an
amino acid sequence of any one of SEQ ID NOs: 1-12, 20-172, and
186-444, preferably of SEQ ID NOs: 163-172 or 436-444, or a
fragment or variant thereof. By a BLyS binding polypeptide that
"enhances the activity of BLyS or a fragment thereof" is meant a
BLyS binding polypeptide that increases the ability of BLyS: to
bind to its receptor (e.g., TACI and BCMA), to stimulate B cell
proliferation, to stimulate immunoglobulin secretion by B cells, to
activate B cells, to increase B cell lifespan and/or to stimulate a
BLyS 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)). Nucleic acid molecules encoding these
BLyS binding polypeptides are also encompassed by the
invention.
[0446] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) useful in the practice of the methods of the
present invention may also be effective to stimulate BLyS mediated
B cell proliferation as determined by any method known in the art,
such as, for example, the assays described in the Examples, infra,
said BLyS binding polypeptides comprising, or alternatively
consisting of, a polypeptide having an amino acid sequence of any
one of SEQ ID NOs: 1-12, 20-172, and 186-444, preferably of SEQ ID
NOs: 163-172 or 436-444, or a fragment or variant thereof. Nucleic
acid molecules encoding these BLyS binding polypeptides are also
encompassed by the invention.
[0447] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) useful in the practice of the methods of the
present invention may include polypeptides effective to
specifically bind to the soluble form of BLyS, polypeptides that
specifically bind to the membrane-bound form of BLyS, and
polypeptides that specifically bind to both the soluble form and
membrane-bound form of BLyS.
[0448] The methods of the present invention may also be carried out
using mixtures of BLyS binding polypeptides (including molecules
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) that specifically bind
to BLyS, wherein the mixture contains at least one, two, three,
four, five or more different BLyS binding polypeptides. In
particular, the invention provides for the use of mixtures of
different BLyS binding polypeptides that specifically bind to the
soluble form of BLyS, the membrane-bound form of BLyS, and/or both
the membrane-bound form and soluble form of BLyS. In specific
embodiments, the methods of the invention utilize mixtures of at
least 2, preferably at least 4, at least 6, at least 8, at least
10, at least 12, at least 15, at least 20, or at least 25 different
BLys binding polypeptides that specifically bind to BLyS, wherein
at least 1, at least 2, at least 4, at least 6, or at least 10,
BLyS binding polypeptides of the mixture are BLyS binding
polypeptides.
[0449] The methods of the present invention may also be carried out
using panels of BLyS binding polypeptides (including molecules
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) that specifically bind
to BLyS, wherein the panel has at least one, two, three, four, five
or more different BLyS binding polypeptides. In particular, the
invention provides for the use of panels of different BLyS binding
polypeptides that specifically bind to the soluble form of BLyS,
the membrane-bound form of BLyS, and/or both the membrane-bound
form and soluble form of BLyS. In specific embodiments, the
invention provides for the use of panels of BLyS binding
polypeptides that have different affinities for BLyS, different
specificities for BLyS, or different dissociation rates. The
invention provides for the use of panels of at least 10, preferably
at least 25, at least 50, at least 75, or at least 100 BLyS binding
polypeptides. Panels of BLyS binding polypeptides can be used, for
example, in 96 well plates for assays such as ELISAs.
[0450] The methods of the present invention may also be carried out
using compositions comprising one or more BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of
BLyS binding polypeptide fragments or variants). In one embodiment,
a composition used in a method of the present invention comprises,
one, two, three, four, five, or more BLyS binding polypeptides that
comprise or alternatively consist of, a polypeptide having an amino
acid sequence of any one or more of the BLyS binding polypeptides
contained in SEQ ID NOs: 1-12, 20-172, and 186-444 as disclosed in
Tables 1-8 and 13, or a variant thereof.
[0451] As discussed in more detail below, a composition useful in
the methods of the invention may be used either alone or in
combination with other compositions. The BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of
BLyS binding polypeptide fragments or variants of the present
invention) 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, BLyS binding
polypeptides 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, polypeptide
linkers, 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 0 396 387.
[0452] Production and Modification of BLyS Binding Polypeptides
[0453] BLyS binding polypeptides useful in practicing the methods
of the present invention may as be produced by chemical synthesis,
semi-synthetic methods, and recombinant DNA methodologies known in
the art.
[0454] In certain embodiments, BLyS binding polypeptides of the
present invention are produced by chemical or semi-synthetic
methodologies known in the art (see, Kelley et al. in Genetic
Engineering Principles and Methods, Setlow, J. K., ed. (Plenum
Press, N.Y., 1990), vol. 12, pp. 1-19; Stewart et al., Solid-Phase
Peptide Synthesis, W. H. Freeman Co., San Francisco, 1989). One
advantage of these methodologies is that they allow for the
incorporation of non-natural amino acid residues into the sequence
of the BLyS binding polypeptide.
[0455] In preferred embodiments, BLyS binding polypeptides are
chemically synthesized (see, e.g., Merrifield, J. Am. Chem. Soc.,
85: 2149 (1963); Houghten, Proc. Natl Acad. Sci. USA, 82: 5132
(1985)). For example, polypeptides can be synthesized by solid
phase techniques, cleaved from the resin, and purified by
preparative high performance liquid chromatography (see, e.g.,
Creighton, Proteins: Structures and Molecular Properties (W. H.
Freeman and Co., N.Y., 1983), pp. 50-60). BLyS binding polypeptides
can also be synthesized by use of a peptide synthesizer. The
composition of the synthetic polypeptides may be confirmed by amino
acid analysis or sequencing (e.g., the Edman degradation procedure;
see Creighton, Proteins: Structures and Molecular Properties (W. H.
Freeman and Co., N.Y., 1983), pp. 34-49). Furthermore, if desired,
BLyS binding polypeptides may contain non-classical amino acids or
chemical amino acid analogs, which can routinely be introduced
during chemical synthesis as a substitution or addition into the
BLyS binding polypeptides. Non-classical amino acids include, but
are not-limited to, the D-isomers of the common amino acids,
2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric
acid (4Abu), 2-aminobutyric acid (Abu), 6-aminohexanoic acid
(epsilon-Ahx), 2-aminoisobutyric acid (Aib), 3-amino propionic
acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,
citrulline, homocitrulline, cysteic acid, t-butylglycine,
t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine
(bAla), fluoro-amino acids, designer amino acids such as
beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl
amino acids, and amino acid analogs in general. Furthermore, the
amino acid can be D (dextrorotary) or L (levorotary).
[0456] Solid phase peptide synthesis begins at the carboxy (C)
terminus of the putative polypeptide by coupling a protected amino
acid to a suitable resin, which reacts with the carboxyl group of
the C-terminal amino acid to form a bond that is readily cleaved
later, for example, a halomethyl resin such as chloromethyl resin,
bromomethyl resin, hydroxymethyl resin, aminomethyl resin,
benzhydrylamine resin, or t-alkyloxycarbonyl-hydrazide resin. After
removal of the .alpha.-amino protecting group with, for example,
trifluoroacetic acid (TFA) in methylene chloride and neutralization
with, for example TEA, the next cycle in the synthesis is ready to
proceed. The remaining .alpha.-amino and, if necessary,
side-chain-protected amino acids are then coupled sequentially in
the desired order by condensation to obtain an intermediate
compound connected to the resin. Alternatively, some amino acids
may be coupled to one another forming an oligopeptide prior to
addition to the growing solid phase polypeptide chain.
[0457] The condensation between two amino acids, or an amino acid
and a peptide, or a peptide and a peptide can be carried out
according to condensation methods known in the art, including but
not limited to, the azide method, mixed acid anhydride method, DCC
(dicyclohexylcarbodiimide) method, active ester method
(p-nitrophenyl ester method, BOP [benzotriazole-1-yl-oxy-tris
(dimethylamino) phosphonium hexafluorophosphate] method,
N-hydroxysuccinic acid imido ester method), and Woodward reagent K
method.
[0458] Common to chemical synthesis of peptides is the protection
or capping (blocking) of the reactive side chain groups of the
various amino acid residues with suitable protecting or capping
groups at that site until the group is ultimately removed after the
polypeptide chain has been completely assembled. Also common is the
protection or capping of the .alpha.-amino group on an amino acid
or a fragment while that entity reacts at the carboxyl group
followed by the selective removal of the .alpha.-amino-protecting
group to allow subsequent reaction to take place at that location.
Accordingly, during synthesis, intermediate compounds are produced
which includes each of the amino acid residues located in the
desired sequence in the peptide chain with various of these
residues having side-chain protecting or capping groups. These
protecting or capping groups on amino acid side chains are then
removed substantially at the same time so as to produce the desired
resultant product following purification.
[0459] The typical protective, capping, or blocking groups for
.alpha.- and .epsilon.-amino side chain groups found in amino acids
are exemplified by benzyloxycarbonyl (Z), isonicotinyloxycarbonyl
(iNOC), O-chlorobenzyloxycarbonyl [Z(NO.sub.2)],
p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc),
t-amyioxycarbonyl (Aoc), isobornyloxycarbonyl, adamatyloxycarbonyl,
2-(4-biphenyl)-2-propyloxycarb- onyl (Bpoc),
9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyiethoxycarbon- yl
(Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl
(NPS), diphenylphosphinothioyl (Ppt), dimethylophosphinothioyl
(Mpt), and the like.
[0460] Protective, capping, or blocking groups for the carboxyl
group of amino acids include, for example, benzyl ester (OBzl),
cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester
(Obut), 4-pyridylmethyl ester (OPic), and the like. It is usually
also desirable that side chain groups of specific amino acids such
as arginine, cysteine, and serine, are protected by a suitable
protective group as occasion demands. For example, the guanidino
group in arginine may be protected with nitro, p-toluenesulfonyl,
benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl,
4-methoxy-2,6-dimethylbenzenesulfonyl (Mds),
1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group
in cysteine may be protected with p-methoxybenzyl, triphenylmethyl,
acetylaminomethyl ethylcarbamoyl, 4-methylbenzyl,
2,4,6-trimethy-benzyl (Tmb), etc., and the hydroxyl group in the
serine can be protected with benzyl, t-butyl, acetyl,
tetrahydropyranyl, etc.
[0461] After the desired amino acid sequence has been completed,
the intermediate polypeptide is removed from the resin support by
treatment with a reagent, such as liquid HF and one or more
thio-containing scavengers, which cleaves the peptide molecule from
the resin and all the remaining side-chain protecting groups.
Following HF cleavage, the protein sequence is washed with ether,
transferred to a large volume of dilute acetic acid, and stirred at
pH adjusted to about 8.0 with ammonium hydroxide. Upon pH
adjustment, the polypeptide takes its desired conformational
arrangement.
[0462] By way of example but not by way of limitation, polypeptides
can be chemically synthesized and purified as follows: Peptides can
be synthesized by employing the N-
alpha-9-fluorenylmethyloxycarbonyl or Fmoc solid phase peptide
synthesis chemistry using a Rainin Symphony Multiplex Peptide
Synthesizer. The standard cycle used for coupling of an amino acid
to the peptide-resin growing chain generally includes: (1) washing
the peptide-resin three times for 30 seconds with
N,N-dimethylformamide (DMF); (2) removing the Fmoc protective group
on the amino terminus by deprotection to with 20% piperdine in DMF
by two washes for 15 minutes each, during which process mixing is
effected by bubbling nitrogen through the reaction vessel for one
second every 10 seconds to prevent peptide-resin settling; (3)
washing the peptide-resin three times for 30 seconds with DMF; (4)
coupling the amino acid to the peptide resin by addition of equal
volumes of a 250 mM solution of the Fmoc derivative of the
appropriate amino acid and an activator mix consisting or 400 mM
N-methylmorpholine and 250 mM
(2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) in DMF; (5) allowing the solution to mix
for 45 minutes; and (6) washing the peptide-resin three times for
30 seconds of DMF. This cycle can be repeated as necessary with the
appropriate amino acids in sequence to produce the desired peptide.
Exceptions to this cycle program are amino acid couplings predicted
to be difficult by nature of their hydrophobicity or predicted
inclusion within a helical formation during synthesis. For these
situations, the above cycle can be modified by repeating step 4 a
second time immediately upon completion of the first 45 minute
coupling step to "double couple" the amino acid of interest.
Additionally, in the first coupling step in peptide synthesis, the
resin can be allowed to swell for more efficient coupling by
increasing the time of mixing in the initial DMF washes to three 15
minute washes rather than three 30 second washes.
[0463] After peptide synthesis, the peptide can be cleaved from the
resin as follows: (1) washing the peptide-resin three times for 30
seconds with DMF; (2) removing the Fmoc protective group on the
amino terminus by washing two times for 15 minutes it 20% piperdine
in DMF; (3) washing the peptide-resin three times for 30 seconds
with DMF; and (4) mixing a cleavage cocktail consisting of 95%
trifluoroacetic acid (TFA), 2.4% water, 2.4% phenol, and 0.2%
triisopropysilane with the peptide-resin for two hours, then
filtering the peptide in the cleavage cocktail away from the resin,
and precipitating the peptide out of solution by addition of two
volumes of ethyl ether. Specifically, to isolate the peptide, the
ether-peptide solution can be allowed to sit at -20.degree. C. for
20 minutes, then centrifuged at 6,000.times.G for 5 minutes to
pellet the peptide, and the peptide can be washed three times with
ethyl ether to remove residual cleavage cocktail ingredients. The
final peptide product can be purified by reversed phase high
pressure liquid chromatography (RP-HPLC) with the primary solvent
consisting of 0.1% TFA and the eluting buffer consisting of 80%
acetonitrile and 0.1% TFA. The purified peptide can then be
lyophilized to a powder.
[0464] In other specific embodiments, branched versions of the BLyS
binding polypeptides described herein are provided, e.g., by
substituting one or more amino acids within the BLyS binding
polypeptide sequence with an amino acid or amino acid analog with a
free side chain capable of forming a peptide bond with one or more
amino acids (and thus capable of forming a "branch").
[0465] Branched peptides may be prepared by any method known in the
art for covalently linking any naturally occurring or synthetic
amino acid to any naturally occurring or synthetic amino acid in a
peptide chain which has a side chain group able to react with the
amino or carboxyl group on the amino acids so as to become
covalently attached to the peptide chain. In particular, amino
acids with a free amino side chain group, such as, but not limited
to, diaminobutyric acid, lysine, arginine, ornithine,
diaminopropionic acid and citrulline, can be incorporated into a
peptide so that an amino acid can form a branch therewith, for
example, by forming a peptide bond to the free amino side group,
from that residue. Alternatively, amino acids with a free carboxyl
side chain group, such as, but not limited to, glutamic acid,
aspartic acid and homocitrulline, can be incorporated into the
peptide so that an amino acid can form a branch therewith, for
example, by forming a peptide bond to the free carboxyl side group,
from that residue. The amino acid forming the branch can be linked
to a side chain group of an amino acid in the peptide chain by any
type of covalent bond, including, but not limited to, peptide
bonds, ester bonds and disulfide bonds. In a specific embodiment,
amino acids, such as those described above, that are capable of
forming a branch point, are substituted for BLyS binding
polypeptide residues within a peptide including a BLyS binding
polypeptide sequence.
[0466] Branched peptides can be prepared by any method known in the
art. For example, but not by way of limitation, branched peptides
can be prepared as follows: (1) the amino acid to be branched from
the main peptide chain can be purchased as an
N-alpha-tert-butyloxycarbonyl (Boc) protected amino acid
pentafluorophenyl (Opfp) ester and the residue within the main
chain to which this branched amino acid will be attached can be an
N-Fmoc-alpha-gamma-diaminobutyric acid; (2) the coupling of the Boc
protected amino acid to diaminobutyric acid can be achieved by
adding 5 grams of each precursor to a flask containing 150 ml DMF,
along with 2.25 ml pyridine and 50 mg dimethylaminopyridine and
allowing the solution to mix for 24 hours; (3) the peptide can then
be extracted from the 150 ml coupling reaction by mixing the
reaction with 400 ml dichlormethane (DCM) and 200 ml 0.12N HCl in a
1 liter separatory funnel, and allowing the phases to separate,
saving the bottom aqueous layer and re-extracting the top layer two
more times with 200 ml 0.12N HCl; (4) the solution containing the
peptide can be dehydrated by adding 2-5 grams magnesium sulfate,
filtering out the magnesium sulfate, and evaporating the remaining
solution to a volume of about 2-5 ml; (5) the dipeptide can then be
precipitated by addition of ethyl acetate and then 2 volumes of
hexanes and then collected by filtration and washed two times with
cold hexanes; and (6) the resulting filtrate can be lyophilized to
achieve a light powder form of the desired dipeptide. Branched
peptides prepared by this method will have a substitution of
diaminobutyric acid at the amino acid position which is branched.
Branched peptides containing an amino acid or amino acid analog
substitution other than diaminobutyric acid can be prepared
analogously to the procedure described above, using the N-Fmoc
coupled form of the amino acid or amino acid analog.
[0467] In a preferred embodiment, the BLyS binding polypeptide is a
cyclic peptide. Cyclization can be, for example, but not by way of
limitation, via a disulfide bond between two cysteine residues or
via an amide linkage. For example, but not by way of limitation,
disulfide bridge formation can be achieved by (1) dissolving the
purified peptide at a concentration of between 0.1-0.5 mg/ml in
0.01 M ammonium acetate, pH 7.5; (2) adding to the dissolved
peptide 0.01 M potassium ferricyanide dropwise until the solution
appears pale yellow in color and allowing this solution to mix for
24 hours; (3) concentrating the cyclized peptide to 5-10 ml of
solution, repurifying the peptide by reverse phase-high pressure
liquid chromatography (RP-HPLC) and finally lyophilizing the
peptide. In a specific embodiment, in which the peptide does not
contain two appropriately situated cysteine residues, cysteine
residues can be introduced at the amino-terminus and/or
carboxy-terminus and/or internally such that the peptide to be
cyclized contains two cysteine residues spaced such that the
residues can form a disulfide bridge. Alternatively, a cyclic
peptide can be obtained by generating an amide linkage using, for
example but not limited to, the following protocol: An allyl
protected amino acid, such as aspartate, glutamate, asparagine or
glutamine, can be incorporated into the peptide as the first amino
acid, and then the remaining amino acids are coupled on. The allyl
protective group can be removed by a two hour mixing of the
peptide-resin with a solution of tetrakistriphenylphosphine
palladium (0) in a solution of chloroform containing 5% acetic acid
and 2.5% N-methylmorpholine. The peptide resin can be washed three
times with 0.5% N,N-diisopropylethylami- ne (DIEA) and 0.5% sodium
diethyldithiocabamate in DMF. The amino terminal Fmoc group on the
peptide chain can be removed by two incubations for 15 minutes each
in 20% piperdine in DMF, and washed three times with DMF for 30
seconds each. The activator mix, N-methylmorpholine and HBTU in
DMF, can be brought onto the column and allowed to couple the free
amino terminal end to the carboxyl group generated by removal of
the allyl group to cyclize the peptide. The peptide can be cleaved
from the resin as described in the general description of chemical
peptide synthesis above and the peptide purified by reverse
phase-high pressure liquid chromatography (RP-HPLC). In a specific
embodiment, in which the peptide to be cyclized does not contain an
allyl protected amino acid, an allyl protected amino acid can be
introduced into the sequence of the peptide, at the amino-terminus,
carboxy-terminus or internally, such that the peptide can be
cyclized.
[0468] In addition, according to certain embodiments, it is
preferable that the BLyS binding polypeptides are produced having
or retaining an amino terminal (N-terminal) and/or a carboxy
terminal (C-terminal) capping group, which may protect the
N-terminal or C-terminal amino acid from undesirable chemical
reactions during use or which may permit further conjugations or
manipulations of the binding polypeptide, for example, in
conjugating the binding polypeptide to a chromatographic support
resin or matrix or to another peptide to tether the binding
polypeptide to a resin or support. Such N-terminal and C-terminal
groups may also be used to label or tag the binding polypeptide to
detect bound complexes or to locate the binding polypeptide
(whether bound or unbound to a BLyS target protein) for example, at
some point in a separation procedure. Accordingly, a BLyS binding
polypeptide synthesized in its final form for use in a detection or
separation procedure may contain an N-terminal and/or a C-terminal
capping group. A particularly preferred N-terminal capping group,
which may be present or retained in binding polypeptides, is an
acetyl group (Ac). A particularly preferred C-terminal capping
group, which may be present or retained in binding polypeptides, is
an amide group. In a further preferred embodiment, the BLyS binding
polypeptides have an acetyl group as an N-terminal capping group
and an amide group as a C-terminal capping group.
[0469] The BLyS binding polypeptides may also be prepared
commercially by companies providing polypeptide synthesis as a
service (e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.;
Quality Controlled Biochemicals, Inc., Hopkinton, Mass.).
[0470] The nucleic acid sequence encoding a BLyS binding
polypeptide can be produced and isolated using well-known
techniques in the art. In one example, nucleic acids encoding the
BLyS binding polypeptides are chemically synthesized based on
knowledge of the amino acid sequence of the BLyS binding
polypeptide (preferably the sequence is codon optimized to the host
system in which the polypeptide will be expressed). In another
example, nucleic acids encoding a BLyS binding polypeptide are
obtained by screening an expression library (e.g., a phage display
library) to identify phage expressing BLyS binding polypeptides,
and isolating BLyS binding polypeptide encoding nucleic acid
sequences from the identified library member (e.g., via polymerase
chain reaction methodology using primers flanking the polypeptide
encoding sequences).
[0471] Thus, BLyS binding polypeptidess can also be obtained by
recombinant expression techniques. (See, e.g., Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Glover, D. M.
(ed.), (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989); DNA Cloning: A Practical Approach (MRL Press, Ltd., Oxford,
U.K., 1985), Vols. I, II.
[0472] To produce a recombinant BLyS binding polypeptide, a nucleic
acid sequence encoding the BLyS binding polypeptide is operatively
linked to a promoter such that the BLyS binding polypeptide is
produced from said sequence. For example, a vector can be
introduced into a cell, within which cell the vector or a portion
thereof is expressed, producing the BLyS binding polypeptides. In a
preferred embodiment, the nucleic acid is DNA if the source of RNA
polymerase is DNA-directed RNA polymerase, but the nucleic acid may
also be RNA if the source of polymerase is RNA-directed RNA
polymerase or if reverse transcriptase is present in the cell or
provided to produce DNA from the RNA. Such a vector can remain
episomal or, become chromosomally integrated, as long as it can be
transcribed to produce the desired RNA. Such vectors can be
constructed by recombinant DNA technology methods standard in the
art. Vectors can be bacteriophage, plasmid, viral, retroviral, or
others known in the art, used for replication and expression in
bacterial, fungal, plant, insect or mammalian cells. Retroviral
vectors may be replication competent or replication defective. In
the latter case, viral propagation generally will occur only in
complementing host cells. 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 well known in the art and are described,
for example, in many standard laboratory manuals, such as Davis et
al., Basic Methods In Molecular Biology (1986).
[0473] The present invention also contemplates the use of BLyS
binding polypeptides (including molecules comprising, or
alternatively consisting of, BLyS binding polypeptide fragments or
variants thereof) that are recombinantly fused or chemically
conjugated (including both covalent and non-covalent conjugations)
to a heterologous polypeptide (or portion thereof, preferably at
least 10, at least 20, at least 30, at least 40, at least 50, at
least 60, at least 70, at least 80, at least 90 or at least 100
amino acids of the heterologous polypeptide) to generate fusion
proteins. The fusion does not necessarily need to be direct, but
may occur through linker sequences. For example, BLyS binding
polypeptides may be used to target heterologous polypeptides to
particular cell types (e.g., cells of monocytic lineage and
B-cells), either in vitro or in vivo, by fusing or conjugating the
heterologous polypeptides to BLyS binding polypeptides that are
specific for particular cell surface antigens (e.g., membrane-bound
BLyS on cells of monocytic lineage) or which bind antigens (i.e.,
BLyS) that bind particular cell surface receptors (e.g., TACI
and/or BCMA located on B cells). BLyS binding polypeptides fused or
conjugated to heterologous polypeptides 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
93/2 1232; EP 439 095; Naramura et al., Immunol. Lett., 39:91-99
(1994); U.S. Pat. No. 5,474,981; Gillies et al., Proc. Nat'l Acad.
Sci. USA, 89:1428-1432 (1992); Fell et al., J. Immunol.,
146:2446-2452 (1991), which are incorporated by reference in their
entireties.
[0474] The present invention further contemplates the use of
compositions comprising, or alternatively consisting of,
heterologous polypeptides fused or conjugated to BLyS binding
polypeptide fragments.
[0475] Fusion proteins useful in the methods of the invention may
be generated through the techniques of gene-shuffling,
motif-shuffling, exon-shuffling, and/or codon-shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be
employed to modulate the activities of BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof), such
methods can be used to generate BLyS binding polypeptides with
altered activity (e.g., BLyS binding polypeptides with higher
affinities and lower dissociation rates). See, generally, U.S. Pat.
Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and
Patten et al., Curr. Opinion Biotechnol., 8:724-33 (1997);
Harayama, Trends Biotechnol., 16(2):76-82 (1998); Hansson, et al.,
J. Mol. Biol., 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques, 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety).
In one embodiment, polynucleotides encoding BLyS binding
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 portions of a polynucleotide encoding a BLyS binding
polypeptide which portions specifically bind to BLyS may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
[0476] Polypeptides of the present invention include 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
may also include an initial modified methionine residue, in some
cases as a result of host-mediated processes.
[0477] The BLyS binding polypeptides that are used in the methods
of the present invention may be modified during or after synthesis
or translation, e.g., by glycosylation, acetylation, benzylation,
phosphorylation, amidation, pegylation, formylation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to an antibody molecule, hydroxylation, iodination, methylation,
myristoylation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation, ubiquitination, etc. (See, for instance, Creighton,
Proteins: Structures and Molecular Properties, 2d Ed. (W. H.
Freeman and Co., N.Y., 1992); Postranslational Covalent
Modification of Proteins, Johnson, ed. (Academic Press, New York,
1983), pp. 1-12; Seifter et al., Meth. Enzymol., 182:626-646
(1990); Rattan et al., Ann. NY Acad. Sci., 663:48-62 (1992).) In
specific embodiments, the peptides are acetylated at the N-terminus
and/or amidated at the C-terminus.
[0478] BLyS binding polypeptides containing two or more residues
that have the potential to interact, such as for example, two
cysteine residues in a polypeptide, may be treated under oxidizing
conditions or other conditions that promote interaction of these
residues (e.g, dislulfide bridge formation).
[0479] Further BLyS binding polypeptide modifications contemplated
herein include, for example, any of numerous chemical modifications
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. Additional
post-translational/post-synthesis modifications that may be
employed 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.
[0480] Chemically modified derivatives of BLyS binding polypetides
may be used which may provide additional advantages such as
increased affinity, decreased off-rate, 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.
[0481] 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.
[0482] 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.
[0483] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the BLyS binding poypeptide with
consideration of effects on functional domains of the polypeptide.
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. In a preferred embodiment, the
polyethylene glycol molecule is attached at an amino group, such as
attachment at the N-terminus or to a lysine side chain amino
group.
[0484] As suggested above, polyethylene glycol may be attached to
polypeptides via linkage to any of a number of amino acid residues.
For example, polyethylene glycol can be linked to a polypeptide 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 polypeptide or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the polypeptide.
[0485] 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 polypeptide molecules in the reaction mix, the
type of pegylation reaction to be performed, and the method of
obtaining the selected N-terminally pegylated polypeptide. 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 polypeptide molecules.
Selective N-terminal modification of proteins may be accomplished
by reductive alkylation which exploits differential reactivity of
different types of primary amino groups (lysine versus the
N-terminus) 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.
[0486] As indicated above, pegylation of the polypeptides 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.
[0487] One system for attaching polyethylene glycol directly to
amino acid residues of polypeptides 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 polyeptide. Thus, the invention includes
polypeptide-polyethylene glycol conjugates produced by reacting
polypeptides with a polyethylene glycol molecule having a
2,2,2-trifluoreothane sulphonyl group.
[0488] Polyethylene glycol can also be attached to polypeptides
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 polypeptides. Polypeptide-polyethylene
glycol conjugates wherein the polyethylene glycol is attached to
the polypeptide by a linker can also be produced by reaction of
polypeptides with compounds such as MPEG-succinimidylsuccinate,
MPEG activated with 1,1'-carbonyldiimidazole,
MPEG-2,4,5-trichlorophenylcarbonate, MPEG-p-nitrophenolcarbonate,
and various MPEG-succinate derivatives. A number of additional
polyethylene glycol derivatives and reaction chemistries for
attaching polyethylene glycol to polypeptides are described in WO
98/32466, the entire disclosure of which is incorporated herein by
reference. Pegylated BLyS binding polypeptide products produced
using the reaction chemistries set out herein are included within
the scope of the invention.
[0489] The number of polyethylene glycol moieties attached to each
polypeptide (i.e., the degree of substitution) may also vary. For
example, the pegylated polypeptides 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 may
range 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 polypeptide 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).
[0490] BLyS Binding Polypeptide Multimers, Conjugates and
Fusions
[0491] The methods of the present invention may also be carried out
using multivalent BLyS binding polypeptides. BLyS binding
polypeptides may be monomeric, dimeric, trimeric, or higher-order
multimers. In a preferred embodiment multivalent BLyS binding
polypeptides are homotrimeric. In another preferred embodiment a
homotrimeric BLyS binding polypeptide binds a single homotrimeric
BLyS.
[0492] In another preferred embodiment, monomeric or multimeric
BLyS binding polypeptides are conjugated with another polypeptide
or other chemical compound. For example, BLyS binding
polypeptide(s) may be conjugated to a radioactive or other toxic
compound so as to target and destroy cells expressing BLyS.
[0493] The present invention also encompasses the use of
heteromeric multimers comprised of one or more BLyS binding
polypeptides and one or more non-BLyS binding polypeptides or other
chemical moieties. Such heteromeric multimers may be monomeric,
dimeric, trimeric, tetrameric, pentameric, or higher-order
multimers. Heteromeric BLyS binding multimers may be used to
target, bind, inhibit, and/or activate responses in cells
expressing BLyS and receptors for the heterologous, non-BLyS
binding polypeptide or other chemical moiety. Such activated
responses may include, for example, apoptosis or other biologically
and chemically mediated forms of cell destruction. Heteromeric BLyS
binding multimers may also be used to target BLyS expressing cells
so as to introduce a desired molecule or compound to the cells. For
example, a heteromeric BLyS binding multimer may be conjugated with
a radioactive or otherwise toxic compound so as to kill BLyS
expressing cells. As an alternative example, a heteromeric BLyS
binding and Adenovirus-binding multimer could be used to
specifically target and introduce adenovirus-mediated gene
therapeutics into BLyS expressing cells.
[0494] BLyS binding polypeptide multimers may be fused or
conjugated as homopolymers and heteropolymers using methods known
in the art. In a preferred embodiment BLyS binding polypeptides are
linked as homomultimers wherein the linker or linkers provide
sufficient length and flexibility such that each BLyS binding
polypeptide may simultaneously bind an individual BLyS molecule. In
another preferred embodiment BLyS binding polypeptides are linked
as heteromultimers wherein the linker or linkers provide sufficient
length and flexibility such that each BLyS binding polypeptide may
simultaneously bind individual BLyS molecules and the heterologous
polypeptide or chemical moiety may simultaneously bind to its
target. Numerous examples of suitable linker molecules are known in
the art. (See, for example, Todorovska et al., J. Immunol. Methods,
248(1-2):47-66 (2001); Mehvar, J. Control Release, 69(1):1-25
(2000); Francis et. al., Int. J. Hematol., 68(1):1-18 (1998).) In
specific embodiments, the linker is a member selected from the
group consisting of: (a) a peptide linker; (b) a glutamate linker;
and (c) a polyethylene glycol linker. The length of linkers to be
used according to the methods of the invention may routinely be
determined using techniques known in the art. In specific
embodiments, the linker is 5-60 angstroms in length. In other
embodiments, the linker is 10-50, 10-40, 10-30, or 10-20 angstroms
in length. In further embodiments, the linker is about 5, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or
100 angstroms in length. In this context "about" includes the
recited length, and/or lengths that are larger or smaller by
several (5, 4, 3, 2, or 1) angstroms. In other embodiments, the
linker is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, or 100 angstroms in length.
[0495] In a preferred embodiment, BLyS binding polypeptides may be
fused with human serum albumin (HA). See, e.g., U.S. application
Ser. No. 09/833,245, filed Apr. 12, 2001, which is hereby
incorporated by reference herein. In one embodiment, the albumin
fusion protein comprises HA as the N-terminal portion, and a BLyS
binding polypeptide as the C-terminal portion. In another
embodiment the albumin fusion protein comprise HA as the C-terminal
portion, and a BLyS binding polypeptide as the N-terminal
portion.
[0496] In other embodiments, the albumin fusion protein has a BLyS
binding polypeptide fused to both the N-terminus and the C-terminus
of albumin. In one preferred embodiment, the BLyS binding
polypeptides fused at the N- and C- termini are the same BLyS
binding polypeptides. In another preferred embodiment, the BLyS
binding polypeptides fused at the N- and C-termini are different
BLyS binding polypeptides. In another preferred embodiment, a BLyS
binding polypeptide is fused at either the N- or C- terminus of HA
and a different (non-BLyS binding) polypeptide is fused at either
the C- or N- terminus, respectively.
[0497] In addition to albumin fusion proteins in which the BLyS
binding polypeptide(s) is (are) fused to the N-terminus and/or
C-terminus of HA, BLyS binding polypeptide/albumin fusion proteins
may also be produced by inserting the BLyS binding polypeptide into
an internal region or regions of HA. For instance, within the
protein sequence of the HA molecule a number of loops or turns
exist between the end and beginning of .alpha.-helices, which are
stabilized by disulphide bonds (see FIGS. 9-11 in U.S. application
Ser. No. 09/833,245). The loops, as determined from the crystal
structure of HA (FIG. 13 of U.S. application Ser. No. 09/833,245)
(PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to 1E71 and 1UOR) for
the most part extend away from the body of the molecule. These
loops are useful for the insertion, or internal fusion, of
therapeutically active peptides (particularly those requiring a
secondary structure to be functional) or therapeutic proteins, to
essentially generate an albumin molecule with specific biological
activity.
[0498] Loops in human albumin structure into which binding
polypeptides may be inserted to generate albumin fusion proteins
include: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His
247-Glu252, Glu 266-Glu277, Glu 280-His288, Ala362-Glu368,
Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys560-Thr566. In
more preferred embodiments, polypeptides are inserted into the
Val54-Asn61, Gln170-Ala176, and/or Lys560-Thr566 loops of mature
human serum albumin (SEQ ID NO: 445).
[0499] In specific embodiments, BLyS binding polypeptides 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 BLyS binding polypeptides is
.sup.111In. In another preferred embodiment, the radiometal ion
associated with the macrocyclic chelator attached to BLyS binding
polypeptides is .sup.90Y. In specific embodiments, the macrocyclic
chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraa-
cetic acid (DOTA). In other specific embodiments, the DOTA is
attached to the BLyS binding polypeptides 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 would be
readily able to adapt the method disclosed therein in order to
conjugate chelating agents to other polypeptides.
[0500] The BLyS binding 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.
[0501] The BLyS binding polypeptides may also be modified with a
detectable label, including, but not limited to, an enzyme,
prosthetic group, fluorescent material, luminescent material,
bioluminescent material, radioactive material, positron emitting
metal, nonradioactive paramagnetic metal ion, and affinity label
for detection and isolation of BLyS target. The detectable
substance may be coupled or conjugated either directly to the
polypeptides or indirectly, through an intermediate (such as, for
example, a linker known in the art) using techniques known in the
art. 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 aequorin; 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.149Pm, .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, 169Yb, .sup.51Cr, 54Mn, .sup.75Se,
.sup.113Sn, and .sup.117Tin.
[0502] In specific embodiments, BLyS binding polypetides are
attached macrocyclic chelators useful for conjugating radiometal
ions, including but not limited to .sup.177Lu, .sup.90Y,
.sup.166Ho, and .sup.153Sm, to polypeptides. 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 BLyS
binding polypeptide 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.
[0503] In a specific embodiment, BLyS binding polypeptides are
labeled with biotin.
[0504] The present invention further encompasses the use of BLyS
binding polypeptides (including molecules comprising, or
alternatively consisting of, BLyS binding polypeptide fragments or
variants thereof), conjugated to a diagnostic or therapeutic agent.
The BLyS binding polypeptides can be used diagnostically to, for
example, monitor or prognose 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 BLyS binding polypeptide to a
detectable substance. Examples of detectable substances include,
but are not limited to, 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 such as, for example, those described
herein. The detectable substance may be coupled or conjugated
either directly to the BLyS binding polypeptide 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 BLyS
binding polypeptides for use as diagnostics according to the
present invention.
[0505] Further, a BLyS binding polypeptide (including a molecule
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants 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, or other
radioisotopes such as, for example, .sup.103Pd, .sup.133Xe,
.sup.131I, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P,
.sup.35S, .sup.90Y, .sup.153Sm, .sup.153Gd, .sup.169Yb, .sup.51Cr,
.sup.54Mn, .sup.75Se, .sup.113Sn, .sup.90Yttrium, .sup.117Tin,
.sup.186Rhenium, .sup.166Holmium, and .sup.188Rhenium. A cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells.
Examples include, but are not limited to, paclitaxol, cytochalasin
B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, thymidine kinase, endonuclease,
RNAse, and puromycin and frragments, variants 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).
[0506] Techniques known in the art may be applied to label BLyS
binding polypeptides. 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).
[0507] The BLyS binding polypeptides which are conjugates 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, but are not limited to, for example, a
toxin such as abrin, ricin A, alpha toxin, pseudomonas exotoxin, or
diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral
protein, alpha-sarcin and cholera 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), 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.
[0508] A BLyS binding polypeptide (including a molecule comprising,
or alternatively consisting of, a BLyS binding polypeptide fragment
or variant thereof), 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.
[0509] Characterization of BLyS Binding Polypeptides
[0510] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) may be characterized in a variety of ways. In
particular, BLyS binding polypeptides and related molecules may be
assayed for the ability to specifically bind to BLyS or a fragment
of BLyS (e.g., to the soluble form or the membrane-bound form of
BLyS) using techniques described herein or routinely modifying
techniques known in the art. BLyS or BLyS fragments that may be
specifically bound by the compositions useful according to the
invention include, but are not limited to, human BLyS (SEQ ID NOs:
173 and/or 174) or BLyS expressed on human monocytes; murine BLyS
(SEQ ID NOs: 175 and/or 176) or BLyS expressed on murine monocytes;
rat BLyS (either the soluble forms as given in SEQ ID NOs: 177,
178, 179 and/or 180 or in a membrane associated form, e.g., on the
surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS
polypeptides of SEQ ID NOS: 181 and/or 182, the soluble form of
monkey BLyS, or BLyS expressed on monkey monocytes) or fragments
thereof. Preferably compositions useful according to the invention
bind human BLyS (SEQ ID NOs: 173 and/or 174) or fragments thereof.
Assays for the ability of the BLyS binding polypeptides to
specifically bind BLyS or a fragment of BLyS may be performed in
solution (e.g., Houghten, Bio/Techniques, 13:412-421(1992)), on
beads (e.g., Lam, Nature, 354:82-84 (1991)), on chips (e.g., Fodor,
Nature, 364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No.
5,223,409), on spores (e.g., Pat. Nos. 5,571,698; 5,403,484; and
5,223,409), on plasmids (e.g., Cull et al., Proc. Natl Acad. Sci.
USA, 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith,
Science, 249:386-390 (1990); Devlin, Science, 249:404-406 (1990);
Cwirla et al., Proc. Natl Acad. Sci. USA, 87:6378-6382 (1990); and
Felici, J. Mol. Biol., 222:301-310 (1991)) (each of these
references is incorporated herein in its entirety by reference).
BLyS binding polypeptides that have been identified to specifically
bind to BLyS or a fragment of BLyS can then be assayed for their
specificity and affinity for BLyS or a fragment of BLyS using or
routinely modifying techniques described herein or otherwise known
in the art.
[0511] The BLyS binding polypeptides may be assayed for specific
binding to BLyS and cross-reactivity with other BLyS-like
polypeptides by any method known in the art. In particular, the
ability of a BLyS binding polypeptide to specifically bind to the
soluble form or membrane-bound form of BLyS and the specificity of
the BLyS binding polypeptide, fragment, or variant for BLyS
polypeptide from a particular species (e.g., murine, monkey or
human, preferably human) may be determined using or routinely
modifying techniques described herein or otherwise known in
art.
[0512] Assays which can be used to analyze specific binding and
cross-reactivity include, but are not limited to, competitive and
non-competitive assay systems using techniques such as western
blots, radioimmunoassay, ELISA (enzyme linked immunosorbent assay),
"sandwich" assays, "immunoprecipitation" assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
radiometric assays, and fluorescent assays, to name but a few. Such
assays are routine and well known in the art (see, e.g., Current
Protocols in Molecular Biology, Vol. 1, Ausubel et al, eds. (John
Wiley & Sons, Inc., New York 1994), which is incorporated by
reference herein in its entirety) and could easily be adapted to
make use of a BLyS binding polypeptide (possibly in conjunction
with an anti-BLyS binding polypeptide antibody) in place of an
anti-BLyS antibody. Exemplary immunoassays that could be modified
to use a BLyS binding polypeptide are described briefly below (but
are not intended by way of limitation).
[0513] 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),
incubating the membrane with BLyS binding polypeptide (the BLyS
binding polypeptide of interest) diluted in blocking buffer,
washing the membrane in washing buffer, incubating the membrane
with a secondary antibody (which recognizes the BLyS binding
polypeptide) conjugated to an enzyme (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. Alternatively,
the BLyS binding polypeptide may be directly conjugated to a
detection molecule (e.g., an enzyme or radiolabel), thereby
omitting the need for a secondary anti-BLyS binding polypeptide
antibody. 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., Current Protocols in Molecular
Biology, Vol. 1, Ausubel et al, eds. (John Wiley & Sons, Inc.,
New York 1994) at 10.8.1.
[0514] ELISAs comprise preparing antigen (e.g., BLyS target),
coating the well of a 96-well microtiter plate with the antigen,
washing away antigen that did not bind the wells, adding the BLyS
binding polypeptide of interest conjugated to a detectable compound
such as an enzyme (e.g., horseradish peroxidase or alkaline
phosphatase) to the wells and incubating for a period of time,
washing away unbound BLyS binding polypeptides or non-specifically
bound BLyS binding polypeptides, and detecting the presence of the
BLyS binding polypeptides specifically bound to the antigen coating
the well. In ELISAs the BLyS binding polypeptide employed in the
assay does not have to be conjugated to a detectable compound;
instead, an antibody that recognizes the BLyS binding polypeptide
and that is conjugated to a detectable compound may be added to the
well. Further, instead of coating the well with the antigen, the
BLyS binding polypeptide may be coated to the well. In this case,
the detectable molecule could be the antigen conjugated to a
detectable compound such as an enzyme (e.g., horseradish peroxidase
or alkaline phosphatase). 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., Current
Protocols in Molecular Biology, Vol. 1, Ausubel et al, eds. (John
Wiley & Sons, Inc., New York 1994) at 11.2.1.
[0515] Immunoprecipitation protocols generally use antibody
molecules to imunopreciptate a protein of interest. A BLyS
preciptation protocol could easily be modified to use a BLyS
binding polypeptide in place of an anti-BLyS antibody.
Immunopreciptation 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 to 4 hours)
at 40 degrees C, adding protein A and/or protein G sepharose beads
to the cell lysate, incubating for about an hour or more at 40
degrees C., washing the beads in lysis buffer and resuspending the
beads in SDS/sample buffer. If one wanted to substitute a BLyS
binding polypeptide for the anti-BLyS antibody one could readily do
so, and then isolate the BLyS-BLyS binding polypeptide complexes
with an antibody that recognizes the BLyS binding polypeptide. Then
the triple complex of BLyS, BLyS binding polypeptide, and anti-BLyS
binding polypeptide antibody could be isolated using protein A
and/or Protein G as described above. Such a protocol may be
desirable if, for example, the anti-BLyS binding polypeptide
antibody has a higher affinity for the BLyS binding polypeptide
than the anti-BLyS antibody may have for BLyS.
[0516] The effectiveness of incorporating a BLyS binding
polypeptide in an immunoprecipitation protocol to precipitate BLyS
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 BLyS binding polypeptide 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., Current Protocols in
Molecular Biology, Vol. 1, Ausubel et al, eds. (John Wiley &
Sons, Inc., New York 1994) at 10.16.1.
[0517] The binding affinity of a BLyS binding polypeptide
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof) to an
antigen and the off-rate of an BLyS binding polypeptide-antigen
interaction can be determined by competitive binding assays. One
example of a competitive binding assay is a modified
radioimmunoassay comprising the incubation of labeled antigen
(e.g., .sup.3H- or .sup.125I-labeled BLyS target) with the BLyS
binding polypeptide of interest in the presence of increasing
amounts of unlabeled antigen, followed by detection of the BLyS
binding polypeptide bound to the labeled antigen. The affinity of
the BLyS binding polypeptide of the present invention for BLyS and
the binding off-rates can be determined from the data by Scatchard
plot analysis. Competition with an anti-BLyS antibody or BLyS
binding polypeptide can also be determined using radioimmunoassays.
In this case, BLyS is incubated with a BLyS binding polypeptide of
the present invention conjugated to a labeled compound (e.g., with
.sup.3H or .sup.125I) in the presence of increasing amounts of an
unlabeled BLyS binding polypeptide or anti-BLyS antibody.
[0518] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of BLyS binding
polypeptides (including molecules comprising, or alternatively
consisting of, BLyS binding polypeptide fragments or variants
thereof) to BLyS, or fragments of BLyS. BIAcore kinetic analysis
comprises analyzing the binding and dissociation of BLyS from chips
with immobilized BLyS binding polypeptides on their surface (see
Example 6, infra).
[0519] The BLyS binding polypeptides (including molecules
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) can also be assayed for
their ability to inhibit, increase, or not significantly alter, the
binding of BLyS to a BLyS receptor (e.g., TACI and BCMA) using
techniques known to those skilled in the art. For example, cells
expressing a receptor for BLyS (e.g., IM9, REH, ARH-77cells,
Namalwa, and RPMI-8226 B cell tumor lines as well as peripheral
CD20+ B cells) can be contacted with BLyS in the presence or
absence of a BLyS binding polypeptide, and the ability of the BLyS
binding polypeptide to inhibit, increase, or not significantly
alter, BLyS binding to the cells can be measured. Alternatively,
the BLyS binding polypeptide may be preincubated with the BLyS
prior to exposure of the BLyS to cells expressing the BLyS
receptor. BLyS binding to cells can be measured by, for example,
flow cytometry or a scintillation assay. BLyS or the BLyS binding
polypeptide can be labeled with a detectable compound such as a
radioactive label (e.g., .sup.32P, .sup.35S, and .sup.125I) or a
fluorescent label (e.g., fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine) to enable detection of an interaction between BLyS
and a BLyS receptor and/or BLyS and a BLyS binding polypeptide.
[0520] The ability of BLyS binding polypeptides to inhibit,
increase, or not significantly alter, BLyS binding to a BLyS
receptor can also be determined in cell-free assays. For example,
native or recombinant BLyS (e.g., having the amino acid sequence of
amino acids 134-285 of SEQ ID NO: 173) or a fragment thereof can be
contacted with a BLyS binding polypeptide and the ability of the
BLyS binding polypeptide to inhibit, increase, or not significantly
alter, BLyS from binding to a BLyS receptor can be determined.
Preferably, the BLyS binding polypeptide or BLyS receptor is
immobilized on a solid support and BLyS or a BLyS fragment is
labeled with a detectable compound. Alternatively, BLyS or a BLyS
fragment is immobilized on a solid support and the BLyS binding
polypeptide is labeled with a detectable compound. BLyS may be
partially or completely purified (e.g., partially or completely
free of other polypeptides) or part of a cell lysate. Further, the
BLyS polypeptide may be a fusion protein comprising BLyS or a
biologically active portion thereof and a domain such as an
Immunoglobulin Fc or glutathionine-S-transferase. Additionally, the
BLyS binding polypeptide and/or BLyS receptor may be a fusion
protein comprising a BLyS binding portion of the polypeptide or
receptor and a domain such as an Immunoglobulin Fc or
glutathionine-S-transferase. For example, amino acid residues 1-154
of TACI (GenBank accesion number AAC51790), or 1-48 of BCMA
(GenBank accession number NP.sub.--001183) may be fused to the Fc
region of an IgG molecule and used in a gab cell free assay to
determine the ability of BLyS binding polypeptides to inhibit,
increase, or not significantly alter, BLyS binding to a BLyS
receptor. Alternatively, BLyS can be biotinylated using techniques
well known to those skilled in the art (e.g., biotinylation kit,
Pierce Chemicals;
[0521] Rockford, Ill.).
[0522] The BLyS binding polypeptides (including molecules
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof), can also be assayed for
their ability to inhibit, stimulate, or not significantly alter,
BLyS-induced B-cell proliferation using techniques known to those
of skill in the art. For example, B-cell proliferation can be
assayed by .sup.3H-thymidine incorporation assays and trypan blue
cell counts (see, e.g., Moore et al., Science, 285: 260-263
(1999)). Further, the BLyS binding polypeptides, or fragments or
variants thereof, can be assayed for their ability to inhibit,
stimulate, or not significantly alter, BLyS-induced activation of
cellular signaling molecules and transcription factors such as
calcium-modulator and cyclophilin ligand (CAML), calcineurin,
nuclear factor of activated T cells transcription factor (NF-AT),
nuclear factor-kappa B (NF-kappa B), SRF, activator protein-1
(AP-1), 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) using techniques known to those of skill
in the art (see, e.g., von Bulow and Bram, Science,
278:138-141(1997)). For example, NF-AT activity can be determined
by electromobility gel shift assays, by detecting the expression of
a protein known to be regulated by NF-AT (e.g., IL-2 expression),
by detecting the induction of a reporter gene (e.g., an NF-AT
regulatory element operably linked to a nucleic acid encoding a
detectable marker such as luciferase, beta-galactosidase or
chloramphenicol acetyltransferase (CAT)), or by detecting a
cellular response (e.g., cellular differentiation, or cell
proliferation).
[0523] The BLyS binding polypeptides, or fragments or variants
thereof can also be assayed for their ability to neutralize,
enhance, or not significantly alter, BLyS activity. For example,
BLyS binding polypeptides or fragments or variants thereof, may be
routinely tested for their ability to inhibit BLyS from binding to
cells expressing the receptor for BLyS.
[0524] Uses of the Binding Polypeptides and Recombinant
Bacteriophage
[0525] The BLyS binding polypeptides described herein are
especially useful to detect, isolate, or remove BLyS target
proteins in solutions. Such solutions may be simple dispersions or
solutions of BLyS and/or BLyS-like polypeptide in water or aqueous
buffer or more complex solutions, such as, a blood and other
biological fluids, tissue homogenates cell extracts, or biopsy
samples, and cell culture media containing BLyS or BLyS-like
polypeptides. Biological fluids include, but are not limited to
sera, plasma, lymph, blood, blood fractions urine, synovial fluid,
spinal fluid, saliva, and mucous.
[0526] In one embodiment, the present invention provides a method
for detecting a BLyS protein and/or a BLyS-like polypeptide in a
solution comprising contacting the solution with a BLyS binding
polypeptide and detecting binding of BLyS or BLyS-like polypeptide
to the BLyS binding polypeptide. The BLyS binding polypeptide may
be either free or immobilized. Preferably, the BLyS binding
polypeptide is a polypeptide immobilized on a solid surface or
chromatographic material or the well of a plastic microtiter assay
dish.
[0527] Another embodiment of the present invention is a method for
isolating BLyS protein and/or BLyS-like polypeptide from a solution
containing it, comprising:
[0528] (a) contacting the solution with a BLyS binding polypeptide
under conditions that permit binding of BLyS and/or BLyS-like
polypeptides to BLyS binding polypeptide, and
[0529] (b) recovering the BLyS and/or BLyS-like polypeptides.
[0530] A further embodiment of the present invention is a method
for isolating BLyS protein and/or BLyS-like polypeptide from a
solution containing it, comprising:
[0531] (a) contacting the solution with a BLyS binding polypeptide
under conditions that permit binding of BLyS and/or BLyS-like
polypeptides to BLyS binding polypeptide, and
[0532] (b) separating the complex(es) formed by the BLyS binding
polypeptide and BLyS and/or BLyS-like polypeptides from other
components of the solution.
[0533] Preferably such method also includes the further steps
of:
[0534] (c) dissociating the BLyS binding polypeptide from the BLyS
and/or BLyS-like polypeptides, and
[0535] (d) recovering the dissociated, BLyS and/or BLyS-like
polypeptide.
[0536] The invention also provides for the use of kits containing a
binding polypeptide for use in methods of detecting or isolating
BLyS and/or BLyS-like polypeptides.
[0537] According to the invention, detection or isolation of BLyS
target proteins comprises contacting a solution containing a BLyS
target protein with a BLyS binding polypeptide. Depending on the
particular application, the BLyS binding polypeptide may be free in
solution or immobilized on a solid support or chromatographic
material. Sufficient time is allowed to permit binding between the
BLyS target protein and the binding polypeptides, and non-binding
components in the solution or mixture are removed or washed away.
The formation of a binding complex between the binding polypeptide
and the BLyS target protein can then be detected, for example, by
detecting the signal from a label on the binding polypeptide, which
is one component of the binding complex. A label may be any label
that generates a signal that can be detected by standard methods,
such as a fluorescent label, a radioactive compound, or an enzyme
that reacts with a substrate to generate a detectable signal.
Suitable such labels are discussed above. A phage binding
polypeptide according to the invention, that is, a recombinant
phage displaying a BLyS binding polypeptide on its surface, may
form a complex with BLyS and/or BLyS-like polypeptides that is
detectable as a precipitate or sediment in a reaction tube, which
can be detected visually after settling or centrifugation.
Alternatively, a sandwich-type assay may be used, wherein a BLyS
binding polypeptide is immobilized on a solid support such as a
plastic tube or well, or a chromatographic support matrix such as
agarose beads, then the solution suspected of containing the BLyS
target is contacted with the immobilized binding polypeptide and
non-binding materials or components are removed or washed away.
[0538] The binding polypeptides according to this invention are
particularly useful for detection and/or isolation of BLyS and/or
BLyS-like polypeptides by affinity chromatography methods. Any
conventional method of chromatography may be employed. Preferably,
a BLyS binding polypeptide will be immobilized on a solid support
suitable, for example, for packing a chromatography column. The
immobilized BLyS binding polypeptide affinity ligand can then be
loaded or contacted with a feed stream under conditions favorable
to formation of binding polypeptide/BLyS (or BLyS-like polypeptide)
complexes. Non-binding materials can be washed away. Examples of
suitable wash conditions can readily be determined by one of skill
in the art and include but are not limited to [PBS/0.01% Tween 20,
pH7.2] and [1M NaCl/10 mM Tris, pH7.5]. Tris wash buffers may be
preferable since phosphates can preciptate in 50% ethylene glycol.
In general, non-limiting terms, wash buffers are pH7.0, optionally
containing 0.0 to 1.5 M NaCl, more preferably 1M NaCl.
Additionally, wash buffers may optionally contain a mild detrgenet,
such as, for example, Tween 20, Tween 80, or NP-80. BLyS or
BLyS-like polypeptide can be eluted from the BLyS binding
polypeptide by introducing solution conditions that favor
dissociation of the binding complex. Suitable elution solutions can
readily be determined by one of skill in the art and include but
are not limited to [50% ethylrne glycol/100 mM NaOAc]. By way of
non-limiting example, useful elution buffers, for the purposes of
the present invention contain 40-60% ethylene glycol, preferably
50% ethylene glycol.; and 50-100 mM NaOAc with a pH in the range of
pH 4-pH7, more preferably, pH 4-pH 6 and most preferably pH 4.5-pH
5.5. Preferably, a fast flow affinity chromatographic technique is
used to bind the molecules and from which purified BLyS or
BLyS-like polypeptides are eluted.
[0539] Alternatively, batch chromatography can be carried out by
mixing a solution containing the BLyS target and the BLyS binding
polypeptide, then isolating complexes of the BLyS target and the
binding polypeptides. For this type of separation, many methods are
known. For example, the binding polypeptide may be immobilized on a
solid support such as beads, then separated from the feed stream
along with the BLyS target by filtration. In another example, the
BLyS binding polypeptide may be modified with its own affinity tag,
such as a polyHis tail or streptavidin binding region, which can be
used to isolate the binding polypeptide after complexes have formed
using an immobilized metal affinity chromatographic resin or
steptavidin-coated substrate. Once separated, the BLyS target can
be released from the binding polypeptide under elution conditions
and recovered in a purified form.
[0540] Methods of producing BLyS or a BLyS-like polypeptides
usually yield BLyS or BLyS-like polypeptides in a feed stream that
additionally contains impurities (with respect to the BLyS target).
One purpose of the present invention is to produce BLyS binding
polypeptides and preparations (such as affinity chromatography
media or surfaces) comprising BLyS binding polypeptides that allow
rapid and highly specific purification of BLyS target proteins from
a feed stream. BLyS binding polypeptides obtained herein may easily
be tailored to isolate BLyS target protein from a particular feed
stream, using or routinely modifying conditions and techniques
known in the art. If an alternate production method for BLyS is
used, producing a different feed stream, a different set of BLyS
binding polypeptides and/or conditions may be necessary to achieve
the same level of purification. The new set of BLyS binding
polypeptides and/or conditions can be readily obtained following or
modifying procedures outlined herein, or otherwise known in the
art.
[0541] Use of BLyS Binding Polypeptides for Epitope Mapping
[0542] The present invention provides BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof), that can
be used to identify epitopes of BLyS. In particular, the BLyS
binding polypeptides of the present invention can be used to
identify epitopes of human BLyS (SEQ ID NOs: 173 and/or 174) or
BLyS expressed on human monocytes; murine BLyS (SEQ ID NOs: 175
and/or 176) or BLyS expressed on murine monocytes; rat BLyS (either
the soluble forms as given in SEQ ID NOs: 177, 178, 179 and/or 180
or in a membrane associated form, e.g., on the surface of rat
monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of
SEQ ID NOS: 181 and/or 182, the soluble form of monkey BLyS, or
BLyS expressed on monkey monocytes)using techniques described
herein or otherwise known in the art. 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.)
[0543] Diagnostic Uses of BLyS Binding Polypeptides
[0544] Labeled and non-labelled BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof) which
specifically bind to BLyS can be used for diagnostic purposes to
detect, diagnose, prognose, or monitor diseases and/or disorders
associated with the aberrant expression and/or activity of BLyS or
BLyS receptor. The invention provides for the detection of aberrant
expression of BLyS comprising: (a) assaying the expression of BLyS
in a biological sample from an individual using one or more BLyS
binding polypeptides that specifically binds to BLyS; and (b)
comparing the level of BLyS with a standard level of BLyS, e.g., in
normal biological samples, whereby an increase or decrease in the
assayed level of BLyS compared to the standard level of BLyS is
indicative of aberrant expression.
[0545] By "biological sample" is intended any fluids and/or cells
obtained from an individual, body fluid, body tissue, body cell,
cell line, tissue culture, or other source which may contain BLyS
protein or niRNA. Body fluids include, but are not limited to,
sera, plasma, urine, synovial fluid, spinal fluid, saliva, and
mucous. Tissues samples may be taken from virtually any tissue in
the body. Tissue samples may also be obtained from autopsy
material. 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.
[0546] The invention also provides for the detection of aberrant
expression of BLyS receptor comprising (a) assaying the expression
of BLyS receptor in a biological sample from an individual using
one or more BLyS binding polypeptides or fragments or variants
thereof that specifically binds only to soluble BLyS, but does not
inhibit BLyS/BLyS receptor binding. Such a BLyS binding
polypeptide, by way of an example that is not to be construed as
limiting, would be one that is able to capture a biotinylated BLyS
from solution, but that would not prevent BLyS from binding to it
receptor expressed, for example on IM-9 cells, and (b) comparing
the level of BLyS receptor with a standard level of BLyS receptor,
e.g., in normal tissue or cell samples, whereby an increase or
decrease in the assayed level of BLyS receptor compared to the
standard level of BLyS receptor is indicative of aberrant
expression.
[0547] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) which specifically bind to BLyS can be used
for diagnostic purposes to detect, diagnose, prognose, or monitor
immune system diseases and disorders, including but not limited to
autoimmune diseases and disorders and/or immunodeficiencies, and/or
diseases, disorders, or conditions associated therewith. The
invention provides for the detection of aberrant expression of BLyS
comprising: (a) assaying the expression of BLyS in a biological
sample from an individual using one or more BLyS binding
polypeptides that specifically binds to BLyS; and (b) comparing the
level of BLyS with a standard level of BLyS, e.g., in normal
biological samples, whereby an increase or decrease in the assayed
level of BLyS compared to the standard level of BLyS is indicative
of an autoimmune disorder or disease and/or an immunodeficiency. In
specific embodiments, an increase in the assayed level of BLyS is
indicative of an autoimmune disorder or disease. In other specific
embodiments, a decrease in the assayed level of BLyS is indicative
of an immunodeficiency.
[0548] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) which specifically bind to BLyS but do not
inhibit BLyS/BLys receptor binding can be used for diagnostic
purposes to detect, diagnose, prognose, or monitor immune system
diseases and disorders, including but not limited to autoimmune
diseases and disorders and/or immunodeficiencies, and/or diseases,
disorders, or conditions associated therewith. The invention
provides for the detection of aberrant expression of BLyS receptor
comprising: (a) assaying the expression of BLyS receptor in a
biological sample from an individual using one or more BLyS binding
polypeptides that specifically binds to BLyS; and (b) comparing the
level of BLyS receptor with a standard level of BLyS receptor,
e.g., in normal biological samples, whereby an increase or decrease
in the assayed level of BLyS receptor compared to the standard
level of BLyS receptor is indicative of an autoimmune disorder or
disease and/or an immunodeficiency. In specific embodiments, an
increase in the assayed level of BLyS receptor is indicative of an
autoimmune disorder or disease. In other specific embodiments, a
decrease in the assayed level of BLyS receptor is indicative of an
immunodeficiency.
[0549] Autoimmune disorders, diseases, or conditions that may be
detected, diagnosed, prognosed, or monitored using the BLyS binding
polypeptides include, but are not limited to, autoimmune hemolytic
anemia, autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia purpura, autoimmune neutropenia,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,
dermatitis, gluten-sensitive enteropathy, 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, myocarditis,
IgA glomerulonephritis, dense deposit disease, rheumatic heart
disease, Guillain-Barre Syndrome, insulin dependent diabetes
mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis,
hypothyroidism (i.e., Hashimoto's thyroiditis), systemic lupus
erhythematosus, 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, schleroderma with anti-collagen BLyS binding
polypeptides, mixed connective tissue disease,
polymyositis/dermatomyositis, pernicious anemia, idiopathic
Addison's disease, infertility, glomerular nephritis such as
primary glomerular nephritis 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.
[0550] In specific embodiments, the present invention encompasses
methods and compositions for detecting, diagnosing, prognosing,
and/or monitoring diseases or disorders associated with
hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some
immunodeficiencies). In other specific embodiments, the present
invention encompasses methods and compositions for detecting,
diagnosing, prognosing, and/or monitoring diseases or disorders
associated with hypogammaglobulinemia (e.g., an
immunodeficiency).
[0551] Immunodeficiencies that may be detected, diagnosed,
prognosed, or monitored using the BLyS binding polypeptides
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.
[0552] Elevated levels of soluble BLyS 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 BLyS, more than 30% of SLE patients had
levels greater than 10 ng/ml, and approximately 10% of SLE patients
had serum BLyS levels greater than 20 ng/ml. In contrast, the
majority of normal controls had BLyS levels less than 5 ng/ml, and
less than 10% had levels higher than 10 ng/ml. The elevated levels
of BLyS 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 BLyS 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 BLyS (unpublished data).
[0553] 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 anaylzed for BLyS 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 BLyS (.about.9 ng/ml BLyS). The
second subgroup, however, which was ANA+ sera from patients who
presented with the clinical impression of SLE, had significantly
increased BLyS levels (.about.15 ng/ml). These results suggest that
an elevated level of BLyS precedes the formal fulfillment of the
ACR criteria. The ACR criteria are desrcibed in Tan et al.,
Arthritis and Rheumatism, 25:1271-1277 (1982).
[0554] Thus, in specific embodiments, BLyS binding polypeptides
which specifically bind to BLyS can be used for diagnostic purposes
to detect, diagnose, prognose, or monitor Systemic Lupus
Erythematosus or conditions associated therewith. The invention
provides for the detection of aberrant expression of BLyS
comprising: (a) assaying the expression of BLyS in a biological
sample (e.g., serum, synovial fluid) of an individual using one or
more BLyS binding polypeptides that specifically binds to BLyS; and
(b) comparing the level of BLyS with a standard level of BLyS,
e.g., in normal biological samples, whereby an increase in the
assayed level of BLyS compared to the standard level of BLyS is
indicative of SLE.
[0555] In additional embodiments, BLyS binding polypeptides which
specifically bind to BLyS can be used for diagnostic purposes to
detect, diagnose, prognose, or monitor Rheumatoid Arthritis. The
invention provides for the detection of aberrant expression of BLyS
comprising: (a) assaying the expression of BLyS in a biological
sample (e.g., serum, synovial fluid) of an individual using one or
more BLyS binding polypeptides that specifically binds to BLyS; and
(b) comparing the level of BLyS with a standard level of BLyS,
e.g., in normal biological samples, whereby an increase in the
assayed level of BLyS compared to the standard level of BLyS is
indicative of Rheumatoid Arthritis.
[0556] In specific embodiments, the present invention encompasses
methods and compositions for detecting, diagnosing and/or
prognosing diseases or disorders of 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.
[0557] In specific embodiments, the present invention encompasses
methods and compositions for detecting, diagnosing, prognosing and
or monitoring growth, progression, and/or metastases of
malignancies and proliferative diseases or disorders associated
with increased cell survival, or the inhibition of apoptosis. For a
review of such disorders, see Fishman et al., Medicine, 2d Ed. (J.
B. Lippincott Co., Philadelphia 1985). An extensive list of
examples of proliferative diseases and disorders is presented below
in the section of this application entitled "Therapeutic Uses of
BLyS Binding Polypeptides." Proliferative diseases and disorders is
also extended to include premalignant conditions (e.g., benign
tumors, hyperproliferative disorders, and benign proliferative
disorders--see below) as well as proliferative disorders of B
cells, monocytes, macrophages, and T cells. Other abnormal growth
conditions that may be treated, diagnosed, prognosed or monitored
include, but are not limited to, hyperplasia, metaplasia, or most
particularly, dysplasia has occurred (for review of such abnormal
growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed.
(W. B. Saunders Co., Philadelphia 1976), pp. 68-79.) Hyperplasia is
a form of controlled cell proliferation involving an increase in
cell number in a tissue or organ, without significant alteration in
structure or function. As but one example, endometrial hyperplasia
often precedes endometrial cancer. Metaplasia is a form of
controlled cell growth in which one type of adult or fully
differentiated cell substitutes for another type of adult cell.
Metaplasia can occur in epithelial or connective tissue cells.
Atypical metaplasia involves a somewhat disorderly metaplastic
epithelium. Dysplasia is frequently a forerunner of cancer, and is
found mainly in the epithelia; it is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and in the architectural orientation of cells.
Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation, and
is often found in the cervix, respiratory passages, oral cavity,
and gall bladder.
[0558] In preferred embodiments, the present invention encompasses
methods and compositions for detecting, diagnosing, prognosing and
or monitoring growth, progression, and/or metastases of
malignancies and proliferative diseases or disorders of monocytic
cells.
[0559] In specific embodiments, the present invention encompasses
methods and compositions for detecting, diagnosing, prognosing and
or monitoring growth, progression, and/or metastases of
malignancies and proliferative diseases or disorders of B
cells.
[0560] The invention provides a diagnostic assay for diagnosing or
prognosing a disease or disorder, comprising: (a) assaying for the
level of BLyS in a biological sample of an individual using one or
more BLyS binding polypeptides that specifically bind to BLyS; and
(b) comparing the level of BLyS with a standard BLyS level, e.g.,
in a biological sample from a patient without the disease or
disorder, whereby an increase or decrease in the assayed BLyS level
compared to the standard level of BLyS is indicative of a
particular disease or disorder. With respect to cancer, the
presence of a relatively high amount of BLyS 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.
[0561] In specific embodiments, the presence of a relatively high
amount of membrane-bound BLyS in a biological sample is indicative
of monocytic cell related leukemias or lymphomas, such as, for
example acute myelogenous leukemia, and/or the severity
thereof.
[0562] In other specific embodiments, the presence of a relatively
high amount of BLyS receptor in a biological sample (as determined
using BLyS binding polypeptides that bind to soluble BLyS, but do
not inhibit BLyS/BLyS receptor binding) is indicative of B cell
related leukemias or lymphomas (e.g., chronic lymphocytic leukemia,
multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's disease),
and/or the severity thereof.
[0563] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) can be used to assay protein levels in a
biological sample using classical immunohistological methods as
described herein or as 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 methods that
can be used for detecting protein gene expression that might
utilize BLyS binding polypeptides or fragments or variants thereof
include, but are not limited to, 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, alkaline phophatase, and horseradish
peroxidase; radioisotopes, such as iodine (.sup.121I, .sup.123I,
.sup.125I, .sup.131I), carbon (.sup.14C), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.111In, .sup.112In, .sup.113mIn,
.sup.115mIn), technetium (.sup.99Tc,.sup.99Tc), thallium
(.sup.201Ti), gallium .sup.68Ga,.sup.67Ga), palladium (.sup.103Pd),
molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F),
.sup.15f3Sm, .sup.177Lu, .sup.159Gd, 149Pm, 140La, .sup.175Yb,
.sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re,
.sup.142Pr, .sup.105Rh, and 97Ru; luminescent labels as luminol;
and fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0564] Certain embodiments of the invention are directed to the
detection and diagnosis of a disease or disorder associated with
aberrant expression of BLyS or BLyS receptor in an animal,
preferably a mammal and most preferably a human. In one embodiment,
diagnosis comprises:
[0565] (a) administering (for example, parenterally,
subcutaneously, or intraperitoneally) to a subject an effective
amount of a labeled BLyS binding polypeptide (including molecules
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) that specifically binds
to BLyS; (b) waiting for a time interval following the
administering for permitting the labeled BLyS binding polypeptide
to preferentially concentrate at sites in the subject where BLyS is
expressed (and for unbound labeled molecule to be cleared to
background level); (c) determining background level; and (d)
detecting the labeled BLyS binding polypeptide in the subject, such
that detection of labeled BLyS binding polypeptide or fragment
thereof above the background level and above or below the level
observed in a person without the disease or disorder indicates that
the subject has a particular disease or disorder associated with
aberrant expression of BLyS or BLyS receptor. 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.
[0566] It will be understood by those skilled 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.99Tc. The labeled BLyS binding
polypeptide will then preferentially accumulate at the location of
cells which contain the specific protein. In vivo tumor imaging is
described in 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).
[0567] 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.
[0568] In an embodiment for monitoring of the disease or disorder,
the method is carried out by repeating the method for diagnosing
the disease or disorder, for example, one month after initial
diagnosis, six months after initial diagnosis, one year after
initial diagnosis, etc. and comparing the results of the successive
tests.
[0569] 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.
[0570] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (see, e.g., 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 patient 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).
[0571] Immunophenotyping Using BLyS Binding Polypeptides
[0572] The BLyS binding polypeptides (including molecules
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) may be utilized for
immunophenotyping of cell lines and biological samples by their
BLyS expression or BLyS receptor expression. Various techniques can
be employed utilizing BLyS binding polypeptides, fragments, or
variants to screen for cellular populations (i.e., immune cells,
particularly monocytic cells or B-cells) expressing BLyS or BLyS
receptor. Such techniques include magnetic separation using BLyS
binding polypeptide-coated magnetic beads, "panning" with BLyS
binding polypeptide 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)).
[0573] 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.
[0574] In one embodiment, BLyS binding polypeptides (including
molecules comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) are used to identify
cells of monocytic or B cell origin.
[0575] Therapeutic Uses of BLyS Binding Polypeptides
[0576] The present invention is further directed to BLyS binding
polypeptide-based therapies which involve administering BLyS
binding polypeptides (including molecules comprising, or
alternatively consisting of, BLyS binding polypeptide fragments or
variants thereof) 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, BLyS binding
polypeptides and nucleic acids encoding BLyS binding polypeptides
and antibodies that bind BLyS binding polypeptides as described
herein. The BLyS binding polypeptides can be used to treat,
ameliorate or prevent diseases, disorders or conditions associated
with aberrant expression and/or activity of BLyS or BLyS receptor,
including, but not limited to, any one or more of the diseases,
disorders, or conditions described herein. The treatment and/or
prevention of diseases, disorders, or conditions associated with
aberrant BLyS expression and/or activity or aberrant BLyS receptor
expression and/or activity includes, but is not limited to,
alleviating symptoms associated with those diseases, disorders or
conditions. BLyS binding polypeptides may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0577] BLyS binding polypeptides of the present invention
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof) that
function as agonists or antagonists of BLyS, preferably of
BLyS-induced signal transduction, can be administered to an animal
to treat, prevent or ameliorate a disease or disorder associated
with aberrant BLyS expression, lack of BLyS function, aberrant BLyS
receptor expression, or lack of BLyS receptor function. For
example, BLyS binding polypeptides which disrupt the interaction
between BLyS and one or more of its receptors may be administered
to an animal to treat, prevent or ameliorate a disease or disorder
associated with aberrant BLyS expression, excessive BLyS function,
aberrant BLyS receptor expression, or excessive BLyS receptor
function. BLyS binding polypeptides which do not prevent BLyS from
binding its receptor but inhibit or downregulate BLyS-induced
signal transduction can be administered to an animal to treat,
prevent or ameliorate a disease or disorder associated with
aberrant BLyS expression, excessive BLyS function, aberrant BLyS
receptor expression, or excessive BLyS receptor function. In
particular, BLyS binding polypeptides of the present invention
which prevent BLyS-induced signal transduction by specifically
recognizing the unbound BLyS, receptor-bound BLyS, or both unbound
and receptor-bound BLyS can be administered to an animal to treat,
prevent or ameliorate a disease or disorder associated with
aberrant BLyS expression, excessive BLyS function, aberrant BLyS
receptor expression, or excessive BLyS receptor function.
[0578] The ability of a BLyS binding polypeptide to inhibit or
downregulate BLyS-induced signal transduction may be determined by
techniques described herein or otherwise known in the art. For
example, BLyS-induced receptor activation and the activation of
signaling molecules can be determined by detecting the
phosphorylation (e.g., tyrosine or serine/threonine) of the
receptor or a signaling molecule by immunoprecipitation followed by
western blot analysis (for example, as described herein).
[0579] In a specific embodiment, a BLyS binding polypeptide of the
present invention (including molecules comprising, or alternatively
consisting of, BLyS binding polypeptide fragments or variants
thereof) that inhibits or reduces BLyS activity 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%, at least 45%, at least 40%, at least
45%, at least 35%, at least 30%, at least 25%, at least 20%, or at
least 10% relative to BLyS activity in the absence of the BLyS
binding polypeptide, is administered to an animal to treat, prevent
or ameliorate a disease or disorder associated with aberrant BLyS
expression, excessive BLyS function, aberrant BLyS receptor
expression, or excessive BLyS receptor function. In another
embodiment, a combination of BLyS binding polypeptides, a
combination of BLyS binding polypeptide fragments, a combination of
BLyS binding polypeptide variants, or a combination of BLyS binding
polypeptides, BLyS binding polypeptide fragments, and/or variants
that inhibit or reduce BLyS activity by 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%, at least 50%, at least 45%, at
least 40%, at least 45%, at least 35%, at least 30%, at least 25%,
at least 20%, or at least 10% relative to BLyS activity in absence
of said BLyS binding polypeptides, BLyS binding polypeptide
fragments, and/or BLyS binding polypeptide variants are
administered to an animal to treat, prevent or ameliorate a disease
or disorder associated with aberrant BLyS expression, excessive
BLyS function, aberrant BLyS receptor expression, or excessive BLyS
receptor function.
[0580] Further, BLyS binding polypeptides of the present invention
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof) which
activate BLyS-induced signal transduction can be administered to an
animal to treat, prevent or ameliorate a disease or disorder
associated with aberrant BLyS expression, lack of BLyS function,
aberrant BLyS receptor expression, or lack of BLyS receptor
function. These BLyS binding polypeptides may potentiate or
activate either all or a subset of the biological activities of
BLyS-mediated receptor activation, for example, by inducing
multimerization of BLyS and/or multimerization of the receptor. The
BLyS binding polypeptides may be administered with or without being
pre-complexed with BLyS. In a specific embodiment, a BLyS binding
polypeptide of the present invention that increases BLyS activity
by at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, or 100% or more relative to BLyS activity in
absence of the BLyS binding polypeptide is administered to an
animal to treat, prevent or ameliorate a disease or disorder
associated with aberrant BLyS expression, lack of BLyS function,
aberrant BLyS receptor expression, or lack of BLyS receptor
function. In another embodiment, a combination of BLyS binding
polypeptides, a combination of BLyS binding polypeptide fragments,
a combination of BLyS binding polypeptide variants, or a
combination of BLyS binding polypeptides, BLyS binding polypeptide
fragments and/or BLyS binding polypeptide variants that increase
BLyS activity by at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 99%, or 100% or more relative to BLyS
activity in absence of the said BLyS binding polypeptides or BLyS
binding polypeptide fragments and/or BLyS binding polypeptide
variants is administered to an animal to treat, prevent or
ameliorate a disease or disorder associated with aberrant BLyS
expression, lack of BLyS function, aberrant BLyS receptor
expression, or lack of BLyS receptor function.
[0581] In a specific embodiment, the present invention provides a
method of treating, preventing or ameliorating a disease or
disorder associated with aberrant BLyS or BLyS receptor expression
or activity, comprising administering to an animal in which such
treatment, prevention or amelioration is desired, a BLyS binding
polypeptide in an amount effective to treat, prevent or ameliorate
the disease or disorder. Diseases and disorders which may be
treated, prevented or ameliorated by this method include, but are
not limited to, immune system diseases and disorders (e.g.,
autoimmune diseases and disorders, immunodeficiencies, lupus,
rheumatoid arthritis, multiple sclerosis, hypogammaglobulinemia and
hypergammaglobulinemia), graft vs. host disease, proliferative
diseases and disorders (e.g., cancer) and infectious diseases and
disorders.
[0582] In a specific embodiment, the present invention provides a
method of treating, preventing or ameliorating a disease or
disorder of cells of hematopoietic origin, comprising administering
to an animal in which such treatment, prevention, or amelioration
is desired, a BLyS binding polypeptide in an amount effective to
treat, prevent or ameliorate the disease or disorder. 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.
[0583] One or more BLyS binding polypeptides of the present
invention (including molecules comprising, or alternatively
consisting of, BLyS binding polypeptide fragments or variants
thereof) that specifically bind to BLyS may be used locally or
systemically in the body as a therapeutic. The BLyS binding
polypeptides (including molecules comprising, or alternatively
consisting of, BLyS binding polypeptide fragments or variants
thereof) may also be advantageously utilized in combination with
monoclonal or chimeric antibodies, lymphokines and/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 BLyS binding polypeptides.
[0584] The BLyS binding polypeptides (including molecules
comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) may be administered
alone or in combination with other types of treatments (e.g.,
radiation therapy, chemotherapy, hormonal therapy, immunotherapy,
anti-tumor agents, anti-angiogenesis and anti-inflammatory
agents).
[0585] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing BLyS binding polypeptides (including
molecules comprising, or alternatively consisting of, BLyS binding
polypeptide fragments or variants thereof) that specifically bind
to BLyS, or polynucleotides encoding BLys binding polypeptides that
specifically bind to BLyS, for both immunoassays directed to and
therapy of disorders related to BLyS polynucleotides or
polypeptides, including fragments thereof. Such BLys binding
polypeptides will preferably have an affinity for BLys and/or BLys
fragments. Preferred binding affinities include those 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, BLyS binding polypeptides bind BLyS target
proteins 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.-8 M, or 10.sup.-8 M. Even more preferably, BLyS
binding polypeptides bind BLyS target proteins 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.-11 M, 5.times.10.sup.-12 M, or 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, or 10.sup.-15 M.
[0586] In a preferred embodiment, BLyS binding polypeptides
neutralize BLyS activity. In another preferred embodiment, BLyS
binding polypeptides inhibit B cell proliferation.
[0587] In a preferred embodiment, BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof) inhibit or
reduce binding of the soluble form of BLyS to a BLyS receptor. In
another preferred embodiment BLyS binding polypeptides inhibit or
reduce B cell proliferation induced by the soluble form of BLyS. In
another preferred embodiment BLyS binding polypeptides inhibit or
reduce immunoglobulin production induced by the soluble form of
BLyS.
[0588] In a preferred embodiment, BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof) inhibit or
reduce binding of membrane-bound BLyS to a BLyS receptor. In
another preferred embodiment, BLyS binding polypeptides inhibit or
reduce B cell proliferation induced by the membrane-bound form of
BLyS. In another preferred embodiment, BLyS binding polypeptides
inhibit or reduce immunoglobulin production induced by the membrane
bound form of BLyS.
[0589] In a preferred embodiment, BLyS binding polypeptides
(including molecules comprising, or alternatively consisting of,
BLyS binding polypeptide fragments or variants thereof) inhibit or
reduce binding of both the soluble and membrane-bound forms of BLyS
to a BLyS receptor. In another preferred embodiment, BLyS binding
polypeptides inhibit or reduce B cell proliferation induced by
either or both forms of BLyS. In another preferred embodiment, BLyS
binding polypeptides inhibit or reduce immunoglobulin production
induced by either or both forms of BLyS.
[0590] In one embodiment, the invention provides a method of
delivering radiolabelled BLyS binding polypeptide and/or BLyS
binding polypeptide conjugates to targeted cells, such as, for
example, monocytic cells expressing the membrane-bound form of
BLyS, or B cells expressing a BLyS receptor.
[0591] 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 BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide inhibits or reduces BLyS mediated
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),
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a BLyS
binding polypeptide in an amount effective to inhibit or reduce
immunoglobulin production.
[0592] 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 BLyS binding
polypeptide with BLyS, wherein the effective amount of the BLyS
binding polypeptide stimulates BLyS mediated immunoglobulin
production. 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 BLyS binding polypeptide in an amount effective to
stimulate immunoglobulin production. 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.
[0593] Receptors belonging to the TNF receptor (TNFR) super family
(e.g., TACI and BCMA) 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 and BCMA do not contain death domains.
[0594] Investigation of BLyS 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 BLyS+SAC treatment
(see Example 12). 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, 1998). 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 BLyS on B cells.
[0595] Additionally, in some samples of human tonsillar B cells
stimulated with BLys and SAC, mRNA for CD25 (IL-2Ralpha) was
upregulated. Nuclear extracts from Human tonsillar B cells treated
with BLyS and from IM-9 cells treated with BLyS 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.
[0596] Thus BLyS 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, BLyS treatment of B cells
induces cellular proliferation immunoglobulin secretion, a
characteristic of activated B cells (Moore et al., Science,
285:260-263, 1999). BLyS binding polypeptides complexed with BLyS
may inhibit, stimulate, or not significantly alter these BLyS
mediated activities.
[0597] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of BLyS binding polypeptide with BLyS, wherein the effective
amount of BLyS binding polypeptide inhibits or reduces BLyS
mediated B cell proliferation. In another embodiment, the invention
provides methods and compositions for inhibiting or reducing B cell
proliferation comprising, or alternatively consisting of,
administering to an animal in which such inhibition or reduction is
desired, a BLyS binding polypeptide in an amount effective to
inhibit or reduce B cell proliferation.
[0598] In one embodiment, the invention provides methods and
compositions for stimulating B cell proliferation, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide stimulates BLyS mediated B cell
proliferation.
[0599] In one embodiment, the invention provides methods and
compositions for stimulating B cell proliferation, comprising, or
alternatively consisting of, administering to an animal in which
such stimulation is desired, a BLyS binding polypeptide in an
amount effective to stimulate B cell proliferation.
[0600] B cell proliferation is most commonly assayed in the art by
measuring tritiated thymidine incorporation (see Examples 7 and 8).
This and other assays are commonly known in the art and may be
routinely adapted for the use of determining the effect of BLys
binding polypeptides on B cell proliferation.
[0601] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of BLyS binding polypeptide with BLyS, wherein the effective
amount of BLyS binding polypeptide inhibits or reduces BLyS
mediated B cell activation.
[0602] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a BLyS
binding polypeptide in an amount effective to inhibit or reduce B
cell activation.
[0603] In one embodiment, the invention provides methods and
compositions for increasing activation of B cells, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide increases BLyS mediated activation of B
cells.
[0604] In one embodiment, the invention provides methods and
compositions for increasing activation of B cells, comprising, or
alternatively consisting of, administering to an animal in which
such increase is desired, a BLyS binding polypeptide in an amount
effective to increase B cell activation.
[0605] 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 (Example 12). 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 may be routinely adapted for the
use of determining the effect of BLys binding polypeptides on B
cell activation.
[0606] In one embodiment, the invention provides methods and
compositions for decreasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide inhibits or reduces BLyS regulated lifespan of
B cells.
[0607] In one embodiment, the invention provides methods and
compositions for decreasing lifespan of B cells, comprising, or
alternatively consisting of, administering to an animal in which
such decrease is desired, a BLyS binding polypeptide in an amount
effective to decrease B cell lifespan.
[0608] In one embodiment, the invention provides methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of BLyS
binding polypeptide with BLyS, wherein the effective amount of BLyS
binding polypeptide increases BLyS regulated lifespan of B
cells.
[0609] In one embodiment, the invention provides methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, administering to an animal in which
such increase is desired, a BLyS binding polypeptide in an amount
effective to increase lifespan of B cells.
[0610] 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 BLyS, B cells
treated with BLyS/BLys binding polypeptide complexes, and untreated
B cells in order to determine the effects of BLyS and BLyS binding
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 BLyS binding
polypeptides on BLyS regulated B cell lifespan.
[0611] In one embodiment, the invention provides a method for the
specific delivery of BLyS binding polypeptides and BLyS binding
polypeptide conjugates to cells by administering molecules 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) in the
targeted cell.
[0612] In another embodiment, the invention provides for a method
of killing cells of hematopoietic origin, comprising, or
alternatively consisting of, contacting BLyS binding polypeptides
with BLyS to form a complex; and contacting the complex 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 BLyS binding 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.
[0613] 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 BLyS binding
polypeptides or BLyS binding polypeptide conjugates (e.g.,
radiolabeled BLyS binding polypetides and/or BLyS binding
polypeptides conjugated with radioisotopes, toxins, or cytotoxic
prodrugs). In a specific embodiment, the invention provides a
method for the specific destruction of cells of monocytic lineage
(e.g., monocytic cell related leukemias or lymphomas, such as, for
example acute myelogenous leukemia) by administering BLyS binding
polypeptides or BLyS binding polypeptide conjugates (e.g., BLyS
binding polypeptides conjugated with radioisotopes, toxins, or
cytotoxic prodrugs) that specifically bind the membrane-bound form
of BLyS. In another 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 (e.g., chronic
lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, and
Hodgkin's disease) by administering BLyS binding polypeptides or
BLyS binding polypeptide conjugates (e.g., BLyS binding
polypeptides conjugated with radioisotopes, toxins, or cytotoxic
prodrugs) that bind soluble BLyS, but do not inhibit BLyS binding
to a BLyS receptor on B cells.
[0614] In another embodiment of the invention, therapeutic or
pharmaceutical compositions are administered to an animal to treat,
prevent or ameliorate diseases and disorders of the immune system.
In a specific embodiment, the invention provides a method of
treating, preventing, or ameliorating an immune system disease or
disorder, comprising, or alternatively consisting of, administering
to an animal in which such treatment, prevention, or amelioration
is desired, a BLyS binding polypeptide in an amount effective to
treat, prevent, or ameliorate the immune system disease or
disorder. Diseases and disorders of the immune system include, but
are not limited to, autoimmune diseases and disorders (e.g.,
arthritis, graft rejection, Hashimoto's thyroiditis,
insulin-dependent diabetes, lupus, rheumatoid arthritisidiopathic
thrombocytopenic purpura, systemic lupus erythramatosus and
multiple sclerosis, and other autoimmune diseases or disorders
named or desdcribed herein), hypogammaglobulinemia,
hypergammaglobulinemia, elective IgA deficiency,
ataxia-telangiectasia, immunodeficiencies (e.g., common variable
immunodeficiency (CVID), X-linked agammaglobulinemia, severe
combined immunodeficiency (SCID), and Wiskott-Aldrich syndrome),
graft vs. host disease, idiopathic hyper-eosinophilic syndrome,
monocytic leukemoid reaction, monocytic leukocytosis, monocytic
leukopenia, monocytopenia, monocytosis, graft or transplant
rejection, as well as infectious diseases (e.g., AIDS and
hepatitis).
[0615] As discussed herein, BLyS binding polypeptides and BLyS
binding polypeptide compositions, may be used to treat, prevent,
ameliorate, diagnose or prognose 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 BLyS binding polypeptides and BLyS binding
polypeptide compositions 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,
BLyS binding polypeptides and BLyS binding polypeptide compositions
are used to treat, prevent, ameliorate, diagnose and/or prognose an
autoimmune disorder, or condition(s) associated with such
disorder.
[0616] Autoimmune disorders and conditions associated with these
disorders that may be treated, prevented, ameliorated, diagnosed
and/or prognosed according to the invention with the therapeutic
and pharmaceutical compositions described herein include, but are
not limited to, autoimmune hemolytic anemia, autoimmune neonatal
thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune
neutropenia, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, gluten-sensitive
enteropathy, 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, myocarditis, IgA glomerulonephritis, dense deposit
disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin
dependent diabetes mellitis, and autoimmune inflammatory eye
disease.
[0617] Additional autoimmune disorders and conditions associated
with these disorders that may be treated, prevented, ameliorated,
diagnosed and/or prognosed according to the present invention with
the therapeutic and pharmaceutical compositions described herein
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 erhythematosus (often characterized,
e.g., by circulating and locally generated immune complexes),
discoid lupus, 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.H
[0618] Additional autoimmune disorders and conditions associated
with these disorders that may be treated, prevented, ameliorated,
diagnosed and/or prognosed according to the present invention with
the therapeutic and pharmaceutical compositions described herein
include, but are not limited to, rheumatoid arthritis (often
characterized, e.g., by immune complexes in joints), schleroderma
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),
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.
[0619] In a preferred embodiment, therapeutic and pharmaceutical
compositions are used to treat, prevent, ameliorate, diagnose or
prognose, a member of the group: autoimmune hemolytic anemia, as
primary glomerulonephritis, IgA glomerulonephritis, Goodpasture's
syndrome, idiopathic thrombocytopenia, Multiple Sclerosis,
Myasthenia Gravis, Pemphigus, polymyositis/dermatomyositis,
relapsing polychondritis, rheumatoid arthritis, Sjogren's syndrome,
systemic lupus erhythematosus, Uveitis, vasculitis,and primary
biliary cirrhosis.
[0620] In another specific preferred embodiment, therapeutic and
pharmaceutical compositions are used to treat, prevent, amelioate,
diagnose or prognose, rheumatoid arthritis and/or medical
conditions associated therewith.
[0621] In a specific preferred embodiment, therapeutic and
pharmaceutical compositions are used to treat, prevent, amelioate,
diagnose or prognose, lupus and/or medical conditions associated
therewith. Lupus-associated conditions that may be treated,
prevented, ameliorated, prognosed and/or diagnosed with the BLyS
binding polypeptides and BLyS binding polypeptide compositions
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
(pleurisy)), 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, therapeutic and
pharmaceutical compositions are used to treat, prevent, ameliorate,
diagnose, or prognose, renal disorders associated with systemic
lupus erythematosus. In a most preferred embodiment, therapeutic
and pharmaceutical compositions are used to treat, prevent,
ameliorate, diagnose, or prognose, nephritis associated with
systemic lupus erythematosus. In another most preferred embodiment,
therapeutic or pharmaceutical compositions are administered to an
animal to treat, prevent or ameliorate lupus or glomerular
nephritis.
[0622] In another embodiment, therapeutic or pharmaceutical
compositions are administered to an animal to treat, prevent or
ameliorate an IgE-mediated allergic reaction or histamine-mediated
allergic reaction. Examples of allergic reactions include, but are
not limited to, asthma, rhinitis, eczema, chronic urticaria, and
atopic dermatitis. In another embodiment, therapeutic or
pharmaceutical compositions are administered to an animal to treat,
prevent, or ameliorate anaphylaxis, hypersensitivity to an
antigenic molecule, or blood group incompatibility. In another
embodiment, therapeutic or pharmaceutical compositions are
administered to an animal to treat, prevent or ameliorate or
modulate inflammation or an inflammatory disorder. Examples of
chronic and acute inflammatory disorders that may be treated
prevented or ameliorated with the therapeutic and pharmaceutical
compositions include, but are not limited to, 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, Crohn's disease, inflammatory bowel disease, chronic and
acute inflammatory pulmonary diseases, bacterial infection,
psoriasis, septicemia, cerebral malaria, arthritis,
gastroenteritis, and glomerular nephritis.
[0623] In another embodiment, therapeutic or pharmaceutical
compositions 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.
[0624] Therapeutic or pharmaceutical compositions may also be
administered 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, therapeutic or pharmaceutical compositions as described
herein may be used to inhibit the proliferation and differentiation
of cells involved in producing anticardiolipin antibodies. These
compositions can be used to treat, prevent, ameliorate, diagnose,
and/or prognose 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.
[0625] Therapeutic or pharmaceutical compositions containing BLyS
binding polypeptides may also be administered to treat, prevent, or
ameliorate 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. Administration of BLyS binding
polypeptides that inhibit an immune response may be an effective
therapy in preventing organ rejection or GVHD. In specific
embodiments the present invention provides a method of inhibiting
or reducing graft rejection, comprising administering to an animal
in which such inhibition or reduction is desired, a BLyS binding
polypeptide in an amount effective to inhibit or reduce graft
rejection.
[0626] In another embodiment, therapeutic or pharmaceutical
compositions are administered to an animal to treat, prevent or
ameliorate a disease or disorder diseases associated with increased
apoptosis including, but not limited to, 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. In
another embodiment, therapeutic or pharmaceutical compositions are
administered to an animal to treat, prevent or ameliorate bone
marrow failure, for example, aplastic anemia and myelodysplastic
syndrome.
[0627] In other embodiment, therapeutic or pharmaceutical
compositions as described herein are used to treat or prevent a
proliferative disorder (e.g., cancer). In preferred embodiments,
therapeutic or pharmaceutical compositions as described herein are
used to treat or prevent proliferative disorders of monocytic
cells. In other preferred embodiments, therapeutic or
pharmaceutical compositions as described herein are used to treat
or prevent a proliferative disorders of B cells (e.g.,
leukemia).
[0628] In another embodiment, therapeutic or pharmaceutical
compositions as described herein are administered to an animal to
treat, prevent or ameliorate growth, progression, and/or metastases
of malignancies and proliferative diseases and disorders associated
with increased cell survival, or the inhibition of apoptosis. In a
specific embodiment, the present invention provides a method of
treating a proliferative disease or disorder, comprising
administering to an animal in which such treatment is desired, a
BLyS binding polypeptide in an amount effective to treat the
proliferative disease or disorder. For a review of such disorders,
see Fishman et al., Medicine, 2d Ed. (J. B. Lippincott Co.,
Philadelphia 1985). Examples of such disorders, include, but are
not limited to, leukemia (e.g., acute leukemia such as acute
lymphocytic leukemia and acute myelocytic leukemia, myeloblastic
leukemia, promyelocytic leukemia, myelomonocytic leukemia,
monocytic leukemia, erythroleukemia, chronic leukemia, chronic
myelocytic (granulocytic) leukemia, and chronic lymphocytic
leukemia), Polycythemia vera, lymphomas (e.g. Hodgkin's lymphoma,
non-Hodgkin's lymphoma) Hodgkin's disease, non-Hodgkin's disease,
multiple myeloma, Waldenstrom's macroglobulinemia, neoplasms,
tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
colorectal 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, nasopharyngeal carcinoma,
bronchogenic carcinoma, esophageal carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, uterine cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
melanoma, neuroblastoma, and retinoblastoma) heavy chain disease,
metastases, or any disease or disorder characterized by
uncontrolled cell growth. This method of treating a proliferative
diseases or disorders can also be used to treat premalignant
conditions (e.g., benign tumors, hyperproliferative disorders, and
benign proliferative disorders--see below) as well as proliferative
disorders of B cells, monocytes, macrophages, and T cells.
[0629] In another embodiment of the present invention, therapeutic
or pharmaceutical compositions as described herein can also be
administered to treat a subset of proliferative disorders, namely,
premalignant conditions (e.g., benign tumors, hyperproliferative
disorders, benign proliferative disorders) and to prevent
progression to a neoplastic or malignant state, including but not
limited to those disorders listed above. Such prophylactic or
therapeutic use is indicated in conditions known or suspected of
preceding progression to neoplasia or cancer, in particular, where
non-neoplastic cell growth consisting of hyperplasia, metaplasia,
or most particularly, dysplasia has occurred (for review of such
abnormal growth conditions, see Robbins and Angell, Basic
Pathology, 2d Ed. (W. B. Saunders Co., Philadelphia 1976), pp.
68-79.) Hyperplasia is a form of controlled cell proliferation
involving an increase in cell number in a tissue or organ, without
significant alteration in structure or function. As but one
example, endometrial hyperplasia often precedes endometrial cancer.
Metaplasia is a form of controlled cell growth in which one type of
adult or fully differentiated cell substitutes for another type of
adult cell. Metaplasia can occur in epithelial or connective tissue
cells. Atypical metaplasia involves a somewhat disorderly
metaplastic epithelium. Dysplasia is frequently a forerunner of
cancer, and is found mainly in the epithelia; it is the most
disorderly form of non-neoplastic cell growth, involving a loss in
individual cell uniformity and in the architectural orientation of
cells. Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation, and
is often found in the cervix, respiratory passages, oral cavity,
and gall bladder.
[0630] Alternatively or in addition to the presence of abnormal
cell growth characterized as hyperplasia, metaplasia, or dysplasia,
the presence of one or more characteristics of a transformed
phenotype, or of a malignant phenotype, displayed in vivo or
displayed in vitro by a cell sample from a patient, can indicate
the desirability of prophylactic/therapeutic administration of a
therapeutic or pharmaceutical composition as described herein.
Characteristics of a transformed phenotype include, but are nor
limited to, morphology changes, looser substratum attachment, loss
of contact inhibition, loss of anchorage dependence, protease
release, increased sugar transport, decreased serum requirement,
expression of fetal antigens, and disappearance of the 250,000
dalton cell surface protein.
[0631] In other embodiments, a patient which exhibits one or more
of the following predisposing factors for malignancy is treated by
administration of an effective amount of a therapeutic or
pharmaceutical composition as described herein: a chromosomal
translocation associated with a malignancy (e.g., the Philadelphia
chromosome for chronic myelogenous leukemia, t(14;18) for
follicular lymphoma, etc.), familial polyposis or Gardner's
syndrome (possible forerunners of colon cancer), benign monoclonal
gammopathy (a possible forerunner of multiple myeloma), and a first
degree kinship with persons having a cancer or precancerous disease
showing a Mendelian (genetic) inheritance pattern (e.g., familial
polyposis of the colon, Gardner's syndrome, hereditary exostosis,
polyendocrine adenomatosis, medullary thyroid carcinoma with
amyloid production and pheochromocytoma, Peutz-Jeghers syndrome,
neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid
body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma,
xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi
syndrome, albinism, Fanconi's aplastic anemia, and Bloom's
syndrome; see Robbins and Angell, supra, pp. 112-113), etc.)
[0632] In a specific embodiment, therapeutic or pharmaceutical
compositions as described herein are used to treat or prevent a
disorder characterized by hypergammagloulinemia (e.g., AIDS,
autoimmune diseases, and some immunodeficiencies).
[0633] In a specific embodiment, therapeutic or pharmaceutical
compositions as described herein are used to treat or prevent a
disorder characterized by deficient serum immunoglobulin
production, recurrent infections, and/or immune system dysfunction.
Moreover, therapeutic or pharmaceutical compositions as described
herein 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, recurrent
bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia,
hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster),
and/or pheumocystis carnii.
[0634] Therapeutic or pharmaceutical compositions as described
herein thereof, may be used to diagnose, prognose, treat or prevent
one or more of the following diseases or disorders, or conditions
associated therewith: primary immuodeficiencies, 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.
[0635] Additionally, therapeutic or pharmaceutical compositions as
described herein 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
mutliple 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.,
hypogamma-globulinemic 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.
[0636] Additional preferred embodiments of the invention include,
but are not limited to, the use of therapeutic or pharmaceutical
compositions as described herein in the following applications:
[0637] 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 induce
higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE),
and/or to increase an immune response. In a specific nonexclusive
embodiment, therapeutic or pharmaceutical compositions as described
herein are administered to boost the immune system to produce
increased quantities of IgG. In another specific nonexclusive
embodiment, BLyS binding polypeptides of the are administered to
boost the immune system to produce increased quantities of IgA. In
another specific non-limiting embodiment, BLyS binding polypeptides
are administered to boost the immune system to produce increased
quantities of IgM.
[0638] 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 applications WO 98/24893,
WO 96/34096, WO 96/33735, and WO 91/10741).
[0639] Additional preferred embodiments of the invention include,
but are not limited to, the use of therapeutic or pharmaceutical
compositions as described herein in the following applications:
[0640] A vaccine adjuvant that enhances immune responsiveness to
specific antigen. In a specific embodiment, the vaccine is a BLyS
binding polypeptide described herein. In another specific
embodiment, the vaccine adjuvant is a polynucleotide described
herein (e.g., a BLyS binding polypeptide polynucleotide genetic
vaccine adjuvant). As discussed herein, therapeutic or
pharmaceutical compositions as described herein 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.
[0641] An adjuvant to enhance tumor-specific immune responses.
[0642] An adjuvant to enhance anti-viral immune responses.
Anti-viral immune responses that may be enhanced using the
compositions as described herein 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 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 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 as described herein are used as an adjuvant to
enhance an immune response to the HIV gpl20 antigen.
[0643] 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 as described herein 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 as described
herein 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 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, Neisseria meningitidis, Streptococcus pneumoniae, Group
B Streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium
(malaria).
[0644] An adjuvant to enhance anti-parasitic immune responses.
Anti-parasitic immune responses that may be enhanced using the
compositions as described herein as an adjuvant, include parasite
and parasite associated diseases or symptoms described herein or
otherwise known in the art. In specific embodiments, the
compositions are used as an adjuvant to enhance an immune response
to a parasite. In another specific embodiment, the compositions are
used as an adjuvant to enhance an immune response to Plasmodium
(malaria).
[0645] As a stimulator of B cell responsiveness to pathogens.
[0646] As an agent that elevates the immune status of an individual
prior to their receipt of immunosuppressive therapies.
[0647] As an agent to induce higher affinity antibodies.
[0648] As an agent to increase serum immunoglobulin
concentrations.
[0649] As an agent to accelerate recovery of immunocompromised
individuals.
[0650] As an agent to boost immunoresponsiveness among aged
populations.
[0651] 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 as described herein may be administered prior to,
concomitant with, and/or after transplantation. In a specific
embodiment, compositions are administered after transplantation,
prior to the beginning of recovery of T cell populations. In
another specific embodiment, compositions are first administered
after transplantation, after the beginning of recovery of T cell
populations, but prior to full recovery of B cell populations.
[0652] 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 BLyS binding polypeptides and/or compositions as
described herein 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.
[0653] In a specific embodiment, BLyS binding polypeptides and/or
compositions are administered to treat or ameliorate selective IgA
deficiency.
[0654] In another specific embodiment, BLyS binding polypeptides
and/or compositions are administered to treat or ameliorate
ataxia-telangiectasia.
[0655] In another specific embodiment BLyS binding polypeptides
and/or compositions are administered to treat or ameliorate common
variable immunodeficiency.
[0656] In another specific embodiment, BLyS binding polypeptides
and/or compositions are administered to treat or ameliorate
X-linked agammaglobulinemia.
[0657] In another specific embodiment, BLyS binding polypeptides
and/or compositions are administered to treat or ameliorate severe
combined immunodeficiency (SCID).
[0658] In another specific embodiment, BLyS binding polypeptides
and/or compositions are administered to treat or ameliorate
Wiskott-Aldrich syndrome.
[0659] In another specific embodiment, BLyS binding polypeptides
and/or compositions are administered to treat or ameliorate
X-linked Ig deficiency with hyper IgM.
[0660] 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 BLyS binding polypeptides and/or
compositions include, but are not limited to, HIV Infection, AIDS,
bone marrow transplant, and B cell chronic lymphocytic leukemia
(CLL).
[0661] 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 BLyS binding polypeptides and/or compositions
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.
[0662] As a regulator of antigen presentation by monocytes,
dendritic cells, T cells and/or B cells. In one embodiment, BLyS
binding polypeptides 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.
[0663] As a mediator of mucosal immune responses. The expression of
BLyS on monocytes, the expression of BLyS receptor on B cells, and
the responsiveness of B cells to BLyS 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.
BLyS binding polypeptides and compositions may therefore be good
regulators 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 BLyS binding
polypeptides or compositions as described herein thereby enhancing
or inhibiting individual's immune status.
[0664] 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.
[0665] 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.
[0666] As a monocyte 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 moncytic cell
populations.
[0667] As a macrophage 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 macrophage cell
populations.
[0668] 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.
[0669] As a means of detecting monocytic 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.
[0670] As a means of detecting macrophage 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.
[0671] 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.
[0672] As a stimulator of B cell production in pathologies such as
AIDS, chronic lymphocyte disorder and/or Common Variable
Immunodificiency.
[0673] As part of a monocyte selection device the function of which
is to isolate monocytes from a heterogenous mixture of cell types.
BLyS binding polypeptides could be coupled to a solid support to
which monocytes would then specifically bind. Unbound cells would
be washed out and the bound cells subsequently eluted. A
non-limiting use of this selection would be to allow purging of
tumor cells from, for example, bone marrow or peripheral blood
prior to transplant.
[0674] As part of a B cell selection device the function of which
is to isolate B cells from a heterogenous mixture of cell types.
BLyS binding polypeptides (that do not inhibit BLyS/BLyS Receptor
intereaction) binding soluble BLyS 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 non-limiting use of this selection would be to allow purging of
tumor cells from, for example, bone marrow or peripheral blood
prior to transplant.
[0675] As a therapy for generation and/or regeneration of lymphoid
tissues following surgery, trauma or genetic defect.
[0676] As a gene-based therapy for genetically inherited disorders
resulting immuno-incompetence such as observed among SCID
patients.
[0677] As an antigen for the generation of antibodies to inhibit or
enhance BLyS mediated responses.
[0678] As a means of activating monocytes/macrophages to defend
against parasitic diseases that effect monocytes such as
Leshmania.
[0679] As pretreatment of bone marrow samples prior to transplant.
Such treatment would increase B cell representation and thus
accelerate recovery.
[0680] As a means of regulating secreted cytokines that are
elicited by BLyS and/or BLyS receptor.
[0681] BLyS binding polypeptides or polynucleotides may be used to
modulate IgE concentrations in vitro or in vivo.
[0682] Additionally, BLyS binding polypeptides or polynucleotides
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.
[0683] In a specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate selective IgA deficiency.
[0684] In another specific embodiment BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate ataxia-telangiectasia.
[0685] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate common variable immunodeficiency.
[0686] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate X-linked agammaglobulinemia.
[0687] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate severe combined immunodeficiency (SCID).
[0688] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate Wiskott-Aldrich syndrome.
[0689] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate X-linked Ig deficiency with hyper IgM. In a
specific embodiment BLyS binding polypeptides or polynucleotides
are administered to treat, prevent, diagnose, and/or ameliorate
X-linked Ig deficiency with hyper IgM.
[0690] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are 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.
[0691] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate monocytic leukemoid reaction, as seen, for
example, with tuberculosis.
[0692] In another specific embodiment, BLyS binding polypeptides or
polynucleotides are administered to treat, prevent, diagnose,
and/or ameliorate monocytic leukocytosis, monocytic leukopenia,
monocytopenia, and/or monocytosis.
[0693] In a specific embodiment, BLyS binding polypeptides or
polynucleotides are used to treat, prevent, detect, and/or diagnose
monocyte disorders and/or diseases, and/or conditions associated
therewith.
[0694] In a specific embodiment, BLyS binding polypeptides or
polynucleotides 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 as described herein include, but are not limited to,
X-Linked Agammaglobulinemia (XLA), severe combined immunodeficiency
disease (SCID), and selective IgA deficiency.
[0695] In a preferred embodiment BLyS binding polypeptides or
polynucleotides 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, BLyS binding polypeptides or
polynucleotides are used to treat, prevent, and/or diagnose
mucositis, especially as associated with chemotherapy.
[0696] In a preferred embodiment, BLyS binding polypeptides or
polynucleotides are used to treat, prevent, and/or diagnose
diseases or disorders affecting or conditions associated with
sinusitis.
[0697] An additional condition, disease or symptom that can be
treated, prevented, and/or diagnosed by BLyS binding polypeptides
or polynucleotides is osteomyelitis.
[0698] An additional condition, disease or symptom that can be
treated, prevented, and/or diagnosed by BLyS binding polypeptides
or polynucleotides is endocarditis.
[0699] All of the above described applications as they may apply to
veterinary medicine.
[0700] BLyS binding polypeptides or polynucleotides may be used to
treat, prevent, and/or diagnose diseases and disorders of the
pulmonary system (e.g., 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.
[0701] In a specific embodiment, BLyS binding polypeptides or
polynucleotides are used to treat, prevent, and/or diagnose chronic
obstructive pulmonary disease (COPD).
[0702] In another embodiment, BLyS binding polypeptides or
polynucleotides are used to treat, prevent, and/or diagnose
fibroses and conditions associated with fibroses, including, 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.
[0703] In another embodiment, therapeutic or pharmaceutical
compositions are administered to an animal to treat, prevent or
ameliorate infectious diseases. Infectious diseases include
diseases associated with yeast, fungal, viral and bacterial
infections. Viruses causing viral infections which can be treated
or prevented in accordance with this invention include, but are not
limited to, retroviruses (e.g., human T-cell lymphotrophic virus
(HTLV) types I and II and human immunodeficiency virus (HIV)),
herpes viruses (e.g., herpes simplex virus (HSV) types I and II,
Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), arenavirues
(e.g., lassa fever virus), paramyxoviruses (e.g., morbillivirus
virus, human respiratory syncytial virus, mumps, and pneumovirus),
adenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses,
filoviruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C
virus (HCV), yellow fever virus, and Japanese encephalitis virus),
hepadnaviruses (e.g., hepatitis B viruses (HBV)), orthomyoviruses
(e.g., influenza viruses A, B and C), papovaviruses (e.g.,
papillomavirues), picornaviruses (e.g., rhinoviruses, enteroviruses
and hepatitis A viruses), poxviruses, reoviruses (e.g.,
rotavirues), togaviruses (e.g., rubella virus), rhabdoviruses
(e.g., rabies virus). Microbial pathogens causing bacterial
infections include, but are not limited to, Streptococcus pyogenes,
Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria
meningitidis, Corynebacterium diphtheriae, Clostridium botulinum,
Clostridium perfringens, Clostridium tetani, Haemophilus
influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella
rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae,
Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio)
fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus,
Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis,
Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella
flexneri, Shigella sonnei, Salmonella typhimurium, Treponema
pallidum, Treponema pertenue, Treponema carateneum, Borrelia
vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,
Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis
carinii, Francisella tularensis, Brucella abortus, Brucella suis,
Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki,
Rickettsia tsutsugumushi, Chlamydia spp., and Helicobacter
pylori.
[0704] Gene Therapy
[0705] In a specific embodiment, nucleic acids comprising sequences
encoding BLyS binding polypeptides or functional derivatives
thereof, are administered to treat, inhibit or prevent a disease or
disorder associated with aberrant expression and/or activity of
BLyS and/or its receptor, 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.
[0706] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0707] 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, 1 1(5):155-215 (1993). Methods commonly known in the
art of recombinant DNA technology which can be used are described
in Current Protocols in Molecular Biology, Ausubel et al., eds.
(John Wiley & Sons, N.Y. 1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual (Stockton Press, N.Y. 1990).
[0708] In a preferred aspect, a composition useful in the methods
of the invention comprises, or alternatively consists of, nucleic
acids encoding a BLyS binding polypeptide, said nucleic acids being
part of an expression vector that expresses the BLyS binding
polypeptide or fragment thereof or chimeric protein including it in
a suitable host. In particular, such nucleic acids have promoters,
preferably heterologous promoters, operably linked to the BLyS
binding polypeptide coding region, said promoter being inducible or
constitutive, and, optionally, tissue-specific. In another
particular embodiment, nucleic acid molecules are used in which the
BLyS binding polypeptide 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 BLyS binding polypeptide
encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci.
USA, 86:8932-8935 (1989); Zijlstra et al., Nature, 342:435-438
(1989).
[0709] 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.
[0710] 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; WO 92/20316; WO 93/14188, 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)).
[0711] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding a BLyS binding polypeptide or
fragments or variants thereof 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 BLyS
binding polypeptide to be used in gene therapy are cloned into one
or more vectors, which facilitates delivery of the gene into a
patient. Additional details concerning retroviral vectors can be
found in Boesen et al., Biotherapy, 6:29 1-302 (1994), which
describes the use of a retroviral vector to deliver the mdr 1 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); Klein 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).
[0712] Other viral vectors that can be used in gene therapy are
adenoviruses. 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. See, Kozarsky and Wilson, Current Opinion in Genetics and
Development, 3:499-503 (1993), presenting 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 WO 94/12649; and Wang
et al., Gene Therapy, 2:775-783 (1995). In a preferred embodiment,
adenovirus vectors are used.
[0713] 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).
[0714] 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.
[0715] 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. Pharma. Ther.,
29:69-92 m (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.
[0716] 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.
[0717] 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.
[0718] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0719] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding a BLyS binding polypeptide
or fragment thereof 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 that 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, 7 1:973-985 (1992); Rheinwald, Meth.
Cell Bio., 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic
Proc., 61:771 (1986)).
[0720] 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.
[0721] Demonstration of Therapeutic or Prophylactic Utility of a
Composition
[0722] The compounds 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 which can be used to
determine whether administration of a specific BLyS binding
polypeptide or composition of the present invention 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 BLyS binding polypeptide or composition of the
present invention, and the effect of such a BLyS binding
polypeptide or composition of the present invention upon the tissue
sample is observed. In various specific embodiments, in vitro
assays can be carried out with representative cells of cell types
involved in a patient's disorder, to determine if a BLyS binding
polypeptide or composition of the present invention has a desired
effect upon such cell types. Preferably, the BLyS binding
polypeptides or compositions are also tested in in vitro assays and
animal model systems prior to administration to humans.
[0723] BLyS binding polypeptides or compositions of the present
invention for use in therapy can be tested for their toxicity in
suitable animal model systems, including but not limited to rats,
mice, chicken, cows, monkeys, and rabbits. For in vivo testing of a
BLyS binding polypeptide or composition's toxicity any animal model
system known in the art may be used.
[0724] Efficacy in treating or preventing viral infection may be
demonstrated by detecting the ability of a BLyS binding polypeptide
or composition to inhibit the replication of the virus, to inhibit
transmission or prevent the virus from establishing itself in its
host, or to prevent, ameliorate or alleviate the symptoms of
disease a progression. The treatment is considered therapeutic if
there is, for example, a reduction in viral load, amelioration of
one or more symptoms, or a decrease in mortality and/or morbidity
following administration of a BLyS binding polypeptide or
composition.
[0725] BLyS binding polypeptides or compositions can be tested for
the ability to induce the expression of cytokines such as
IFN-.gamma., by contacting cells, preferably human cells, with a
BLyS binding polypeptide or composition or a control BLyS binding
polypeptide or control composition and determining the ability of
the BLyS binding polypeptide or composition to induce one or more
cytokines. Techniques known to those skilled in the art can be used
to measure the level of expression of cytokines. For example, the
level of expression of cytokines can be measured by analyzing the
level of RNA of cytokines by, for example, RT-PCR and Northern blot
analysis, and by analyzing the level of cytokines by, for example,
immunoprecipitation followed by western blot analysis and ELISA. In
a preferred embodiment, a compound is tested for its ability to
induce the expression of IFN-.gamma..
[0726] BLyS binding polypeptides or compositions can be tested for
their ability to modulate the biological activity of immune cells
by contacting immune cells, preferably human immune cells (e.g., T
cells, B cells, and Natural Killer cells), with a BLyS binding
polypeptide or composition or a control compound and determining
the ability of the BLyS binding polypeptide or compostion to
modulate (i.e, increase or decrease) the biological activity of
immune cells. The ability of a BLyS binding polypeptide or
composition to modulate the biological activity of immune cells can
be assessed by detecting the expression of antigens, detecting the
proliferation of immune cells (i.e., B cell proliferation),
detecting the activation of signaling molecules, detecting the
effector function of immune cells, or detecting the differentiation
of immune cells. Techniques known to those of skill in the art can
be used for measuring these activities. For example, cellular
proliferation can be assayed by .sup.3H-thymidine incorporation
assays and trypan blue cell counts. Antigen expression can be
assayed, for example, by immunoassays including, but not limited
to, competitive and non-competitive assay systems using techniques
such as western blots, immunohistochemistry 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 and FACS analysis. The
activation of signaling molecules can be assayed, for example, by
kinase assays and electrophoretic shift assays (EMSAs). In a
preferred embodiment, the ability of a BLyS binding polypeptide or
composition to induce B cell proliferation is measured. In another
preferred embodiment, the ability of a BLyS binding polypeptide or
composition to modulate immunoglobulin expression is measured.
[0727] BLyS binding polypeptides or compositions can be tested for
their ability to reduce tumor formation in in vitro, ex vivo and in
vivo assays. BLyS binding polypeptides or compositions can also be
tested for their ability to inhibit viral replication or reduce
viral load in in vitro and in vivo assays. BLyS binding
polypeptides or compositions can also be tested for their ability
to reduce bacterial numbers in in vitro and in vivo assays known to
those of skill in the art. BLyS binding polypeptides or
compositions can also be tested for their ability to alleviate of
one or more symptoms associated with cancer, an immune disorder
(e.g., an inflammatory disease), a neurological disorder or an
infectious disease. BLyS binding polypeptides or compositions can
also be tested for their ability to decrease the time course of the
infectious disease. Further, BLyS binding polypeptides or
compositions can be tested for their ability to increase the
survival period of animals suffering from disease or disorder,
including cancer, an immune disorder or an infectious disease.
Techniques known to those of skill in the art can be used to
analyze the function of the BLyS binding polypeptides or
compositions in vivo.
[0728] Therapeutic/Prophylactic Compositions and Administration
[0729] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of BLyS binding polypeptide (or fragment or variant thereof) or
pharmaceutical composition, preferably a BLyS binding polypeptide.
In a preferred aspect, a BLyS binding polypeptide or fragment or
variant thereof is substantially purified (i.e., 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 a
human.
[0730] 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.
[0731] Various delivery systems are known and can be used to
administer BLyS binding polypeptide or fragment or variant thereof,
e.g., encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the BLyS binding
polypeptide or BLyS binding polypeptide fragment, 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 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 compositions 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.
[0732] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions 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 sialastic
membranes, or fibers. Preferably, when administering a protein,
including a BLyS binding polypeptide, care must be taken to use
materials to which the protein does not absorb.
[0733] In another embodiment, the 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 1989), pp. 353-365; Lopez-Berestein, ibid., pp.
317-327; see, generally, ibid.).
[0734] In yet another embodiment, the 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, Macromol. Sci. Rev. Macromol. Chem., 23:61
(1983); see also Levy et al., Science, 228:190 (1985); During et
al., Ann. Neurol., 25:35 1 (1989); Howard et al., J. Neurosurg., 7
1:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, e.g.,
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)). Other controlled release
systems are discussed in the review by Langer (Science,
249:1527-1533 (1990)).
[0735] In a specific embodiment where the composition to be used in
the method 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.
[0736] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a BLyS binding polypeptide or a fragment
thereof, 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, 18th Ed., Gennaro, ed. (Mack Publishing
Co., 1990). Such compositions will contain a therapeutically
effective amount of the BLyS binding polypeptide or fragment
thereof, 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.
[0737] 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 lignocamne 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.
[0738] The compositions for use in the methods 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-ethylaminoethanol, histidine, procaine, etc.
[0739] The amount of the composition 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 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.
[0740] For BLyS binding polypeptides, 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.
Further, the dosage and frequency of administration of therapeutic
or pharmaceutical compositions may be reduced by enhancing uptake
and tissue penetration (e.g., into the brain) of the BLyS binding
polypeptides by modifications such as, for example, lipidation.
[0741] The BLyS binding polypeptides and BLyS binding polypeptide
compositions may be administered alone or in combination with other
molecules including BLyS. In further embodiments of the invention,
the BLyS binding polypeptides are administered in complex with
BLyS. Preferably the BLyS binding polypeptide is radiolabelled or
in complex with a radioisotope, toxin, or prodrug. 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.
[0742] The BLyS binding polypeptides and BLyS binding polypeptide
compositions may be administered alone or in combination with other
adjuvants. Adjuvants that may be administered with the BLyS binding
polypeptides and BLyS binding polypeptide compositions 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, BLyS binding polypeptides and BLyS binding
polypeptide compositions are administered in combination with alum.
In another specific embodiment, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with QS-21. Further adjuvants that may be administered with the
BLyS binding polypeptides and BLyS binding polypeptide compositions
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 BLyS binding polypeptides and BLyS binding
polypeptide compositions 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..
[0743] In another specific embodiment, BLyS binding polypeptides
and BLyS binding polypeptide compositions 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, BLyS binding polypeptides and BLyS
binding polypeptide compositions 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, BLyS binding
polypeptides and BLyS binding polypeptide compositions 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, BLyS binding
polypeptides and BLyS binding polypeptide compositions 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, BLyS binding polypeptides and BLyS binding
polypeptide compositions 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.
[0744] The BLyS binding polypeptides and BLyS binding polypeptide
compositions 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.
[0745] In one embodiment, the BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with other members of the TNF family. TNF, TNF-related or TNF-like
molecules that may be administered with the BLyS binding
polypeptides and BLyS binding polypeptide compositions 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 (PCT publication WO 96/14328),
TRAIL, AIM-II (PCT publication WO 97/34911), APRIL (J. Exp. Med.,
188(6):1185-1190 (1998)), endokine-alpha (PCT publication WO
98/07880), Neutrokine-alpha (PCT publication WO 98/18921), OPG,
OX40, and nerve growth factor (NGF), and soluble forms of fas,
CD30, CD27, CD40 and 4-IBB, TR2 (PCT publication WO 96/34095), DR3
(PCT publication WO 97/33904), DR4 (PCT publication WO 98/32856),
TR5 (PCT publication WO 98/30693), TR6 (PCT publication WO
98/30694), TR7 (PCT publication WO 98/41629), TRANK, TR9 (PCT
publication WO 98/56892), 312C2 (PCT publication WO 98/06842), and
TR12, and soluble forms CD154, CD70, and CD153.
[0746] In a preferred embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered in
combination with CD40 ligand (CD40L), a soluble form of CD40L
(e.g., AVREND.TM.), bioloigically 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).
[0747] In an additional embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered alone or
in combination with an anti-angiogenic agent(s). Anti-angiogenic
agents that may be administered with the BLyS binding polypeptides
and BLyS binding polypeptide compositions 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.
[0748] 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.
[0749] 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.
[0750] 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.
[0751] 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.
[0752] 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 (Brem and
Folkman, J. Pediatr. Surg., 28:445-51 (1993)); an integrin alpha v
beta 3 antagonist (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 (ICRF 187); 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.
[0753] Anti-angiogenic agents that may be administered in
combination with the compounds 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 BLyS binding polypeptides and
BLyS binding polypeptide compositions 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
BLyS binding polypeptides and BLyS binding polypeptide compositions
include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt,
Germany) and Vitaxin (Ixsys, La Jolla, CA/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 BLyS binding polypeptides and
BLyS binding polypeptide compositions include, but are not limited
to, Angiozyme (Ribozyme, Boulder, Col.), Anti-VEGF BLyS binding
polypeptide (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 BLyS binding polypeptides and BLyS binding polypeptide
compositions 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.).
[0754] In particular embodiments, the use of BLyS binding
polypeptides and BLyS binding polypeptide compositions 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.
[0755] In a particular embodiment, the use of BLyS binding
polypeptides and BLyS binding polypeptide compositions in
combination with anti-angiogenic agents is contemplated for the
treatment, prevention, and/or amelioration of arthritis. In a more
particular embodiment, the use of BLyS binding polypeptides and
BLyS binding polypeptide compositions in combination with
anti-angiogenic agents is contemplated for the treatment,
prevention, and/or amelioration of rheumatoid arthritis.
[0756] In another embodiment, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with an anticoagulant. Anticoagulants that may be administered with
the BLyS binding polypeptides and BLyS binding polypeptide
compositions include, but are not limited to, heparin, warfarin,
and aspirin. In a specific embodiment, BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered in
combination with heparin and/or warfarin. In another specific
embodiment, BLyS binding polypeptides and BLyS binding polypeptide
compositions are administered in combination with warfarin. In
another specific embodiment, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with warfarin and aspirin. In another specific embodiment, BLyS
binding polypeptides and BLyS binding polypeptide compositions are
administered in combination with heparin. In another specific
embodiment, BLyS binding polypeptides and BLyS binding polypeptide
compositions are administered in combination with heparin and
aspirin.
[0757] In another embodiment, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with an agent that suppresses the production of anticardiolipin
antibodies. In specific embodiments, the polypeptides 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).
[0758] In certain embodiments, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with anti-retroviral 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 BLyS binding
polypeptides and BLyS binding polypeptide compositions, 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 BLyS
binding polypeptides and BLyS binding polypeptide compositions,
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 BLyS
binding polypeptides and BLyS binding polypeptide compositions,
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 BLyS binding polypeptides and BLyS binding
polypeptide compositions to treat, prevent, and/or diagnose AIDS
and/or to treat, prevent, and/or diagnose HIV infection.
[0759] In other embodiments, BLyS binding polypeptides and BLyS
binding polypeptide compositions may be administered in combination
with anti-opportunistic infection agents. Anti-opportunistic agents
that may be administered in combination with the BLyS binding
polypeptides and BLyS binding polypeptide compositions, 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., FAMCICLOVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
BLyS binding polypeptides and BLyS binding polypeptide compositions
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, BLyS binding polypeptides and BLyS binding polypeptide
compositions 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, BLyS binding polypeptides and BLyS binding polypeptide
compositions 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, BLyS
binding polypeptides and BLyS binding polypeptide compositions 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, BLyS binding polypeptides and BLyS binding polypeptide
compositions 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, BLyS binding polypeptides and BLyS
binding polypeptide compositions are used in any combination with
ACYCLOVIR.TM. and/or FAMCICLOVIR.TM. to prophylactically treat,
prevent, and/or diagnose an opportunistic herpes simplex virus type
I and/or type II infection. In another specific embodiment, BLyS
binding polypeptides and BLyS binding polypeptide compositions 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, BLyS binding polypeptides and BLyS binding polypeptide
compositions are used in any combination with LEUCOVORIN.TM. and/or
NEUPOGEN.TM. to prophylactically treat, prevent, and/or diagnose an
opportunistic bacterial infection.
[0760] In a further embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered in
combination with an antiviral agent. Antiviral agents that may be
administered with the BLyS binding polypeptides and BLyS binding
polypeptide compositions include, but are not limited to,
acyclovir, ribavirin, amantadine, and remantidine.
[0761] In a further embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered in
combination with an antibiotic agent. Antibiotic agents that may be
administered with the BLyS binding polypeptides and BLyS binding
polypeptide compositions 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.
[0762] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the BLyS binding polypeptides
and BLyS binding polypeptide compositions 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.
[0763] In specific embodiments, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with immunosuppressants. Immunosuppressant preparations that may be
administered with the BLyS binding polypeptides and BLyS binding
polypeptide compositions include, but are not limited to,
ORTHOCLONE.TM. (OKT3), SANDIMMUNE.TM./NEORAL.TM./SANGDYA.TM.
(cyclosporin), PROGRAF.TM. (tacrolimus), CELLCEPT.TM.
(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE.TM.
(sirolimus). In a specific embodiment, immunosuppressants may be
used to prevent rejection of organ or bone marrow
transplantation.
[0764] In a preferred embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered in
combination with steroid therapy. Steroids that may be administered
in combination with the BLyS binding polypeptides and BLyS binding
polypeptide compositions, include, but are not limited to, oral
corticosteroids, prednisone, and methylprednisolone (e.g., IV
methylprednisolone). In a specific embodiment, BLyS binding
polypeptides and BLyS binding polypeptide compositions are
administered in combination with prednisone. In a further specific
embodiment, the BLyS binding polypeptides and BLyS binding
polypeptide compositions are administered in combination with
prednisone and an immunosuppressive agent. Immunosuppressive agents
that may be administered with the BLyS binding polypeptides and
BLyS binding polypeptide compositions and prednisone are those
described herein, and include, but are not limited to,
azathioprine, cylophosphamide, and cyclophosphamide IV. In a
another specific embodiment, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with methylprednisolone. In a further specific embodiment, the BLyS
binding polypeptides and BLyS binding polypeptide compositions are
administered in combination with methylprednisolone and an
immunosuppressive agent. Immunosuppressive agents that may be
administered with the BLyS binding polypeptides and BLyS binding
polypeptide compositions and methylprednisolone are those described
herein, and include, but are not limited to, azathioprine,
cylophosphamide, and cyclophosphamide IV.
[0765] In a preferred embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered in
combination with an antimalarial. Antimalarials that may be
administered with the BLyS binding polypeptides and BLyS binding
polypeptide compositions include, but are not limited to,
hydroxychloroquine, chloroquine, and/or quinacrine.
[0766] In a preferred embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered in
combination with an NSAID.
[0767] In a nonexclusive embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions 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), Leuko Vax
(Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), and butixocort
propionate (WarnerLambert).
[0768] In a preferred embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions 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.
[0769] In a more preferred embodiment, the BLyS binding
polypeptides and BLyS binding polypeptide compositions are
administered in combination with an antimalarial, methotrexate,
anti-TNF antibody, REMICADE.TM., ENBREL.TM. and/or suflasalazine.
In one embodiment, the BLyS binding polypeptides and BLyS binding
polypeptide compositions are administered in combination with
methotrexate. In another embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered in
combination with anti-TNF antibody. In another embodiment, the BLyS
binding polypeptides and BLyS binding polypeptide compositions are
administered in combination with methotrexate and anti-TNF
antibody. In another embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered in
combination with suflasalazine. In another specific embodiment, the
BLyS binding polypeptides and BLyS binding polypeptide compositions
are administered in combination with methotrexate, anti-TNF
antibody, and suflasalazine. In another embodiment, the BLyS
binding polypeptides and BLyS binding polypeptide compositions are
administered in combination ENBREL.TM.. In another embodiment, the
BLyS binding polypeptides and BLyS binding polypeptide compositions
are administered in combination with ENBREL.TM. and methotrexate.
In another embodiment, the BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with ENBREL.TM., methotrexate and suflasalazine. In another
embodiment, the BLyS binding polypeptides and BLyS binding
polypeptide compositions are administered in combination with
ENBREL.TM., methotrexate and suflasalazine. In other embodiments,
one or more antimalarials is combined with one of the above-recited
combinations. In a specfic embodiment, the BLyS binding
polypeptides and BLyS binding polypeptide compositions are
administered in combination with an antimalarial (e.g.,
hydroxychloroquine), ENBREL.TM., methotrexate and suflasalazine. In
another specfic embodiment, the BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with an antimalarial (e.g., hydroxychloroquine), sulfasalazine,
anti-TNF antibody, and methotrexate.
[0770] In an additional embodiment, BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered alone or in
combination with one or more intravenous immune globulin
preparations. Intravenous immune globulin preparations that may be
administered with the BLyS binding polypeptides and BLyS binding
polypeptide compositions include, but not limited to, GAMMAR.TM.,
IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM., and
GAMIMUNE.TM.. In a specific embodiment, BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered in
combination with intravenous immune globulin preparations in
transplantation therapy (e.g., bone marrow transplant).
[0771] 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).
[0772] In an additional embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered alone or
in combination with an anti-inflammatory agent. Anti-inflammatory
agents that may be administered with the BLyS binding polypeptides
and BLyS binding polypeptide compositions 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.
[0773] In another embodiment, compostions are administered in
combination with a chemotherapeutic agent. Chemotherapeutic agents
that may be administered with the BLyS binding polypeptides and
BLyS binding polypeptide compositions 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).
[0774] In a specific embodiment, BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone) or any combination of the components of CHOP. In
another embodiment, BLyS binding polypeptides and BLyS binding
polypeptide compositions are administered in combination with
Rituximab. In a further embodiment, BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered with
Rituxmab and CHOP, or Rituxmab and any combination of the
components of CHOP.
[0775] In an additional embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered in
combination with cytokines. Cytokines that may be administered with
the BLyS binding polypeptides and BLyS binding polypeptide
compositions include, but are not limited to, GM-CSF, G-CSF, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-1 0, IL-12, IL13, IL-15,
anti-CD40, CD40L, IFN-alpha (IFN-.alpha.), IFN-beta (IFN-.beta.),
IFN-gamma (IFN-.gamma.), TNF-alpha (TNF-.alpha.), and TNF-beta
(TNF-.beta.). In preferred embodiments, BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered with
BLyS (e.g., amino acids 134-285 of SEQ ID NO: 173). In another
embodiment, BLyS binding polypeptides and BLyS binding polypeptide
compositions 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 BLyS binding polypeptides and BLyS binding
polypeptide compositions are administered in combination with IL-4
and IL-1 0.
[0776] In one embodiment, the BLyS binding polypeptides and BLyS
binding polypeptide compositions are administered in combination
with one or more chemokines. In specific embodiments, the BLyS
binding polypeptides and BLyS binding polypeptide compositions are
administered in combination with an .alpha.(CxC) chemokine selected
from the group consisting of gamma-interferon inducible protein-10
(.gamma.IP-10), interleukin-8 (IL-8), platelet factor-4 (PF4),
neutrophil activating protein (NAP-2), GRO-.alpha., GRO-.beta.,
GRO-.gamma., neutrophil-activating peptide (ENA-78), granulocyte
chemoattractant protein-2 (GCP-2), and stromal cell-derived
factor-1 (SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or a
.beta.(CC) chemokine selected from the group consisting of: RANTES
(regulated on activation, normal T expressed and secreted),
macrophage inflammatory protein-1 alpha (MIP-1.alpha.), macrophage
inflammatory protein-1 beta (MIP-1.beta.), monocyte chemotactic
protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2), monocyte
chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4
(MCP-4) macrophage inflammatory protein-l gamma (MIP-1.gamma.),
macrophage inflammatory protein-3 alpha (MIP-3.alpha.), macrophage
inflammatory protein-3 beta (MIP-3.beta.), macrophage inflammatory
protein-4 (MIP-4/DC-CK-1I/PARC), eotaxin, Exodus, and I-309; and/or
the .gamma.(C) chemokine, lymphotactin.
[0777] In another embodiment, the BLyS binding polypeptides and
BLyS binding polypeptide compositions are administered with
chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory
protein-4. In a preferred embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered with
chemokine beta-8.
[0778] In an additional embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered in
combination with an IL-4 antagonist. IL-4 antagonists that may be
administered with the BLyS binding polypeptides and BLyS binding
polypeptide compositions 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 BLyS binding polypeptide fragments, such as,
for example, those described herein).
[0779] The invention also encompasses combining the polynucleotides
and/or polypeptides (and/or agonists or antagonists thereof) with
other proposed or conventional hematopoietic therapies. Thus, for
example, the polynucleotides and/or polypeptides (and/or agonists
or antagonists thereof) can be combined with compounds that singly
exhibit erythropoietic stimulatory effects, such as erythropoietin,
testosterone, progenitor cell stimulators, insulin-like growth
factor, prostaglandins, serotonin, cyclic AMP, prolactin, and
triiodothyzonine. Also encompassed are combinations of the BLyS
binding polypeptides and BLyS binding polypeptide compositions with
compounds generally used to treat aplastic anemia, such as, for
example, methenolene, stanozolol, and nandrolone; to treat
iron-deficiency anemia, such as, for example, iron preparations; to
treat malignant anemia, such as, for example, vitamin B.sub.2
and/or folic acid; and to treat hemolytic anemia, such as, for
example, adrenocortical steroids, e.g., corticoids. See, e.g.,
Resegotti et al., Panminerva Medica, 23:243-248 (1981); Kurtz, FEBS
Letters, 14a: 105-108 (1982); McGonigle et al., Kidney Int.,
25:437-444 (1984); and Pavlovic-Kantera, Expt. Hematol., 8(supp.
8):283-291 (1980), the contents of each of which are hereby
incorporated by reference in their entireties.
[0780] Compounds that enhance the effects of or synergize with
erythropoietin are also useful as adjuvants herein, and include but
are not limited to, adrenergic agonists, thyroid hormones,
androgens, hepatic erythropoietic factors, erythrotropins, and
erythrogenins. See, for example, Dunn, Current Concepts in
Erythropoiesis (John Wiley and Sons, Chichester, England 1983);
Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, New Eng. J. Med.,
289:72-80 (1973); Urabe et al., J. Exp. Med., 149:1314-1325 (1979);
Billat et al., Expt. Hematol., 10:133-140 (1982); Naughton et al.,
Acta Haemat., 69:171-179 (1983); Cognote et al., in abstract 364,
Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec,
Jul. 1-7, 1984); and Rothman et al., J. Surg. Oncol., 20:105-108
(1982). Methods for stimulating hematopoiesis comprise
administering a hematopoietically effective amount (i.e., an amount
which effects the formation of blood cells) of a pharmaceutical
composition containing polynucleotides and/or poylpeptides (and/or
agonists or antagonists thereof) to a patient. The polynucleotides
and/or polypeptides and/or agonists or antagonists thereof are
administered to the patient by any suitable technique, including
but not limited to parenteral, sublingual, topical, intrapulmonary
and intranasal, and those techniques further discussed herein. The
pharmaceutical composition optionally contains one or more members
of the group consisting of erythropoietin, testosterone, progenitor
cell stimulators, insulin-like growth factor, prostaglandins,
serotonin, cyclic AMP, prolactin, triiodothyzonine, methenolene,
stanozolol, and nandrolone, iron preparations, vitamin B12, folic
acid and/or adrenocortical steroids.
[0781] In an additional embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered in
combination with hematopoietic growth factors. Hematopoietic growth
factors that may be administered with the BLyS binding polypeptides
and BLyS binding polypeptide compositions include, but are not
limited to, LEUKINE.TM. (sargramostim) and NEUPOGEN.TM.
(filgrastim).
[0782] In an additional embodiment, the BLyS binding polypeptides
and BLyS binding polypeptide compositions are administered in
combination with fibroblast growth factors. Fibroblast growth
factors that may be administered with the BLyS binding polypeptides
and BLyS binding polypeptide compositions 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-l l, FGF-12, FGF-13, FGF-14, and FGF-15.
[0783] Additionally, the BLyS binding polypeptides and BLyS binding
polypeptide compositions 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.
[0784] Kits for Detecting and/or Quantitating BLyS or BLyS-like
Polypeptides
[0785] The present invention is also directed to an assay kit which
can be useful in screening for the presence of BLyS and/or
quantitating BLyS concentrations in a fluid, such as, for example,
a biological fluid (e.g., blood, serum, synovial fluid).
[0786] In a particular embodiment of the present invention, an
assay kit is contemplated which comprises in one or more containers
one or more BLyS binding polypeptides and optionally, a detection
means for determining the presence of a BLyS-BLyS binding
polypeptide interaction or the absence thereof. The kit further
optionally contains BLyS protein that may be used, for example as a
control. The BLyS binding polypeptide may be free or expressed on
the surface of a phage.
[0787] In a specific embodiment, either the BLyS binding
polypeptide or the BLyS protein is labeled. As further discussed
herein, a wide range of labels can be used accordance with the
present invention, including but not limited to conjugating the
recognition unit to biotin by conventional means. Alternatively,
the label may comprise, e.g., a fluorogen, an enzyme, an epitope, a
chromogen, or a radionuclide. Preferably, the biotin is conjugated
by covalent attachment to either the BLyS binding polypeptide or
the BLyS protein. Preferably, the BLyS binding polypeptide is
immobilized on a solid support. The detection means employed to
detect the label will depend on the nature of the label and can be
any known in the art, e.g., film to detect a radionuclide, an
enzyme substrate that gives rise to a detectable signal to detect
the presence of an enzyme, antibody to detect the presence of an
epitope, etc.
[0788] 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. 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. In one preferred embodiment the kit comprises
a vial containing BLyS binding polypeptides conjugated to a toxin
or a label (as described herein). Such conjugated binding
polypeptide may be used to kill a particular population of cells or
to quantitate a particular population of cells. In a preferred
embodiment, such conjugated BLyS binding polypeptides are used to
kill monocyte cells expressing the membrane-bound form of BLyS. In
another preferred embodiment, such conjugated BLyS binding
polypeptides are used to quantitate monocyte cells expressing the
membrane-bound form of BLyS. In another preferred embodiment, such
conjugated BLyS binding polypeptides are used to kill B cells
expressing BLyS receptor on their surface. In another preferred
embodiment, such conjugated BLyS binding polypeptides are used to
quantitate B cells expressing BLyS receptor on their surface.
[0789] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises a BLyS binding
polypeptide, preferably a purified BLyS binding polypeptide, in one
or more containers. In an alterative embodiment, a kit comprises a
BLyS binding polypeptide fragment that specifically binds to BLyS.
In a specific embodiment, the kits of the present invention contain
a substantially isolated BLyS polypeptide as a control. Preferably,
the kits of the present invention further comprise a control
binding polypeptide which does not react with BLyS. In another
specific embodiment, the kits of the present invention contain a
means for detecting the binding of a BLyS binding polypeptide to
BLyS (e.g., the BLyS binding polypeptide 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 BLyS binding polypeptide may
be conjugated to a detectable substrate). In specific embodiments,
the kit may include a recombinantly produced or chemically
synthesized BLyS. The BLyS provided in the kit may also be attached
to a solid support. In a more specific embodiment the detecting
means of the above-described kit includes a solid support to which
BLyS is attached. Such a kit may also include a non-attached
reporter-labeled anti-BLyS binding polypeptide antibody. In this
embodiment, binding of the BLyS binding polypeptide to BLyS can be
detected by binding of the said reporter-labeled antibody.
Alternatively, or in addition, the detecting means may include a
labeled, competing antigen.
[0790] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing BLyS or
BLyS-like polypeptides. The diagnostic kit includes a substantially
isolated BLyS binding polypeptide specifically reactive with BLyS
target, and means for detecting the binding of BLyS target to the
BLyS binding polypeptide. In one embodiment, the BLyS binding
polypeptide is attached to a solid support.
[0791] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound BLyS binding
polypeptide according to the present invention. After BLyS binds to
a specific BLyS binding polypeptide, the unbound serum components
are removed by washing, reporter-labeled anti-BLyS binding
polypeptide antibody is added, unbound anti-BLyS binding
polypeptide antibody is removed by washing, and a reagent is
reacted with reporter-labeled anti-BLyS binding polypeptide
antibody to bind reporter to the reagent in proportion to the
amount of bound BLyS binding polypeptide on the solid support.
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.
[0792] 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 BLyS binding polypeptides.
[0793] 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 BLyS, and a reporter-labeled
anti-BLyS binding polypeptide antibody for detecting surface-bound
anti-BLyS binding polypeptide.
[0794] Methods of Screening for BLyS Binding Molecules
[0795] The invention also encompasses screening methods for
identifying polypeptides and nonpolypeptides that bind BLyS, and
the BLyS binding molecules identified thereby. This method
comprises the steps of:
[0796] (a) contacting BLyS or BLyS-like polypeptide with a
plurality of molecules; and
[0797] (b) identifying molecule(s) that binds the BLyS or BLyS-like
polypeptide.
[0798] The step of contacting the BLyS protein or BLyS-like protein
with the plurality of molecules may be effected in a number of
ways. For example, one may contemplate immobilizing BLyS target on
a solid support and bringing a solution of the plurality of
molecules in contact with the immobilized BLyS target. Such a
procedure would be akin to an affinity chromatographic process,
with the affinity matrix being comprised of the immobilized BLyS
protein or BLyS-like polypeptide. The molecules having a selective
affinity for the BLyS or BLyS-like polypeptide can then be purified
by affinity selection. The nature of the solid support, process for
attachment of the BLyS or BLyS-like polypeptide to the solid
support, solvent, and conditions of the affinity isolation or
selection are largely conventional and well known to those of
ordinary skill in the art.
[0799] Alternatively, one may also separate a plurality of
polypeptides into substantially separate fractions comprising a
subset of or individual polypeptides. For instance, one can
separate the plurality of polypeptides by gel electrophoresis,
column chromatography, or like method known to those of ordinary
skill for the separation of polypeptides. The individual
polypeptides can also be produced by a transformed host cell in
such a way as to be expressed on or about its outer surface (e.g.,
a recombinant phage). Individual isolates can then be "probed"
using a BLyS target protein, optionally in the presence of an
inducer should one be required for expression, to determine if any
selective affinity interaction takes place between the BLyS target
protein and the individual clone. Prior to contacting the BLyS
target protein with each fraction comprising individual
polypeptides, the polypeptides could first be transferred to a
solid support for additional convenience. Such a solid support may
simply be a piece of filter membrane, such as one made of
nitrocellulose or nylon. In this manner, positive clones could be
identified from a collection of transformed host cells of an
expression library, which harbor a DNA construct encoding a
polypeptide having a selective affinity for BLyS or BLyS-like
polypeptide. Furthermore, the amino acid sequence of the
polypeptide having a selective affinity for the BLyS protein or
BLyS-like protein can be determined directly by conventional means,
or the coding sequence of the DNA encoding the polypeptide can
frequently be determined more conveniently. The primary amino acid
sequence can then be deduced from the corresponding DNA sequence.
If the amino acid sequence is to be determined from the polypeptide
itself, one may use microsequencing techniques. The sequencing
technique may include mass spectroscopy.
[0800] In certain situations, it may be desirable to wash away any
BLyS or BLyS-like polypeptide, or alternatively, unbound
polypeptides, from a mixture of BLyS or BLyS-like polypeptide and
the plurality of polypeptides prior to attempting to determine or
to detect the presence of a selective affinity interaction. One or
more such a wash steps may be particularly desirable when the BLyS
or BLyS-like polypeptide or the plurality of polypeptides is bound
to a solid support.
[0801] The plurality of molecules provided according to this method
may be provided by way of diversity libraries, such as random or
combinatorial peptide or non-peptide libraries which can be
screened for molecules that specifically bind to BLyS. Peptide
libraries may be designed such that the polypeptides encoded by the
libraries are automatically fused to a polypeptide linker moiety,
for example. Many libraries are known in the art that can be used,
e.g., chemically synthesized libraries, recombinant (e.g., phage
display libraries), and in vitro translation-based libraries.
Examples of chemically synthesized libraries are described in Fodor
et al., Science, 251:767-773 (1991); Houghten et al., Nature,
354:84-86 (1991); Lam et al., Nature, 354:82-84 (1991); Medynski,
Bio/Technology, 12:709-710 (1994); Gallop et al., J. Medicinal
Chemistry, 37(9):1233-1251 (1994); Ohlmeyer et al., Proc. Natl.
Acad. Sci. USA, 90:10922-10926 (1993); Erb et al., Proc. Natl.
Acad. Sci. USA, 91:11422-11426 (1994); Houghten et al.,
Biotechniques, 13:412 (1992); Jayawickreme et al., Proc. Natl.
Acad. Sci. USA, 91:1614-1618 (1994); Salmon et al., Proc. Natl.
Acad. Sci. USA, 90:11708-11712 (1993); PCT publication WO 93/20242;
and Brenner and Lerner, Proc. Natl. Acad. Sci. USA, 89:5381-5383
(1992).
[0802] Examples of phage display libraries are described in Scott
and Smith, Science, 249:386-390 (1990); Devlin et al., Science,
249:404-406 (1990); Christian et al., J. Mol. Biol., 227:711-718
(1992); Lenstra, J. Immunol. Meth., 152:149-157 (1992); Kay et al.,
Gene, 128:59-65 (1993); and PCT publication WO 94/18318.
[0803] In vitro translation-based libraries include but are not
limited to those described in PCT publication WO 91/05058 and
Mattheakis et al., Proc. Natl. Acad. Sci. USA, 91:9022-9026
(1994).
[0804] By way of examples of non-peptide libraries, a
benzodiazepine library (see, e.g., Bunin et al., Proc. Natl. Acad.
Sci. USA, 91:4708-4712 (1994) can be adapted for use. Peptoid
libraries (Simon et al., Proc. Natl. Acad. Sci. USA, 89:9367-9371
(1992)) can also be used. Another example of a library that can be
used, in which the amide functionalities in peptides have been
permethylated to generate a chemically transformed combinatorial
library, is described by Ostresh et al. (Proc. Natl. Acad. Sci.
USA, 91:11138-11142 (1994)).
[0805] The variety of non-peptide libraries that are useful in the
present invention is great. For example, Ecker and Crooke,
Bio/Technology, 13:351-360 (1995) list benzodiazepines, hydantoins,
piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones,
arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines,
aminimides, and oxazolones as among the chemical species that form
the basis of various libraries.
[0806] Non-peptide libraries can be classified broadly into two
types: decorated monomers and oligomers. Decorated monomer
libraries employ a relatively simple scaffold structure upon which
a variety functional groups is added. Often the scaffold will be a
molecule with a known useful pharmacological activity. For example,
the scaffold might be the benzodiazepine structure.
[0807] Non-peptide oligomer libraries utilize a large number of
monomers that are assembled together in ways that create new shapes
that depend on the order of the monomers. Among the monomer units
that have been used are carbamates, pyrrolinones, and morpholinos.
Peptoids, peptide-like oligomers in which the side chain is
attached to the alpha amino group rather than the alpha carbon,
form the basis of another version of non-peptide oligomer
libraries. The first non-peptide oligomer libraries utilized a
single type of monomer and thus contained a repeating backbone.
Recent libraries have utilized more than one monomer, giving the
libraries added flexibility.
[0808] Screening the libraries can be accomplished by any of a
variety of commonly known methods. See, e.g., the following
references, which disclose screening of peptide libraries: Parmley
and Smith, Adv. Exp. Med. Biol., 251:215-218 (1989); Scott and
Smith, Science, 249:386-390 (1990); Fowlkes et al., Bio Techniques,
13:422-427 (1992); Oldenburg et al., Proc. Natl. Acad. Sci. USA,
89:5393-5397 (1992); Yu et al., Cell, 76:933-945 (1994); Staudt et
al., Science, 241:577-580 (1988); Bock et al., Nature, 355:564-566
(1992); Tuerk et al., Proc. Natl. Acad. Sci. USA, 89:6988-6992
(1992); Ellington et al., Nature, 355:850-852 (1992); U.S. Pat. No.
5,096,815; U.S. Pat. No. 5,223,409; and U.S. Pat. No. 5,198,346,
all to Ladner et al.; Rebar and Pabo, Science, 263:671-673 (1993);
and PCT publication WO 94/18318.
[0809] In a specific embodiment, screening to identify a molecule
that binds BLyS can be carried out by contacting the library
members with BLyS or BLyS-like polypeptide immobilized on a solid
phase and harvesting those library members that bind to the BLyS or
BLyS-like polypeptide. Examples of such screening methods, termed
"panning" techniques are described by way of example in Parmley and
Smith, Gene, 73:305-318 (1998); Fowlkes et al., Bio Techniques,
13:422-427 (1992); PCT publication WO 94/18318; and in references
cited therein.
[0810] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields and Song, Nature, 340:245-246
(1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582
(1991)) can be used to identify molecules that specifically bind to
BLyS or BLyS-like polypeptides.
[0811] Where the BLyS binding molecule is a polypeptide, the
polypeptide can be conveniently selected from any peptide library,
including random peptide libraries, combinatorial peptide
libraries, or biased peptide libraries. The term "biased" is used
herein to mean that the method of generating the library is
manipulated so as to restrict one or more parameters that govern
the diversity of the resulting collection of molecules, in this
case peptides.
[0812] Thus, a truly random peptide library would generate a
collection of peptides in which the probability of finding a
particular amino acid at a given position of the peptide is the
same for all 20 amino acids. A bias can be introduced into the
library, however, by specifying, for example, that a lysine occur
every fifth amino acid, that certain amino acid positions in a
peptide remain fixed (e.g., as cysteine), or that positions 4, 8,
and 9, for example, of a decapeptide library be limited to permit
several but less than all of the twenty naturally-occurring amino
acids. Clearly, many types of biases can be contemplated, and the
present invention is not restricted to any particular bias.
Furthermore, the present invention contemplates specific types of
peptide libraries, such as phage displayed peptide libraries and
those that utilize a DNA construct comprising a lambda phage vector
with a DNA insert.
[0813] As mentioned above, in the case of a BLyS binding molecule
that is a polypeptide, the polypeptide may have about 6 to less
than about 60 amino acid residues, preferably about 6 to about 10
amino acid residues, and most preferably, about 6 to about 22 amino
acids. In another embodiment, a BLyS binding polypeptide has in the
range of 15-100 amino acids, or 20-50 amino acids.
[0814] The selected BLyS binding polypeptide can be obtained by
chemical synthesis or recombinant expression.
[0815] The specific BLyS binding polypeptides disclosed herein were
isolated using phage display technology, to identify BLyS binding
polypeptides exhibiting particular preselected binding properties.
These BLyS binding polypeptides were isolated initially by
screening nine phage display libraries, that is, populations of
recombinant bacteriophage transformed to express an exogenous
recombinant polypeptide on their surface. In order to isolate new
polypeptide binding moieties for a particular target, such as BLyS,
screening of peptide libraries, for example using phage display
techniques, is especially advantageous, in that very large numbers
(e.g., 5.times.10.sup.9) of potential binders can be tested and
successful binders isolated in a short period of time.
[0816] In order to prepare a phage library of potential binding
polypeptides to screen for members of the library that are BLyS
binding polypeptides, a candidate binding domain is selected to
serve as a structural template for the polypeptides to be displayed
in the library. The phage library is made up of polypeptide
analogues of this template or "parental binding domain." The
parental binding domain is a polypeptide molecule that may be a
naturally occurring or synthetic protein or polypeptide, or
polypeptide region or domain of a protein. The parental binding
domain may be selected based on knowledge of a known interaction
between the parental binding domain and a target protein, but this
is not critical. In fact, it is not essential that the parental
binding domain have any affinity for a target at all because its
purpose is to provide a structure from which a multiplicity of
polypeptide analogues (a "library") can be generated, which
multiplicity of polypeptide analogues will include one or more
binding polypeptides that exhibit the desired binding and release
properties with respect to BLyS target proteins (and any other
properties selected).
[0817] Knowledge of the exact polypeptide that will serve as the
parental binding domain, or knowledge of a class of proteins or
domains to which the parental binding domain belongs can be useful
in determining the conditions under which BLyS binding polypeptides
optimally bind BLyS target proteins as well as the conditions under
which BLyS binding polypeptides optimally release BLyS target
proteins. Similarly, the binding and/or release conditions may be
selected with regard to known interactions between a binding domain
and the BLyS target protein, for example, to favor the interaction
under the binding and/or release conditions, or they may be
selected without regard to such known interactions. Likewise, the
parental binding domain can be selected taking into account a
desired binding and/or release condition or not. It is understood
that if the binding domain analogues of a library are unstable
under a proposed or desired binding or release condition, no useful
binding polypeptides may be obtained.
[0818] In selecting the parental binding domain, the most important
consideration is how the analogue domains will be presented to the
BLyS target protein, that is, in what conformations the BLyS target
and the polypeptide analogues will contact one another. In
preferred embodiments, for example, the polypeptide analogues will
be generated by insertion of synthetic DNA encoding the polypeptide
analogue into a replicable genetic package, resulting in display of
the domain on the surface of a microorganism, such as M13 phage,
using techniques as described in Kay et al., Phage Display of
Peptides and Proteins: A Laboratory Manual (Academic Press, Inc.;
San Diego 1996) and U.S. Pat. No. 5,223,409 (Ladner et al.),
incorporated herein by reference. For formation of phage display
libraries, it is preferred to use structured polypeptides as the
parental binding domain or template, as opposed to unstructured,
linear peptides. Mutation of surface residues in a protein domain
or polypeptide molecule will usually have little effect on the
overall structure or general properties (such as size, stability,
and temperature of denaturation) of the protein; while at the same
time mutation of surface residues may profoundly affect the binding
properties of the molecule. The more tightly a polypeptide segment
is constrained, the less likely it is to bind to any particular
target. If it does bind, however, the binding is likely to be
tighter and more specific. Thus, it is preferred to select a
parental binding domain wherein the parental polypetide has
structure and, thereby in turn, select a structure for the
polypeptide analogues of the library, which is constrained within a
framework having some degree of rigidity.
[0819] Preferably the protein domain that is used as the template
or parental domain for generating the library of domain analogues
will be a peptide molecule that is a relatively small protein or
polypeptide. Small polypeptides offer several advantages over large
proteins: First, the mass per binding site is reduced. Highly
stable protein domains having low molecular weights, for example,
Kunitz domains (.about.7 kilodaltons, kDa), Kazal domains (.about.7
kDa), Cucurbida maxima trypsin inhibitor (CMTI) domains (.about.3.5
kDa), and endothelin (-2 kDa), can show much higher binding per
gram than do antibodies (150 kDa) or single chain scFv antibodies
(30 kDa). Second, the possibility of non-specific binding is
reduced because there is less molecular surface available for
nonspecific binding. Third, small polypeptides can be engineered to
have unique tethering sites in a way that is impracticable for
larger proteins or antibodies. For example, small proteins and
polypeptides can be engineered to have lysines only at sites
suitable for tethering to a chromatography matrix. This is not
feasible for antibodies. Fourth, a constrained polypeptide
structure is more likely to retain its functionality when
transferred (with the structural domain intact) from one framework
to another. For instance, the binding domain structure is likely to
be transferable from the framework used for presentation in a
library, such as displayed on a phage, to an isolated protein
removed from the presentation framework or immobilized on a
chromatographic substrate.
[0820] In specific embodiments, the BLyS binding polypeptides are
immobilized. BLyS binding polypeptide molecules according to the
invention may be immobilized, for example, on chromatographic
support materials to form efficient BLyS separation or affinity
chromatographic media. Immobilized BLyS binding polypeptides have
uses that include, but are not limted to, detecting, isolating or
removing BLyS target proteins from solutions. One strategy for
generating BLyS binding polypeptide molecules that can be
immobilized, for example, on matrices, resins, or supports,
involves selecting appropriate binding domain templates such that
BLyS binding polypeptide molecules are generated that have one or
more amino acids that may be used to covalently link the BLyS
binding polypeptide to a chromatographic resin or substrate to form
an affinity resin. Similarly, the N-terminal amino group or the
C-terminal carboxyl group of a peptide molecule may be modified by
adding a capping group to render it inert or a functional group,
which permits linkage to a support medium. For example, the
C-terminal carboxyl group of a protein domain may be converted to
an amide or a hydrazide (--NH--NH.sub.2) group for reaction with an
aldehyde-functional substrate or other amine-reactive substrate.
This technique is preferred. Another preferred modification of BLyS
binding polypeptides useful for linking a BLyS binding polypeptide
molecule to a chromatography material is a polypeptide linker
comprising, or alternatively consisting of, the amino acid sequence
Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 13).
[0821] In one non-limiting example of a screening procedure to
obtain BLyS binding polypeptides encompassed by the invention, the
phage in a phage display library are contacted with and allowed to
bind a BLyS target protein that is immobilized on a solid support.
Those phage that display non-binding polypeptides are separated
from those that bind the BLyS target protein. Any of various
techniques known in the art may be applied to dissociate the bound
phage from the immobilized BLyS protein, and to collect and/or
amplify the phage and/or their nucleic acid contents. Using these
techniques it is possible to identify a BLyS binding phage that is
about 1 in 20 million in the population. Libraries, displaying
10-20 million or more potential binding peptide molecules each, are
rapidly screened to find high-affinity BLyS binding
polypeptides.
[0822] In each round of screening, the diversity of a population
falls until only efficient binding polypeptides remain, that is,
the process converges. Typically, a phage display library will
contain several closely related binding polypeptides (10 to 50
different binding polypeptides out of 10 million). Indications of
convergence include increased binding (measured by phage titers)
and recovery of closely related sequences. After a first set of
binding polypeptide molecules is identified, the sequence
information can be used to design other libraries biased for
members having additional desired properties, for example,
discrimination between different forms of BLyS (e.g., the membrane
form and the soluble form of BLyS) and fragments thereof, or
discrimination between BLyS and closely related impurities in a
feed stream.
[0823] Such techniques make it possible not only to screen a large
number of potential binding polypeptides, but make it practical to
repeat the binding and elution cycles and to build secondary,
biased libraries for screening polypeptide analogue-displaying
phage that meet specific criteria. Using these techniques, a
polypeptide analogue biased library may be screened to reveal
members that bind tightly, that is, have high affinity for BLyS
target protein, under the screening conditions.
[0824] In the present invention target BLyS protein molecules were
biotinylated and then bound to streptavidin-coated magnetic
particles. Nine phage display libraries of different design were
screened for the ability to bind the immobilized BLyS. Each library
was characterized by M13 phage displaying variegated peptides of
different lengths and overall structure: A library designated TN6/6
(2.times.10.sup.8 variants) displayed a variegated 12-mer with two
internal invariant cyteines to form a hexamer loop structure. A
library designated TN7/4 (2.3.times.10.sup.9 variants) presented a
variegated 13-mer having two internal invariant cyteines to form a
heptamer loop structure. A library designated TN8/9
(5.times.10.sup.9 variants) displayed a variegated 14-mer with two
internal invariant cyteines to form an octamer loop structure. A
library designated TN9/4 (3.2.times.10.sup.9 variants) presented a
variegated 16-mer having two internal invariant cyteines to form a
nonamer loop structure. A library designated TN10/9
(2.5.times.10.sup.9 variants) displayed a variegated 16-mer with
two internal invariant cyteines to form a decamer loop structure. A
library designated TN12/1 (1.4.times.10.sup.9 variants) presented a
variegated 18-mer having two internal invariant cyteines to form a
dodecamer loop structure. A library designated as Substrate Phage
Library #2, having a diversity of about 2.times.10.sup.8 amino acid
sequences, was designed to include a linear peptide-variegated
region in the display polypeptide consisting of 13 consecutive
amino acids, and the display polypeptide design allowed any amino
acid residue except cysteine to occur at each position. Finally,
two commercially available linear phage display libraries were also
screened, designated PhD 7 and PhD 12, respectively (New England
Biolabs). The PhD 7 library displayed a linear random-sequence
7-mer; the PhD 12 libary displayed a random-sequence 12-mer.
[0825] BLyS binding phage were isolated and collected from all of
the libraries except PhD 7.
[0826] After analysis of the sequences isolated from the library
screenings, several families of BLyS binding peptides were defined
(see, consensus sequences A-G and H-L, above). The amino acid
sequences of the BLyS-binding "hits" from the first rounds of
screening are set forth in Tables 1-8 (infra).
[0827] In order to obtain BLyS binding polypeptides having an even
higher affinity for BLyS targets, a specialized library was
prepared, i.e., a BLyS affinity maturation library, based on
variegation of high affinity examplars of the PhD 12 library (see
Example 6). This library was designed to provide a population
enriched with polypeptides likely to show high affinity for BLyS.
The selections from this library were performed to eliminate, by
prolonged competition with soluble eluants of soluble BLyS or other
BLyS binding polypeptides, all but the polypeptides having the
highest affinity for BLyS. A large family of high affinity BLyS
binding polypeptides was isolated from four rounds of screening the
affinity maturation library, and their amino acid sequences appear
in Table 13 (infra).
[0828] As it within the scope of the present invention to screen
phage libraries that bind one or more of the various forms of BLyS,
the following outlines some assays that may be used in screening
for BLyS binding polypeptides that bind the soluble form of BLyS,
the membrane-bound form of BLyS, or both the soluble and the
membrane-bound forms of BLyS. Assays to determine the specificity
of binding polypeptides for different forms of a protein are
commonly known in the art and may be readily adapted for
determining the specificity of BLyS binding polypeptides for
different forms of BLyS.
[0829] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) may be screened in a variety of assays to
identify those BLyS binding polypeptides that specifically bind to
the soluble form of BLyS. BLyS binding polypeptides may be assayed
in neutralization assays described herein (see Examples 7 and 8) or
otherwise known in the art. For example, BLyS binding polypeptides
may be tested for their ability to inhibit soluble BLyS from
binding a BLyS receptor. The BLyS receptor used in these assays may
be an isolated BLyS receptor (e.g., BLyS receptor conjugated to
agaorose beads) or may be present on the cell surface of cell lines
that express BLyS receptors which include, but are not limited to,
peripheral CD20+B cells, IM-9, REH, ARH-77, Namalwa, and RPMI-8226
B cell tumor lines.
[0830] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants thereof) may be screened in a variety of assays
commonly known in the art to identify those BLyS binding
polypeptides that specifically bind to the membrane-bound form of
BLyS. For example, BLyS binding polypeptides may be assayed for
binding BLyS protein present on cell membranes of cells that
express BLyS. Cell lines that express BLyS that might be useful for
testing BLyS binding polypeptide binding to membrane-bound form of
BLyS include, K-562, HL-60, THP-1, and U937 cells.
[0831] Aditionally, BLyS binding polypeptides may be screened
against cells engineered to express an "uncleavable" form of BLyS
in order to determine their specificity for the membrane-bound form
of BLyS. Mutations in BLyS which may achieve this result include,
but are not limited to, the mutation or deletion of amino acid
residues Lys-132 and/or Arg-133 of the BLyS sequence shown in SEQ
ID NO: 173. A typical mutagenesis might include mutation of one or
both of residues Lys-132 or Arg-133 to alanine residues. Cells
expressing such an "uncleavable" form of BLyS provide a profound
reagent to use in assaying the ability of BLyS binding polypeptides
to bind the membrane-bound form of BLyS.
[0832] BLyS binding polypeptides (including molecules comprising,
or alternatively consisting of, BLyS binding polypeptide fragments
or variants) may be screened in a variety of assays to identify
those BLyS binding polypeptides or BLyS binding polypeptide
fragments or variants that specifically bind to the soluble form
and membrane-bound form of BLyS. This can readily be determined by
performing assays to distinguish binding to the soluble form and
assays to distinguish binding to the membrane-bound form (such as
the assays described herein or otherwise known in the art), and
identifying the BLyS binding polypeptides that bind both forms.
[0833] Additionally, BLyS binding polypeptides may be screened for
the ability to inhibit, stimulate or not significantly alter BLyS
activity, e.g., the ability of BLyS: to bind to its receptor (e.g.,
TACI and BCMA), to stimulate B cell proliferation, to stimulate
immunoglobulin secretion by B cells, to activate B cells, to
increase B cell lifespan and/or to stimulate a BLyS receptor
signaling 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"; 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)). Assays that may be used to screen
for the effects on BLyS activity are described herein (see, for
example, Examples 7, 8, and 12) and are commonly known in the
art.
[0834] Anti-BLyS Binding Polypeptide Antibodies
[0835] Further polypeptides useful herein relate to antibodies and
T-cell antigen receptors (TCR) which immunospecifically bind a BLyS
binding polypeptide (as determined by immunoassays well known in
the art for assaying specific antibody-antigen binding). Antibodies
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), and
epitope-binding fragments of any of the above. The term "antibody,"
as used herein, refers to immunoglobulin molecules and
immunologically active portions of no immunoglobulin molecules,
i.e., molecules that contain an antigen binding site that
immunospecifically binds an antigen. The immunoglobulin molecules
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. Immunoglobulins may have both a heavy and
light chain. In specific embodiments, the immunoglobulin molecules
are IgG1. In other specific embodiments, the immunoglobulin
molecules are IgG4. 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.
[0836] 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 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 to Kucherlapati et al.
[0837] 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, WO 91/00360, 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).
[0838] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a BLyS
binding polypeptide of the present invention which they recognize
or specifically bind. 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 BLyS binding polypeptides of the present
invention, and allows for the exclusion of the same.
[0839] In further preferred, nonexclusive embodiments, the
antibodies (e.g., anti-idiotypic antibodies) inhibit one or more
biological activities of BLyS through specific binding to BLyS. In
more preferred embodiments, the antibody inhibits BLyS-mediated B
cell proliferation.
[0840] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other BLyS binding polypeptide 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 BLyS binding polypeptide of the present invention are also
included in the present invention. 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 BLyS binding
polypeptide of the present invention are also included in the
present invention. Further included in the present invention are
antibodies which bind polypeptides encoded by polynucleotides, the
complement of which hybridize to a polynucleotides of the present
invention under stringent hybridization conditions (as described
herein). Antibodies of the present invention may also be described
or specified in terms of their binding affinity to a BLyS binding
polypeptide. 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.-6M, 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, or 10.sup.-15 M.
[0841] The invention also provides antibodies that competitively
inhibit binding of an antibody to a BLyS binding polypeptide as
determined by any method known in the art for determining
competitive binding. In preferred embodiments, the antibody
competitively inhibits binding to the BLyS binding polypeptide 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%.
[0842] Antibodies of the present invention (e.g., anti-idiotypic
antibodies) may act as agonists or antagonists of BLyS or
alternatively may not significantly alter BLyS mediated activity.
For example, the present invention includes antibodies (e.g.,
anti-idiotypic antibodies) which disrupt BLyS/BLyS receptor (e.g.,
TACI and BCMA) interactions either partially or fully. In another
example, antibodies of the invention enhance BLyS/BLyS receptor
interactions either partially or fully. Such activity may be the
result of, for example, the antibody binding to a BLyS binding
polypeptide, or alternatively as a result of direct binding of the
antibody (e.g., an anti-idiotypic antibody to BLyS).
[0843] Preferrably, antibodies of the present invention bind a BLyS
binding polypeptide disclosed herein, a portion thereof, or an
antibody that binds a BLyS binding polypeptide disclosed herein, or
a portion thereof. The invention features both BLyS binding
polypeptide-specific antibodies and antibodies that are specific to
BLyS binding polypeptide/BLyS complexes. The invention features
antibodies that enhance BLyS/BLyS binding polypeptide binding
and/or BLyS/BLyS receptor binding. The invention also features
antibodies that do not inhibit or reduce BLyS/BLyS binding
polypeptide binding and/or BLyS/BLyS receptor binding. The
invention also features BLyS binding polypeptide specific
antibodies that inhibit binding of the BLyS binding polypeptide to
BLyS or BLyS binding to BLyS receptor. In specific embodiments,
antibodies are provided that inhibit BLyS activity or BLyS 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. 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).
[0844] The antibodies of the present invention may be used, for
purposes including, but not limited to, purify, detect, and target
the BLyS binding 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 Lip BLyS in biological
samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1988).
[0845] 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
conjugated) 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.
[0846] 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, phosphorylation, 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
[0847] 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 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.
[0848] According to certain embodiments of the invention,
multivalent BLyS binding polypeptides are administered to the host
animal. Multivalent BLyS binding polypeptide complexes may be
prepared using techniques and materials known in the art such as,
for example, by cross-linking the polypeptide to a carrier protein
(e.g., bovine serum albumin (BSA), human albumin, keyhole limpet
hemocyanin (KLH), or succinylated KLH) by use of conventional
cross-linking reagents.
[0849] In specific embodiments multivalent BLyS binding
polypeptides are administered in the form of multiple antigen
peptides (MAP) (Tam, J. Imm. Meth., 124:53-61 (1989); Tam, Proc.
Natl. Acad. Sci. USA, 85:5409-5413 (1988)). In this form, the
multivalent BLyS binding polypeptide is synthesized on a branching
lysyl matrix using solid-phase peptide synthesis methods.
Recognition units in the form of MAP may be prepared by methods
known in the art (Tam, 1989, supra; Tam, 1988, supra), or, for
example, by a stepwise solid-phase procedure on MAP resins (Applied
Biosystems), utilizing methodology established by the manufacturer.
MAP peptides may be synthesized comprising (BLyS binding
polypeptide).sub.2 Lys, (BLyS binding polypeptide).sub.4 Lys.sub.3,
(BLyS binding polypeptide).sub.8 Lys.sub.6 or more levels of
branching.
[0850] 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, Cold Spring Harbor 1988);
Hammerling et al., in Monoclonal Antibodies and T-Cell Hybridomas
(Elsevier, N.Y. 1981), pp. 563-681 (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.
[0851] A "monoclonal antibody" may comprise, or alternatively
consist of, two proteins, i.e., a heavy and a light chain.
[0852] 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 or a
cell expressing such peptide. Once an immune response is detected,
e.g., antibodies specific for the antigen are art 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 American Type Culture Collection (ATCC), to
form hybridoma cells. 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. Ascites fluid, which generally contains high levels of
antibodies, can be generated by immunizing mice with positive
hybridoma clones.
[0853] 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 according to 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 BLyS
binding polypeptide.
[0854] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.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').sub.2
fragments). F(ab').sub.2 fragments contain the variable region, the
light chain constant region and the CH1 domain of the heavy
chain.
[0855] 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 that 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 international 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.
[0856] 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').sub.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., Bio Techniques, 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 herein by
reference in their entireties).
[0857] 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., Proc. Natl. Acad. Sci.
USA, 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., Bio Techniques, 4:214 (1986); Gillies
et al., J. Immunol. Methods, 125:191-202 (1989); U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816397, which are incorporated herein
by reference in their entirety. A humanized antibody is an antibody
molecule made using one or more complementarity determining regions
(CDRs) from a non-human species antibody that binds the desired
antigen and framework regions 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.,
Proc. Natl. Acad. Sci. USA, 91:969-973 (1994)), and chain shuffling
(U.S. Pat. No. 5,565,332).
[0858] 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.
[0859] 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 that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a binding polypeptide. 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 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, each of which is incorporated by
reference herein in its 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.
[0860] Completely human antibodies that 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. (See,
Jespers et al., Bio/technology, 12:899-903 (1988).)
[0861] Further, antibodies to the BLyS binding polypeptides can, in
turn, be utilized to generate anti-idiotype antibodies that "mimic"
BLyS binding polypeptides, 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 the binding of BLyS binding polypeptide to BLyS can be used
to generate anti-idiotypes that "mimic" the BLyS/BLyS binding
polypeptide binding domain and, as a consequence, bind to and
neutralize or enhance BLyS binding to BLyS receptor (e.g., TACI and
BCMA). Such neutralizing anti-idiotypes or Fab fragments of such
anti-idiotypes can be used in therapeutic regimens to bind BLyS
and/or neutralize or enhance BLyS mediated acitivity. In a specific
embodiment, anti-idiotypic antibodies can be used to bind BLyS, and
thereby block its biological activity. In another specific
embodiment, anti-idiotypic antibodies can be used to bind BLyS, and
thereby enhance its biological activity (e.g., via multimerization
of BLyS).
[0862] Polynucleotides Encoding Antibodies
[0863] 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 hybridization conditions, e.g., as
defined supra, to polynucleotides that encode an antibody,
preferably, that specifically binds to BLyS or a BLyS binding
polypeptide.
[0864] 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., Bio Techniques, 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.
[0865] 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 known in the art.
[0866] 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., Molecular Cloning: A
Laboratory Manual, 2d Ed. (Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. 1990) and Current Protocols in Molecular
Biology, Ausubel et al., eds. (John Wiley & Sons, N.Y. 1993),
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.
[0867] 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 BLyS
or a BLyS binding polypeptide. 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.
[0868] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. USA,
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 antibody and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0869] 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)).
[0870] Methods of Producing Antibodies
[0871] 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.
[0872] Recombinant expression of an antibody, or fragment,
derivative or analog thereof, (e.g., a heavy or light chain of an
antibody or a single chain antibody), 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) 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.
[0873] 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, 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.
[0874] 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. Thus, the
invention includes host cells containing a polynucleotide encoding
an antibody, or a heavy or light chain thereof, or a single chain
antibody, 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.
[0875] A variety of host-expression vector systems may be utilized
to express the antibody molecules. 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 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)).
[0876] 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 lacZ 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.
[0877] 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).
[0878] 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 El or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. See, e.g., 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)).
[0879] 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, NSO, 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.
[0880] 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.
[0881] 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., Proc. 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; 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 Current Protocols in
Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,
N.Y. 1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual (Stockton Press, N.Y. 1990); and Current Protocols in Human
Genetics, Dracopoli et al., eds. (John Wiley & Sons, N.Y.
1994), Chapters 12 and 13; Colberre-Garapin et al., J. Mol. Biol.,
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0882] 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)).
[0883] The host cell may be co-transfected with two expression
vectors, 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.
[0884] Once an antibody molecule 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.
[0885] 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 BLyS binding polypeptides 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., Proc. Natl. Acad.
Sci. USA, 89:1428-1432 (1992); Fell et al., J. Immunol.,
146:2446-2452(1991), which are incorporated by reference in their
entireties.
[0886] 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).
[0887] As discussed, supra, the polypeptides corresponding to a
BLyS binding polypeptide 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 BLyS binding polypeptides 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 (see, 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).
[0888] 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.
[0889] 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 aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.111In or .sup.99Tc.
[0890] 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. 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, colchicin, 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), cyclothosphamide, 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).
[0891] The conjugates 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,PCT publication WO
97/33899), AIM II (See, PCT publication WO 97/34911), Fas Ligand
(Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
PCT publication 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-l ("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.
[0892] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., eds.
(Alan R. Liss, Inc. 1985), pp. 243-56; Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al., eds. (Marcel Dekker, Inc. 1987), pp. 623-53;
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. (Academic
Press 1985), pp. 303-16; and Thorpe et al., "The Preparation And
Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev.,
62:119-58 (1982).
[0893] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the BLyS
binding polypeptide. Such solid supports include, but are not
limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,
polyvinyl chloride or polypropylene.
[0894] 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.
[0895] 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.
[0896] Assays For Antibody Binding
[0897] 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., Current Protocols in Molecular
Biology, Ausubel et al., eds. (John Wiley & Sons, N.Y. 1993),
which is incorporated by reference herein in its entirety).
Exemplary immunoassays are described briefly below (but are not
intended by way of limitation).
[0898] 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., Current Protocols in Molecular Biology,
Ausubel et al., eds. (John Wiley & Sons, N.Y. 1993) at
10.16.1.
[0899] 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., Current Protocols in Molecular Biology,
Ausubel et al., eds. (John Wiley & Sons, N.Y. 1993) at
10.8.1.
[0900] 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., Current Protocols in Molecular Biology,
Ausubel et al., eds. (John Wiley & Sons, N.Y. 1993) at
11.2.1.
[0901] 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.
[0902] Therapeutic Uses of Antibodies
[0903] 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 diseases, disorders, or
conditions disclosed herein. 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 BLyS expression
and/or activity, including, but not limited to, any one or more of
the diseases, disorders, or conditions described herein.
[0904] The treatment and/or prevention of diseases, disorders, or
conditions associated with aberrant expression and/or activity of
BLyS or BLyS receptor 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 BLyS on their surface and/or cells having BLyS
bound to their surface. This targeting may be the result of binding
of the antibody to BLyS binding polypeptides that have been
coadministered, or alternatively, the result of direct binding of
the antibody to BLyS. Antibodies of the invention may be provided
in pharmaceutically acceptable compositions as known in the art or
as described herein.
[0905] Non-limiting examples of the ways in which the antibodies of
the present invention may be used therapeutically includes binding
BLyS binding polypeptides of the present invention that have been
coadministered in order to bind or neutralize BLyS, or by direct
cytotoxicity of the antibody, e.g., as mediated by complement (CDC)
or by effector cells (ADCC). BLyS binding polypeptides and
anti-BLyS binding polypeptide antibodies may be administered either
locally or systemically. 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.
[0906] 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.
[0907] 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.
[0908] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides of
the present invention, fragments or regions thereof, for both
immunoassays directed to and therapy of disorders related to
polypeptides, including fragments thereof, of the present
invention. Such antibodies, fragments, or regions, will preferably
have an affinity for polypeptides, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or K.sub.D 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.
[0909] Demonstration of Therapeutic or Prophylactic Activity of
Antibodies
[0910] 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.
[0911] Therapeutic and/or Prophylactic Administration and
Composition
[0912] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a BLyS binding compound or pharmaceutical composition,
preferably an antibody. 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.
[0913] 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.
[0914] Various delivery systems are known and can be used to
administer a compound, 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 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.
[0915] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions 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 sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, care must be taken
to use materials to which the protein does not absorb.
[0916] 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 1989), pp. 353-365; Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[0917] 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, thus requiring only a fraction of the systemic
dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, Langer and Wise, eds. (CRC Press, Boca Raton, Fla. 1974),
vol. 2, pp. 115-138 (1984)).
[0918] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0919] In a specific embodiment where the compound 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.
[0920] 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, 18th Ed.,
Gennaro, ed. (Mack Publishing Co., 1990). 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.
[0921] 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.
[0922] The compounds for use in the methods 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.
[0923] The amount of the compound used 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 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.
[0924] 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 may be reduced by
enhancing uptake and tissue penetration (e.g., into the brain) of
the antibodies by modifications such as, for example,
lipidation.
[0925] 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. 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.
[0926] Diagnosis and Imaging
[0927] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a BLyS binding 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 BLyS. The invention provides for the detection
of aberrant expression of BLyS, comprising (a) contacting cells or
body fluid with a BLyS binding polypeptide; (b) assaying the
expression of BLyS in cells or body fluid of an individual using
one or more antibodies specific to the BLyS binding polypeptide and
(c) comparing the level of BLyS expression with a standard BLyS
expression level, whereby an increase or decrease in the assayed
BLyS expression level compared to the standard expression level is
indicative of aberrant expression.
[0928] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) contacting cells or body fluid with a BLyS
binding polypeptide; (b) assaying the expression of BLyS in cells
or body fluid of an individual using one or more antibodies
specific to the BLyS binding polypeptide of interest and (c)
comparing the level of BLyS expression with a standard BLyS
expression level, whereby an increase or decrease in the assayed
BLyS 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 BLyS 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.
[0929] Antibodies can be used to assay BLyS protein levels in a
biological sample using or routinely modifying 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 (131I, .sup.125I,
.sup.123I, .sup.121I), carbon (.sup.14C), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.115In, .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.149Pm,
.sup.140La, .sup.175,Yb, .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.
[0930] Techniques known in the art may be applied to label
antibodies. 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).
[0931] One embodiment of the invention is the detection and
diagnosis of a disease or disorder associated with aberrant
expression of BLyS 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 BLyS (e.g., a BLyS binding
polyptide) or which specifically binds to a molecule that
specifically binds to BLyS (e.g., an anti-BLyS binding polypeptide
antibody); (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 to quantitate or qualitate concentrations of cells of B
cell lineage or cells of monocytic lineage.
[0932] It will be understood by those skilled 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 polypeptide. 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).
[0933] 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.
[0934] In a further embodiment, monitoring of the disease or
disorder is carried out by repeating the method for diagnosing the
disease or disorder, for example, one month after initial
diagnosis, six months after initial diagnosis, one year after
initial diagnosis, etc. and comparing the results.
[0935] 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.
[0936] 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).
[0937] Antibody Kits
[0938] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody,
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 a polypeptide according to 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).
[0939] 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.
[0940] 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.
[0941] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide. 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.
[0942] 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 calorimetric substrate (Sigma, St.
Louis, Mo.).
[0943] 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 protein(s).
[0944] 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.
[0945] In another specific embodiment, any of the antibodies listed
above are conjugated to a toxin or a label (as described supra).
Such conjugated antibodies are used to kill a particular population
o of cells or to quantitate a particular population of cells. In a
preferred embodiment, such conjugated antibodies are used to kill B
cells expressing BLyS receptor on their surface. In another
preferred embodiment, such conjugated antibodies are used to
quantitate B cells expressing BLyS receptor on their surface.
[0946] In another specific embodiment, any of the antibodies listed
above are conjugated to a toxin or a label (as described supra).
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
monocyte cells expressing the membrane-bound form of BLyS. In
another preferred embodiment, such conjugated antibodies are used
to quantitate monocyte cells expressing the membrane-bound form of
BLyS.
[0947] 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 BLyS 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 fix complement. In other
specific embodiments, as further described herein, the antibodies
(or fragments thereof) are associated with heterologous
polypeptides or nucleic acids (e.g. toxins, such as, compounds that
bind and activate endogenous cytotoxic effecter systems, and
radioisotopes; and cytotoxic prodrugs).
[0948] As discussed above, antibodies to the BLyS binding
polypeptides can, in turn, be utilized to generate anti-idiotype
antibodies that "mimic" the BLyS binding polypeptide, 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 BLyS binding polypeptides and competitively inhibit
BLyS/BLyS binding polypeptide binding can be used to generate
anti-idiotypes that "mimic" the BLyS binding polypeptide/BLyS
binding domain and, as a consequence, bind to and, for example,
neutralize BLyS. Such neutralizing anti-idiotypes or Fab fragments
of such anti-idiotypes can be used in therapeutic regimens to
neutralize BLyS. For example, such anti-idiotypic antibodies can be
used to bind BLyS and thereby block BLyS mediated B cell
activation, proliferation, survival and/or differentiation.
EXAMPLES
[0949] Isolation of BLyS binding polypeptides and their use in
accordance with this invention will be further illustrated below.
The specific parameters included in the following examples are
intended to illustrate the practice of the invention, and they are
not presented to in any way limit the scope of the invention.
Example 1
[0950] Screening of Phage Display Libraries
[0951] Streptavidin-coated magnetic beads (Dynal M-280) were chosen
for presentation of the target during screening because of their
superior binding capacity compared to that of a 96 well plate. The
binding capacity of the beads for biotinylated antibodies was 5-10
.mu.g/mg of beads as stated by the manufacturer. For this study and
the screening to follow, 5 .mu.g of biotinylated recombinant BLyS
(obtained from Human Genome Sciences, Inc.) was allowed for each mg
of beads. This amount of biotinylated BLyS represents a 10-fold
excess of target, for saturation of the beads. Unbound BLyS was
washed away. Bound biotinylated BLyS was confirmed with detection
using Mab 16C9 (murine anti-BLyS, Human Genome Sciences) primary
antibody and a goat anti-mouse HRP conjugate as the secondary
antibody. An irrelevant monoclonal antibody (anti-TNF.alpha.) was
used to probe a second set of beads to control for nonspecific
binding. The color reagent TMB was used and the assay read at OD
630 nm.
[0952] Nine phage display libraries, TN6/6, TN7/4, TN8/9, TN9/4,
TN10/9, TN12/1, and Substrate Phage #2 (Dyax Corp., Cambridge,
Mass. (US)), and PhD7 and PhD12 (New England Biolabs), were
screened for BLyS binders. The makeup of these libraries was as
follows:
[0953] The TN6/6 phage display library was composed of recombinant
M13 phage displaying variegated peptides with the potential to form
loop structures based on a polypeptide template having the
structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID
NO: 14) and providing 2.0.times.10.sup.8 peptide diversity.
[0954] The TN7/4 phage display library was composed of recombinant
M13 phage displaying variegated peptides with the potential to form
loop structures based on a polypeptide template having the
structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ
ID NO: 15) and providing 2.3.times.10.sup.9 peptide diversity.
[0955] The TN8/9 phage display library was composed of recombinant
M13 phage displaying variegated peptides with the potential to form
loop structures based on a polypeptide template having the
structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa
(SEQ ID NO: 16) and providing about 5.times.10.sup.9 peptide
diversity.
[0956] The TN9/4 phage display library was composed of recombinant
M13 phage displaying variegated peptides with the potential to form
loop structures based on a polypeptide template having the
structure Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-
Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO: 17) and providing about
3.2.times.10.sup.9 peptide diversity.
[0957] The TN10/9 phage display library was composed of recombinant
M13 phage displaying variegated peptides with the potential to form
loop structures based on a polypeptide template having the
structure
Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa
(SEQ ID NO: 18) and providing 2.5.times.10.sup.9 peptide
diversity.
[0958] The TN12/1 phage display library was composed of recombinant
M13 phage displaying variegated peptides with the potential to form
loop structures based on a polypeptide template having the
structure
Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa
(SEQ ID NO: 19) and providing 1.4.times.10.sup.9 peptide
diversity.
[0959] Substrate Phage Library #2 was composed of recombinant M13
phage displaying a polypeptide insert of approximately 80 amino
acids, having two streptavidin binding domains, a linear variegated
segment of thirteen amino acids where all amino acids except Cys
were permitted at each position, and a Factor Xa cleavage site,
linked together with peptide linkers. This library provided a
diversity of 2.times.10.sup.8 display polypeptides.
[0960] Libraries PhD7 and PhD12 were composed of recombinant M13
phage displaying randomized linear seven- and twelve-amino acid
peptides, respectively.
[0961] Screening was performed as described in PCT/USO1/[ ],
entitled "Binding Polypeptides for B Lymphocyte Stimulator Protein
(BLyS)", filed concurrently herewith.
[0962] At the conclusion of screening individual phage isolates
were randomly selected and tested by ELISA for binding to BLyS. The
same isolates were submitted for DNA sequence analysis to identify
the nucleotide and deduced amino acid sequence of the displayed
peptide. Isolates were also tested for their ability to bind to
recombinant BLyS in feed streams of CHO supernatant and Sf9
supernatant (supplied by Human Genome Sciences, Inc.).
[0963] Each isolate was tested for binding to BLyS by standard
ELISA techniques where bound phage were detected with a monoclonal
anti-phage antibody/HRP conjugate.
[0964] Amino acid sequences of the displayed peptides were derived
from sequencing the phage isolate DNA inserts. Sequence data from
the phage isolates were grouped by library and sorted according to
the degree of similarity. The BLyS binding phage isolate peptides
are shown in Tables 1-8 below. These peptides represent the
translation of the DNA sequences across the varied regions of the
genes encoding the phage display fusion/peptide.
1TABLE 1 TN6/6 Library BLyS-binding Sequences Phage Isolate Amino
Acid Sequence SEQ ID NO: 453-01-B06 HLRCWSTNCRYD 20 453-01-A04
VMDCLINRCDTV 21
[0965]
2TABLE 2 TN7/4 Library BLyS-binding Sequences Phage Isolate Amino
Acid Sequence SEQ ID NO: 453-01-B04 KSKCFFPWECQQA 22 453-01-D11
AMKCYFPWECANG 23 453-01-A05 NVACYFPWECHHP 24 453-01-D01
NAPCYFPWECFSI 25 453-01-D03 SVNCWFPWECVGN 26 453-01-A08
KEPCYFYWECAVS 27
[0966]
3TABLE 3 TN8/9 Library BLyS-binding Sequences Phage Isolate Amino
Acid Sequence SEQ ID NO: 453-01-D04 DTNCDLLTKMCGPQ 28 453-01-C06
GTPCDLLTKLCLLW 29 453-01-D10 MSECDLLTKICLMG 30 453-01-B07
VPFCDLLTKHCFEA 31 453-01-B09 VPFCDLLTKHCFEA 32 453-01-C02
WSACDLLTKQCVQV 33 453-01-A06 -DGCDELTKICGMK 34 453-01-B03
KSWCDELTKVCFDP 35 453-01-B11 KWMCDELTKQCQYV 36 453-01-A02
MKYCDELTKICVGW 37 453-01-B05 YFQCDELTKMCWQK 38 453-01-A11
AMHCDKLTKHCKFH 39 453-01-A03 VPYCDKLTKICQW- 40 453-01-A07
EVFCDVLTKVCFHD 41 453-01-C09 KPKCDVLTKMCDWL 42 453-01-B02
TQHCDVLTKQCFTI 43 453-01-C01 GHFCDRLTKYCFEP 44 453-01-A09
HIQCDRLTKSCLSV 45 453-01-D05 IKACDILTKVCWPP 46 453-01-A01
QFDCDPLTKYCGEF 47 453-01-C07 KMYCDHLTGYCWPE 48 453-01-C11
MQSCDILTGYCFKR 49 453-01-D12 GPWCDILTGFCLAQ 50 453-01-C04
SVRCDLLTGWCPVW 51 453-01-B10 PADCDPLTNICFWK 52 453-01-D02
TNVCDPLTNVCFMN 53 453-01-C05 EHWCDDLTHLCFRL 54 453-01-D08
GYWCDVLTNNCWKI 55 453-01-C10 LYNCDYLTRLCFEP 56 453-01-C08
HVDCLLHPKACYKY 57 453-01-D07 VQDCLLHPKACQMQ 58 453-01-D09
KFDCLLKPMFCSNH 59 453-01-C12 FADCLIHPKSCKPL 60 453-01-D06
HGNCYPFPWECESK 61 453-01-B01 MIIVLLLLRFAISR 62 453-01-A12
SLLVIFLLIGAGSL 63
[0967]
4TABLE 4 TN9/4 Library BLyS-binding Sequences Phage Isolate Amino
Acid Sequence SEQ ID NO: 453-01-G06 FHPCDMLTGIWCQPN 64 453-01-H01
SKRCDLLTKMWCETE 65 453-01-F02 TKFCDRLTMPKCVWK 66 453-01-E03
NTFCPDPLTGRCVNP 67 453-01-E11 DWTCDPLFHRECIFE 68 453-01-H09
PQPCDLLFEKKCSIK 69 453-01-H02 RWHCDMLINPSCLPD 70 453-01-E04
KIQCDIVNLSSCVYP 71 453-01-G11 LNACDIVHPNYCSGM 72 453-01-F01
AKACSIVNLESCEYL 73 453-01-H06 RQACSIITPWGCPIP 74 453-01-F10
ADNCTVATLDFCYWT 75 453-01-E05 KPECNITKPQFCFGE 76
[0968]
5TABLE 5 TN10 Library BLyS-binding Sequences Phage Isolate Amino
Acid Sequence SEQ ID NO: 453-01-H07 -NNCQWDELTSMCDPF 77 453-01-F05
SRLCHMDELTHVCVHF 78 453-01-F09 SRPCQIDELTKACFYN 79 453-01-G09
DRVCKLDFLTYNCLNH 80 453-01-F04 HSNCIMDLLTNRCFYD 81 453-01-H03
PFNCFHDPLTGLCLHS 82 453-01-F03 YDSCTYDRLTKQCYPS 83 453-01-F07
FHDCMYDALLGYCLPY 84 453-01-G08 NRSCDPLTRPKSCGL 85 453-01-G04
LSNCDWDDLIRQCLHD 86 453-01-E01 FWDCLFHPNSRYCVLS 87 453-01-E10
SRDCLLSPAMAWCGLD 88
[0969]
6TABLE 6 TN12/1 Library BLyS-binding Sequences Phage Isolate Amino
Acid Sequence SEQ ID NO: 453-01-H05 GGNCYTDSLTKLHFCMGD 89
453-01-H04 --MCPRDPLTKAKLCNWH 90 453-01-G03 PNQCQDDLTKQWYSCHYH 91
453-01-F11 FDMCFDALTKQNFYCRFH 92 453-01-F06 RNMCVDRLTKLQHGCEGA 93
453-01-G07 DPECLTSFDRLTKMCWPW 94 453-01-H11 DDECHYDYLTHYMRCDYR 95
453-01-G05 FGGCNIDLLTNTMMCHRN 96 453-01-G10 HGPCYWDELTMQWHCNHH 97
453-01-H12 GAMCVDLLTYTFRPCMYA 98 453-01-E07 SNKCWDELTHAWAECGRF 99
453-01-E09 RPVCYKGYDILTTQCMPW 100 453-01-G01 PSRCWFDLLFNKFVCKRN 101
453-01-H08 RSGCVYDMLLMTMYCPSN 102 453-01-H10 SNRCEGDQLMRPPSCRHL 103
453-01-F08 YRMCWWDDLLRGFVCDFH 104 453-01-E06 HDGCYDELLYRWTRCEHR 105
453-01-E08 WAWCFDELVQRYFTCFDH 106 453-01-E02 LPECRQYFPWEKQVCSYW
107
[0970]
7TABLE 7 PhD 12 Library BLyS-binding Sequences Phage Isolate Amino
Acid Sequence SEQ ID NO: 453-02-B05 VHYDSLTKMWTR 108 453-02-D09
FTDPLTKMSLHS 109 453-02-C12 GYDVLTKLYFVP 110 453-02-A05
YYDRLTKLYSSM 111 453-02-B06 L?KDPLTKLYIS 112 453-02-A04
GYDVLTKL?FVP 113 453-02-B03 RLYDPLTKLVLS 114 453-02-B01
MFDPLTKIAFPA 115 453-02-D04 FYDSLTKTNLRD 116 453-02-B02
GIYDKLTRAWLP 117 453-02-B08 KYDPLTRAR?PL 118 453-02-D06
YIDQLTRLSLPS 119 453-02-A09 HqTFDILTRLHF 120 453-02-B04
WQFDVLTRSWTP 121 453-02-A02 GAAYDHLTRTWL 122 453-02-D05
YFDQLTHLSIKK 123 453-02-A06 AWDPLTMLVLPW 124 453-02-D03
ALWMDPLTGLAF 125 453-02-B12 WQFDVLT?SWTP 126 453-02-A01
WTDPLTHMEIYH 127 453-02-C04 WTDSLTGLWFPD 128 453-02-C05
YTDPLTGIV?PF 129 453-02-D08 YWDKLTMLHLGV 130 453-02-D02
YYDFLTRTVLPS 131 453-02-A03 RLDPLSKNDFPR 132 453-02-A11
LRYDPLLKS?IY 133 453-02-D07 LRYDPLLKSYIY 134 453-02-A07
YFDQFTHLSIKK 135 453-02-C08 YFDQ?THLSIKK 136
[0971]
8TABLE 8 Substrate Phage Library BLyS-binding Sequences Phage
Isolate Amino Acid Sequence SEQ ID NO: 453-02-E04 EHYYTDPLTGARI 137
453-02-F01 EHY?TDPLTGARI 138 453-02-E09 EHYSTDPLTGARI 139
453-02-E07 EHYYTDPL?G?RI 140 453-02-G05 EHYYTDPL?G?R? 141
453-02-G09 EHYYTDPL?GAR? 142 453-02-E06 EH?YTDPLNGAR? 143
453-02-E05 EHYYNDPLNGAR? 144 453-02-F04 ?H?YNDPLNGAR? 145
453-02-G07 KPYYDPITKMTHH 146 453-02-F06 KPYYDPITKMSHH 147
453-02-E08 KPYYDPISKMTHH 148 453-02-G08 KPISKMTHH 149 453-02-E01
QIGYDELTKAWVT 150 453-02-G02 QLGYDELTKAWVT 151 453-02-H06
KIDEL?MQNIIIW 152 453-02-F08 DHTDPLIQGLTKR 153 453-02-H01
WHDPLKHMHFHHE 154 453-02-F03 KHIDMETGLILQN 155 453-02-G03
MQVDPETGLKYEH 156 453-02-E03 ?LDQHVYQS 157 453-02-F10 TGAR? 158
453-02-F02 GPYNI?RL?GEr? 159 453-02-E02 HIKMLHQGSFVGV 160
453-02-H08 HPTNQ?VYS 161 453-02-H05 HRGQNGMv? 162 ? = amino acid
unknown (all tables) lower case = amino acid identity probable but
not completely characterized
Example 2
[0972] Immobilization of BLyS Binding Polypeptides on Sepharose-4FF
Beads
[0973] On the basis of the above results, six display phage
sequences were chosen for further study:
[0974] TN7-01-A08 (SEQ ID NO: 27), TN8-01-B07 (SEQ ID NO: 31),
TN10-01-F05 (SEQ ID NO: 78) TN12-01-H05 (SEQ ID NO: 89), PhD-02-C04
(SEQ ID NO: 128), and PhD-02-C12 (SEQ ID NO: 110). In order to
develop a suitable BLyS affinity ligand, the identified display
peptides were synthesized to order by a commercial vendor, with
slight modifications:
[0975] Two amino acids of leader were added to each binding peptide
at the N-terminus, in order to avoid leaving a free amine at the
first amino acid of the sequence corresponding to the variegated
region of the phage display template; the N-terminus was acetylated
to prevent immobilization of the peptide to the chromatographic
matrix through that position; a C-terminal linker was added (i.e.,
-PGPEGGGK; SEQ ID NO: 13); and any internal lysines in the peptide
were blocked with the group: ivDde (i.e.,
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methyl
butyl-L-lysine). This group was intact on the finished synthesized
peptides and was removed after immobilization or fluorescein
labeling. As an alternative modification, peptides with internal
lysines were also synthesized with C-terminal hydrazide functional
groups, which could be immobilized onto activated aldehyde
chromatographic media.
[0976] The peptides were immobilized onto NHS-activated SEPHAROSE-4
Fast Flow agarose media (Pharmaceia) at ligand densities targeted
to 2 .mu.mol/ml. Actual ligand densities of peptides on the media
ranged from 0.76 .mu.mol/ml to 1.98 .mu.mol/ml, as determined by
amino acid analysis of immobilized peptide. All but one peptide was
immobilized in aqueous conditions of 100 mM KH.sub.2PO.sub.4/150 mM
NaCl/0.05% Tween 20, pH 7.5. For solubility reasons, the peptide
DX217 (see, Table 9, below) was immobilized in 30% dimethyl
formamide(DMF)/100 mM KH2PO4/150 mM NaCl/0.05% Tween 20. pH 7.5.
Immobilization reactions were allowed to proceed for 2 hours at
ambient temperature, followed by brief washing with pH 7.5 buffer.
The Fast Flow SEPHAROSE media was then allowed to tumble at ambient
temperature overnight to hydrolyze remaining NHS esters after which
the media was washed to remove any unbound peptide. A solution of
2% hydrazine/DMF was used to de-block ligands containing
ivDde-lysine. Media was then further washed with aqueous buffers
and stored at 4.degree. C. until packed into columns. Table 9 shows
the sequences of the synthesized peptides and their measured
densities on the agarose media.
9TABLE 9 BLyS Binding Peptides Synthesizes as Affinity Li- gands
Pep- Sequence SEQ tide Isolate (potential disulfide ID Name source
loop underlined) NO: DX212 01-A08 Ac-AGKEPCYFYWECAVSGPGPEGGGK 163
DX214 01-B07 Ac-AGVPFCDLLTKHCFEAGPGPEGGGK 164 DX216 01-F-5
Ac-GSSRLCHMDELTHVCVHFAPPGPEGGGK 165 DX217 01-H05
Ac-GDGGNCYTDSLTKLHFCMGDEPGPEGGGK 166 DX219 02-C12
Ac-GYDVLTKLYFVPGGPGPEGGGK 167 DX221 02-C04
Ac-WTDSLTGLWFPDGGPGPEGGGK 168 Ac denotes N-terminal acetylation
[0977] BLyS-Ligand Affinity Determination (Overview of
Procedure)
[0978] Dissociation constants between the synthetic peptides and
BLyS (free in solution) were measured by fluorescence anisotropy
(FA). In these experiments, the concentration of the
fluorescein-labeled peptide is held constant and the BLyS protein
concentration was varied. The observed change in anisotropy is fit
to the following equation via nonlinear regression to obtain the
apparent K.sub.D. 1 Peptide + BLyS K D Peptide BLyS r obs = r free
+ ( r bound - r free ) ( K D + BLYS + P ) - ( K D + BLYS + P ) 2 -
4 BLYS P 2 P
[0979] where:
[0980] r.sub.obs=observed anisotrpy, r.sub.free=anisotropy of free
peptide, r.sub.bound=anisotropy of bound peptide,
K.sub.D=dissociation constant, BLyS=total BLyS concentration, and
P=total fluorescein labeled peptide concentration.
[0981] Binding reactions containing 50 nM fluorescein-labeled
peptide and a varied concentration of BLyS in a volume between 10
and 20 .mu.L per well were performed in 384 well microplates.
Reactions were assayed using a Tecan Polarion fluorescence
polarization plate reader. Cross-competition studies between
peptides were performed using 50 nM fluorescein-labeled peptide and
1-2 .mu.M BLyS in the presence and absence of 100 .mu.M unlabeled
peptide. The influence of pH on the observed K.sub.D was
investigated at pH 6.0 using the primary binding buffer [15 mM
sodium citrate, 120 mM NaCl, 0.01% Tween 20] and at pH 9.0 using
200 mM sodium bicarbonate, 125 mM sodium chloride. Other buffers in
which dissociation constants of BLyS Binding polypeptides were
detremined include: [pH 6.0, 0.01% Tween], [pH 6.0, 0.1% gelatin],
[pH5.0, 0.01% Tween], [pH9.0, 0.1% Tween], [pH6.0, 15% ethylene
glycol, 0.01 % Tween], ], [pH5.0, 15% ethylene glycol, 0.01 %
Tween], and [pH9.0, 15% ethylene glycol, 0.01 % Tween]. All six of
the peptides (DX212, DX214, DX216, DX217, DX219, and DX221) bound
BLyS in solution with approximately the same affinity
(K.sub.D=0.5-2 .mu.M). Cross-competition studies demonstrated that
all peptides compete with each other for BLyS binding, which
suggests that they all bind to the same site on BLyS.
Example 3
[0982] Design of modified BLyS Binding Peptides
[0983] Once a promising BLyS binding polypeptide has been isolated,
improvements to that polypeptide can be made by changing, adding or
removing individual or multiple amino acid residues from the
polypeptide. Amino acid substitutions can be conservative or non
conservative. Conservative amino acids exchanges include, for
example, the exchange of aromatic residues (e.g., phenylalanine,
tryptophan, and tyrosine) for one another, the exchange of p
hydrophobic residues (e.g, leucine, isoleucine, and valine) for one
another, the exchange of polar residues (e.g., glutamine and
asparagine) for one another, the exchange of acidic residues (e.g.,
arginine, lysine, and histidine) for one another, and the exchange
of small residues (e.g., alanine, serine, threonine, methionine,
and glycine) for one another, the exchange of aromatic residues for
one another. Additionally, nonclassical amino acids, chemical amino
acid analogs, or chemically modified classical amino acids can be
introduced as a substitution or addition to a BLyS binding
polypeptide of the invention. Non-classical amino acids include,
but are not limited to, the D-isomers of the common amino acids,
2,4-diaminobutyric acid (Dbu), 4-aminobutyric acid (bAbu),
2-aminobutyric acid (Abu), 6-amino hexanoic acid (epsilon-Ahx),
2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib),
3-aminopropanoic acid (bAla), ornithine (Orn), norleucine (Nle),
norvaline (Nva), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp),
sarcosine (MeGly), citrulline, homocitrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
fluoro-amino acids, designer amino acids such as .beta.-methyl
amino acids, C.alpha.-methyl amino acids, N.alpha.-methyl amino
acids, and amino acid analogs in general. By way of example, four
modified peptides based on the DX212 sequence have been
designed:
[0984] 1. Ac-AGK(Ac)EPCYFYWECAVSGPGPEGGGK (SEQ ID NO:
169)--internal lysine side chain acetylated;
[0985] 2. Ac-AGREPCYFYWECAVSGPGPEGGGK (SEQ ID NO: 170)--arginine
substitution;
[0986] 3. Ac-AGQEPCYFYWECAVSGPGPEGGGK (SEQ ID NO: 171)--glutamine
substitution;
[0987] 4. Ac-AGNleEPCYFYWECAVSGPGPEGGGK (SEQ ID NO:
172)--norleucine substitution.
[0988] Ac denotes N-terminal acetylation.
Example 4
[0989] Biacore Analysis of the Affinity of BLyS Binding
Polypeptides
[0990] Binding of BLyS binding polypeptides to BLyS, for example,
can be analyzed by BIAcore analysis. Either BLyS (or another
antigen for which one wants to know the affinity of a BLyS binding
polypeptide) or BLyS binding polpeptide can be covalently
immobilized to a BIAcore sensor chip (CM5 chip) via amine groups
using N-ethyl-N'-(dimethylaminopropyl)carbodi-
imide/N-hydroxysuccinimide chemistry. Various dilutions of BLyS
binding polypeptides or BLyS (or other antigen for which one wants
to know the affinity of a BLyS binding polypeptide), respectively
are flowed over the derivatized CM5 chip in flow cells at 15
microlters/min. for a total volume of 50 microliters. The amount of
bound protein is determined during washing of the flow cell with
HBS buffer (10 mM HEPES, pH7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005%
surfactant P20). Binding specificty for the protein of inerest is
determined by competition with soluble competitor in the presence
the protein of ineterest.
[0991] The flow cell surface can be regenerated by displacing bound
protein by washing with 20 microliters of 10 mM glycine-HCl, pH2.3.
For kinetic analysis, the flow cells are tested at different flow
rates and different polypetide densities on the CM5 chip. The
on-rates and off-rates can be determined using the kinetic
evaluation program in BIAevaluation 3 software.
Example 5
[0992] BLyS Binding Polypeptide Neutralization of Murine Splenocyte
Proliferation
[0993] To determine if an BLyS binding polypeptide inhibits BLyS
mediated B cell proliferation, a splenocyte proliferation assay can
be performed Briefly, murine splenocytes are isolated by flushing
spleen with complete medium using a 25 g needle and 10 ml of
complete medium (RPMI 1640 with 10% FBS containing 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, 4 mM glutamine,
5.times.10.sup.-5M .beta.-mercaptoethanol). The cells are passed
through a 100 micron nylon filter to remove cell clumps. The cell
suspension is then separated by gradient centrifugation at
400.times.g for 25 minutes at room temperature (one 15 ml conical
tube/spleen; 3 ml Ficol, 10 ml cell suspension/spleen; Ficol 1083
from Sigma). The recovered cells are washed 3 times in complete
medium and counted. Recovered cells are then diluted to a
concentration of 3.times.10.sup.6/ml in complete medium containing
a 3.times. concentration of SAC (3.times.=1:33,333 dilution of
stock Staph. aureus Cowan strain; Calbiochem).
[0994] For each BLyS binding polypeptide, 50 microliters of
dilutions at 30 .mu.g/ml, 3.0 .mu.g/ml, and 0.3 .mu.g/ml
concentrations are aliquotted into individual wells of a 96 well
plate in triplicate. Suitable positive controls, such as, for
example monoclonal antibody 15C10, can also be used. Medium
containing no BLyS binding polpeptide is used as negative control.
BLyS protein is diluted in complete medium to concentrations of 300
ng/ml, 90 ng/ml and 30ng/ml. 50 microliters of each of the BLyS
dilutions were then added to the BLyS binding polypeptide dilution
series in the plates. The plate containing the BLyS binding
polypeptide and BLyS dilutions are then incubated for 30 minutes at
37.degree. C., 5% CO.sub.2, after which 50 microliters of the
splenocyte cell suspension containing SAC is added to all wells.
The plates are then incubated for 72 hours (37.degree. C., 5%
CO.sub.2).
[0995] After 72 hours, each well is supplemented with 50 .mu.l of
complete medium containing 0.5 .mu.Ci of .sup.3H-thymidine (6.7
Ci/mM; Amersham) and cells are incubated for an additional 20-24
hours at (37.degree. C., 5% CO.sub.2). Following incubation cells
are harvested using a Tomtec Cell Harvester and filters counted in
a TopCount Scintillation counter (Packard).
Example 6
[0996] In Vitro Screening of BLyS Antagonists
[0997] The bioassay for assessing the effects of putative BLyS
antagonists is performed in triplicate in 96 well format by mixing
equal volumes of BLyS, responder cells, and putative antagonist
each of which is prepared as a 3.times. stock reagent.
[0998] B-lymphocytes are purified from human tonsil by MACS
(anti-CD3 depletion), washed, and resuspended in complete medium
(CM) (RPMI 1640 with 10% FBS containing 100 U/ml penicillin, 100
.mu.g/ml streptomycin, 4 mM glutamine, 5.times.10E-5 M
beta-mercaptoethanol) at a concentration of 3.times.10e6 cells/mL.
Staphylococcus aureus, Cowan I (SAC, CalBiochem) is added to cells
at 3.times. concentration (3.times.=1:33,333 dilution of
stock).
[0999] Meanwhile, eight serial dilutions (3-fold) of potential
antagonists are prepared in CM such that the diluted antagonists
are at 3.times. the final concentrations to be tested in the assay.
BLyS binding polypeptides are routinely tested starting at a final
concentration of 10 .mu.g/mL and going down to about 1.5 ng/mL.
[1000] Human rBLyS was prepared in CM to 3.times. concentration
(3.times.=300 ng/mL, 30 ng/mL, and 3 ng/mL) in CM. Potential
inhibitors are routinely tested at several concentrations of BLyS
to avoid false negatives due to unexpectedly low affinity or
antagonist concentration.
[1001] Fifty microliters of diluted antagonist and 50 .mu.L of
diluted BLyS are added to the putative antagonist dilution series.
Cells are then incubated for 72 hours (37.degree. C., 5% CO.sub.2)
in a fully humidified chamber. After 72 hrs., the cells are
supplemented with 0.5 .mu.Ci/well 3H-thymidine (e.g., 6.7 Ci/mmol)
and incubated for an additional 24 hours. Plates are harvested
using a Tomtec Cell Harvester and filters counted in a TopCount
Scintillation counter (Packard).
Example 7
[1002] Protein Fusions of BLyS Binding Polypeptides
[1003] BLyS binding polypeptides of the invention are optionally
fused to other proteins. These fusion proteins can be used for a
variety of applications. For example, fusion of BLyS binding
polypeptides to His-tag, HA-tag, protein A, IgG domains, and
maltose binding protein facilitates purification. (See, EP A 394
827; Traunecker et al., Nature, 331:84-86 (1988)). Similarly,
fusion to IgG-1, IgG-3, and albumin increases the half-life time in
vivo. Nuclear localization signals fused to BLyS binding
polypeptides can target the protein to a specific subcellular
localization, while covalent heterodimer or homodimers can increase
or decrease the activity of a fusion protein. Fusion proteins can
also create chimeric molecules having more than one function.
Finally, fusion proteins can increase solubility and/or stability
of the fused protein compared to the non-fused protein. All of the
types of fusion proteins described above can be made using
techniques known in the art or by using or routinely modifying the
following protocol, which outlines the fusion of a polypeptide to
an IgG molecule.
[1004] Briefly, the human Fc portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the
sequence described below (SEQ ID NO: 447). These primers also
preferably contain convenient restriction enzyme sites that will
facilitate cloning into an expression vector, preferably a
mammalian expression vector.
[1005] For example, if the pC4 (Accession No. 209646) expression
vector is used, the human Fc portion can be ligated into the BamHI
cloning site. Note that the 3' BamHI site should be destroyed.
Next, the vector containing the human Fc portion is re-restricted
with BamHI, linearizing the vector, and BLyS binding polynucleotide
is ligated into this BamHI site. Note that the polynucleotide is
cloned without a stop codon, otherwise a fusion protein will not be
produced.
[1006] If the naturally occurring signal sequence is used to
produce the secreted protein, pC4 does not need a second signal
peptide. Alternatively, if the naturally occurring signal sequence
is not used, the vector can be modified to include a heterologous
signal sequence. (See, e.g., WO 96/34891.)
[1007] Human IgG Fc region:
[1008]
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGA-
GGGTGCACCG
TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTC-
ACATGCGTGGTGGT
GGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG-
TGCATAATGCCAAGACAA
AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGT-
CCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATC-
GAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG-
AGCTGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTG-
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAG
ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC-
CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG-
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT
CTCCGGGTAAATGAGTGCGACGGCCGCGACT- CTAGAGGAT (SEQ ID NO: 449)
Example 8
[1009] Isolation of scFV Molecules Recognizing BLyS Binding
Polypeptides
[1010] Naturally occuring V-genes isolated from human PBLs are
constructed into a large library of antibody fragments which
contain reactivities against polypeptides of the present invention
to which the donor may or may not have been exposed (see, e.g.,
U.S. Pat. No. 5,885,793, incorporated herein by reference in its
entirety).
[1011] Rescue of the Library
[1012] A library of scFvs is constructed from the RNA of human PBLs
as described in WO 92/01047. To rescue phage displaying antibody
fragments, approximately 10.sup.9 E. coli harbouring the phagemid
are used to inoculate 50 ml of 2.times.TY containing 1% glucose and
100 .mu.g/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
innoculate 50 ml of 2.times.TY-AMP-GLU, 2.times.108 TU of .DELTA.
gene 3 helper phage (M13 .DELTA. gene III, see WO 92/01047) 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 minutes
and the pellet resuspended in 2 liters of 2.times.TY containing 100
.mu.g/ml ampicillin and 50 .mu.g/ml kanamycin and grown overnight.
Phage are prepared as described in WO92/01047.
[1013] 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 are pelleted (IEC-Centra 8, 4000 revs/min. for 10
min.), resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g 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 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[1014] Panning of the Library
[1015] Immunotubes (Nunc) are coated overnight in PBS with 4 ml of
either 100 mg/ml or 10 mg/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 are 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.OM 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 .mu.g/ml
ampicillin. The resulting bacterial library is then rescued with
.DELTA. gene III 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.
[1016] Characterization of Binders
[1017] Eluted phage from the 3rd and 4th 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 microtitre plates coated with either 10 pg/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., WO 92/01047) and then by sequencing.
[1018] Additionaly, scFvs may be converted to complete Ig molecules
using techniques which are commonly known in the art.
Example 9
[1019] Production of an anti-BLyS Binding Polypeptide Antibody
[1020] a) Hybridoma Technology
[1021] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing BLyS binding polypeptides
are administered to an animal to induce the production of sera
containing polyclonal antibodies. In a preferred method, a
preparation of BLyS binding polypeptide is prepared and purified to
render it substantially free of natural contaminants which is then
conjugated to a carrier molecule such as keyhole limpet hemocyanin
(KLH), suucinylated KLH, or chicken gamma globulin (CGG). Such a
preparation is then introduced into an animal in order to produce
polyclonal antisera of greater specific activity.
[1022] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or BLyS protein binding
fragments thereof). Such monoclonal antibodies can be prepared
using hybridoma technology. (Kohler et al., Nature, 256:495 (1975);
Kohler et al., Eur. J. Immunol., 6:511 (1976); Kohler et al., Eur.
J. Immunol., 6:292 (1976); Hammerling et al., in Monoclonal
Antibodies and T-Cell Hybridomas (Elsevier, N.Y. 1981), pp.
563-681.) In general, such procedures involve immunizing an animal
(preferably a mouse) with BLyS binding polypeptide or, more
preferably, with a secreted BLyS binding polypeptide-expressing
cell. Such cells may be cultured in any suitable tissue culture
medium; however, it is preferable to culture cells in Earle's
modified Eagle's medium supplemented with 10% fetal bovine serum
(inactivated at about 56.degree. C.), and supplemented with about
10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin,
and about 100 .mu.g/ml of streptomycin.
[1023] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP2/0),
available from the ATCC. After fusion, the resulting hybridoma
cells are selectively maintained in HAT medium, and then cloned by
limiting dilution as described by Wands et al. (Gastroenterology,
80:225-232 (1981).) The hybridoma cells obtained through such a
selection are then assayed to identify clones which secrete
antibodies capable of binding the BLyS binding polypeptide.
[1024] Alternatively, additional antibodies capable of binding to
BLyS binding polypeptide can be produced in a two-step procedure
using anti-idiotypic antibodies. Such a method makes use of the
fact that antibodies are themselves antigens, and therefore, it is
possible to obtain an antibody which binds to a second antibody. In
accordance with this method, protein specific antibodies are used
to immunize an animal, preferably a mouse. The splenocytes of such
an animal are then used to produce hybridoma cells, and the
hybridoma cells are screened to identify clones which produce an
antibody whose ability to bind to the BLyS binding
polypeptide-specific antibody can be blocked by BLyS binding
polypeptide. Such antibodies comprise anti-idiotypic antibodies to
the BLyS binding protein-specific antibody and can be used to
immunize an animal to induce formation of further BLyS binding
polypeptide-specific antibodies.
[1025] It will be appreciated that Fab and F(ab').sub.2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments). Alternatively, secreted BLyS binding
protein-binding fragments can be produced through the application
of recombinant DNA technology or through synthetic chemistry.
[1026] For in vivo use of antibodies in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. 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 171 496; Morrison et al., EP 173
494; Neuberger et al., WO 86/01533; Robinson et al., WO 87/02671;
Boulianne et al., Nature, 312:643 (1984); Neuberger et al., Nature,
314:268 (1985).)
Example 10
[1027] BLyS-Induced Signalling in B cells
[1028] Total RNA was prepared from tonsillar B cells unstimulated
or stimulated with SAC or SAC plus BLyS (100 ng/mL) for 12 hours.
Messenger RNA levels of ERK-l and PLK was determined by real time
quantitaive 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 probe as endogenous
reference. Expression levels were characterized relative to
observed levels in unstimulated B-cells.
Example 11
[1029] Affinity Maturation of BLyS Binding Polypeptides
[1030] In order to identify high affinity BLyS-binding
polypeptides, a BLyS Affinity Maturation Library (BAML) was
designed around a 14-mer linear peptide template sequence having
fixed amino acid residues at 5 of the 14 positions. 3 of the 5
fixed residues corresponded to a highly conserved DxLT tetrapeptide
amino acid motif (SEQ ID NO: 446) isolated from both the
constrained and linear peptide libraries. The design of the 14-mer
allowed for some amino acid variation at each of the remaining 9
positions, however, preference was given for a particular amino
acid at each of these positions. Analysis of binding affinity of
the newly isolated peptides for BLyS was evaluated by direct and
indirect phage ELISA and fluorescence anisotropy.
[1031] BAML was designed on a 14-mer linear (non-constrained)
template peptide sequence having fixed residues at positions 1
(Ala), 5 (Asp), 7 (Leu), 8 (Thr), and 10 (Leu). The amino acid
sequence of positions 3-14 in the BAML template most closely
resembles a binding polypeptide isolated from the PhD 12 linear
polypeptide library (see Table 7, supra). Residues at position 1
(fixed Ala) and position 2 (variable) were included to extend the
length and presentation of the BLyS-binding sequence. Positions 5-8
correspond to the DxLT motif found in peptide isolates from both
the constrained and linear peptide libraries (see Tables 1-8,
supra). Since hydrophobic amino acids (L, M, I, A, and G) were
found at position 10 in 85% of the original isolates, a Leu
residue, occurring in 42% of the isolates, was fixed at that
position in the BAML template peptide.
[1032] Table 10 shows the design of the 14-mer BAML template
sequence.
10TABLE 10 BAML template sequence (14-mer) SEQ ID amino acid
position NO: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A n w y D s L T k L w
l p d 184
[1033] Referring to Table 10, the upper case letters indicate the
fixed residues at positions 1, 5, 7, 8, and 10 of the template.
Lower case letters designate preferred amino acids at those
positions, however the design of the variegated DNA template
encoding the 14-mer allows for some sequence variation at these
positions.
[1034] Table 11 shows the design of the variegated DNA template
used to generate the BAML peptides.
11TABLE 11 BAML DNA template sequence (14-mer) codon position 1 2 3
4 5 6 7 8 9 10 11 12 13 14 codons* GCT eez zjj zez GAT zqz CTT ACT
eej CTC zjj qzz qqz jez *The sequence of codons is SEQ ID
NO:185.
[1035] Referring to Table 11, the nucleotide coding sequences for
the fixed amino acids in the BAML 14-mer template are shown in
upper case letters. The letters "e", "j", "q", and "z" in the
variegated DNA template each represent a particular mixture of
nucleoside bases present in the input dNTPs for each position:
[1036] j=79% guanine, 7% cytosine, 7% adenine, 7% thymine
[1037] q=7% guanine, 79% cytosine, 7% adenine, 7% thymine
[1038] e=7% guanine, 7% cytosine, 79% adenine, 7% thymine
[1039] z=7% guanine, 7% cytosine, 7% adenine, 79% thymine.
[1040] The codons of the DNA template were designed to skew the
encoded variable amino acid toward the preferred amino acid at each
position shown in SEQ ID NO: 184 (Table 10, lower case). Later
sequencing of phage isolates showed that, at any particular
position, preferred residues occurred at a frequency of from 44% to
70%.
[1041] Synthetic DNA sequences fitting the DNA template were
amplified by large scale PCR.
[1042] The amplified DNAs were restriction digested for insertion
into a M13 phage expression vector (MANP vector, Dyax Corp.,
Cambridge, Mass.), and vectors bearing the inserts were used to
transform M13 phage by electroporation, to produce the BAML.
[1043] Recombinant phage were collected and purified by PEG
precipitation and titered. A total of 3.2.times.10.sup.13 PFU were
amplified in BAML from 1.6.times.10.sup.9 transformants.
[1044] Screening BAML
[1045] As outlined in Table 12 below, a two-step competition
method, starting with the original BAML library, was used over 4
rounds of screening to isolate the highest affinity BLyS-binding
polypeptides from the BAML. Prior to screening, the amplified BAML
was contacted with Seradyn streptavidin-coated magnetic beads
(MG-SA, Seradyn, Indianapolis, Id.), to remove bead- and
streptavidin-binding phage.
[1046] For screening BAML, phage were incubated in solution with
biotinylated BLyS (b-BLyS) in 200 .mu.l PBS, pH 7.4, Tween-20
(0.1%), to form phage/b-BLyS binding complexes. For the first
competition step, unlabeled BLyS (1-2 .mu.M) was added to the
phage/b-BLyS binding complex mixture in solution and incubated for
1-20 hrs. (See Table 12.) The phage/b-BLyS complexes remaining in
solution after incubation with unlabeled BLyS were captured by
brief (10 min. on rotator) incubation with MG-SA streptavidin beads
(50 .mu.l). After capture of the phage/b-BLyS complexes on
streptavidin beads, the unbound fraction was removed and beads were
washed 15-20 times with 1 ml PBS-Tween 20 prior to the second
competition step. The phage/unlabeled BLyS complexes from the round
1 competition step only, were collected and used as a fraction of
the input phage for the second round of screening along with the
bead-captured phage/b-BLyS complexes, however, in each subsequent
round of screening only the bead-associated phage were collected
after the first competition step for further screening, and the
phage/unlabeled BLyS complexes were discarded.
[1047] For the second competition step, the competitor peptide was
a polypeptide (DX221; SEQ ID NO: 168) based on a BLyS-binding
polypeptide isolated from the PhD 12 library in the initial
screenings described above. The phage/b-BLyS complexes bound to
streptavidin, collected after lo the first competition incubation
step, were serially diluted with 50 .mu.M DX221 BLyS-binding
peptide (K.sub.D=3 .mu.M) in 300 .mu.l PBS-Tween-20 (0.1%). A
series of short incubations (3-4 per round, for 1 hour) of the
phage/b-BLyS complexes with DX221 followed by a final incubation of
from overnight (O/N, for rounds 1, 2, and 4) to 3 days (for round
3). (See Table 12.) The second competition step in round 4 included
an incubation with 67 nM BLyS for 1 hour prior to incubation with
DX221. The streptavidin bead-associated phage/b-BLyS binding
complexes remaining after the DX221 competition step in round 4
were collected for further analysis.
12TABLE 12 BLyS affinity maturation library (BAML) screening
conditions First Second Competition Competition Screening Input
Incubation Competitor Incubation Peptide Round phage.sup.1
b-BLyS.sup.2 Time (hrs) (BLyS) Time (hrs) Elutions 11 1.5 .times.
10.sup.11 100 nM 2 2 .mu.M 1 50 .mu.M DX221, 4 .times. 1 hr, then
O/N 2 2 .times. 10.sup.10 100 nM 1 1 .mu.M 20 50 .mu.M DX221, 3
.times. 1 hr, then O/N 3 6.5 .times. 10.sup.10 100 pM 16 1 .mu.M 3
50 .mu.M DX221, 4 .times. 1 hr, then 3 days 4 6.0 .times. 10.sup.10
10 pM 16 1 .mu.M 2 67 nM BLyS, 1 hr; 50 .mu.M DX221 + 67 nM BLyS 3
.times. 1 hr, O/N, then add'l 4 hrs .sup.1Input phage for round 1
was original BAML; for round 2 was amplified output phage from
overnight (final) peptide elution and bead-associated phage from
round 1; for round 3 was amplified bead-associated output phage
from round 2; and for round 4 was amplified bead-associated output
phage from round 3. All amplified phage samples were pre-cleared on
streptavidin beads before incubation with biotin-BLyS in solution.
.sup.2b-BLyS = biotinylated BLyS
[1048] ELISA Analysis
[1049] Approximately four hundred BAML isolates from rounds 2, 3
and 4 of the above screening were analyzed by direct and indirect
phage ELISA assays.
[1050] For indirect phage ELISA, Immulon-2HB plates (Dynex
Technologies, Inc., Chantilly, Va.) were coated with 100 .mu.l of 1
.mu.g/ml Immunopure streptavidin (Pierce, Rockford, Ill.) diluted
in PBS. 100 .mu.l of a series of 10-fold dilutions of b-BLyS (0-0.1
.mu.g/ml in PBS) were immobilized in the streptavidin-coated wells
(1 hr, 37.degree. C.). After washing, 1-25 .mu.l of overnight
culture of E. coli infected with the individual phage plaques were
added to the appropriate wells and incubated for 1 hour, followed
by 10 washes with PBS-Tween-20. Anti-M13 antibody conjugated to
horseradish peroxidase (1:10,000 in PBS-Tween-20) was added to the
wells (30 min., room temperature), the color reagent TMB was used
and the plates read at OD 630 nm.
[1051] Individual phage isolates binding to immobilized BLyS were
sequenced and the sequences analyzed. The unique sequences of the
BAML BLyS-binding 14-mer display peptides are shown in Table
13.
[1052] Analysis of the peptides reveals a significant sequence
"collapse" around one motif: W.sub.3YDPLTKLWL.sub.12 (SEQ ID NO:
436) (subscripts indicate amino acid position in the 14-mer display
peptide sequence). This most numerous core motif includes the four
fixed residues from the original BAML template, i.e., Asp (D) at
position 5, Leu (L) at position 7, Thr (T) at position 8, and Leu
(L) at position 10. In addition, 5 of the 6 preferred residues from
the original BAML template sequence were included in this motif
(see Table 10).
[1053] 73% (143 of 197) of the round 4 isolates included this core
motif (SEQ ID NO: 436). Single residue substitutions within the
10-mer core motif centered on positions 4 (Y.fwdarw.F) and 12
(L.fwdarw.F, I, or V), with the substitutions at position 12 being
alternative hydrophobic residues for Leu.
[1054] For the three remaining variable positions (i.e., 2, 13, and
14), selection was not as stringent, although some preferences were
apparent, being either built into the library or persisting through
rounds of selection. For example, in round 4 isolates, 51 %
included Asn at position 2; 77% included Pro at position 13; and
32% included Asp at position 14. The presence of Val (27%) or Glu
(19%) at position 14 was among the most highly selected in the
round 4 isolates, in comparison to their theoretical proportion (4%
each) at position 14 in BAML.
[1055] The sequences in Table 13 are grouped according to their
degree of difference from the core sequence (SEQ ID NO: 436).
13TABLE 13 Sequences of BAML Phage Isolates (from Rounds 2, 3, 4)
SEQ ID NO: 14-mer amino acid position consensus; 184 1 2 3 4 5 6 7
8 9 10 11 12 13 14 A n w y D s L T k L w 1 p d A N W Y D P L T K L
W L P D 186 A N W Y D P L T K L W L P E 187 A N W Y D P L T K L W L
P G 188 A N W Y D P L T K L W L P V 189 A N W Y D P L T K L W L S D
190 A N W Y D P L T K L W L N D 191 A N W Y D P L T K L W L P T 192
A N W Y D P L T K L W L P A 193 A N W Y D P L T K L W L P N 194 A N
W Y D P L T K L W L V D 195 A N W Y D P L T K L W L H D 196 A N W Y
D P L T K L W L T D 197 A N W Y D P L T K L W L P H 198 A N W Y D P
L T K L W L T V 199 A N W Y D P L T K L W L L D 200 A N W Y D P L T
K L W L L E 201 A N W Y D P L T K L W L H E 202 A N W Y D P L T K L
W L P R 203 A N W Y D P L T K L W L A D 204 A N W Y D P L T K L W L
P Y 205 A N W Y D P L T K L W L P I 206 A N W Y D P L T K L W L I D
207 A N W Y D P L T K L W L R D 208 A Y W Y D P L T K L W L P D 209
A Y W Y D P L T K L W L L E 210 A Y W Y D P L T K L W L R V 211 A Y
W Y D P L T K L W L P E 212 A Y W Y D P L T K L W L P V 213 A Y W Y
D P L T K L W L H Q 214 A Y W Y D P L T K L W L P A 215 A Y W Y D P
L T K L W L R V 216 A Y W Y D P L T K L W L P G 217 A Y W Y D P L T
K L W L R Y 218 A Y W Y D P L T K L W L P Y 219 A Y W Y D P L T K L
W L L Y 220 A Y W Y D P L T K L W L R D 221 A Y W Y D P L T K L W L
P V 222 A Y W Y D P L T K L W L L G 223 A Y W Y D P L T K L W L T H
224 A Y W Y D P L T K L W L P T 225 A Y W Y D P L T K L W L L V 226
A Y W Y D P L T K L W L Y Y 227 A Y W Y D P L T K L W L S D 228 A S
W Y D P L T K L W L P A 229 A S W Y D P L T K L W L H D 230 A S W Y
D P L T K L W L P G 231 A S W Y D P L T K L W L P Q 232 A S W Y D P
L T K L W L P Y 233 A S W Y D P L T K L W L P H 234 A S W Y D P L T
K L W L P V 235 A S W Y D P L T K L W L P I 236 A S W Y D P L T K L
W L P E 237 A F W Y D P L T K L W L R V 238 A F W Y D P L T K L W L
P E 239 A F W Y D P L T K L W L L E 240 A F W Y D P L T K L W L P V
241 A I W Y D P L T K L W L P E 242 A I W Y D P L T K L W L P D 243
A I W Y D P L T K L W L H D 244 A I W Y D P L T K L W L T D 245 A I
W Y D P L T K L W L P F 246 A I W Y D P L T K L W L L D 247 A I W Y
D P L T K L W L P R 248 A I W Y D P L T K L W L P A 249 A I W Y D P
L T K L W L T A 250 A I W Y D P L T K L W L A V 251 A I W Y D P L T
K L W L P G 252 A I W Y D P L T K L W L R V 253 A I W Y D P L T K L
W L P H 254 A I W Y D P L T K L W L R E 255 A I W Y D P L T K L W L
S D 256 A T W Y D P L T K L W L P A 257 A T W Y D P L T K L W L A D
258 A T W Y D P L T K L W L T S 259 A T W Y D P L T K L W L P G 260
A T W Y D P L T K L W L P Y 261 A T W Y D P L T K L W L S G 262 A T
W Y D P L T K L W L P V 263 A T W Y D P L T K L W L P D 264 A D W Y
D P L T K L W L P V 265 A D W Y D P L T K L W L P K 266 A D W Y D P
L T K L W L P D 267 A D W Y D P L T K L W L P E 268 A D W Y D P L T
K L W L H Q 269 A E W Y D P L T K L W L R D 270 A E W Y D P L T K L
W L P D 271 A E W Y D P L T K L W L P Y 272 A L W Y D P L T K L W L
P A 273 A L W Y D P L T K L W L P D 274 A L W Y D P L T K L W L R G
275 A L W Y D P L T K L W L L G 276 A M W Y D P L T K L W L P A 277
A M W Y D P L T K L W L Q V 278 A M W Y D P L T K L W L L G 279 A A
W Y D P L T K L W L P D 280 A A W Y D P L T K L W L A D 281 A A W Y
D P L T K L W L L D 282 A H W Y D P L T K L W L T D 283 A H W Y D P
L T K L W L P V 284 A H W Y D P L T K L W L H D 285 A H W Y D P L T
K L W L P D 286 A P W Y D P L T K L W L H D 287 A P W Y D P L T K L
W L P V 288 A Q W Y D P L T K L W L P E 289 A Q W Y D P L T K L W L
P Y 290 A Q W Y D P L T K L W L P R 291 A K W Y D P L T K L W L P D
292 A K W Y D P L T K L W L P V 293 A K W Y D P L T K L W L P V 294
A K W Y D P L T K L W L N G 295 A W W Y D P L T K L W L P A 296 A V
W Y D P L T K L W L T D 297 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A N W
Y D P L T K L W L P D 186 A Y E Y D P L T K L W L L Y 298 A T K Y D
P L T K L W L P D 299 A T L Y D P L T K L W L P G 300 A I R Y D P L
T K L W L P Y 301 A E R Y D P L T K L W L P H 302 A D R Y D P L T K
L W L P Q 303 A N S Y D P L T K L W L P E 304 A I L Y D P L T K L W
L P D 305 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A N W Y D P L T K L W L
P D 186 A N W F D P L T K L W L P Q 306 A N W F D P L T K L W L P V
307 A N W F D P L T K L W L T D 308 A N W F D P L T K L W L P D 309
A N W F D P L T K L W L P G 310 A N W F D P L T K L W L P E 311 A N
W F D P L T K L W L P A 312 A N W F D P L T K L W L P N 313 A N W F
D P L T K L W L S E 314 A N W F D P L T K L W L H D 315 A N W F D P
L T K L W L V D 316 A Y W F D P L T K L W L P D 317 A Y W F D P L T
K L W L P V 318 A Y W F D P L T K L W L P A 319 A Q W F D P L T K L
W L P D 320 A H W F D P L T K L W L P D 321 A T W F D P L T K L W L
P V 322 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A N W Y D P L T K L W L P
D 186 A Y W Y D S L T K L W L P V 323 A Y W Y D S L T K L W L H D
324 A N W Y D S L T K L W L P D 325 A N W Y D S L T K L W L P V 326
A N W Y D S L T K L W L P D 327 A N W Y D S L T K L W L A D 328 A N
W Y D S L T K L W L P A 329 A N W Y D S L T K L W L Y E 330 1 2 3 4
5 6 7 8 9 10 11 12 13 14 A N W Y D P L T K L W L P D 186 A G W Y D
S L T K L W L P D 331 A V W Y D S L T K L W L T D 332 A N W Y D A L
T K L W L P V 333 A Y W Y D T L T K L W L P N 334 1 2 3 4 5 6 7 8 9
10 11 12 13 14 A N W Y D P L T K L W L P D 186 A F W Y D P L T N L
W L L E 335 A Y W Y D P L T G L W L L V 336 A Y W Y D P L T G L W L
L Y 337 A Y W Y D P L T G L W L R V 338 A Y W Y D P L T E L W L R L
339 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A N W Y D P L T K L W L P D
186 A M W Y D P L T K L S L P D 340 A Y W Y D P L T K L S L L V 341
A I W Y D P L T K L S L T V 342 A I W Y D P L T K L S L L V 343 A D
W Y D P L T K L S L L L 344 A Y W Y D P L T K L R L L E 345 A D W Y
D P L T K L R L L V 346 A D W Y D P L T K L R L I V 347 A I W Y D P
L T K L Y L P D 348 A I W Y D P L T K L G L L V 349 A N W Y D P L T
K L T L L V 350 A N W Y D P L T K L L L P N 351 1 2 3 4 5 6 7 8 9
10 11 12 13 14 A N W Y D P L T K L W L P D 186 A S W Y D P L T K L
W L P D 352 A N W Y D P L T K L W F P D 353 A N W Y D P L T K L W F
S D 354 A S W Y D P L T K L W F P V 355 A D W Y D P L T K L W F P V
356 A S W Y D P L T K L W F P K 357 A K W Y D P L T K L W F P D 358
A S W Y D P L T K L W F L E 359 A N W Y D P L T K L W F P A 360 A T
W Y D P L T K L W F P D 361 A I W Y D P L T K L W F P E 362 A I W Y
D P L T K L W F P D 363 A I W Y D P L T K L W F P G 364 A Y W Y D P
L T K L W F P H 365 A N W Y D P L T K L W F P V 366 A Y W Y D P L T
K L W F P D 367 A G W Y D P L T K L W F P D 368 A I W Y D P L T K L
W F P T 369 A K W Y D P L T K L W F P A 370 A Y W Y D P L T K L W F
F D 371 A N W Y D P L T K L W F A D 372 1 2 3 4 5 6 7 8 9 10 11 12
13 14 A N W Y D P L T K L W L P D 186 A N W Y D P L T K L W F P Y
373 A D W Y D P L T K L W F R D 374 A N W Y D P L T K L W V P D 375
A D W Y D P L T K L W V P A 376 A N W Y D P L T K L W V P N 377 A N
W Y D P L T K L W V P E 378 A N W Y D P L T K L W V P Q 379 A E W Y
D P L T K L W V P K 380 A Q W Y D P L T K L W V P V 381 A N W Y D P
L T K L W V P Y 382 A L W Y D P L T K L W V P Y 383 A N W Y D P L T
K L W V P G 384 A S W Y D P L T K L W I P Y 385 A D W Y D P L T K L
W I P G 386 A N W Y D P L T K L W I P Y 387 A K W Y D P L T K L W I
P Y 388 A I W Y D P L T K L W I P N 389 A T W Y D P L T K L W I P Q
390 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A N W Y D P L T K L W L P D
186 A S W Y D P L T N L W V P D 391 A Y E Y D P L T N L W L L Y 392
A Y W Y D P L T N L S L L V 393 A Y W Y D P L T K L S I L E 394 A N
W Y D S L T K L W I P Y 395 A H W F D P L T Q L K I R V 396 A Y W C
D P L T K L C I L E 397 A N S Y D P L T K L W F P Y 398 A N L Y D P
L T K L W V P Y 399 A N W Y D A L T K L W L H D 400 A N W Y D S L T
K L W F P D 401 A T S Y D S L T K L W L P A 402 A C W Y D S L T K L
C H R E 403 A I G N D P L T K L W I P Y 404 A N W Q D C L T K L C L
A G 405 A Y W F D P L T N L W L L E 406 A Y W Y D P L T N L S L L V
407 A N C F D S L T R L W L C D 408 A C A Y D A L T K L C L P A 409
A N W Y D P L T N L S L L L 410 A Y W Y D P L T Q L S L L V 411 A Y
R Y D A L T G L W L L Y 412 A Y W N D P L T K L K L R L 413 A Y W Y
D P L T Q L S L L V 414 A Y R Y D A L T G L W L L Y 415 A Y R Y D S
L T N L W L L Y 416 A Y W Y D P L T K L S I L E 417 A S C Y D P L T
K L C F P V 418 A F W F D P L T G L W L L E 419 1 2 3 4 5 6 7 8 9
10 11 12 13 14 A N W Y D P L T K L W L P D 186 A H W Y D P L T K L
S I R V 420 A P W Y D S L T K L W F P S 421 A N C Y D T L T K L W L
T C 422 A N W Y D S L T K L S L P D 423 A Y A Y D F L T Q L S L P D
424 A F R Y D S L T G L W L R Y 425 A N C Y D S L T K L W L P C 426
A N G Y D L L T N L S V S D 427 A N W Y D P L T R L W I P V 428 A L
K F D Y L T K L W L P D 429 A Y R Y D S L T K L W L P G 430 A Y C Y
D S L T K L W I P D 431 A S W E D S L T K L W L S K 432 A Y W Y D S
L T G L S L L V 433 A Y W Y D P L T Y L R L R V 434 A K C Y D S L T
N L W L C D 435
[1056] Nearly all of the ELISA signals of the BAML isolates were
higher than those isolated in the initial screen (see Example 1).
For comparison, peptide 453-01-B07 (SEQ ID NO: 31) (K.sub.D=700 nM)
was used as a reference (positive control). Negative control MAEX
(M13 phage with no insert) did not bind b-BLyS at any concentration
tested.
[1057] For direct phage ELISA, the signal measured is a reflection
of the ability of a set number of phage to bind to various
concentrations of b-BLyS. Peptides tested by the direct phage ELISA
assay were chosen based on high affinity for BLyS as determined in
the indirect phage ELISA assay. For this assay, Immulon-2HB plates
were coated with 0 or 1000 ng anti-Fd antibody (Sigma, St. Louis,
Mo.). After washing (PBS-Tween-20), phage dilutions were added to
saturate the available antibody and incubated for 1 hour, washed,
then incubated with 100 .mu.l of 10-fold dilutions of b-BLyS (0-1
.mu.g/ml) for 1 hour at room temperature. Streptavidin-HRP (1:1000
in PBS-tween-20; Endogen, Woburn, Mass.) was added to the wells and
incubated for 1 hour, developed using TMB and reading at OD 630
nm.
[1058] Determination of BAML Peptide K.sub.D by Fluoresence
Anisotropy
[1059] Several peptides containing the 10-mer core structural motif
or single-position variants of that motif identified by sequence
analysis were synthesized with a short Gly-Gly-Lys linker sequence
and the C-terminal lysine was labeled with fluorescein. These
peptides, shown in Table 14, below, were synthesized by solid phase
synthesis for determination of dissociation constant with respect
to BLyS. The DX815 and DX876 polypeptides were derived from DX814
(SEQ ID NO: 186) by deletion of two N-terminal amino acids or the
two amino acids N-terminal and C-terminal to the core peptide at
(positions 3-12). DX816, DX817, DX819, and DX822 correspond to
other BAML isolates (SEQ ID NOs:189, 309, 353, 327, respectively).
DX818 corresponds to isolate SEQ ID NO: 340, except that Asn has
been substituted for Met at position 2. The K.sub.D of several BLyS
binding BAML peptides was determined by fluorescence anisotropy,
performed as previously described. The sequence of DX822 without
the--GGK linker (see SEQ ID NO: 327) matches the BAML template
sequence (see Table 10). The BAML consensus sequence found in DX822
resulted in a more than 10-fold improvement in binding affinity for
BLyS, as compared to one of the highest affinity binders isolated
in the initial screen (453-01-B07, SEQ ID NO: 31).
14TABLE 14 Dissociation Constants of Synthetic BLyS-binding
Polypeptides SEQ Peptide Sequence ID NO: K.sub.D (nM) DX814
Ac-ANWYDPLTKLWLPDGGK-fitc 437 26 .+-. 7 DX815
Ac-WYDPLTKLWLPDGGK-fitc 438 31 .+-. 13 DX876 Ac-WYDPLTKLWLGGK-fitc
439 171 .+-. 90 DX816 Ac-ANWYDPLTKLWLPVGGK-fitc 440 44 .+-. 15
DX817 Ac-ANWFDPLTKLWLPDGGK-fitc 441 32 .+-. 26 DX818
Ac-ANWYDPLTKLSLPDGGK-fitc 442 342 .+-. 108 DX819
Ac-ANWYDPLTKLWFPDGGK-fitc 443 69 .+-. 38 DX822
Ac-ANWYDSLTKLWLPDGGK-fitc 444 79 .+-. 54
[1060] Analysis of the BAML isolates revealed a lack of sequence
conservation at position 2 (varied in the BAML template, see Table
10). To examine whether the N-terminal residues at positions 1 and
2 in the BAML sequence were necessary for binding to BLyS, a
truncated version of DX814 comprising only residues 3-14 (DX815;
see Table 14) was synthesized and analyzed by fluorescence
anisotropy. The K.sub.D for DX815 was indistinguishable from that
of DX814, suggesting that residues 1-2 are not required for high
affinity binding to BLyS. Further truncation of DX814 to the
minimal core (residues 1-10, DX876) increased the K.sub.D to 171
nM, indicating a contribution from Pro at position 13 and/or Asp at
position 14 of the 14-mer to high affinity BLyS binding.
Substitution of Val in DX816 at that position had little effect on
the K.sub.D (see Table 14). In comparing the BLyS-binding
polypeptide DX221 (Ac-WTDSLTGLWFPDGGPGPEGGGK; K.sub.D=3 .mu.M; SEQ
ID NO: 168) with the BAML peptide closest in sequence (DX819,
Ac-ANWYDPLTKLWFPDGGK; K.sub.D=69 nM; SEQ ID NO: 443), differences
are seen at three positions 4 (T.fwdarw.Y), 6 (S.fwdarw.P), and 9
(G.fwdarw.K), indicating the contribution of these residues in
binding affinity.
[1061] The synthesized BAML peptides exhibited K.sub.D values in
the low nanomolar range, two orders of magnitude lower than primary
isolate-derived peptides (see Example 1). Phenylalanine
substitutions (F.sub.4.fwdarw.Y.sub.4; F.sub.12.fwdarw.L.sub.12;
Table 14) were the most common minor variations to the core
sequence and these changes failed to significantly affect the
dissociation constants of the synthesized peptides. A change at
position 11 (W.sub.11.fwdarw.S.sub.11; DX818), however, resulted in
an approximately 10-fold decrease in affinity compared to
DX814.
[1062] Following the foregoing description, the characteristics
important for using various ;) affinity binding polypeptides for
targeting of BLyS or BLyS-like polypeptides (BLyS target protein)
in vitro or in vivo can be appreciated. Additional binding
polypeptide uses of the invention and alternative methods adapted
to a particular use will be evident from studying the foregoing
description. For instance, any spacer or linker sequences
associated with BLyS binding polypeptides discussed above may be
removed or substituted to yield additional BLyS binding
polypeptides for use in the methods of this invention. All such
embodiments and obvious alternatives are intended to be within the
scope of this invention, as defined by the claims that follow.
[1063] Publications referred to above are hereby incorporated by
reference.
Sequence CWU 1
1
458 1 13 PRT Artificial Sequence BLyS binding polypeptide 1 Xaa Xaa
Xaa Cys Xaa Pro Xaa Thr Gly Cys Xaa Xaa Xaa 1 5 10 2 14 PRT
Artificial Sequence BLyS binding polypeptide 2 Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 3 15 PRT Artificial
Sequence BLyS binding polypeptide 3 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa 10 15 4 16 PRT Artificial Sequence BLyS binding polypeptide
4 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1
5 10 15 5 18 PRT Artificial Sequence BLyS binding polypeptide 5 Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10
15 Xaa Xaa 6 12 PRT Artificial Sequence BLyS binding polypeptide 6
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 7 13 PRT
Artificial Sequence BLyS binding polypeptide 7 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 8 7 PRT Artificial Sequence
BLyS binding polypeptide 8 Cys Xaa Pro Xaa Thr Gly Cys 1 5 9 8 PRT
Artificial Sequence BLyS binding polypeptide 9 Cys Xaa Xaa Xaa Xaa
Xaa Xaa Cys 1 5 10 9 PRT Artificial Sequence BLyS binding
polypeptide 10 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 11 10 PRT
Artificial Sequence BLyS binding polypeptide 11 Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys 1 5 10 12 12 PRT Artificial Sequence BLyS
binding polypeptide 12 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys 1 5 10 13 8 PRT Artificial Sequence c-terminal linker 13 Pro
Gly Pro Glu Gly Gly Gly Lys 1 5 14 12 PRT Artificial Sequence phage
display library template 14 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa 1 5 10 15 13 PRT Artificial Sequence phage display library
template 15 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5
10 16 14 PRT Artificial Sequence phage display library template 16
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 17
15 PRT Artificial Sequence phage display library template 17 Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15
18 16 PRT Artificial Sequence phage display library template 18 Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10
15 19 18 PRT Artificial Sequence phage display library template 19
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5
10 15 Xaa Xaa 20 12 PRT Artificial Sequence BLyS binding
polypeptide 20 His Leu Arg Cys Trp Ser Thr Asn Cys Arg Tyr Asp 1 5
10 21 12 PRT Artificial Sequence BLyS binding polypeptide 21 Val
Met Asp Cys Leu Ile Asn Arg Cys Asp Thr Val 1 5 10 22 13 PRT
Artificial Sequence BLyS binding polypeptide 22 Lys Ser Lys Cys Phe
Phe Pro Trp Glu Cys Gln Gln Ala 1 5 10 23 13 PRT Artificial
Sequence BLyS binding polypeptide 23 Ala Met Lys Cys Tyr Phe Pro
Trp Glu Cys Ala Asn Gly 1 5 10 24 14 PRT Artificial Sequence BLyS
binding polypeptide 24 Glu Asn Val Ala Cys Tyr Phe Pro Trp Glu Cys
His His Pro 1 5 10 25 13 PRT Artificial Sequence BLyS binding
polypeptide 25 Asn Ala Pro Cys Tyr Phe Pro Trp Glu Cys Phe Ser Ile
1 5 10 26 13 PRT Artificial Sequence BLyS binding polypeptide 26
Ser Val Asn Cys Trp Phe Pro Trp Glu Cys Val Gly Asn 1 5 10 27 13
PRT Artificial Sequence BLyS binding polypeptide 27 Lys Glu Pro Cys
Tyr Phe Tyr Trp Glu Cys Ala Val Ser 1 5 10 28 14 PRT Artificial
Sequence BLyS binding polypeptide 28 Asp Thr Asn Cys Asp Leu Leu
Thr Lys Met Cys Gly Pro Gln 1 5 10 29 14 PRT Artificial Sequence
BLyS binding polypeptide 29 Gly Thr Pro Cys Asp Leu Leu Thr Lys Leu
Cys Leu Leu Trp 1 5 10 30 14 PRT Artificial Sequence BLyS binding
polypeptide 30 Met Ser Glu Cys Asp Leu Leu Thr Lys Ile Cys Leu Met
Gly 1 5 10 31 14 PRT Artificial Sequence BLyS binding polypeptide
31 Val Pro Phe Cys Asp Leu Leu Thr Lys His Cys Phe Glu Ala 1 5 10
32 14 PRT Artificial Sequence BLyS binding polypeptide 32 Val Pro
Phe Cys Asp Leu Leu Thr Lys His Cys Phe Glu Ala 1 5 10 33 14 PRT
Artificial Sequence BLyS binding polypeptide 33 Trp Ser Ala Cys Asp
Leu Leu Thr Lys Gln Cys Val Gln Val 1 5 10 34 13 PRT Artificial
Sequence BLyS binding polypeptide 34 Asp Gly Cys Asp Glu Leu Thr
Lys Ile Cys Gly Met Lys 1 5 10 35 14 PRT Artificial Sequence BLyS
binding polypeptide 35 Lys Ser Trp Cys Asp Glu Leu Thr Lys Val Cys
Phe Asp Pro 1 5 10 36 14 PRT Artificial Sequence BLyS binding
polypeptide 36 Lys Trp Met Cys Asp Glu Leu Thr Lys Gln Cys Gln Tyr
Val 1 5 10 37 14 PRT Artificial Sequence BLyS binding polypeptide
37 Met Lys Tyr Cys Asp Glu Leu Thr Lys Ile Cys Val Gly Trp 1 5 10
38 14 PRT Artificial Sequence BLyS binding polypeptide 38 Tyr Phe
Gln Cys Asp Glu Leu Thr Lys Met Cys Trp Gln Lys 1 5 10 39 14 PRT
Artificial Sequence BLyS binding polypeptide 39 Ala Met His Cys Asp
Lys Leu Thr Lys His Cys Lys Phe His 1 5 10 40 13 PRT Artificial
Sequence BLyS binding polypeptide 40 Val Pro Tyr Cys Asp Lys Leu
Thr Lys Ile Cys Gln Trp 1 5 10 41 14 PRT Artificial Sequence BLyS
binding polypeptide 41 Glu Val Phe Cys Asp Val Leu Thr Lys Val Cys
Phe His Asp 1 5 10 42 14 PRT Artificial Sequence BLyS binding
polypeptide 42 Lys Pro Lys Cys Asp Val Leu Thr Lys Met Cys Asp Trp
Leu 1 5 10 43 14 PRT Artificial Sequence BLyS binding polypeptide
43 Thr Gln His Cys Asp Val Leu Thr Lys Gln Cys Phe Thr Ile 1 5 10
44 14 PRT Artificial Sequence BLyS binding polypeptide 44 Gly His
Phe Cys Asp Arg Leu Thr Lys Tyr Cys Phe Glu Pro 1 5 10 45 14 PRT
Artificial Sequence BLyS binding polypeptide 45 His Ile Gln Cys Asp
Arg Leu Thr Lys Ser Cys Leu Ser Val 1 5 10 46 14 PRT Artificial
Sequence BLyS binding polypeptide 46 Ile Lys Ala Cys Asp Ile Leu
Thr Lys Val Cys Trp Pro Pro 1 5 10 47 14 PRT Artificial Sequence
BLyS binding polypeptide 47 Gln Phe Asp Cys Asp Pro Leu Thr Lys Tyr
Cys Gly Glu Phe 1 5 10 48 14 PRT Artificial Sequence BLyS binding
polypeptide 48 Lys Met Tyr Cys Asp His Leu Thr Gly Tyr Cys Trp Pro
Glu 1 5 10 49 14 PRT Artificial Sequence BLyS binding polypeptide
49 Met Gln Ser Cys Asp Ile Leu Thr Gly Tyr Cys Phe Lys Arg 1 5 10
50 14 PRT Artificial Sequence BLyS binding polypeptide 50 Gly Pro
Trp Cys Asp Ile Leu Thr Gly Phe Cys Leu Ala Gln 1 5 10 51 14 PRT
Artificial Sequence BLyS binding polypeptide 51 Ser Val Arg Cys Asp
Leu Leu Thr Gly Trp Cys Pro Val Trp 1 5 10 52 14 PRT Artificial
Sequence BLyS binding polypeptide 52 Pro Ala Asp Cys Asp Pro Leu
Thr Asn Ile Cys Phe Trp Lys 1 5 10 53 14 PRT Artificial Sequence
BLyS binding polypeptide 53 Thr Asn Val Cys Asp Pro Leu Thr Asn Val
Cys Phe Met Asn 1 5 10 54 14 PRT Artificial Sequence BLyS binding
polypeptide 54 Glu His Trp Cys Asp Asp Leu Thr His Leu Cys Phe Arg
Leu 1 5 10 55 14 PRT Artificial Sequence BLyS binding polypeptide
55 Gly Tyr Trp Cys Asp Val Leu Thr Asn Asn Cys Trp Lys Ile 1 5 10
56 14 PRT Artificial Sequence BLyS binding polypeptide 56 Leu Tyr
Asn Cys Asp Tyr Leu Thr Arg Leu Cys Phe Glu Pro 1 5 10 57 14 PRT
Artificial Sequence BLyS binding polypeptide 57 His Val Asp Cys Leu
Leu His Pro Lys Ala Cys Tyr Lys Tyr 1 5 10 58 14 PRT Artificial
Sequence BLyS binding polypeptide 58 Val Gln Asp Cys Leu Leu His
Pro Lys Ala Cys Gln Met Gln 1 5 10 59 14 PRT Artificial Sequence
BLyS binding polypeptide 59 Lys Phe Asp Cys Leu Leu Lys Pro Met Phe
Cys Ser Asn His 1 5 10 60 14 PRT Artificial Sequence BLyS binding
polypeptide 60 Phe Ala Asp Cys Leu Ile His Pro Lys Ser Cys Lys Pro
Leu 1 5 10 61 14 PRT Artificial Sequence BLyS binding polypeptide
61 His Gly Asn Cys Tyr Pro Phe Pro Trp Glu Cys Glu Ser Lys 1 5 10
62 14 PRT Artificial Sequence BLyS binding polypeptide 62 Met Ile
Ile Val Leu Leu Leu Leu Arg Phe Ala Ile Ser Arg 1 5 10 63 14 PRT
Artificial Sequence BLyS binding polypeptide 63 Ser Leu Leu Val Ile
Phe Leu Leu Ile Gly Ala Gly Ser Leu 1 5 10 64 15 PRT Artificial
Sequence BLyS binding polypeptide 64 Phe His Pro Cys Asp Met Leu
Thr Gly Ile Trp Cys Gln Pro Asn 1 5 10 15 65 15 PRT Artificial
Sequence BLyS binding polypeptide 65 Ser Lys Arg Cys Asp Leu Leu
Thr Lys Met Trp Cys Glu Thr Glu 1 5 10 15 66 15 PRT Artificial
Sequence BLyS binding polypeptide 66 Thr Lys Phe Cys Asp Arg Leu
Thr Met Pro Lys Cys Val Trp Lys 1 5 10 15 67 15 PRT Artificial
Sequence BLyS binding polypeptide 67 Asn Thr Phe Cys Pro Asp Pro
Leu Thr Gly Arg Cys Val Asn Pro 1 5 10 15 68 15 PRT Artificial
Sequence BLyS binding polypeptide 68 Asp Trp Thr Cys Asp Pro Leu
Phe His Arg Glu Cys Ile Phe Glu 1 5 10 15 69 15 PRT Artificial
Sequence BLyS binding polypeptide 69 Pro Gln Pro Cys Asp Leu Leu
Phe Glu Lys Lys Cys Ser Ile Lys 1 5 10 15 70 15 PRT Artificial
Sequence BLyS binding polypeptide 70 Arg Trp His Cys Asp Met Leu
Ile Asn Pro Ser Cys Leu Pro Asp 1 5 10 15 71 15 PRT Artificial
Sequence BLyS binding polypeptide 71 Lys Ile Gln Cys Asp Ile Val
Asn Leu Ser Ser Cys Val Tyr Pro 1 5 10 15 72 15 PRT Artificial
Sequence BLyS binding polypeptide 72 Leu Asn Ala Cys Asp Ile Val
His Pro Asn Tyr Cys Ser Gly Met 1 5 10 15 73 15 PRT Artificial
Sequence BLyS binding polypeptide 73 Ala Lys Ala Cys Ser Ile Val
Asn Leu Glu Ser Cys Glu Tyr Leu 1 5 10 15 74 15 PRT Artificial
Sequence BLyS binding polypeptide 74 Arg Gln Ala Cys Ser Ile Ile
Thr Pro Trp Gly Cys Pro Ile Pro 1 5 10 15 75 15 PRT Artificial
Sequence BLyS binding polypeptide 75 Ala Asp Asn Cys Thr Val Ala
Thr Leu Asp Phe Cys Tyr Trp Thr 1 5 10 15 76 15 PRT Artificial
Sequence BLyS binding polypeptide 76 Lys Pro Glu Cys Asn Ile Thr
Lys Pro Gln Phe Cys Phe Gly Glu 1 5 10 15 77 15 PRT Artificial
Sequence BLyS binding polypeptide 77 Asn Asn Cys Gln Trp Asp Glu
Leu Thr Ser Met Cys Asp Pro Phe 1 5 10 15 78 16 PRT Artificial
Sequence BLyS binding polypeptide 78 Ser Arg Leu Cys His Met Asp
Glu Leu Thr His Val Cys Val His Phe 1 5 10 15 79 16 PRT Artificial
Sequence BLyS binding polypeptide 79 Ser Arg Pro Cys Gln Ile Asp
Glu Leu Thr Lys Ala Cys Phe Tyr Asn 1 5 10 15 80 16 PRT Artificial
Sequence BLyS binding polypeptide 80 Asp Arg Val Cys Lys Leu Asp
Phe Leu Thr Tyr Asn Cys Leu Asn His 1 5 10 15 81 16 PRT Artificial
Sequence BLyS binding polypeptide 81 His Ser Asn Cys Ile Met Asp
Leu Leu Thr Asn Arg Cys Phe Tyr Asp 1 5 10 15 82 16 PRT Artificial
Sequence BLyS binding polypeptide 82 Pro Phe Asn Cys Phe His Asp
Pro Leu Thr Gly Leu Cys Leu His Ser 1 5 10 15 83 16 PRT Artificial
Sequence BLyS binding polypeptide 83 Tyr Asp Ser Cys Thr Tyr Asp
Arg Leu Thr Lys Gln Cys Tyr Pro Ser 1 5 10 15 84 16 PRT Artificial
Sequence BLyS binding polypeptide 84 Phe His Asp Cys Met Tyr Asp
Ala Leu Leu Gly Tyr Cys Leu Pro Tyr 1 5 10 15 85 15 PRT Artificial
Sequence BLyS binding polypeptide 85 Asn Arg Ser Cys Asp Pro Leu
Thr Arg Pro Lys Ser Cys Gly Leu 1 5 10 15 86 16 PRT Artificial
Sequence BLyS binding polypeptide 86 Leu Ser Asn Cys Asp Trp Asp
Asp Leu Ile Arg Gln Cys Leu His Asp 1 5 10 15 87 16 PRT Artificial
Sequence BLyS binding polypeptide 87 Phe Trp Asp Cys Leu Phe His
Pro Asn Ser Arg Tyr Cys Val Leu Ser 1 5 10 15 88 16 PRT Artificial
Sequence BLyS binding polypeptide 88 Ser Arg Asp Cys Leu Leu Ser
Pro Ala Met Ala Trp Cys Gly Leu Asp 1 5 10 15 89 18 PRT Artificial
Sequence BLyS binding polypeptide 89 Gly Gly Asn Cys Tyr Thr Asp
Ser Leu Thr Lys Leu His Phe Cys Met 1 5 10 15 Gly Asp 90 16 PRT
Artificial Sequence BLyS binding polypeptide 90 Met Cys Pro Arg Asp
Pro Leu Thr Lys Ala Lys Leu Cys Asn Trp His 1 5 10 15 91 18 PRT
Artificial Sequence BLyS binding polypeptide 91 Pro Asn Gln Cys Gln
Asp Asp Leu Thr Lys Gln Trp Tyr Ser Cys His 1 5 10 15 Tyr His 92 18
PRT Artificial Sequence BLyS binding polypeptide 92 Phe Asp Met Cys
Phe Asp Ala Leu Thr Lys Gln Asn Phe Tyr Cys Arg 1 5 10 15 Phe His
93 18 PRT Artificial Sequence BLyS binding polypeptide 93 Arg Asn
Met Cys Val Asp Arg Leu Thr Lys Leu Gln His Gly Cys Glu 1 5 10 15
Gly Ala 94 18 PRT Artificial Sequence BLyS binding polypeptide 94
Asp Pro Glu Cys Leu Thr Ser Phe Asp Arg Leu Thr Lys Met Cys Trp 1 5
10 15 Pro Trp 95 18 PRT Artificial Sequence BLyS binding
polypeptide 95 Asp Asp Glu Cys His Tyr Asp Tyr Leu Thr His Tyr Met
Arg Cys Asp 1 5 10 15 Tyr Arg 96 18 PRT Artificial Sequence BLyS
binding polypeptide 96 Phe Gly Gly Cys Asn Ile Asp Leu Leu Thr Asn
Thr Met Met Cys His 1 5 10 15 Arg Asn 97 18 PRT Artificial Sequence
BLyS binding polypeptide 97 His Gly Pro Cys Tyr Trp Asp Glu Leu Thr
Met Gln Trp His Cys Asn 1 5 10 15 His His 98 18 PRT Artificial
Sequence BLyS binding polypeptide 98 Gly Ala Met Cys Val Asp Leu
Leu Thr Tyr Thr Phe Arg Pro Cys Met 1 5 10 15 Tyr Ala 99 18 PRT
Artificial Sequence BLyS binding polypeptide 99 Ser Asn Lys Cys Trp
Asp Glu Leu Thr His Ala Trp Ala Glu Cys Gly 1 5 10 15 Arg Phe 100
18 PRT Artificial Sequence BLyS binding polypeptide 100 Arg Pro Val
Cys Tyr Lys Gly Tyr Asp Ile Leu Thr Thr Gln Cys Met 1 5 10 15 Pro
Trp 101 18 PRT Artificial Sequence BLyS binding polypeptide 101 Pro
Ser Arg Cys Trp Phe Asp Leu Leu Phe Asn Lys Phe Val Cys Lys 1 5 10
15 Arg Asn 102 18 PRT Artificial Sequence BLyS binding polypeptide
102 Arg Ser Gly Cys Val Tyr Asp Met Leu Leu Met Thr Met Tyr Cys Pro
1 5 10 15 Ser Asn 103 18 PRT Artificial Sequence BLyS binding
polypeptide 103 Ser Asn Arg Cys Glu Gly Asp Gln Leu Met Arg Pro Pro
Ser Cys Arg 1 5 10 15 His Leu 104 18 PRT Artificial Sequence BLyS
binding polypeptide 104 Tyr Arg Met Cys Trp Trp Asp Asp Leu Leu Arg
Gly Phe Val Cys Asp 1 5 10 15 Phe His 105 18 PRT Artificial
Sequence BLyS binding polypeptide 105 His Asp Gly Cys Tyr Asp Glu
Leu Leu Tyr Arg Trp Thr Arg Cys Glu 1 5 10 15 His Arg 106 18 PRT
Artificial Sequence BLyS binding polypeptide 106 Trp Ala Trp Cys
Phe Asp Glu Leu Val Gln Arg Tyr Phe Thr Cys Phe 1 5 10 15 Asp His
107 18 PRT Artificial Sequence BLyS binding polypeptide 107 Leu Pro
Glu Cys Arg Gln Tyr Phe Pro Trp Glu Lys Gln Val Cys Ser 1 5 10 15
Tyr Trp 108 12 PRT Artificial Sequence BLyS binding polypeptide 108
Val His Tyr Asp Ser Leu Thr Lys Met Trp Thr Arg 1 5 10 109 12 PRT
Artificial Sequence BLyS binding polypeptide 109 Phe Thr Asp Pro
Leu Thr Lys Met Ser Leu His Ser 1 5 10 110 12 PRT Artificial
Sequence BLyS binding polypeptide 110 Gly Tyr Asp Val Leu Thr
Lys Leu Tyr Phe Val Pro 1 5 10 111 12 PRT Artificial Sequence BLyS
binding polypeptide 111 Tyr Tyr Asp Arg Leu Thr Lys Leu Tyr Ser Ser
Met 1 5 10 112 12 PRT Artificial Sequence BLyS binding polypeptide
112 Leu Xaa Lys Asp Pro Leu Thr Lys Leu Tyr Ile Ser 1 5 10 113 12
PRT Artificial Sequence BLyS binding polypeptide 113 Gly Tyr Asp
Val Leu Thr Lys Leu Xaa Phe Val Pro 1 5 10 114 12 PRT Artificial
Sequence BLyS binding polypeptide 114 Arg Leu Tyr Asp Pro Leu Thr
Lys Leu Val Leu Ser 1 5 10 115 12 PRT Artificial Sequence BLyS
binding polypeptide 115 Met Phe Asp Pro Leu Thr Lys Ile Ala Phe Pro
Ala 1 5 10 116 12 PRT Artificial Sequence BLyS binding polypeptide
116 Phe Tyr Asp Ser Leu Thr Lys Thr Asn Leu Arg Asp 1 5 10 117 12
PRT Artificial Sequence BLyS binding polypeptide 117 Gly Ile Tyr
Asp Lys Leu Thr Arg Ala Trp Leu Pro 1 5 10 118 12 PRT Artificial
Sequence BLyS binding polypeptide 118 Lys Tyr Asp Pro Leu Thr Arg
Ala Arg Xaa Pro Leu 1 5 10 119 12 PRT Artificial Sequence BLyS
binding polypeptide 119 Tyr Ile Asp Gln Leu Thr Arg Leu Ser Leu Pro
Ser 1 5 10 120 12 PRT Artificial Sequence BLyS binding polypeptide
120 His Gln Thr Phe Asp Ile Leu Thr Arg Leu His Phe 1 5 10 121 12
PRT Artificial Sequence BLyS binding polypeptide 121 Trp Gln Phe
Asp Val Leu Thr Arg Ser Trp Thr Pro 1 5 10 122 12 PRT Artificial
Sequence BLyS binding polypeptide 122 Gly Ala Ala Tyr Asp His Leu
Thr Arg Thr Trp Leu 1 5 10 123 12 PRT Artificial Sequence BLyS
binding polypeptide 123 Tyr Phe Asp Gln Leu Thr His Leu Ser Ile Lys
Lys 1 5 10 124 12 PRT Artificial Sequence BLyS binding polypeptide
124 Ala Trp Asp Pro Leu Thr Met Leu Val Leu Pro Trp 1 5 10 125 12
PRT Artificial Sequence BLyS binding polypeptide 125 Ala Leu Trp
Met Asp Pro Leu Thr Gly Leu Ala Phe 1 5 10 126 12 PRT Artificial
Sequence BLyS binding polypeptide 126 Trp Gln Phe Asp Val Leu Thr
Xaa Ser Trp Thr Pro 1 5 10 127 12 PRT Artificial Sequence BLyS
binding polypeptide 127 Trp Thr Asp Pro Leu Thr His Met Glu Ile Tyr
His 1 5 10 128 12 PRT Artificial Sequence BLyS binding polypeptide
128 Trp Thr Asp Ser Leu Thr Gly Leu Trp Phe Pro Asp 1 5 10 129 12
PRT Artificial Sequence BLyS binding polypeptide 129 Tyr Thr Asp
Pro Leu Thr Gly Ile Val Xaa Pro Phe 1 5 10 130 12 PRT Artificial
Sequence BLyS binding polypeptide 130 Tyr Trp Asp Lys Leu Thr Met
Leu His Leu Gly Val 1 5 10 131 12 PRT Artificial Sequence BLyS
binding polypeptide 131 Tyr Tyr Asp Phe Leu Thr Arg Thr Val Leu Pro
Ser 1 5 10 132 12 PRT Artificial Sequence BLyS binding polypeptide
132 Arg Leu Asp Pro Leu Ser Lys Asn Asp Phe Pro Arg 1 5 10 133 12
PRT Artificial Sequence BLyS binding polypeptide 133 Leu Arg Tyr
Asp Pro Leu Leu Lys Ser Xaa Ile Tyr 1 5 10 134 12 PRT Artificial
Sequence BLyS binding polypeptide 134 Leu Arg Tyr Asp Pro Leu Leu
Lys Ser Tyr Ile Tyr 1 5 10 135 12 PRT Artificial Sequence BLyS
binding polypeptide 135 Tyr Phe Asp Gln Phe Thr His Leu Ser Ile Lys
Lys 1 5 10 136 12 PRT Artificial Sequence BLyS binding polypeptide
136 Tyr Phe Asp Gln Xaa Thr His Leu Ser Ile Lys Lys 1 5 10 137 13
PRT Artificial Sequence BLyS binding polypeptide 137 Glu His Tyr
Tyr Thr Asp Pro Leu Thr Gly Ala Arg Ile 1 5 10 138 13 PRT
Artificial Sequence BLyS binding polypeptide 138 Glu His Tyr Xaa
Thr Asp Pro Leu Thr Gly Ala Arg Ile 1 5 10 139 13 PRT Artificial
Sequence BLyS binding polypeptide 139 Glu His Tyr Ser Thr Asp Pro
Leu Thr Gly Ala Arg Ile 1 5 10 140 13 PRT Artificial Sequence BLyS
binding polypeptide 140 Glu His Tyr Tyr Thr Asp Pro Leu Xaa Gly Xaa
Arg Ile 1 5 10 141 13 PRT Artificial Sequence BLyS binding
polypeptide 141 Glu His Tyr Tyr Thr Asp Pro Leu Xaa Gly Xaa Arg Xaa
1 5 10 142 13 PRT Artificial Sequence BLyS binding polypeptide 142
Glu His Tyr Tyr Thr Asp Pro Leu Xaa Gly Ala Arg Xaa 1 5 10 143 13
PRT Artificial Sequence BLyS binding polypeptide 143 Glu His Xaa
Tyr Thr Asp Pro Leu Asn Gly Ala Arg Xaa 1 5 10 144 13 PRT
Artificial Sequence BLyS binding polypeptide 144 Glu His Tyr Tyr
Asn Asp Pro Leu Asn Gly Ala Arg Xaa 1 5 10 145 13 PRT Artificial
Sequence BLyS binding polypeptide 145 Xaa His Xaa Tyr Asn Asp Pro
Leu Asn Gly Ala Arg Xaa 1 5 10 146 13 PRT Artificial Sequence BLyS
binding polypeptide 146 Lys Pro Tyr Tyr Asp Pro Ile Thr Lys Met Thr
His His 1 5 10 147 13 PRT Artificial Sequence BLyS binding
polypeptide 147 Lys Pro Tyr Tyr Asp Pro Ile Thr Lys Met Ser His His
1 5 10 148 13 PRT Artificial Sequence BLyS binding polypeptide 148
Lys Pro Tyr Tyr Asp Pro Ile Ser Lys Met Thr His His 1 5 10 149 13
PRT Artificial Sequence BLyS binding polypeptide 149 Lys Pro Xaa
Xaa Asp Pro Ile Ser Lys Met Thr His His 1 5 10 150 13 PRT
Artificial Sequence BLyS binding polypeptide 150 Gln Ile Gly Tyr
Asp Glu Leu Thr Lys Ala Trp Val Thr 1 5 10 151 13 PRT Artificial
Sequence BLyS binding polypeptide 151 Gln Leu Gly Tyr Asp Glu Leu
Thr Lys Ala Trp Val Thr 1 5 10 152 13 PRT Artificial Sequence BLyS
binding polypeptide 152 Lys Ile Asp Glu Leu Xaa Met Gln Asn Ile Ile
Ile Trp 1 5 10 153 13 PRT Artificial Sequence BLyS binding
polypeptide 153 Asp His Thr Asp Pro Leu Ile Gln Gly Leu Thr Lys Arg
1 5 10 154 13 PRT Artificial Sequence BLyS binding polypeptide 154
Trp His Asp Pro Leu Lys His Met His Phe His His Glu 1 5 10 155 13
PRT Artificial Sequence BLyS binding polypeptide 155 Lys His Ile
Asp Met Glu Thr Gly Leu Ile Leu Gln Asn 1 5 10 156 13 PRT
Artificial Sequence BLyS binding polypeptide 156 Met Gln Val Asp
Pro Glu Thr Gly Leu Lys Tyr Glu His 1 5 10 157 13 PRT Artificial
Sequence BLyS binding polypeptide 157 Xaa Leu Asp Gln His Val Asn
Xaa Xaa Xaa Tyr Gln Ser 1 5 10 158 13 PRT Artificial Sequence BLyS
binding polypeptide 158 Glu Xaa Xaa Xaa Thr Xaa Xaa Leu Thr Gly Ala
Arg Xaa 1 5 10 159 13 PRT Artificial Sequence BLyS binding
polypeptide 159 Gly Pro Tyr Asn Ile Xaa Arg Leu Xaa Gly Glu Arg Xaa
1 5 10 160 13 PRT Artificial Sequence BLyS binding polypeptide 160
His Ile Lys Met Leu His Gln Gly Ser Phe Val Gly Val 1 5 10 161 13
PRT Artificial Sequence BLyS binding polypeptide 161 His Pro Thr
Asn Thr Xaa Xaa His Gln Xaa Val Tyr Ser 1 5 10 162 13 PRT
Artificial Sequence BLyS binding polypeptide 162 His Arg Gly Gln
Val Xaa Xaa Leu Asn Gly Met Val Xaa 1 5 10 163 24 PRT Artificial
Sequence BLyS binding polypeptide 163 Ala Gly Lys Glu Pro Cys Tyr
Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15 Pro Gly Pro Glu Gly
Gly Gly Lys 20 164 25 PRT Artificial Sequence BLyS binding
polypeptide 164 Ala Gly Val Pro Phe Cys Asp Leu Leu Thr Lys His Cys
Phe Glu Ala 1 5 10 15 Gly Pro Gly Pro Glu Gly Gly Gly Lys 20 25 165
28 PRT Artificial Sequence BLyS binding polypeptide 165 Gly Ser Ser
Arg Leu Cys His Met Asp Glu Leu Thr His Val Cys Val 1 5 10 15 His
Phe Ala Pro Pro Gly Pro Glu Gly Gly Gly Lys 20 25 166 29 PRT
Artificial Sequence BLyS binding polypeptide 166 Gly Asp Gly Gly
Asn Cys Tyr Thr Asp Ser Leu Thr Lys Leu His Phe 1 5 10 15 Cys Met
Gly Asp Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 167 22 PRT
Artificial Sequence BLyS binding polypeptide 167 Gly Tyr Asp Val
Leu Thr Lys Leu Tyr Phe Val Pro Gly Gly Pro Gly 1 5 10 15 Pro Glu
Gly Gly Gly Lys 20 168 22 PRT Artificial Sequence BLyS binding
polypeptide 168 Trp Thr Asp Ser Leu Thr Gly Leu Trp Phe Pro Asp Gly
Gly Pro Gly 1 5 10 15 Pro Glu Gly Gly Gly Lys 20 169 24 PRT
Artificial Sequence modified BLyS binding polypeptide 169 Ala Gly
Lys Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15
Pro Gly Pro Glu Gly Gly Gly Lys 20 170 24 PRT Artificial Sequence
modified BLyS binding polypeptide 170 Ala Gly Arg Glu Pro Cys Tyr
Phe Tyr Trp Glu Cys Ala Val Ser Gly 1 5 10 15 Pro Gly Pro Glu Gly
Gly Gly Lys 20 171 24 PRT Artificial Sequence modified BLyS binding
polypeptide 171 Ala Gly Gln Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala
Val Ser Gly 1 5 10 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 172 25 PRT
Artificial Sequence modified BLyS binding polypeptide 172 Ala Gly
Asn Xaa Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala Val Ser 1 5 10 15
Gly Pro Gly Pro Glu Gly Gly Gly Lys 20 25 173 285 PRT Homo Sapiens
173 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu
1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile
Leu Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp
Gly Lys Leu Leu 35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser
Cys Cys Leu Thr Val Val 50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu
Gln Gly Asp Leu Ala Ser Leu Arg 65 70 75 80 Ala Glu Leu Gln Gly His
His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95 Ala Pro Lys Ala
Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105 110 Lys Ile
Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125
Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln 130
135 140 Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln
Lys 145 150 155 160 Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe
Lys Arg Gly Ser 165 170 175 Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu
Val Lys Glu Thr Gly Tyr 180 185 190 Phe Phe Ile Tyr Gly Gln Val Leu
Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205 Gly His Leu Ile Gln Arg
Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220 Ser Leu Val Thr
Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu 225 230 235 240 Pro
Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250
255 Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu
260 265 270 Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275
280 285 174 266 PRT Homo Sapiens 174 Met Asp Asp Ser Thr Glu Arg
Glu Gln Ser Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu
Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys Glu
Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45 Ala
Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55
60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg
65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly
Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val
Thr Ala Gly Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu
Gly Asn Ser Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln
Gly Pro Glu Glu Thr Gly Ser Tyr 130 135 140 Thr Phe Val Pro Trp Leu
Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu 145 150 155 160 Glu Lys Glu
Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile 165 170 175 Tyr
Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu 180 185
190 Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val
195 200 205 Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro
Asn Asn 210 215 220 Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu
Gly Asp Glu Leu 225 230 235 240 Gln Leu Ala Ile Pro Arg Glu Asn Ala
Gln Ile Ser Leu Asp Gly Asp 245 250 255 Val Thr Phe Phe Gly Ala Leu
Lys Leu Leu 260 265 175 309 PRT mouse 175 Met Asp Glu Ser Ala Lys
Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15 Ser Glu Lys Gly
Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30 Gln Lys
Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu 35 40 45
Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala 50
55 60 Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu Met Asn
Leu 65 70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala Thr Pro
Ala Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala Gly Val Lys Leu
Leu Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn Ser Ser Arg Gly
His Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro Glu Glu Thr Glu
Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140 Ala Pro Cys Leu Pro
Gly Cys Arg His Ser Gln His Asp Asp Asn Gly 145 150 155 160 Met Asn
Leu Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp 165 170 175
Ser Asp Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp 180
185 190 Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn
Lys 195 200 205 Ile Val Val Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser
Gln Val Leu 210 215 220 Tyr Thr Asp Pro Ile Phe Ala Met Gly His Val
Ile Gln Arg Lys Lys 225 230 235 240 Val His Val Phe Gly Asp Glu Leu
Ser Leu Val Thr Leu Phe Arg Cys 245 250 255 Ile Gln Asn Met Pro Lys
Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala 260 265 270 Gly Ile Ala Arg
Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro 275 280 285 Arg Glu
Asn Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly 290 295 300
Ala Leu Lys Leu Leu 305 176 290 PRT mouse 176 Met Asp Glu Ser Ala
Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15 Ser Glu Lys
Gly Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30 Gln
Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu 35 40
45 Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala
50 55 60 Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu Met
Asn Leu 65 70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala Thr
Pro Ala Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala Gly Val Lys
Leu Leu Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn Ser Ser Arg
Gly His Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro Glu Glu Thr
Glu Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140 Ala Pro Cys Leu
Pro Gly Cys Arg His Ser Gln His Asp Asp Asn Gly 145 150 155 160 Met
Asn Leu Arg Asn Arg Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser 165 170
175 Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys Ile Val Val
180 185 190 Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln
Val Leu Tyr Thr Asp 195 200 205 Pro Ile Phe Ala Met Gly His Val Ile
Gln Arg Lys Lys Val His Val 210 215 220 Phe Gly Asp Glu Leu Ser Leu
Val Thr Leu Phe Arg Cys Ile Gln Asn 225 230 235 240 Met Pro Lys Thr
Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala 245 250 255 Arg Leu
Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn 260 265 270
Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys 275
280 285 Leu Leu 290 177 239 PRT rat 177 Ala Val Gln Ala Asp Leu Met
Ser Leu Arg Met Glu Leu Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala Thr
Pro Ala Ala Pro Gly Ala Pro Gly Leu Ser Ala 20 25 30 Gly Val Lys
Leu Pro Thr Pro Ala Ala Pro Gly Pro His Asn Ser Ser 35 40 45 Arg
Gly Gln Arg Asn Arg Arg Ala Phe Gln Gly Pro Glu Glu Thr Glu 50 55
60 Gln Asp Val Asp Leu Ser Ala Thr Pro Ala Pro Ser Leu Pro Gly Asn
65 70 75 80 Cys His Ala Ser His His Asp Glu Asn Gly Leu Asn Leu Arg
Thr Ile 85 90 95 Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Asn
Thr Pro Thr Ile 100 105 110 Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp
Leu Leu Ser Phe Lys Arg 115 120 125 Gly Asn Ala Leu Glu Glu Lys Glu
Asn Lys Ile Val Val Arg Gln Thr 130 135 140 Gly Tyr Phe Phe Ile Tyr
Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe 145 150 155 160 Ala Met Gly
His Val Ile Gln Arg Lys Lys Ile His Val Phe Gly Asp 165 170 175 Glu
Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys 180 185
190 Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu
195 200 205 Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala
Gln Ile 210 215 220 Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu
Lys Leu Leu 225 230 235 178 220 PRT rat 178 Ala Val Gln Ala Asp Leu
Met Ser Leu Arg Met Glu Leu Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala
Thr Pro Ala Ala Pro Gly Ala Pro Gly Leu Ser Ala 20 25 30 Gly Val
Lys Leu Pro Thr Pro Ala Ala Pro Gly Pro His Asn Ser Ser 35 40 45
Arg Gly Gln Arg Asn Arg Arg Ala Phe Gln Gly Pro Glu Glu Thr Glu 50
55 60 Gln Asp Val Asp Leu Ser Ala Thr Pro Val Pro Ser Leu Pro Gly
Asn 65 70 75 80 Cys His Ala Ser His His Asp Glu Asn Gly Leu Asn Leu
Arg Thr Arg 85 90 95 Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe
Lys Arg Gly Asn Ala 100 105 110 Leu Glu Glu Lys Glu Asn Lys Ile Val
Val Arg Gln Thr Gly Tyr Phe 115 120 125 Phe Ile Tyr Ser Gln Val Leu
Tyr Thr Asp Pro Ile Phe Ala Met Gly 130 135 140 His Val Ile Gln Arg
Lys Lys Ile His Val Phe Gly Asp Glu Leu Ser 145 150 155 160 Leu Val
Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro 165 170 175
Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp 180
185 190 Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg
Asn 195 200 205 Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 210
215 220 179 207 PRT rat 179 Ala Val Gln Ala Asp Leu Met Ser Leu Arg
Met Glu Leu Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala Thr Pro Ala Ala
Pro Gly Ala Pro Gly Leu Ser Ala 20 25 30 Gly Val Lys Leu Pro Thr
Pro Ala Ala Pro Gly Pro His Asn Ser Ser 35 40 45 Arg Gly Gln Arg
Asn Arg Arg Ala Phe Gln Gly Pro Glu Glu Thr Val 50 55 60 Ile Gln
Asp Cys Leu Gln Leu Ile Ala Asp Ser Asn Thr Pro Thr Ile 65 70 75 80
Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg 85
90 95 Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln
Thr 100 105 110 Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp
Pro Ile Phe 115 120 125 Ala Met Gly His Val Ile Gln Arg Lys Lys Ile
His Val Phe Gly Asp 130 135 140 Glu Leu Ser Leu Val Thr Leu Phe Arg
Cys Ile Gln Asn Met Pro Lys 145 150 155 160 Thr Leu Pro Asn Asn Ser
Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu 165 170 175 Glu Gly Asp Glu
Val Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile 180 185 190 Ser Arg
Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 195 200 205 180
188 PRT rat 180 Ala Val Gln Ala Asp Leu Met Ser Leu Arg Met Glu Leu
Gln Ser Tyr 1 5 10 15 Arg Ser Ser Ala Thr Pro Ala Ala Pro Gly Ala
Pro Gly Leu Ser Ala 20 25 30 Gly Val Lys Leu Pro Thr Pro Ala Ala
Pro Gly Pro His Asn Ser Ser 35 40 45 Arg Gly Gln Arg Asn Arg Arg
Ala Phe Gln Gly Pro Glu Glu Thr Gly 50 55 60 Thr Tyr Thr Phe Val
Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala 65 70 75 80 Leu Glu Glu
Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe 85 90 95 Phe
Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly 100 105
110 His Val Ile Gln Arg Lys Lys Ile His Val Phe Gly Asp Glu Leu Ser
115 120 125 Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr
Leu Pro 130 135 140 Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu
Glu Glu Gly Asp 145 150 155 160 Glu Ile Gln Leu Ala Ile Pro Arg Glu
Asn Ala Gln Ile Ser Arg Asn 165 170 175 Gly Asp Asp Thr Phe Phe Gly
Ala Leu Lys Leu Leu 180 185 181 243 PRT monkey 181 Lys Asp Arg Lys
Leu Leu Ala Ala Ala Leu Leu Leu Ala Leu Leu Ser 1 5 10 15 Cys Cys
Leu Met Val Val Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly 20 25 30
Asp Leu Ala Ser Leu Arg Ala Glu Leu Gln Gly His His Ala Glu Lys 35
40 45 Leu Pro Ala Arg Ala Arg Ala Pro Lys Ala Gly Leu Gly Glu Ala
Pro 50 55 60 Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala
Pro Gly Glu 65 70 75 80 Gly Asn Ser Ser Gln Ser Ser Arg Asn Lys Arg
Ala Ile Gln Gly Ala 85 90 95 Glu Glu Thr Val Ile Gln Asp Cys Leu
Gln Leu Ile Ala Asp Ser Glu 100 105 110 Thr Pro Thr Ile Gln Lys Gly
Ser Tyr Thr Phe Val Pro Trp Leu Leu 115 120 125 Ser Phe Lys Arg Gly
Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu 130 135 140 Val Lys Glu
Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr 145 150 155 160
Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val His 165
170 175 Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile
Gln 180 185 190 Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser
Ala Gly Ile 195 200 205 Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu
Ala Ile Pro Arg Glu 210 215 220 Asn Ala Gln Ile Ser Leu Asp Gly Asp
Val Thr Phe Phe Gly Ala Leu 225 230 235 240 Lys Leu Leu 182 219 PRT
monkey 182 Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg
Ala Glu 1 5 10 15 Leu Gln Ser His His Ala Glu Lys Leu Pro Ala Arg
Ala Arg Ala Pro 20 25 30 Lys Ala Gly Leu Gly Glu Ala Pro Ala Val
Thr Ala Gly Leu Lys Ile 35 40 45 Phe Glu Pro Pro Ala Pro Gly Glu
Gly Asn Ser Ser Gln Ser Ser Arg 50 55 60 Asn Lys Arg Ala Ile Gln
Gly Ala Glu Glu Thr Val Ile Gln Asp Cys 65 70 75 80 Leu Gln Leu Ile
Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser 85 90 95 Tyr Thr
Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu 100 105 110
Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe 115
120 125 Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly
His 130 135 140 Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu
Leu Ser Leu 145 150 155 160 Val Thr Leu Phe Arg Cys Ile Gln Asn Met
Pro Glu Thr Leu Pro Asn 165 170 175 Asn Ser Cys Tyr Ser Ala Gly Ile
Ala Lys Leu Glu Glu Gly Asp Glu 180 185 190 Leu Gln Leu Ala Ile Pro
Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly 195 200 205 Asp Val Thr Phe
Phe Gly Ala Leu Lys Leu Leu 210 215 183 8 PRT Artificial Sequence
epitope tag 183 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 184 14 PRT
Artificial Sequence concensus BLyS binding polypeptide 184 Ala Asn
Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 185 42 DNA
Artificial Sequence coding sequence for BLyS affinity maturation
library template 185 gctnnnnnnn nngatnnnct tactnnnctc nnnnnnnnnn nn
42 186 14 PRT Artificial Sequence BLyS binding polypeptide 186 Ala
Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 187 14
PRT Artificial Sequence BLyS binding polypeptide 187 Ala Asn Trp
Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 188 14 PRT
Artificial Sequence BLyS binding polypeptide 188 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 189 14 PRT
Artificial Sequence BLyS binding polypeptide 189 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 190 14 PRT
Artificial Sequence BLyS binding polypeptide 190 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ser Asp 1 5 10 191 14 PRT
Artificial Sequence BLyS binding polypeptide 191 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Asn Asp 1 5 10 192 14 PRT
Artificial Sequence BLyS binding polypeptide 192 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Thr 1 5 10 193 14 PRT
Artificial Sequence BLyS binding polypeptide 193 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 194 14 PRT
Artificial Sequence BLyS binding polypeptide 194 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asn 1 5 10 195 14 PRT
Artificial Sequence BLyS binding polypeptide 195 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Val Asp 1 5 10 196 14 PRT
Artificial Sequence BLyS binding polypeptide 196 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 197 14 PRT
Artificial Sequence BLyS binding polypeptide 197 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 198 14 PRT
Artificial Sequence BLyS binding polypeptide 198 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 199 14 PRT
Artificial Sequence BLyS binding polypeptide 199 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr Val 1 5 10 200 14 PRT
Artificial Sequence BLyS binding polypeptide 200 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Asp 1 5 10 201 14 PRT
Artificial Sequence BLyS binding polypeptide 201 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Glu 1 5 10 202 14 PRT
Artificial Sequence BLyS binding polypeptide 202 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Glu 1 5 10 203 14 PRT
Artificial Sequence BLyS binding polypeptide 203 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Arg 1 5 10 204 14 PRT
Artificial Sequence BLyS binding polypeptide 204 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 205 14 PRT
Artificial Sequence BLyS binding polypeptide 205 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 206 14 PRT
Artificial Sequence BLyS binding polypeptide 206 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ile 1 5 10 207 14 PRT
Artificial Sequence BLyS binding polypeptide 207 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ile Asp 1 5 10 208 14 PRT
Artificial Sequence BLyS binding polypeptide 208 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Asp 1 5 10 209 14 PRT
Artificial Sequence BLyS binding polypeptide 209 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 210 14 PRT
Artificial Sequence BLyS binding polypeptide 210 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Glu 1 5 10 211 14 PRT
Artificial Sequence BLyS binding polypeptide 211 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 212 14 PRT
Artificial Sequence BLyS binding polypeptide 212 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 213 14 PRT
Artificial Sequence BLyS binding polypeptide 213 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 214 14 PRT
Artificial Sequence BLyS binding polypeptide 214 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Gln 1 5 10 215 14 PRT
Artificial Sequence BLyS binding polypeptide 215 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 216 14 PRT
Artificial Sequence BLyS binding polypeptide 216 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 217 14 PRT
Artificial Sequence BLyS binding polypeptide 217 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 218 14 PRT
Artificial Sequence BLyS binding polypeptide 218 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Tyr 1 5 10 219 14 PRT
Artificial Sequence BLyS binding polypeptide 219 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 220 14 PRT
Artificial Sequence BLyS binding polypeptide 220 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Tyr 1 5 10 221 14 PRT
Artificial Sequence BLyS binding polypeptide 221 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Asp 1 5 10 222 14 PRT
Artificial Sequence BLyS binding polypeptide 222 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 223 14 PRT
Artificial Sequence BLyS binding polypeptide 223 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Gly 1 5 10 224 14 PRT
Artificial Sequence BLyS binding polypeptide 224 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr His 1 5 10 225 14 PRT
Artificial Sequence BLyS binding polypeptide 225 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Thr 1 5 10 226 14 PRT
Artificial Sequence BLyS binding polypeptide 226 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Val 1 5 10 227 14 PRT
Artificial Sequence BLyS binding polypeptide 227 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Tyr Tyr 1 5 10 228 14 PRT
Artificial Sequence BLyS binding polypeptide 228 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ser Asp 1 5 10 229 14 PRT
Artificial Sequence BLyS binding polypeptide 229 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 230 14 PRT
Artificial Sequence BLyS binding polypeptide 230 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 231 14 PRT
Artificial Sequence BLyS binding polypeptide 231 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 232 14 PRT
Artificial Sequence BLyS binding polypeptide
232 Ala Ser Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Gln 1 5 10
233 14 PRT Artificial Sequence BLyS binding polypeptide 233 Ala Ser
Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 234 14 PRT
Artificial Sequence BLyS binding polypeptide 234 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 235 14 PRT
Artificial Sequence BLyS binding polypeptide 235 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 236 14 PRT
Artificial Sequence BLyS binding polypeptide 236 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ile 1 5 10 237 14 PRT
Artificial Sequence BLyS binding polypeptide 237 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 238 14 PRT
Artificial Sequence BLyS binding polypeptide 238 Ala Phe Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 239 14 PRT
Artificial Sequence BLyS binding polypeptide 239 Ala Phe Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 240 14 PRT
Artificial Sequence BLyS binding polypeptide 240 Ala Phe Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Glu 1 5 10 241 14 PRT
Artificial Sequence BLyS binding polypeptide 241 Ala Phe Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 242 14 PRT
Artificial Sequence BLyS binding polypeptide 242 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 243 14 PRT
Artificial Sequence BLyS binding polypeptide 243 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 244 14 PRT
Artificial Sequence BLyS binding polypeptide 244 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 245 14 PRT
Artificial Sequence BLyS binding polypeptide 245 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 246 14 PRT
Artificial Sequence BLyS binding polypeptide 246 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Phe 1 5 10 247 14 PRT
Artificial Sequence BLyS binding polypeptide 247 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Asp 1 5 10 248 14 PRT
Artificial Sequence BLyS binding polypeptide 248 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Arg 1 5 10 249 14 PRT
Artificial Sequence BLyS binding polypeptide 249 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 250 14 PRT
Artificial Sequence BLyS binding polypeptide 250 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr Ala 1 5 10 251 14 PRT
Artificial Sequence BLyS binding polypeptide 251 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ala Val 1 5 10 252 14 PRT
Artificial Sequence BLyS binding polypeptide 252 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 253 14 PRT
Artificial Sequence BLyS binding polypeptide 253 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Val 1 5 10 254 14 PRT
Artificial Sequence BLyS binding polypeptide 254 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 255 14 PRT
Artificial Sequence BLyS binding polypeptide 255 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Glu 1 5 10 256 14 PRT
Artificial Sequence BLyS binding polypeptide 256 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ser Asp 1 5 10 257 14 PRT
Artificial Sequence BLyS binding polypeptide 257 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 258 14 PRT
Artificial Sequence BLyS binding polypeptide 258 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 259 14 PRT
Artificial Sequence BLyS binding polypeptide 259 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr Ser 1 5 10 260 14 PRT
Artificial Sequence BLyS binding polypeptide 260 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 261 14 PRT
Artificial Sequence BLyS binding polypeptide 261 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 262 14 PRT
Artificial Sequence BLyS binding polypeptide 262 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ser Gly 1 5 10 263 14 PRT
Artificial Sequence BLyS binding polypeptide 263 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 264 14 PRT
Artificial Sequence BLyS binding polypeptide 264 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 265 14 PRT
Artificial Sequence BLyS binding polypeptide 265 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 266 14 PRT
Artificial Sequence BLyS binding polypeptide 266 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Lys 1 5 10 267 14 PRT
Artificial Sequence BLyS binding polypeptide 267 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 268 14 PRT
Artificial Sequence BLyS binding polypeptide 268 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 269 14 PRT
Artificial Sequence BLyS binding polypeptide 269 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Gln 1 5 10 270 14 PRT
Artificial Sequence BLyS binding polypeptide 270 Ala Glu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Asp 1 5 10 271 14 PRT
Artificial Sequence BLyS binding polypeptide 271 Ala Glu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 272 14 PRT
Artificial Sequence BLyS binding polypeptide 272 Ala Glu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 273 14 PRT
Artificial Sequence BLyS binding polypeptide 273 Ala Leu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 274 14 PRT
Artificial Sequence BLyS binding polypeptide 274 Ala Leu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 275 14 PRT
Artificial Sequence BLyS binding polypeptide 275 Ala Leu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Arg Gly 1 5 10 276 14 PRT
Artificial Sequence BLyS binding polypeptide 276 Ala Leu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Gly 1 5 10 277 14 PRT
Artificial Sequence BLyS binding polypeptide 277 Ala Met Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 278 14 PRT
Artificial Sequence BLyS binding polypeptide 278 Ala Met Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Gln Val 1 5 10 279 14 PRT
Artificial Sequence BLyS binding polypeptide 279 Ala Met Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Gly 1 5 10 280 14 PRT
Artificial Sequence BLyS binding polypeptide 280 Ala Ala Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 281 14 PRT
Artificial Sequence BLyS binding polypeptide 281 Ala Ala Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 282 14 PRT
Artificial Sequence BLyS binding polypeptide 282 Ala Ala Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Asp 1 5 10 283 14 PRT
Artificial Sequence BLyS binding polypeptide 283 Ala His Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 284 14 PRT
Artificial Sequence BLyS binding polypeptide 284 Ala His Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 285 14 PRT
Artificial Sequence BLyS binding polypeptide 285 Ala His Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 286 14 PRT
Artificial Sequence BLyS binding polypeptide 286 Ala His Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 287 14 PRT
Artificial Sequence BLyS binding polypeptide 287 Ala Pro Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 288 14 PRT
Artificial Sequence BLyS binding polypeptide 288 Ala Pro Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 289 14 PRT
Artificial Sequence BLyS binding polypeptide 289 Ala Gln Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 290 14 PRT
Artificial Sequence BLyS binding polypeptide 290 Ala Gln Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 291 14 PRT
Artificial Sequence BLyS binding polypeptide 291 Ala Gln Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Arg 1 5 10 292 14 PRT
Artificial Sequence BLyS binding polypeptide 292 Ala Lys Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 293 14 PRT
Artificial Sequence BLyS binding polypeptide 293 Ala Lys Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 294 14 PRT
Artificial Sequence BLyS binding polypeptide 294 Ala Lys Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 295 14 PRT
Artificial Sequence BLyS binding polypeptide 295 Ala Lys Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Asn Gly 1 5 10 296 14 PRT
Artificial Sequence BLyS binding polypeptide 296 Ala Trp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 297 14 PRT
Artificial Sequence BLyS binding polypeptide 297 Ala Val Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 298 14 PRT
Artificial Sequence BLyS binding polypeptide 298 Ala Tyr Glu Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Leu Tyr 1 5 10 299 14 PRT
Artificial Sequence BLyS binding polypeptide 299 Ala Thr Lys Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 300 14 PRT
Artificial Sequence BLyS binding polypeptide 300 Ala Thr Leu Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 301 14 PRT
Artificial Sequence BLyS binding polypeptide 301 Ala Ile Arg Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Tyr 1 5 10 302 14 PRT
Artificial Sequence BLyS binding polypeptide 302 Ala Glu Arg Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro His 1 5 10 303 14 PRT
Artificial Sequence BLyS binding polypeptide 303 Ala Asp Arg Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gln 1 5 10 304 14 PRT
Artificial Sequence BLyS binding polypeptide 304 Ala Asn Ser Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 305 14 PRT
Artificial Sequence BLyS binding polypeptide 305 Ala Ile Leu Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 306 14 PRT
Artificial Sequence BLyS binding polypeptide 306 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gln 1 5 10 307 14 PRT
Artificial Sequence BLyS binding polypeptide 307 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 308 14 PRT
Artificial Sequence BLyS binding polypeptide 308 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 309 14 PRT
Artificial Sequence BLyS binding polypeptide 309 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 310 14 PRT
Artificial Sequence BLyS binding polypeptide 310 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 311 14 PRT
Artificial Sequence BLyS binding polypeptide 311 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Glu 1 5 10 312 14 PRT
Artificial Sequence BLyS binding polypeptide 312 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 313 14 PRT
Artificial Sequence BLyS binding polypeptide 313 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asn 1 5 10 314 14 PRT
Artificial Sequence BLyS binding polypeptide 314 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Ser Glu 1 5 10 315 14 PRT
Artificial Sequence BLyS binding polypeptide 315 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu His Asp 1 5 10 316 14 PRT
Artificial Sequence BLyS binding polypeptide 316 Ala Asn Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Val Asp 1 5 10 317 14 PRT
Artificial Sequence BLyS binding polypeptide 317 Ala Tyr Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 318 14 PRT
Artificial Sequence BLyS binding polypeptide 318 Ala Tyr Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 319 14 PRT
Artificial Sequence BLyS binding polypeptide 319 Ala Tyr Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 320 14 PRT
Artificial Sequence BLyS binding polypeptide 320 Ala Gln Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 321 14 PRT
Artificial Sequence BLyS binding polypeptide 321 Ala His Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 322 14 PRT
Artificial Sequence BLyS binding polypeptide 322 Ala Thr Trp Phe
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 323 14 PRT
Artificial Sequence BLyS binding polypeptide 323 Ala Tyr Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 324 14 PRT
Artificial Sequence BLyS binding polypeptide 324 Ala Tyr Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu His Asp 1 5 10 325 14 PRT
Artificial Sequence BLyS binding polypeptide 325 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Ile Pro Asp 1 5 10 326 14 PRT
Artificial Sequence BLyS binding polypeptide 326 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 327 14 PRT
Artificial Sequence BLyS binding polypeptide 327 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 328 14 PRT
Artificial Sequence BLyS binding polypeptide 328 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Ala Asp 1 5 10 329 14 PRT
Artificial Sequence BLyS binding polypeptide 329 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 330 14 PRT
Artificial Sequence BLyS binding polypeptide 330 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Tyr Glu 1 5 10 331 14 PRT
Artificial Sequence BLyS binding polypeptide 331 Ala Gly Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 332 14 PRT
Artificial Sequence BLyS binding polypeptide 332 Ala Val Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Thr Asp 1 5 10 333 14 PRT
Artificial Sequence BLyS binding polypeptide 333 Ala Asn Trp Tyr
Asp Ala Leu Thr Lys Leu Trp Leu Pro Val 1 5 10 334 14 PRT
Artificial Sequence BLyS binding polypeptide 334 Ala Tyr Trp Tyr
Asp Thr Leu Thr Lys Leu Trp Leu Pro Asn 1 5 10 335 14 PRT
Artificial Sequence BLyS binding polypeptide 335 Ala Phe Trp Tyr
Asp Pro Leu Thr Asn Leu Trp Leu Leu Glu 1 5 10 336 14 PRT
Artificial Sequence BLyS binding polypeptide 336 Ala Tyr Trp Tyr
Asp Pro Leu Thr Gly Leu Trp Leu Leu Val 1 5 10 337 14 PRT
Artificial Sequence BLyS binding polypeptide 337 Ala Tyr Trp Tyr
Asp Pro Leu Thr Gly Leu Trp Leu Leu Tyr 1 5 10 338 14 PRT
Artificial Sequence BLyS binding polypeptide 338 Ala Tyr Trp Tyr
Asp Pro Leu Thr Gly Leu Trp Leu Arg Val 1 5 10 339 14 PRT
Artificial Sequence BLyS binding polypeptide 339 Ala Tyr Trp Tyr
Asp Pro Leu Thr Glu Leu Trp Leu Arg Leu 1 5 10 340 14 PRT
Artificial Sequence BLyS binding polypeptide 340 Ala Met Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Leu Pro Asp 1 5 10 341 14 PRT
Artificial Sequence BLyS binding polypeptide 341 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Leu Leu Val 1 5 10 342 14 PRT
Artificial Sequence BLyS binding polypeptide 342 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Leu Thr Val 1 5 10 343 14 PRT
Artificial Sequence BLyS binding polypeptide 343 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Leu Leu Val 1 5 10 344 14 PRT
Artificial Sequence BLyS binding polypeptide 344 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Leu Leu Leu 1 5 10 345 14 PRT
Artificial Sequence BLyS binding polypeptide 345 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Arg Leu Leu Glu 1 5 10 346 14 PRT
Artificial Sequence BLyS binding polypeptide 346 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Arg Leu Leu Val 1 5 10 347 14 PRT
Artificial Sequence BLyS binding polypeptide 347 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Arg Leu Ile Val 1 5 10 348 14 PRT
Artificial Sequence BLyS binding polypeptide 348 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Tyr Leu Pro Asp 1 5 10 349 14 PRT
Artificial Sequence BLyS binding polypeptide 349 Ala Ile Trp
Tyr Asp Pro Leu Thr Lys Leu Gly Leu Leu Val 1 5 10 350 14 PRT
Artificial Sequence BLyS binding polypeptide 350 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Thr Leu Leu Val 1 5 10 351 14 PRT
Artificial Sequence BLyS binding polypeptide 351 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Leu Leu Pro Asn 1 5 10 352 14 PRT
Artificial Sequence BLyS binding polypeptide 352 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 353 14 PRT
Artificial Sequence BLyS binding polypeptide 353 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 354 14 PRT
Artificial Sequence BLyS binding polypeptide 354 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Ser Asp 1 5 10 355 14 PRT
Artificial Sequence BLyS binding polypeptide 355 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Val 1 5 10 356 14 PRT
Artificial Sequence BLyS binding polypeptide 356 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Val 1 5 10 357 14 PRT
Artificial Sequence BLyS binding polypeptide 357 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Lys 1 5 10 358 14 PRT
Artificial Sequence BLyS binding polypeptide 358 Ala Lys Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 359 14 PRT
Artificial Sequence BLyS binding polypeptide 359 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Leu Glu 1 5 10 360 14 PRT
Artificial Sequence BLyS binding polypeptide 360 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Ala 1 5 10 361 14 PRT
Artificial Sequence BLyS binding polypeptide 361 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 362 14 PRT
Artificial Sequence BLyS binding polypeptide 362 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Glu 1 5 10 363 14 PRT
Artificial Sequence BLyS binding polypeptide 363 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 364 14 PRT
Artificial Sequence BLyS binding polypeptide 364 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Gly 1 5 10 365 14 PRT
Artificial Sequence BLyS binding polypeptide 365 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro His 1 5 10 366 14 PRT
Artificial Sequence BLyS binding polypeptide 366 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Val 1 5 10 367 14 PRT
Artificial Sequence BLyS binding polypeptide 367 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 368 14 PRT
Artificial Sequence BLyS binding polypeptide 368 Ala Gly Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 369 14 PRT
Artificial Sequence BLyS binding polypeptide 369 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Thr 1 5 10 370 14 PRT
Artificial Sequence BLyS binding polypeptide 370 Ala Lys Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Ala 1 5 10 371 14 PRT
Artificial Sequence BLyS binding polypeptide 371 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Phe Asp 1 5 10 372 14 PRT
Artificial Sequence BLyS binding polypeptide 372 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Ala Asp 1 5 10 373 14 PRT
Artificial Sequence BLyS binding polypeptide 373 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Tyr 1 5 10 374 14 PRT
Artificial Sequence BLyS binding polypeptide 374 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Arg Asp 1 5 10 375 14 PRT
Artificial Sequence BLyS binding polypeptide 375 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Asp 1 5 10 376 14 PRT
Artificial Sequence BLyS binding polypeptide 376 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Ala 1 5 10 377 14 PRT
Artificial Sequence BLyS binding polypeptide 377 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Asn 1 5 10 378 14 PRT
Artificial Sequence BLyS binding polypeptide 378 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Glu 1 5 10 379 14 PRT
Artificial Sequence BLyS binding polypeptide 379 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Gln 1 5 10 380 14 PRT
Artificial Sequence BLyS binding polypeptide 380 Ala Glu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Lys 1 5 10 381 14 PRT
Artificial Sequence BLyS binding polypeptide 381 Ala Gln Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Val 1 5 10 382 14 PRT
Artificial Sequence BLyS binding polypeptide 382 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Tyr 1 5 10 383 14 PRT
Artificial Sequence BLyS binding polypeptide 383 Ala Leu Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Tyr 1 5 10 384 14 PRT
Artificial Sequence BLyS binding polypeptide 384 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Gly 1 5 10 385 14 PRT
Artificial Sequence BLyS binding polypeptide 385 Ala Ser Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 386 14 PRT
Artificial Sequence BLyS binding polypeptide 386 Ala Asp Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Ile Pro Gly 1 5 10 387 14 PRT
Artificial Sequence BLyS binding polypeptide 387 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 388 14 PRT
Artificial Sequence BLyS binding polypeptide 388 Ala Lys Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 389 14 PRT
Artificial Sequence BLyS binding polypeptide 389 Ala Ile Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Ile Pro Asn 1 5 10 390 14 PRT
Artificial Sequence BLyS binding polypeptide 390 Ala Thr Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Ile Pro Gln 1 5 10 391 14 PRT
Artificial Sequence BLyS binding polypeptide 391 Ala Ser Trp Tyr
Asp Pro Leu Thr Asn Leu Trp Val Pro Asp 1 5 10 392 14 PRT
Artificial Sequence BLyS binding polypeptide 392 Ala Tyr Glu Tyr
Asp Pro Leu Thr Asn Leu Trp Leu Leu Tyr 1 5 10 393 14 PRT
Artificial Sequence BLyS binding polypeptide 393 Ala Tyr Trp Tyr
Asp Pro Leu Thr Asn Leu Ser Leu Leu Val 1 5 10 394 14 PRT
Artificial Sequence BLyS binding polypeptide 394 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Ile Leu Glu 1 5 10 395 14 PRT
Artificial Sequence BLyS binding polypeptide 395 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 396 14 PRT
Artificial Sequence BLyS binding polypeptide 396 Ala His Trp Phe
Asp Pro Leu Thr Gln Leu Lys Ile Arg Val 1 5 10 397 14 PRT
Artificial Sequence BLyS binding polypeptide 397 Ala Tyr Trp Cys
Asp Pro Leu Thr Lys Leu Cys Ile Leu Glu 1 5 10 398 14 PRT
Artificial Sequence BLyS binding polypeptide 398 Ala Asn Ser Tyr
Asp Pro Leu Thr Lys Leu Trp Phe Pro Tyr 1 5 10 399 14 PRT
Artificial Sequence BLyS binding polypeptide 399 Ala Asn Leu Tyr
Asp Pro Leu Thr Lys Leu Trp Val Pro Tyr 1 5 10 400 14 PRT
Artificial Sequence BLyS binding polypeptide 400 Ala Asn Trp Tyr
Asp Ala Leu Thr Lys Leu Trp Leu His Asp 1 5 10 401 14 PRT
Artificial Sequence BLyS binding polypeptide 401 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Phe Pro Asp 1 5 10 402 14 PRT
Artificial Sequence BLyS binding polypeptide 402 Ala Thr Ser Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Ala 1 5 10 403 14 PRT
Artificial Sequence BLyS binding polypeptide 403 Ala Cys Trp Tyr
Asp Ser Leu Thr Lys Leu Cys His Arg Glu 1 5 10 404 14 PRT
Artificial Sequence BLyS binding polypeptide 404 Ala Ile Gly Asn
Asp Pro Leu Thr Lys Leu Trp Ile Pro Tyr 1 5 10 405 14 PRT
Artificial Sequence BLyS binding polypeptide 405 Ala Asn Trp Gln
Asp Cys Leu Thr Lys Leu Cys Leu Ala Gly 1 5 10 406 14 PRT
Artificial Sequence BLyS binding polypeptide 406 Ala Tyr Trp Phe
Asp Pro Leu Thr Asn Leu Trp Leu Leu Glu 1 5 10 407 14 PRT
Artificial Sequence BLyS binding polypeptide 407 Ala Tyr Trp Tyr
Asp Pro Leu Thr Asn Leu Ser Leu Leu Val 1 5 10 408 14 PRT
Artificial Sequence BLyS binding polypeptide 408 Ala Asn Cys Phe
Asp Ser Leu Thr Arg Leu Trp Leu Cys Asp 1 5 10 409 14 PRT
Artificial Sequence BLyS binding polypeptide 409 Ala Cys Ala Tyr
Asp Ala Leu Thr Lys Leu Cys Leu Pro Ala 1 5 10 410 14 PRT
Artificial Sequence BLyS binding polypeptide 410 Ala Asn Trp Tyr
Asp Pro Leu Thr Asn Leu Ser Leu Leu Leu 1 5 10 411 14 PRT
Artificial Sequence BLyS binding polypeptide 411 Ala Tyr Trp Tyr
Asp Pro Leu Thr Gln Leu Ser Leu Leu Val 1 5 10 412 14 PRT
Artificial Sequence BLyS binding polypeptide 412 Ala Tyr Arg Tyr
Asp Ala Leu Thr Gly Leu Trp Leu Leu Tyr 1 5 10 413 14 PRT
Artificial Sequence BLyS binding polypeptide 413 Ala Tyr Trp Asn
Asp Pro Leu Thr Lys Leu Lys Leu Arg Leu 1 5 10 414 14 PRT
Artificial Sequence BLyS binding polypeptide 414 Ala Tyr Trp Tyr
Asp Pro Leu Thr Gln Leu Ser Leu Leu Val 1 5 10 415 14 PRT
Artificial Sequence BLyS binding polypeptide 415 Ala Tyr Arg Tyr
Asp Ala Leu Thr Gly Leu Trp Leu Leu Tyr 1 5 10 416 14 PRT
Artificial Sequence BLyS binding polypeptide 416 Ala Tyr Arg Tyr
Asp Ser Leu Thr Asn Leu Trp Leu Leu Tyr 1 5 10 417 14 PRT
Artificial Sequence BLyS binding polypeptide 417 Ala Tyr Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Ile Leu Glu 1 5 10 418 14 PRT
Artificial Sequence BLyS binding polypeptide 418 Ala Ser Cys Tyr
Asp Pro Leu Thr Lys Leu Cys Phe Pro Val 1 5 10 419 14 PRT
Artificial Sequence BLyS binding polypeptide 419 Ala Phe Trp Phe
Asp Pro Leu Thr Gly Leu Trp Leu Leu Glu 1 5 10 420 14 PRT
Artificial Sequence BLyS binding polypeptide 420 Ala His Trp Tyr
Asp Pro Leu Thr Lys Leu Ser Ile Arg Val 1 5 10 421 14 PRT
Artificial Sequence BLyS binding polypeptide 421 Ala Pro Trp Tyr
Asp Ser Leu Thr Lys Leu Trp Phe Pro Ser 1 5 10 422 14 PRT
Artificial Sequence BLyS binding polypeptide 422 Ala Asn Cys Tyr
Asp Thr Leu Thr Lys Leu Trp Leu Thr Cys 1 5 10 423 14 PRT
Artificial Sequence BLyS binding polypeptide 423 Ala Asn Trp Tyr
Asp Ser Leu Thr Lys Leu Ser Leu Pro Asp 1 5 10 424 14 PRT
Artificial Sequence BLyS binding polypeptide 424 Ala Tyr Ala Tyr
Asp Phe Leu Thr Gln Leu Ser Leu Pro Asp 1 5 10 425 14 PRT
Artificial Sequence BLyS binding polypeptide 425 Ala Phe Arg Tyr
Asp Ser Leu Thr Gly Leu Trp Leu Arg Tyr 1 5 10 426 14 PRT
Artificial Sequence BLyS binding polypeptide 426 Ala Asn Cys Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Cys 1 5 10 427 14 PRT
Artificial Sequence BLyS binding polypeptide 427 Ala Asn Gly Tyr
Asp Leu Leu Thr Asn Leu Ser Val Ser Asp 1 5 10 428 14 PRT
Artificial Sequence BLyS binding polypeptide 428 Ala Asn Trp Tyr
Asp Pro Leu Thr Arg Leu Trp Ile Pro Val 1 5 10 429 14 PRT
Artificial Sequence BLyS binding polypeptide 429 Ala Leu Lys Phe
Asp Tyr Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 430 14 PRT
Artificial Sequence BLyS binding polypeptide 430 Ala Tyr Arg Tyr
Asp Ser Leu Thr Lys Leu Trp Leu Pro Gly 1 5 10 431 14 PRT
Artificial Sequence BLyS binding polypeptide 431 Ala Tyr Cys Tyr
Asp Ser Leu Thr Lys Leu Trp Ile Pro Asp 1 5 10 432 14 PRT
Artificial Sequence BLyS binding polypeptide 432 Ala Ser Trp Glu
Asp Ser Leu Thr Lys Leu Trp Leu Ser Lys 1 5 10 433 14 PRT
Artificial Sequence BLyS binding polypeptide 433 Ala Tyr Trp Tyr
Asp Ser Leu Thr Gly Leu Ser Leu Leu Val 1 5 10 434 14 PRT
Artificial Sequence BLyS binding polypeptide 434 Ala Tyr Trp Tyr
Asp Pro Leu Thr Tyr Leu Arg Leu Arg Val 1 5 10 435 14 PRT
Artificial Sequence BLyS binding polypeptide 435 Ala Lys Cys Tyr
Asp Ser Leu Thr Asn Leu Trp Leu Cys Asp 1 5 10 436 10 PRT
Artificial Sequence core peptide of high affinity BLyS binders 436
Trp Tyr Asp Pro Leu Thr Lys Leu Trp Leu 1 5 10 437 17 PRT
Artificial Sequence BLyS binding polypeptide 437 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly 1 5 10 15 Lys 438
15 PRT Artificial Sequence BLyS binding polypeptide 438 Trp Tyr Asp
Pro Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly Lys 1 5 10 15 439 13
PRT Artificial Sequence BLyS binding polypeptide 439 Trp Tyr Asp
Pro Leu Thr Lys Leu Trp Leu Gly Gly Lys 1 5 10 440 17 PRT
Artificial Sequence BLyS binding polypeptide 440 Ala Asn Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Val Gly Gly 1 5 10 15 Lys 441
17 PRT Artificial Sequence BLyS binding polypeptide 441 Ala Asn Trp
Phe Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly 1 5 10 15 Lys
442 17 PRT Artificial Sequence BLyS binding polypeptide 442 Ala Asn
Trp Tyr Asp Pro Leu Thr Lys Leu Ser Leu Pro Asp Gly Gly 1 5 10 15
Lys 443 17 PRT Artificial Sequence BLyS binding polypeptide 443 Ala
Asn Trp Tyr Asp Pro Leu Thr Lys Leu Trp Phe Pro Asp Gly Gly 1 5 10
15 Lys 444 17 PRT Artificial Sequence BLyS binding polypeptide 444
Ala Asn Trp Tyr Asp Ser Leu Thr Lys Leu Trp Leu Pro Asp Gly Gly 1 5
10 15 Lys 445 585 PRT HomoSapiens 445 Asp Ala His Lys Ser Glu Val
Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro
Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310
315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val
Ser Thr Pro Thr Leu
Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys
Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp
Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu
Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470
475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro
Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe
Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys
Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala
Ser Gln Ala Ala Leu Gly Leu 580 585 446 4 PRT Artificial Sequence
recurring structural motif of BLyS binding polypeptides 446 Asp Xaa
Leu Thr 1 447 14 PRT Artificial Sequence BLyS binding polypeptide
447 Ala Xaa Xaa Xaa Asp Xaa Leu Thr Xaa Leu Xaa Xaa Xaa Xaa 1 5 10
448 10 PRT Artificial Sequence BLyS binding polypeptide 448 Xaa Xaa
Asp Xaa Leu Thr Xaa Leu Xaa Xaa 1 5 10 449 733 DNA Homo Sapiens 449
gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg
60 aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac
accctcatga 120 tctcccggac tcctgaggtc acatgcgtgg tggtggacgt
aagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg 240 aggagcagta caacagcacg
taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300 ggctgaatgg
caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360
agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc
420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct 480 atccaagcga catcgccgtg gagtgggaga gcaatgggca
gccggagaac aactacaaga 540 ccacgcctcc cgtgctggac tccgacggct
ccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcag gtggcagcag
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660 acaaccacta
cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720
gactctagag gat 733 450 16 PRT Artificial Sequence BLyS binding
polypeptide 450 Ala Gly Lys Glu Pro Cys Tyr Phe Tyr Trp Glu Cys Ala
Val Ser Gly 1 5 10 15 451 17 PRT Artificial Sequence BLyS binding
polypeptide 451 Ala Gly Val Pro Phe Cys Asp Leu Leu Thr Lys His Cys
Phe Glu Ala 1 5 10 15 Gly 452 20 PRT Artificial Sequence BLyS
binding polypeptide 452 Gly Ser Ser Arg Leu Cys His Met Asp Glu Leu
Thr His Val Cys Val 1 5 10 15 His Phe Ala Pro 20 453 21 PRT
Artificial Sequence BLyS binding polypeptide 453 Gly Asp Gly Gly
Asn Cys Tyr Thr Asp Ser Leu Thr Lys Leu His Phe 1 5 10 15 Cys Met
Gly Asp Glu 20 454 14 PRT Artificial Sequence BLyS binding
polypeptide 454 Gly Tyr Asp Val Leu Thr Lys Leu Tyr Phe Val Pro Gly
Gly 1 5 10 455 14 PRT Artificial Sequence BLyS binding polypeptide
455 Trp Thr Asp Ser Leu Thr Gly Leu Trp Phe Pro Asp Gly Gly 1 5 10
456 12 PRT Artificial Sequence BLyS binding polypeptide 456 Trp Tyr
Asp Pro Leu Thr Lys Leu Trp Leu Pro Asp 1 5 10 457 10 PRT
Artificial Sequence BLyS binding polypeptide 457 Trp Tyr Asp Pro
Leu Thr Lys Leu Trp Leu 1 5 10 458 14 PRT Artificial Sequence BLyS
binding polypeptide 458 Ala Asn Trp Tyr Asp Pro Leu Thr Lys Leu Ser
Leu Pro Asp 1 5 10
* * * * *
References