U.S. patent application number 11/932823 was filed with the patent office on 2008-08-14 for albumin fusion proteins.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Adam Bell, Jason B. Bock, William A. Haseltine, David LaFleur, Michael W. Laird, Paul A. Moore, Craig A. Rosen, Yanggu Shi, Mani Subramanian, Douglas B. Woods.
Application Number | 20080194481 11/932823 |
Document ID | / |
Family ID | 40591364 |
Filed Date | 2008-08-14 |
United States Patent
Application |
20080194481 |
Kind Code |
A1 |
Rosen; Craig A. ; et
al. |
August 14, 2008 |
Albumin Fusion Proteins
Abstract
The present invention encompasses albumin fusion proteins.
Nucleic acid molecules encoding the albumin fusion proteins of the
invention are also encompassed by the invention, as are vectors
containing these nucleic acids, host cells transformed with these
nucleic acids vectors, and methods of making the albumin fusion
proteins of the invention and using these nucleic acids, vectors,
and/or host cells. Additionally the present invention encompasses
pharmaceutical compositions comprising albumin fusion proteins and
methods of treating, preventing, or ameliorating diseases,
disorders or conditions using albumin fusion proteins of the
invention.
Inventors: |
Rosen; Craig A.;
(Laytonsville, MD) ; Bell; Adam; (Germantown,
MD) ; Moore; Paul A.; (North Bethesda, MD) ;
Shi; Yanggu; (Gaithersburg, MD) ; LaFleur; David;
(Washington, DC) ; Bock; Jason B.; (North Potomac,
MD) ; Laird; Michael W.; (San Ramon, CA) ;
Haseltine; William A.; (Washington, DC) ; Woods;
Douglas B.; (Gaithersburg, MD) ; Subramanian;
Mani; (Ellicott City, MD) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Human Genome Sciences, Inc.
|
Family ID: |
40591364 |
Appl. No.: |
11/932823 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11495624 |
Jul 31, 2006 |
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11932823 |
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PCT/US2005/004041 |
Feb 9, 2005 |
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11495624 |
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11175690 |
Jul 7, 2005 |
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11495624 |
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PCT/US2004/001369 |
Jan 20, 2004 |
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11175690 |
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11429276 |
May 8, 2006 |
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11495624 |
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10775204 |
Feb 11, 2004 |
7141547 |
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11429276 |
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PCT/US2002/040891 |
Dec 23, 2002 |
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10775204 |
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60707521 |
Aug 12, 2005 |
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60712386 |
Aug 31, 2005 |
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60732724 |
Nov 3, 2005 |
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60776914 |
Feb 28, 2006 |
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60781361 |
Mar 13, 2006 |
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60810182 |
Jun 2, 2006 |
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60813682 |
Jun 15, 2006 |
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60542274 |
Feb 9, 2004 |
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60549901 |
Mar 5, 2004 |
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60556906 |
Mar 29, 2004 |
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60636603 |
Dec 17, 2004 |
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60441305 |
Jan 22, 2003 |
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60453201 |
Mar 11, 2003 |
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May 2, 2003 |
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May 23, 2003 |
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Jun 6, 2003 |
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Current U.S.
Class: |
514/6.9 ;
435/252.3; 435/254.2; 435/320.1; 435/325; 435/69.7; 530/362;
536/23.4 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
1/16 20180101; A61P 25/00 20180101; A61P 35/00 20180101; A61P 9/00
20180101; A61P 37/08 20180101; A61P 9/04 20180101; A61P 25/36
20180101; A61P 13/12 20180101; A61P 25/30 20180101; A61P 37/02
20180101; A61P 11/00 20180101; A61P 31/12 20180101; A61P 9/10
20180101; A61P 3/00 20180101; A61P 3/10 20180101; A61P 9/08
20180101; C07K 14/58 20130101; C07K 2319/01 20130101; A61P 1/00
20180101; C07K 14/765 20130101; A61P 3/04 20180101; A61P 25/28
20180101 |
Class at
Publication: |
514/12 ; 530/362;
536/23.4; 435/320.1; 435/325; 435/254.2; 435/252.3; 435/69.7 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C07K 14/00 20060101 C07K014/00; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 5/06 20060101
C12N005/06; A61P 9/00 20060101 A61P009/00; A61P 1/00 20060101
A61P001/00; A61P 25/00 20060101 A61P025/00; C12N 1/00 20060101
C12N001/00; C12N 1/20 20060101 C12N001/20; C12P 21/04 20060101
C12P021/04 |
Claims
1-30. (canceled)
31. An albumin fusion protein comprising an atrial natriuretic
polypeptide (ANP) fused to albumin, wherein the fusion protein has
ANP activity, and wherein: (a) the ANP polypeptide is selected from
a wild-type ANP, an ANP fragment, and an ANP variant, and (b) the
albumin is selected from a wild-type albumin, an albumin fragment,
and an albumin variant, wherein the albumin increases the serum
plasma half-life of the ANP polypeptide.
32. The albumin fusion protein of claim 31, wherein the ANP
polypeptide comprises the amino acid sequence selected from SEQ ID
NO: 302 and SEQ ID NO: 560.
33. The albumin fusion protein of claim 31, wherein the albumin is
selected from: a) human albumin; b) cow albumin; c) sheep albumin;
d) pig albumin; e) hen albumin; f) salmon albumin; j) an albumin
fragment; k) an albumin variant; l) SEQ ID NO:1; m) a fragment of
albumin consisting of amino acids 1-194 of SEQ ID NO:1; n) a
fragment of albumin consisting of amino acids 195-387 of SEQ ID
NO:1; o) a fragment of albumin consisting of amino acids 388-585 of
SEQ ID NO:1; p) a fragment of albumin consisting of amino acids
1-387 of SEQ ID NO:1; q) a fragment of albumin consisting of amino
acids 195-585 of SEQ ID NO:1; r) a fragment of albumin consisting
of amino acids 1-105 of SEQ ID NO:1; s) a fragment of albumin
consisting of amino acids 120-194 of SEQ ID NO:1; t) a fragment of
albumin consisting of amino acids 195-291 of SEQ ID NO:1; u) a
fragment of albumin consisting of amino acids 316-387 of SEQ ID
NO:1; v) a fragment of albumin consisting of amino acids 388-491 of
SEQ ID NO:1; w) a fragment of albumin consisting of amino acids
512-585 of SEQ ID NO:1; x) a fragment of albumin that is 10, 15,
20, 25, 30, 50, 100, or 150 amino acids in length; y) a fragment of
albumin consisting of one or more domains of albumin; and z) a
variant of SEQ ID NO:1 selected from i) L407A; ii) L408V; iii)
V409A; iv) R410A; v) K413Q; and vi) K414Q.
34. The albumin fusion protein of claim 31, wherein the albumin
fusion protein further comprises a leader sequence.
35. The albumin fusion protein of claim 34, wherein the leader
sequence is selected from: a) HSA; b) kex2; and c) a fusion of HSA
and kex2.
36. The albumin fusion protein of claim 31, further comprising one
or more additional therapeutic polypeptides.
37. The albumin fusion protein of claim 31 comprising the amino
acid sequence selected from SEQ ID NO: 227 and SEQ ID NO: 524.
38. The albumin fusion protein of claim 31 consisting of the amino
acid sequence selected from SEQ ID NO: 227 and SEQ ID NO: 524.
39. A nucleotide sequence encoding the albumin fusion protein of
claim 37.
40. The nucleotide sequence of claim 39, comprising a ANP
nucleotide sequence selected from SEQ ID NO: 152 and SEQ ID NO:
488.
41. A construct expressing the albumin fusion protein of claim
31.
42. The construct of claim 41 selected from construct ID 3484 and
construct ID 4174.
43. A host cell expressing the albumin fusion protein of claim
31.
44. The host cell of claim 43, wherein the host cell is a mammalian
cell, a yeast cell or a prokaryotic cell.
45. A method for expressing an albumin fusion protein comprising
culturing the host cell of claim 43 under conditions suitable for
the expression of the albumin fusion protein and recovering the
albumin fusion protein.
46. An albumin fusion protein expressed by the host cell of claim
43, wherein the albumin fusion protein is glycosylated,
non-glycosylated or a glycosylation isomer.
47. A composition comprising the albumin fusion protein of claim 31
and a pharmaceutically acceptable carrier.
48. A kit comprising the composition of claim 47 and instructions
for the use thereof.
49. A method of treating, preventing, diagnosing or ameliorating a
disease, disorder or condition in a subject in need thereof
comprising administering an effective amount of an albumin fusion
protein comprising a ANP polypeptide fused to albumin, wherein the
fusion protein has ANP activity, and wherein: (a) the ANP
polypeptide is selected from a wild-type ANP, an ANP fragment, and
an ANP variant, and (b) the albumin is selected from a wild-type
albumin, an albumin fragment, and an albumin variant, wherein the
albumin increases the serum plasma half-life of the ANP
polypeptide.
50. The method of claim 49, wherein the disease, disorder or
condition is selected from hypertension; salt-sensitive
hypertension; congestive heart failure; angina pectoris; peripheral
artery disease; diabetic nephropathy; stroke; kidney failure; acute
and/or chronic renal failure; acute tubular necrosis; acute renal
failure; renal disease; renal glomerular disease; excess fluid in
tissues; hypotension; cardiac volume overload; cardiac
decompensation; left ventricular dysfunction; dyspnea; treatment
for elevated aldosterone levels; vasoconstriction; impaired cardiac
output and/or hypertension; cardiovascular disease; cardiac
failure; myocardial reperfusion injury; left ventricular remodeling
and post-myocardial infarction.
51. An albumin fusion protein comprising a butyrylcholinesterase
polypeptide (BChE) fused to albumin, wherein the fusion protein has
BChE activity, and wherein: (a) the BChE polypeptide is selected
from a wild-type BChE, a BChE fragment, and a BChE variant, and (b)
the albumin is selected from a wild-type albumin, an albumin
fragment, and an albumin variant, wherein the albumin increases the
serum plasma half-life of the BChE polypeptide.
52. The albumin fusion protein of claim 51, wherein the BChE
polypeptide comprises the amino acid sequence selected from SEQ ID
NO: 708 and SEQ ID NO: 709.
53. The albumin fusion protein of claim 51, wherein the albumin is
selected from: a) human albumin; b) cow albumin; c) sheep albumin;
d) pig albumin; e) hen albumin; f) salmon albumin; j) an albumin
fragment; k) an albumin variant; l) SEQ ID NO:1; m) a fragment of
albumin consisting of amino acids 1-194 of SEQ ID NO:1; n) a
fragment of albumin consisting of amino acids 195-387 of SEQ ID
NO:1; o) a fragment of albumin consisting of amino acids 388-585 of
SEQ ID NO:1; p) a fragment of albumin consisting of amino acids
1-387 of SEQ ID NO:1; q) a fragment of albumin consisting of amino
acids 195-585 of SEQ ID NO:1; r) a fragment of albumin consisting
of amino acids 1-105 of SEQ ID NO:1; s) a fragment of albumin
consisting of amino acids 120-194 of SEQ ID NO:1; t) a fragment of
albumin consisting of amino acids 195-291 of SEQ ID NO:1; u) a
fragment of albumin consisting of amino acids 316-387 of SEQ ID
NO:1; v) a fragment of albumin consisting of amino acids 388-491 of
SEQ ID NO:1; w) a fragment of albumin consisting of amino acids
512-585 of SEQ ID NO:1; x) a fragment of albumin that is 10, 15,
20, 25, 30, 50, 100, or 150 amino acids in length; y) a fragment of
albumin consisting of one or more domains of albumin; and z) a
variant of SEQ ID NO:1 selected from i) L407A; ii) L408V; iii)
V409A; iv) R410A; v) K413Q; and vi) K414Q.
54. The albumin fusion protein of claim 51, wherein the albumin
fusion protein further comprises a leader sequence.
55. The albumin fusion protein of claim 54, wherein the leader
sequence is selected from: a) HSA; b) kex2; c) a consensus signal
sequence; and d) a fusion of at least one of the leader sequences
selected from HSA, kex2, and a consensus signal sequence.
56. The albumin fusion protein of claim 51, further comprising one
or more additional therapeutic polypeptides.
57. The albumin fusion protein of claim 51 comprising the amino
acid sequence selected from SEQ ID NO: 669 and SEQ ID NO: 670.
58. The albumin fusion protein of claim 51 consisting of the amino
acid sequence selected from SEQ ID NO: 669 and SEQ ID NO: 670.
59. A nucleotide sequence encoding the albumin fusion protein of
claim 57.
60. The nucleotide sequence of claim 59, comprising a BChE
nucleotide sequence selected from SEQ ID NO: 630 and SEQ ID NO:
631.
61. A construct expressing the albumin fusion protein of claim
51.
62. The construct of claim 41 selected from construct ID 4258 and
construct ID 4259.
63. A host cell expressing the albumin fusion protein of claim
51.
64. The host cell of claim 63, wherein the host cell is a mammalian
cell, a yeast cell or a prokaryotic cell.
65. A method for expressing an albumin fusion protein comprising
culturing the host cell of claim 63 under conditions suitable for
the expression of the albumin fusion protein and recovering the
albumin fusion protein.
66. An albumin fusion protein expressed by the host cell of claim
63, wherein the albumin fusion protein is glycosylated,
non-glycosylated or a glycosylation isomer.
67. A composition comprising the albumin fusion protein of claim 51
and a pharmaceutically acceptable carrier.
68. A kit comprising the composition of claim 67 and instructions
for the use thereof.
69. A method of treating, preventing, diagnosing or ameliorating a
disease, disorder or condition in a subject in need thereof
comprising administering an effective amount of an albumin fusion
protein comprising a BChE polypeptide fused to albumin, wherein the
fusion protein has BChE activity, and wherein: (a) the BChE
polypeptide is selected from a wild-type BChE, a BChE fragment, and
a BChE variant, and (b) the albumin is selected from a wild-type
albumin, an albumin fragment, and an albumin variant, wherein the
albumin increases the serum plasma half-life of the BChE
polypeptide.
70. The method of claim 69, wherein the disease, disorder or
condition is selected from detoxification for cocaine overdose,
suxamethonium sensitivity and apnea.
71. The method of claim 70, wherein the disease, disorder or
condition is cocaine overdose.
72. The method of claim 70, wherein the BChE polypeptide comprises
the following amino acid substitutions: A227S, S315G, A356W and
Y360G.
73. The albumin fusion of claim 52, wherein the BChE polypeptide
BChE polypeptide comprises the following amino acid substitutions:
A227S, S315G, A356W and Y360G.
74. A method of treating, preventing, diagnosing or ameliorating a
disease, disorder or condition in a subject in need thereof
comprising administering an effective amount of an albumin fusion
protein, wherein the disease, disorder or condition comprises
Alzheimer's disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. application Ser. No. 11/495,624, which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application Nos.
60/707,521, filed Aug. 12, 2005; 60/712,386, filed Aug. 31, 2005;
60/732,724, filed Nov. 3, 2005; 60/776,914, filed Feb. 28, 2006;
60/781,361, filed Mar. 13, 2006; 60/810,182, filed Jun. 2, 2006;
and 60/813,682, filed Jun. 15, 2006. U.S. application Ser. No.
11/495,624 is also a continuation-in-part of International
Application No. PCT/US2005/004041, filed Feb. 9, 2005, which claims
benefit under 119(e) of U.S. Provisional Application Nos.
60/542,274, filed Feb. 9, 2004, 60/549,901, filed Mar. 5, 2004,
60/556,906, filed Mar. 29, 2004, and 60/636,603, filed Dec. 17,
2004. U.S. application Ser. No. 11/495,624 is also a
continuation-in-part of U.S. application Ser. No. 11/175,690, filed
Jul. 7, 2005, which is a continuation of International Application
No. PCT/2004/001369, filed Jan. 20, 2004, which claims benefit
under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Application Nos.
60/441,305, filed Jan. 22, 2003; 60/453,201, filed Mar. 11, 2003;
60/467,222, filed May 2, 2003; 60/472,816, filed May 23, 2003;
60/476,267, filed Jun. 6, 2003; 60/505,172, filed Sep. 24, 2003;
and 60/506,746, filed Sep. 30, 2003. U.S. application Ser. No.
11/495,624 is also a continuation-in-part of U.S. application Ser.
No. 11/429,276, filed May 8, 2006, which is a continuation of U.S.
application Ser. No. 10/775,204, filed Feb. 11, 2004, which is a
continuation of International Application No. PCT/US2002/40891,
filed Dec. 23, 2002, which claims benefit under 35 U.S.C. .sctn.
119(e) U.S. Provisional Application Nos. 60/341,811, filed Dec. 21,
2001; 60/350,358, filed Jan. 24, 2002; 60/351,360, filed Jan. 28,
2002; 60/359,370, filed Feb. 26, 2002; 60/360,000, filed Feb. 28,
2002; 60/367,500, filed Mar. 27, 2002; 60/370,227, filed Apr. 8,
2002; 60/378,950, filed May 10, 2002; 60/382,617, filed May 24,
2002; 60/383,123, filed May 28, 2002; 60/385,708, filed Jun. 5,
2002; 60/394,625, filed Jul. 10, 2002; 60/398,008, filed Jul. 24,
2002; 60/402,131, filed Aug. 9, 2002; 60/402,708, filed Aug. 13,
2002; 60/411,426, filed Sep. 18, 2002; 60/411,355, filed Sep. 18,
2002; 60/414,984, filed Oct. 2, 2002; 60/417,611, filed Oct. 11,
2002; 60/420,246, filed Oct. 23, 2002; and 60/423,623, filed Nov.
5, 2002. All of the above listed applications are incorporated by
reference herein in their entireties.
REFERENCE TO SEQUENCE LISTING ON COMPACT DISC
[0002] This application refers to a "Sequence Listing," which was
provided with U.S. application Ser. No. 11/495,624 as an electronic
document on three identical compact discs (CD-R), labeled "Copy 1,"
"Copy 2," and "CRF." These compact discs each contain the file
"PF617 Sequence Listing.txt" (1,193,482 bytes, created on Jul. 28,
2006), which is incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The invention relates generally to Therapeutic proteins
(including, but not limited to, at least one polypeptide, antibody,
peptide, or fragment and variant thereof) fused to albumin or
fragments or variants of albumin. The invention encompasses
polynucleotides encoding therapeutic albumin fusion proteins,
therapeutic albumin fusion proteins, compositions, pharmaceutical
compositions, formulations and kits. Host cells transformed with
the polynucleotides encoding therapeutic albumin fusion proteins
are also encompassed by the invention, as are methods of making the
albumin fusion proteins of the invention using these
polynucleotides, and/or host cells.
[0004] Human serum albumin (HSA, or HA), a protein of 585 amino
acids in its mature form (as shown in FIG. 1 (SEQ ID NO:1)), is
responsible for a significant proportion of the osmotic pressure of
serum and also functions as a carrier of endogenous and exogenous
ligands. At present, HA for clinical use is produced by extraction
from human blood. The production of recombinant HA (rHA) in
microorganisms has been disclosed in EP 330 451 and EP 361 991.
[0005] Therapeutic proteins in their native state or when
recombinantly produced, such as interferons and growth hormones,
are typically labile molecules exhibiting short shelf-lives,
particularly when formulated in aqueous solutions. The instability
in these molecules when formulated for administration dictates that
many of the molecules must be lyophilized and refrigerated at all
times during storage, thereby rendering the molecules difficult to
transport and/or store. Storage problems are particularly acute
when pharmaceutical formulations must be stored and dispensed
outside of the hospital environment.
[0006] Few practical solutions to the storage problems of labile
protein molecules have been proposed. Accordingly, there is a need
for stabilized, long lasting formulations of proteinaceous
therapeutic molecules that are easily dispensed, preferably with a
simple formulation requiring minimal post-storage manipulation.
[0007] Upon in vivo administration, therapeutic proteins in their
native state or when recombinantly produced, such as interferons
and growth hormones, exhibit a short plasma stability due to rapid
clearance from the bloodstream. Accordingly, the therapeutic
effects provided by these proteins are also short-lived. Thus, in
order to sustain their desired therapeutic effect in vivo, the
rapid clearance of these proteins from the blood dictates that the
therapeutic molecules must be administered more frequently or at a
higher dose. However, increasing the dosing schedule for
administration of the therapeutic protein often results in an
increase in injection site reactions, side-effects, and toxicity in
the patient. Similarly, administration of the therapeutic protein
at a higher dose also commonly results in an increase in toxicity
and side-effects in the patient.
[0008] The few practical solutions to increasing plasma stability
of therapeutic molecules that have been proposed, including
chemical conjugation, have provided limited benefit to the patient.
Generally, in most cases, these chemically modified therapeutic
molecules are still administered on a frequent dosing schedule,
retaining significant injection site reactions, side-effects, and
toxicity in patients. Accordingly, there is a need for an
stabilized form of therapeutic molecules that retains a higher
plasma stability in vivo than the native or recombinantly produced
therapeutic alone and can be administered less frequently, thereby
decreasing potential side-effects to the patient.
SUMMARY OF THE INVENTION
[0009] The present invention encompasses albumin fusion proteins
comprising a Therapeutic protein (e.g., a polypeptide, antibody, or
peptide, or fragment or variant thereof) fused to albumin or a
fragment (portion) or variant of albumin. The present invention
also encompasses polynucleotides comprising, or alternatively
consisting of, nucleic acid molecules encoding a Therapeutic
protein (e.g., a polypeptide, antibody, or peptide, or fragment or
variant thereof) fused to albumin or a fragment (portion) or
variant of albumin. The present invention also encompasses
polynucleotides, comprising, or alternatively consisting of,
nucleic acid molecules encoding proteins comprising a Therapeutic
protein (e.g., a polypeptide, antibody, or peptide, or fragment or
variant thereof) fused to albumin or a fragment (portion) or
variant of albumin, that is sufficient to prolong the shelf life of
the Therapeutic protein, to increase the plasma stability of the
Therapeutic protein compared to its unfused state, and/or stabilize
the Therapeutic protein and/or its activity in solution (or in a
pharmaceutical composition) in vitro and/or in vivo. Albumin fusion
proteins encoded by a polynucleotide of the invention are also
encompassed by the invention, as are host cells transformed with
polynucleotides of the invention, and methods of making the albumin
fusion proteins of the invention and using these polynucleotides of
the invention, and/or host cells.
[0010] In a preferred aspect of the invention, albumin fusion
proteins include, but are not limited to, those described in Table
2 and the polynucleotides encoding such proteins.
[0011] The invention also encompasses pharmaceutical formulations
comprising an albumin fusion protein of the invention and a
pharmaceutically acceptable diluent or carrier. Such formulations
may be in a kit or container. Such kit or container may be packaged
with instructions pertaining to the extended shelf life of the
Therapeutic protein. Such formulations may be used in methods of
treating, preventing, ameliorating or diagnosing a disease or
disease symptom in a patient, preferably a mammal, most preferably
a human, comprising the step of administering the pharmaceutical
formulation to the patient.
[0012] In other embodiments, the present invention encompasses
methods of preventing, treating, or ameliorating a disease or
disorder. In preferred embodiments, the present invention
encompasses a method of treating a disease or disorder listed in
the "Preferred Indication: Y" column of Table 1 comprising
administering to a patient in which such treatment, prevention or
amelioration is desired an albumin fusion protein of the invention
that comprises a Therapeutic protein or portion corresponding to a
Therapeutic protein (or fragment or variant thereof) disclosed in
the "Therapeutic Protein: X" column of Table 1 (in the same row as
the disease or disorder to be treated as listed in the "Preferred
Indication: Y" column of Table 1) in an amount effective to treat,
prevent or ameliorate the disease or disorder.
[0013] In one embodiment, an albumin fusion protein described in
Table 1 or 2 has extended shelf life.
[0014] In a second embodiment, an albumin fusion protein described
in Table 1 or 2 is more stable than the corresponding unfused
Therapeutic molecule described in Table 1.
[0015] The present invention further includes transgenic organisms
modified to contain the nucleic acid molecules of the invention
(including, but not limited to, the polynucleotides described in
Tables 1 and 2), preferably modified to express an albumin fusion
protein of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A-D shows the amino acid sequence of the mature form
of human albumin (SEQ ID NO:1) and a polynucleotide encoding it
(SEQ ID NO:2). Nucleotides 1 to 1755 of SEQ ID NO:2 encode the
mature form of human albumin (SEQ ID NO:1).
[0017] FIG. 2 shows the restriction map of the pPPC0005 cloning
vector ATCC deposit PTA-3278.
[0018] FIG. 3 shows the restriction map of the pSAC35 yeast S.
cerevisiae expression vector (Sleep et al., BioTechnology 8:42
(1990)).
[0019] FIG. 4 compares the anti-proliferative activity of IFN
albumin fusion protein encoded by CID 3165 (CID 3165 protein) and
recombinant IFNa (rIFNa) on Hs294T melanoma cells. The cells were
cultured with varying concentrations of either CID 3165 protein or
rIFNa and proliferation was measured by BrdU incorporation after 3
days of culture. CID 3165 protein caused measurable inhibition of
cell proliferation at concentrations above 10 ng/ml with 50%
inhibition achieved at approximately 200 ng/ml. (.box-solid.)=CID
3165 protein, (.diamond-solid.)=rIFNa.
[0020] FIG. 5 shows the effect of various dilutions of IFNa albumin
fusion proteins on SEAP activity in the ISRE-SEAP/293F reporter
cells. One preparation of IFNa fused upstream of albumin
(.diamond-solid.) was tested, as well as two different preparations
of IFNa fused downstream of albumin ( ) and (.box-solid.).
[0021] FIG. 6 shows the effect of time and dose of IFNa albumin
fusion protein encoded by DNA comprised in construct 2249 (CID 2249
protein) on the mRNA level of OAS (p41) in treated monkeys (see
Example 76). Per time point: first bar=Vehicle control, 2.sup.nd
bar=30 ug/kg CID 2249 protein day 1 iv, third bar=30 ug/kg CID 2249
protein day 1 sc, 4.sup.th bar=300 ug/kg CID 2249 protein day 1 sc,
5.sup.th bar=40 ug/kg recombinant IFNa day 1, 3 and 5 sc.
[0022] FIG. 7 shows the dose-response relationship of BNP albumin
fusion proteins encoded by DNA comprised in constructs CID 3691 and
3618 (CID 3691 and 3618 protein) on activating cGMP formation in
NPR-A/293F reporter cells (see Examples 78 and 79). Both BNP
peptide (.box-solid.), as well as, two different preparations of
BNP fused upstream of albumin (.quadrature.) and ( ) were
tested.
[0023] FIG. 8 shows the effect of BNP albumin fusion protein on
mean arterial pressure in spontaneously hypertensive rats (see
Example 78). Vehicle (.quadrature.), BNP peptide ( ), or BNP
albumin fusion protein (.largecircle.) were delivered via tail vein
injection. Systolic and diastolic blood pressures were recorded by
cuff-tail method.
[0024] FIG. 9 shows the plasma cGMP levels in eleven- to
12-week-old male C57/BL6 mice after intravenous injection of
recombinant BNP peptide ( ) or BNP albumin fusion protein (O (see
Example 78). cGMP levels were determined from plasma prepared from
tail bleeds collected at several time points after intravenous
injection.
[0025] FIG. 10 shows the dose-response relationship of BNP peptide
and BNP albumin fusion proteins encoded by DNA comprised in
constructs CID 3796 and 3959 on activating cGMP formation in
NPR-A/293F reporter cells (see Example 80). Both BNP peptide
(.box-solid.), as well as, two different preparations comprising
BNP fused downstream of albumin, (.quadrature.) and (.diamond.)
were tested.
[0026] FIG. 11A shows the dose-response relationship of BNP and ANP
peptides with or without treatment of neprilysin for 24 hours on
activating cGMP formation in NPR-A/293F reporter cells (see Example
81).
[0027] FIG. 11B shows the dose-response relationship of ANP peptide
on activating cGMP formation in NPR-A/293F reporter cells following
treatment of neprilysin or control MES buffer for 20 minutes, 1
hour, or 24 hours (see Example 81).
[0028] FIG. 11C shows the dose-response relationship of ANP albumin
fusion protein comprising ANP fused upstream of albumin and encoded
by DNA comprised in construct CID3484 on activating cGMP formation
in NPR-A/293F reporter cells following treatment of neprilysin or
control MES buffer for 20 minutes, 1 hour, or 24 hours (see Example
81).
[0029] FIG. 11D shows the percentage of intact natriuretic peptides
following treatment with neprilysin for the specified time. Both
ANP and BNP peptides, as well as, two albumin fusion proteins
comprising BNP fused upstream of albumin via tripartite glycines
(CID 3809) and ANP fused upstream to albumin (CID 3484) were tested
(see Example 81).
[0030] FIG. 12 shows the reduction in HCV RNA titer, as measured by
median HCV RNA change (log.sub.10 IU/ml), in patients infected with
chronic hepatitis C genotype 1 and who have previously failed to
respond to at least one treatment regimen of pegylated interferon
alpha in combination with ribavirin (PEG-RBV) (nonresponders)
following treatment with HSA-IFN.alpha.2b in combination with
ribavirin for 0 to 24 weeks.
[0031] FIGS. 13A and B show the effect of HSA-BNP (Construct ID
#3959) on plasma and urine cGMP levels, respectively following
administration of an 5 mg/kg IV bolus in normal healthy pigs
(n=4-6/group). Asterisks indicate significant differences in cGMP
levels from vehicle (p<0.05).
[0032] FIG. 14A shows the effect of administration of an
intravenous bolus of 2 mg/kg or 6 mg/kg HSA-BNP (Construct ID
#3959) on end-diastolic diameter change in a porcine experimental
heart failure model (n=10/group). Heart failure was induced in the
pig by ventricular pacing. End diastolic diameter was measure by
echocardiography. Significant changes (p<0.05) from vehicle or
baseline are indicated (& and #, respectively).
[0033] FIG. 14B shows the effect of administration of an
intravenous bolus of 2 mg/kg or 6 mg/kg HSA-BNP (Construct ID
#3959) on fractional shortening in a porcine experimental heart
failure model (n=10/group). Heart failure was induced in the pig by
ventricular pacing. Significant changes (p<0.05) from vehicle or
baseline are indicated (& and #, respectively).
[0034] FIGS. 15A-H show the hemodynamic effects of HSA-BNP
(Construct ID #3959) administered via a single intravenous bolus at
0.5 mg/kg or 5 mg/kg in a normal dog model. Cardiac output (CO),
mean arterial pressure (MAP), pulmonary capillary wedge pressure
(PCWP) and pulmonary arterial pressure (PAP) were measured at
baseline prior to intravenous bolus of 0.5 mg/kg or 5 mg/kg HSA-BNP
(Construct ID #3959) and at 30, 60, 90, 150, 210, and 270
post-infusion in anesthesized normal mongrels (n=8/group).
Asterisks indicate statistically significant changes from baseline
(p<0.05).
[0035] FIGS. 16A-H show the renal effects of HSA-BNP (Construct ID
#3959) administered via a single intravenous bolus at 0.5 mg/kg or
5 mg/kg in a normal dog model. Urine flow (rate/30 minute
collection), sodium excretion, renal blood flow, and glomerular
filtration rate (GFR) were measured at baseline prior to
intravenous bolus of 0.5 mg/kg or 5 mg/kg HSA-BNP (Construct ID
#3959) and at 30, 60, 90, 150, 210, and 270 post-infusion in
anesthesized normal mongrels (n=8/group). Asterisks indicate
statistically significant changes from baseline (p<0.05).
[0036] FIGS. 17A-F show the hormonal effects of HSA-BNP (Construct
ID #3959) administered via a single intravenous bolus at 0.5 mg/kg
or 5 mg/kg in a normal dog model. Plasma aldosterone, renin, and
angiotensin II levels were measured at baseline prior to
intravenous bolus of 0.5 mg/kg or 5 mg/kg HSA-BNP (Construct ID
#3959) and at 30, 60, 90, 150, 210, and 270 post-infusion in
anesthesized normal mongrels (n=8/group). Asterisks indicate
statistically significant changes from baseline (p<0.05).
[0037] FIGS. 18A-C show the effect of a single intravenous bolus of
5 mg/kg HSA-BNP (Construct ID #3959) on systolic and mean arterial
blood pressure in normal, healthy, awake beagles surgically
implanted with a Data Sciences International radiotelemetry
transmitter, which had systemic arterial blood pressure, heart rate
and ECG data collection capabilities. Change from baseline of
systolic blood pressure (FIG. 18A), difference in mean systolic
blood pressure (FIG. 18B), and change from baseline in mean
arterial pressures (FIG. 18C) over 48 hours of continuous data
recording following infusion are presented. Asterisks indicate a
statistically significant difference in baseline-adjusted mean
values for 5 mg/kg HSA-BNP (Construct ID #3959) compared to vehicle
(p<0.05).
[0038] FIGS. 19A and B show a comparison of the effect of an
intravenous bolus of 0.02 mg/kg unfused BNP peptide and a
subcutaneous injection of 10 mg/kg HSA-BNP (Construct ID #3959) on
systemic blood pressure in normal, healthy, awake beagles
surgically implanted with a Data Sciences International
radiotelemetry transmitter, which had systemic arterial blood
pressure, heart rate and ECG data collection capabilities. Change
from baseline of systolic blood pressure over 48 hours of
continuous data recording following administration of BNP (FIG.
19A) and HSA-BNP (Construct ID #3959) are presented. Asterisks
indicate a statistically significant difference in
baseline-adjusted mean values for 5 mg/kg HSA-BNP (Construct ID
#3959) compared to vehicle (p<0.05).
[0039] FIG. 20 shows the nucleic acid sequence and amino acid
sequence of a BChE-albumin fusion. The fusion is discussed in
Example 90.
[0040] FIG. 21. Catalytic power of wild-type BChE (WT) and cocaine
hydrolases derived from this enzyme. Values are expressed as kcat
(molecules of natural, (-)-cocaine hydrolyzed per min per molecule
of enzyme). Amino acid substitutions in the BChE mutants are:
A328W/Y332A (CocE, Sun et al., 2002); F227A/S287G/A328W/Y332A
(AME359, Pancook et al 2003); A328W/Y332G/S287G/A199S (CocH, Pan et
al, 2004).
[0041] FIG. 22. Purification and titration of Albu-CocH. A)
Coomassie-Blue stained SDS electrophoresis gel of final product
(M=markers, R=sample under reducing conditions, NR sample under
non-reducing conditions). B) Assessment of purify by size-exclusion
chromatography (SEC-HPLC) and N-terminal sequencing. C) Active site
titration. Residual BChE activity was reduced in linear fashion
after overnight incubation with increasing sub-stoichiometric
amounts of the irreversible organophosphate cholinesterase
inhibitor, di-isopropylfluorophosphate (DFP). The X-axis intercept
with this typical batch (one of three) indicates approximately 7.7
pmol of active site serine residues (the putative DFP target). The
amount of enzyme protein was 0.72 .mu.g, equivalent to 8.5 pmol.
Thus, over 90% of the purified material was enzymatically
active.
[0042] FIG. 23. Stability of Albu-CocH in vivo. Representative time
course of plasma cocaine hydrolase activity in 1 of 5 rats injected
at zero-time with Albu-CocH, 3 mg/kg i.v. These data, fitted to a
double exponential decay equation, indicated a terminal elimination
half-life of 7.9 hr. The higher slope at early times suggests a
preliminary redistribution phase, which might represent enzyme
binding to tissue components, metabolic destruction, or limited
transcapillary passage into extracellular fluid.
[0043] FIG. 24. Blunting of cocaine-induced hypertension. Rats were
anesthetized with urethane (1.45 g/kg) for arterial cannulation.
Subsequently Albu-CocH was administered (filled circles, 3 mg/kg,
i.v.) or saline (open circles), followed by atropine (1 mg/kg) to
reduce vagal reflexes, and baseline pressure was recorded for 10
min. At zero time, the rats were challenged with cocaine (3.5
mg/kg) and at 10 min with norepinephrine (NE, 6 .mu.g/kg). Changes
in mean blood pressure are shown (mean .+-.SEM, 5 rats per
group).
[0044] FIG. 25. Prevention and rescue from cocaine overdose. A:
Percent incidence of arousal and seizures when cocaine (100 mg/kg
i.p.) was given 10 min after i.v. saline (n=10), Albu-CocH (n=6 per
dose), or wild type BChE (WT, n=3). B: effect of Albu-CocH (10
mg/kg) on the dose-response curve for seizures from cocaine
administered 10 min later (n=6 per group).
[0045] FIG. 26. Accelerated cocaine clearance. Plasma cocaine
levels are shown as a function of time after injection of cocaine
(30 .mu.Ci, 3.5 mg/kg, i.v.) into rats that 10 min earlier had
received Albu-CocH (3 mg/kg i.v--filled symbols, n=4) or saline
(empty symbols, n=4). Blood samples were drawn from the femoral
artery beginning 30 seconds (sec) after cocaine and were assayed
radiometrically. As shown here, plasma cocaine levels in control
rats declined slowly but in Albu-CocH-treated rats they dropped
nearly to the detection limit by the earliest sampling point (30
sec after drug injection).
[0046] FIG. 27. Reduced tissue accumulation of cocaine. Rats (n=4
per group) received 3H-cocaine (30 .mu.Ci, 3.5 mg/kg, i.v.) 10 min
after treatment with Albu-CocH (3 mg/kg, i.v.) or saline. Ten min
after the cocaine injections, brains, hearts, and plasma were
collected for analysis of cocaine and its metabolite, benzoic acid.
Treatment with Albu-CocH greatly lowered tissue burden. Intact
cocaine was nearly undetectable in hearts and plasma from the
enzyme-treated rats, where it was quantitatively replaced by the
metabolite, benzoic acid. The treatment effect was substantial in
brain as well, but smaller, consistent with the fact that nervous
tissue is a preferred site for cocaine uptake.
[0047] FIG. 28. Selective block of cocaine-primed reinstatement of
drug-seeking behavior. Fifteen rats that had previously
self-administered cocaine and extinguished when cocaine was
replaced with saline were primed with an i.v. injection of saline
(S), cocaine (C, 10 mg/kg) or amphetamine (A, 2 mg/kg) just before
each of twelve daily, 2-hr sessions. On days 4 and 6, they received
Albu-CocH enzyme (E), 2 mg/kg i.v., 2 hr beforehand. Data shown are
mean .+-.SEM of total responses on the previously active lever
(which had no consequences). Horizontal brackets indicate
statistical comparisons (* p<0.05; ** p<0.01).
DETAILED DESCRIPTION
Definitions
[0048] The following definitions are provided to facilitate
understanding of certain terms used throughout this
specification.
[0049] As used herein, "polynucleotide" refers to a nucleic acid
molecule having a nucleotide sequence encoding a fusion protein
comprising, or alternatively consisting of, at least one molecule
of albumin (or a fragment or variant thereof) joined in frame to at
least one Therapeutic protein X (or fragment or variant thereof); a
nucleic acid molecule having a nucleotide sequence encoding a
fusion protein comprising, or alternatively consisting of, the
amino acid sequence of SEQ ID NO:Y (as described in column 6 of
Table 2) or a fragment or variant thereof; a nucleic acid molecule
having a nucleotide sequence comprising or alternatively consisting
of the sequence shown in SEQ ID NO:X; a nucleic acid molecule
having a nucleotide sequence encoding a fusion protein comprising,
or alternatively consisting of, the amino acid sequence of SEQ ID
NO:Z; a nucleic acid molecule having a nucleotide sequence encoding
an albumin fusion protein of the invention generated as described
in Table 2 or in the Examples; a nucleic acid molecule having a
nucleotide sequence encoding a Therapeutic albumin fusion protein
of the invention, a nucleic acid molecule having a nucleotide
sequence contained in an albumin fusion construct described in
Table 2, or a nucleic acid molecule having a nucleotide sequence
contained in an albumin fusion construct deposited with the ATCC
(as described in Table 3).
[0050] As used herein, "albumin fusion construct" refers to a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of a Therapeutic
protein (or fragment or variant thereof); a nucleic acid molecule
comprising, or alternatively consisting of, a polynucleotide
encoding at least one molecule of albumin (or a fragment or variant
thereof) joined in frame to at least one polynucleotide encoding at
least one molecule of a Therapeutic protein (or fragment or variant
thereof) generated as described in Table 2 or in the Examples; or a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of a Therapeutic
protein (or fragment or variant thereof), further comprising, for
example, one or more of the following elements: (1) a functional
self-replicating vector (including but not limited to, a shuttle
vector, an expression vector, an integration vector, and/or a
replication system), (2) a region for initiation of transcription
(e.g., a promoter region, such as for example, a regulatable or
inducible promoter, a constitutive promoter), (3) a region for
termination of transcription, (4) a leader sequence, and (5) a
selectable marker. The polynucleotide encoding the Therapeutic
protein and albumin protein, once part of the albumin fusion
construct, may each be referred to as a "portion," "region" or
"moiety" of the albumin fusion construct.
[0051] The present invention relates generally to polynucleotides
encoding albumin fusion proteins; albumin fusion proteins; and
methods of treating, preventing, or ameliorating diseases or
disorders using albumin fusion proteins or polynucleotides encoding
albumin fusion proteins. As used herein, "albumin fusion protein"
refers to a protein formed by the fusion of at least one molecule
of albumin (or a fragment or variant thereof) to at least one
molecule of a Therapeutic protein (or fragment or variant thereof).
An albumin fusion protein of the invention comprises at least a
fragment or variant of a Therapeutic protein and at least a
fragment or variant of human serum albumin, which are associated
with one another by genetic fusion (i.e., the albumin fusion
protein is generated by translation of a nucleic acid in which a
polynucleotide encoding all or a portion of a Therapeutic protein
is joined in-frame with a polynucleotide encoding all or a portion
of albumin). The Therapeutic protein and albumin protein, once part
of the albumin fusion protein, may each be referred to as a
"portion", "region" or "moiety" of the albumin fusion protein
(e.g., a "Therapeutic protein portion" or an "albumin protein
portion"). In a highly preferred embodiment, an albumin fusion
protein of the invention comprises at least one molecule of a
Therapeutic protein X or fragment or variant of thereof (including,
but not limited to a mature form of the Therapeutic protein X) and
at least one molecule of albumin or fragment or variant thereof
(including but not limited to a mature form of albumin).
[0052] In a further preferred embodiment, an albumin fusion protein
of the invention is processed by a host cell and secreted into the
surrounding culture medium. Processing of the nascent albumin
fusion protein that occurs in the secretory pathways of the host
used for expression may include, but is not limited to signal
peptide cleavage; formation of disulfide bonds; proper folding;
addition and processing of carbohydrates (such as for example, N-
and O-linked glycosylation); specific proteolytic cleavages; and
assembly into multimeric proteins. An albumin fusion protein of the
invention is preferably in the processed form. In a most preferred
embodiment, the "processed form of an albumin fusion protein"
refers to an albumin fusion protein product which has undergone
N-terminal signal peptide cleavage, herein also referred to as a
"mature albumin fusion protein".
[0053] In several instances, a representative clone containing an
albumin fusion construct of the invention was deposited with the
American Type Culture Collection (herein referred to as
"ATCC.RTM."). Furthermore, it is possible to retrieve a given
albumin fusion construct from the deposit by techniques known in
the art and described elsewhere herein. The ATCC.RTM. is located at
10801 University Boulevard, Manassas, Va. 20110-2209, USA. The
ATCC.RTM. deposits were made pursuant to the terms of the Budapest
Treaty on the international recognition of the deposit of
microorganisms for the purposes of patent procedure.
[0054] In one embodiment, the invention provides a polynucleotide
encoding an albumin fusion protein comprising, or alternatively
consisting of, a Therapeutic protein and a serum albumin protein.
In a further embodiment, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, a Therapeutic
protein and a serum albumin protein. In a preferred embodiment, the
invention provides an albumin fusion protein comprising, or
alternatively consisting of, a Therapeutic protein and a serum
albumin protein encoded by a polynucleotide described in Table 2.
In a further preferred embodiment, the invention provides a
polynucleotide encoding an albumin fusion protein whose sequence is
shown as SEQ ID NO:Y in Table 2. In other embodiments, the
invention provides an albumin fusion protein comprising, or
alternatively consisting of, a biologically active and/or
therapeutically active fragment of a Therapeutic protein and a
serum albumin protein. In other embodiments, the invention provides
an albumin fusion protein comprising, or alternatively consisting
of, a biologically active and/or therapeutically active variant of
a Therapeutic protein and a serum albumin protein. In preferred
embodiments, the serum albumin protein component of the albumin
fusion protein is the mature portion of serum albumin. The
invention further encompasses polynucleotides encoding these
albumin fusion proteins.
[0055] In further embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
Therapeutic protein, and a biologically active and/or
therapeutically active fragment of serum albumin. In further
embodiments, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, a Therapeutic protein
and a biologically active and/or therapeutically active variant of
serum albumin. In preferred embodiments, the Therapeutic protein
portion of the albumin fusion protein is the mature portion of the
Therapeutic protein. In a further preferred embodiment, the
Therapeutic protein portion of the albumin fusion protein is the
extracellular soluble domain of the Therapeutic protein. In an
alternative embodiment, the Therapeutic protein portion of the
albumin fusion protein is the active form of the Therapeutic
protein. The invention further encompasses polynucleotides encoding
these albumin fusion proteins.
[0056] In further embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
biologically active and/or therapeutically active fragment or
variant of a Therapeutic protein and a biologically active and/or
therapeutically active fragment or variant of serum albumin. In
preferred embodiments, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, the mature
portion of a Therapeutic protein and the mature portion of serum
albumin. The invention further encompasses polynucleotides encoding
these albumin fusion proteins.
[0057] Therapeutic Proteins
[0058] As stated above, a polynucleotide of the invention encodes a
protein comprising or alternatively consisting of, at least a
fragment or variant of a Therapeutic protein and at least a
fragment or variant of human serum albumin, which are associated
with one another, preferably by genetic fusion.
[0059] An additional embodiment includes a polynucleotide encoding
a protein comprising or alternatively consisting of at least a
fragment or variant of a Therapeutic protein and at least a
fragment or variant of human serum albumin, which are linked with
one another by chemical conjugation.
[0060] As used herein, "Therapeutic protein" refers to proteins,
polypeptides, antibodies, peptides or fragments or variants
thereof, having one or more therapeutic and/or biological
activities. Therapeutic proteins encompassed by the invention
include but are not limited to, proteins, polypeptides, peptides,
antibodies, and biologics. (The terms peptides, proteins, and
polypeptides are used interchangeably herein.) It is specifically
contemplated that the term "Therapeutic protein" encompasses
antibodies and fragments and variants thereof. Thus a protein of
the invention may contain at least a fragment or variant of a
Therapeutic protein, and/or at least a fragment or variant of an
antibody. Additionally, the term "Therapeutic protein" may refer to
the endogenous or naturally occurring correlate of a Therapeutic
protein.
[0061] By a polypeptide displaying a "therapeutic activity" or a
protein that is "therapeutically active" is meant a polypeptide
that possesses one or more known biological and/or therapeutic
activities associated with a therapeutic protein such as one or
more of the Therapeutic proteins described herein or otherwise
known in the art. As a non-limiting example, a "Therapeutic
protein" is a protein that is useful to treat, prevent or
ameliorate a disease, condition or disorder. As a non-limiting
example, a "Therapeutic protein" may be one that binds specifically
to a particular cell type (normal (e.g., lymphocytes) or abnormal
e.g., (cancer cells)) and therefore may be used to target a
compound (drug, or cytotoxic agent) to that cell type
specifically.
[0062] For example, a non-exhaustive list of "Therapeutic protein"
portions which may be comprised by an albumin fusion protein of the
invention includes, but is not limited to, IFN.alpha., ANP, BNP,
LANP, VDP, KUP, CNP, DNP, HCC-1, beta defensin-2, fractalkine,
oxyntomodulin, killer toxin peptide, TIMP-4, PYY, adrenomedullin,
ghrelin, CGRP, IGF-1, neuraminidase, hemagglutinin,
butyrylcholinesterase, endothelin, and mechano growth factor.
[0063] Interferon hybrids may also be fused to the amino or carboxy
terminus of albumin to form an interferon hybrid albumin fusion
protein. Interferon hybrid albumin fusion protein may have
enhanced, or alternatively, suppressed interferon activity, such as
antiviral responses, regulation of cell growth, and modulation of
immune response (Lebleu et al., PNAS USA, 73:3107-3111 (1976);
Gresser et al., Nature, 251:543-545 (1974); and Johnson, Texas
Reports Biol Med, 35:357-369 (1977)). Each interferon hybrid
albumin fusion protein can be used to treat, prevent, or ameliorate
viral infections (e.g., hepatitis (e.g., HCV); or HIV), multiple
sclerosis, or cancer.
[0064] In one embodiment, the interferon hybrid portion of the
interferon hybrid albumin fusion protein comprises an interferon
alpha-interferon alpha hybrid (herein referred to as an alpha-alpha
hybrid). For example, the alpha-alpha hybrid portion of the
interferon hybrid albumin fusion protein consists, or alternatively
comprises, of interferon alpha A fused to interferon alpha D. In a
further embodiment, the A/D hybrid is fused at the common BgIII
restriction site to interferon alpha D, wherein the N-terminal
portion of the A/D hybrid corresponds to amino acids 1-62 of
interferon alpha A and the C-terminal portion corresponds to amino
acids 64-166 of interferon alpha D. For example, this A/D hybrid
would comprise the amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRX.sub.1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLF-
TTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNX.sub.2DSILAVKKYFRRITLYLTEKKY-
SPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO:99), wherein the X.sub.1
is R or K and the X.sub.2 is A or V. In an additional embodiment,
the A/D hybrid is fused at the common PvuIII restriction site,
wherein the N-terminal portion of the A/D hybrid corresponds to
amino acids 1-91 of interferon alpha A and the C-terminal portion
corresponds to amino acids 93-166 of interferon alpha D. For
example, this A/D hybrid would comprise the amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRX.sub.1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLF-
STKDSSAAWDETLLDKFYTELYQQLNDLEACVMQEERVGETPLMNX.sub.2DSILAVKKYFRRITLYLTEKKY-
SPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO:100), wherein the X.sub.1
is R or K and the second X.sub.2 is A or V. These hybrids are
further described in U.S. Pat. No. 4,414,510, which is hereby
incorporated by reference in its entirety.
[0065] In an additional embodiment, the alpha-alpha hybrid portion
of the interferon hybrid albumin fusion protein consists, or
alternatively comprises, of interferon alpha A fused to interferon
alpha F. In a further embodiment, the A/F hybrid is fused at the
common PvuIII restriction site, wherein the N-terminal portion of
the A/F hybrid corresponds to amino acids 1-91 of interferon alpha
A and the C-terminal portion corresponds to amino acids 93-166 of
interferon alpha F. For example, this A/F hybrid would comprise the
amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRXISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSS-
AAWDETLLDKFYTELYQQLNDMEACVIQEVGVEETPLMNVDSILAVK
KYFQRrTLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE (SEQ ID NO:101),
wherein X is either R or K. These hybrids are further described in
U.S. Pat. No. 4,414,510, which is hereby incorporated by reference
in its entirety. In a further embodiment, the alpha-alpha hybrid
portion of the interferon hybrid albumin fusion protein consists,
or alternatively comprises, of interferon alpha A fused to
interferon alpha B. In an additional embodiment, the A/B hybrid is
fused at the common PvuIII restriction site, wherein the N-terminal
portion of the A/B hybrid corresponds to amino acids 1-91 of
interferon alpha A and the C-terminal portion corresponds to amino
acids 93-166 of interferon alpha B. For example, this A/B hybrid
would comprise an amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRX.sub.1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLF-
STKDSSAAWDETLLDKFYTELYQQLNDLEX.sub.2X.sub.3X.sub.4X.sub.5QEVGVIESPLMYEDSIL-
AVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE (SEQ ID NO:102),
wherein the X.sub.1 is R or K and X.sub.2 through X.sub.5 is SCVM
or VLCD. These hybrids are further described in U.S. Pat. No.
4,414,510, which is hereby incorporated by reference in its
entirety.
[0066] In another embodiment, the interferon hybrid portion of the
interferon hybrid albumin fusion protein comprises an interferon
beta-interferon alpha hybrid (herein referred to as a beta-alpha
hybrid). For example, the beta-alpha hybrid portion of the
interferon hybrid albumin fusion protein consists, or alternatively
comprises, of interferon beta-1 fused to interferon alpha D (also
referred to as interferon alpha-1). In a further embodiment, the
beta-1/alpha D hybrid is fused wherein the N-terminal portion
corresponds to amino acids 1-73 of interferon beta-1 and the
C-terminal portion corresponds to amino acids 74-167 of interferon
alpha D. For example, this beta-1/alpha D hybrid would comprise an
amino acid sequence:
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQD-
SSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNXDSILAVKKYFRRITLYLTEKKYSPCAWEVVRA-
EIMRSLSLSTNLQERLRRKE (SEQ ID NO:103), wherein X is A or V. These
hybrids are further described in U.S. Pat. No. 4,758,428, which is
hereby incorporated by reference in its entirety.
[0067] In another embodiment, the interferon hybrid portion of the
interferon hybrid albumin fusion protein comprises an interferon
alpha-interferon beta hybrid (herein referred to as a alpha-beta
hybrid). For example, the alpha-beta hybrid portion of the
interferon hybrid albumin fusion protein consists, or alternatively
comprises, of interferon alpha D (also referred to as interferon
alpha-1) fused to interferon beta-1. In a further embodiment, the
alpha D/beta-1 hybrid is fused wherein the N-terminal portion
corresponds to amino acids 1-73 of interferon alpha D and the
C-terminal portion corresponds to amino acids 74-166 of interferon
beta-1. For example, this alpha D/beta-1 hybrid would have an amino
acid sequence:
MCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKD-
SSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVR-
VEILRNFYFINRLTGYLRN (SEQ ID NO:104). These hybrids are further
described in U.S. Pat. No. 4,758,428, which is hereby incorporated
by reference in its entirety.
[0068] In further embodiments, the interferon hybrid portion of the
interferon hybrid albumin fusion proteins may comprise additional
combinations of alpha-alpha interferon hybrids, alpha-beta
interferon hybrids, and beta-alpha interferon hybrids. In
additional embodiments, the interferon hybrid portion of the
interferon hybrid albumin fusion protein may be modified to include
mutations, substitutions, deletions, or additions to the amino acid
sequence of the interferon hybrid. Such modifications to the
interferon hybrid albumin fusion proteins may be made, for example,
to improve levels of production, increase stability, increase or
decrease activity, or confer new biological properties.
[0069] The above-described interferon hybrid albumin fusion
proteins are encompassed by the invention, as are host cells and
vectors containing polynucleotides encoding the polypeptides. In
one embodiment, a interferon hybrid albumin fusion protein encoded
by a polynucleotide as described above has extended shelf life. In
an additional embodiment, a interferon hybrid albumin fusion
protein encoded by a polynucleotide described above has a longer
serum half-life and/or more stabilized activity in solution (or in
a pharmaceutical composition) in vitro and/or in vivo than the
corresponding unfused interferon hybrid molecule.
[0070] In another non-limiting example, a "Therapeutic protein" is
a protein that has a biological activity, and in particular, a
biological activity that is useful for treating, preventing or
ameliorating a disease. A non-inclusive list of biological
activities that may be possessed by a Therapeutic protein includes,
inhibition of HIV-1 infection of cells, stimulation of intestinal
epithelial cell proliferation, reducing intestinal epithelial cell
permeability, stimulating insulin secretion, induction of
bronchodilation and vasodilation, inhibition of aldosterone and
renin secretion, blood pressure regulation, promoting neuronal
growth, enhancing an immune response, enhancing inflammation,
suppression of appetite, or any one or more of the biological
activities described in the "Biological Activities" section below
and/or as disclosed for a given Therapeutic protein in Table 1
(column 2).
[0071] In one embodiment, IFN-alpha-HSA fusions are used to inhibit
viral agents classified under Category A-Filo (Ebola), Arena
(Pichende), Category B-Toga (VEE) or Category C-Bunya (Punto toro),
Flavi (Yellow fever, West Nile). For example, CPE inhibition,
neutral red staining and virus yield assays were employed to
evaluate the antiviral activities of INF-alpha fused downstream of
HSA (CID 3165 protein). The pharmacokinetics and pharmacodynamic
activity of CID 3165 protein in cynomolgus monkeys and human
subjects were evaluated. The results indicate that antiviral
activity was achieved against all the RNA viruses evaluated with a
favorable safety index. The IC50 values ranged from <0.1 ng/ml
(Punta Toro A) to 19 ng/ml (VEE) in the CPE assay. In cynomolgus
monkeys, the half-life of CID 3165 protein was 90 hours and was
detectable up to 14 days post-dose. In human subjects, CID 3165
protein was safe and well tolerated. C.sub.max following single
injection doses was dose-proportional. The mean C.sub.max in the
500 ug cohort was 22 ng/ml, and the mean t.sub.1/2 was 150 hours.
Dosing once every 2-4 weeks or more is supported by the
pharmacokinetics. Antiviral response against Hepatitis C was
observed in the majority of subjects in the single injection
cohorts (120-500 ug).
[0072] In a further embodiment, IFN-alpha-HSA fusions are used to
treat patients with chronic Hepatitis C infection (HCV). Interferon
alpha, also known as interferon alfa or leukocyte interferon, is
the standard of care for treatment of patients infected with HCV.
The term "interferon alpha" refers to a family of highly homologous
related polypeptides with anti-viral activity. The interferon alpha
portion of the IFN-alpha-HSA fusion consists or alternatively
comprises any interferon alpha or fragment thereof known in the
art. Non-limiting examples of the interferon alpha portion of the
IFN-alpha-HSA fusion proteins of the invention include, but are not
limited to, the interferon alpha proteins disclosed in the
Therapeutic protein column of Table 1. In particular embodiments,
the interferon alpha portion consists or alternatively comprises
interferon alpha-2a, interferon alpha-2b, interferon alpha-2c,
consensus interferon, interferon alfacon-1, interferon alpha-n1,
interferon alpha-n3, any commercially available form of interferon
alpha, such as, for example, INTRON.RTM. A (Schereing Corp.,
Kenilworth, N.J.), ROFERON.RTM. A (Hoffman-La Roche, Nutley, N.J.),
Berofor alpha inteferon (Boehringer Ingelheim Pharmaceutical, Inc.,
Ridgefied, Conn.), OMNIFERON.TM. (Viragen, Inc., Plantation, Fla.),
MULTIFERON.TM. (Viragen, Inc., Plantation, Fla.) WELLFERON.RTM.
(GlaxoSmithKline, London, Great Britian), INFERGEN.RTM. (Amgen,
Inc., Thousands Oaks, Calif.), SUMIFERON.RTM. (Sumitomo, Japan),
BELEROFON.RTM. (Nautilus Biotech, France), MAXY-ALPHA.TM. (Maxygen,
Redwood City, Calif./Hoffman-La Roche, Nutley, N.J.), or any
purified interferon alpha product or a fragment thereof. In further
embodiments, the interferon alpha portion of the IFN-alpha-HSA
fusion protein consists or alternatively comprises interferon alpha
modified or formulated for extended or controlled release. For
example, the interferon alpha portion consists, or alternatively
comprises commercially available extended release or controlled
release interferon alpha, including, but not limited to
interferon-alpha-XL (Flamel Technologies, France) and LOCTERON.TM.
(BioLex Therapeutics/OctoPlus, Pittsboro, N.C.). In additional
embodiments, the interferon alpha portion of the IFN-alpha-HSA
fusion protein may be modified by the attachment of chemical
moieties. For example, the inteferon alpha portion may be modified
by pegylation. Accordingly, in additional embodiments, the
interferon alpha portion of the IFN-alpha-HSA fusion protein
consists or alternatively comprises pegylated forms of interferon
alpha-2a,2b, or consensus interferon and include, but are not
limited to, a commercially available pegylated interferon alpha,
such as, for example, PEG-INTRON.RTM. (Schereing Corp., Kenilworth,
N.J.), PEGASYS.RTM. (Hoffman-La Roche, Nutley, N.J.),
PEG-OMNIFERON.TM. (Viragen, Inc., Plantation, Fla.) or a fragment
thereof. However, as used herein, "IFN-alpha-HSA" fusions refers to
the HSA fused to any of the interferon alpha proteins known in the
art or a fragment thereof. Patients infected with HCV may fall
within two categories based on previous exposure to an interferon
regimen for treatment of the HCV infection. "Treatment-naive
patients" or "naive patients" are those patients who have never
been treated with an interferon regimen. "Treatment-experienced
patients" or "experienced patients" are those patients who have
been treated or are currently being treated with an interferon
regimen. "Non-responders" are experienced patients who have been
previously treated with an interferon regimen but have failed to
meet the primary endpoint of treatment such as an early viral load
reduction (EVR) or an end-of-treatment response (ETR). "Relapsers"
are experienced patients who have previously been treated with an
interferon regimen and have a achieved primary endpoint of
treatment such as EVR or ETR, but become subsequently positive for
HCV at later time points. However, as used herein, an "HCV patient"
refers to a patient who is infected with HCV and who is either
naive or experienced. In addition, as used herein, an "HCV patient"
who is "experienced" is either a non-responder or a relapser.
[0073] In addition, the Hepatitis C virus can be classified into
numerous genotypes, with four genotypes, genotype 1, 2, 3, or 4,
being the most prevalent. Generally, the Hepatitis C virus that
infects an HCV patient comprises a single genotype. However, the
Hepatitis virus can comprise a combination of two or more
genotypes. In addition, the genotype of Hepatitis C virus may also
be a variant of one of the known HCV genotypes. In a further
embodiment, the Hepatitis C virus of the HCV patient is genotype 1
or a variant thereof. However, as used herein, "HCV" refers to the
Hepatitis C virus of any genotype, or combination or variants
thereof.
[0074] The standard treatment regimen for patients with HCV
involves treatment with interferon alpha in combination with an
antiviral agent, such as, ribavirin. In general, the interferon
alpha is administered daily, twice-a-week, or weekly and the
ribavirin is administered daily. However, recent studies have also
used inteferon alpha in combination with other antiviral agents
known in the art for the treatment of HCV. Thus, in a further
embodiment the IFN-alpha-HSA fusion may be administered to the HCV
patient either alone or in combination with an antiviral agent,
such as, for example, ribavirin. In a more preferred embodiment,
IFN-alpha-HSA fusion may be administered to the HCV patient in
combination with one, two three, or more antiviral agents, such as,
for example, ribavirin and an additional antiviral agent.
[0075] As noted above, pharmokinetics of the CID 3165 protein
support a dosing schedule of once every 2-4 weeks or greater. Thus,
in a further embodiment, the HCV patients are treated with an
IFN-alpha-HSA fusion by administration once every 2-4 weeks alone
or in combination with an effective amount of an antiviral agent.
In a preferred embodiment, the HCV patients are treated with an
IFN-alpha-HSA fusion by administration once every 2-4 weeks in
combination with an effective amount of one, two three, or more
antiviral agents. In an additional preferred embodiment, the
IFN-alpha-HSA fusion is administered to the HCV patient once every
4 weeks. In an additional preferred embodiment, the IFN-alpha-HSA
fusion is administered to the HCV patient more than once every 4
weeks. In additional embodiments, the IFN-alpha-HSA fusion is
adminstered once every 4 weeks or more to an HCV patient, wherein
the treatment also includes administration of an effective amount
of one, two three, or more antiviral agents.
[0076] In a another embodiment, IFN-alpha-HSA fusions may be used
as a low-dose monotherapy for maintenance therapy of HCV. In a
further additional embodiment, IFN-alpha-HSA fusions may used in
combination with ribavirin and one or more other antiviral agents
for the treatment of HCV. Alternatively, in another embodiment,
IFN-alpha-HSA fusions may be used in combination with one, two,
three, or more antiviral agents, other than ribavirin, for the
treatment of HCV.
[0077] In an additional embodiment, IFN-alpha-HSA fusions may be
used for the treatment of other viral infections. For example, in
one embodiment, IFN-alpha-HSA fusions may be used for the treatment
of Hepatitis B (HBV). In an additional embodiment, IFN-alpha-HSA
fusions may be used for the treatment of Human Papilloma Virus
(HPV). In a further embodiment, IFN-alpha-HSA fusions may be used
in the treatment of cancer, including, but not limited to hairy
cell leukemia, malignant melanoma, follicular lymphoma, chronic
myelogenous leukemia, AIDS related Kaposi's Sarcoma, multiple
myeloma, or renal cell cancer.
[0078] In another embodiment, HSA fusions with natriuretic
peptides, including but not limited to ANP-HSA fusions or BNP-HSA
fusions, may be used for the treatment of cardiovascular disorders.
For example, in a preferred embodiment, HSA fusions with
natriuretic peptides, including but not limited to ANP-HSA fusions
or BNP-HSA fusions, may be used for the treatment of congestive
heart failure. In an additional preferred embodiment, HSA fusions
with natriuretic peptides, including but not limited to ANP-HSA
fusions or BNP-HSA fusions, may be used in the treatment of
post-myocardial infarction. In additional embodiments, HSA fusions
with natriuretic peptides, including but not limited to ANP-HSA
fusions or BNP-HSA fusions, may be used to additional
cardiovascular disorders, including, but not limited to
hypertension, salt-sensitive hypertension, angina pectoris,
peripheral artery disease, hypotension, cardiac volume overload,
cardiac decompensation, cardiac failure, left ventricular
dysfunction, dyspnea, myocardial reperfusion injury, or left
ventricular remodeling. In another embodiment, HSA fusions with
natriuretic peptides, including but not limited to ANP-HSA fusions
or BNP-HSA fusions, may be used in the treatment for elevated
aldosterone levels, which can lead to vasoconstriction, impaired
cardiac output and/or hypertension. In further embodiments, HSA
fusions with natriuretic peptides, including but not limited to
ANP-HSA fusions or BNP-HSA fusions, may be used in the treatment of
renal diseases, including, but not limited to diabetic nephropathy;
glomerular hypertrophy, glomerular injury, renal glomerular
disease, acute and/or chronic renal failure. In an additional
embodiment, HSA fusions with natriuretic peptides, including but
not limited to ANP-HSA fusions or BNP-HSA fusions, may be used to
treat stroke or excess fluid in tissues.
[0079] In an additional embodiment, HSA may be fused with
natriuretic peptide variants including, but not limited to, BNP-HSA
fusions wherein the BNP component of the fusion protein is BNP
amino acid residues 1-29. In one embodiment, the BNP component of
the HSA fusion protein consists of two BNP variants (e.g., BNP
amino acid residues 1-29) in tandem. In another embodiment, the BNP
component of the HSA fusion protein consists of three, four, five
or more BNP variants (e.g., BNP amino acid residues 1-29) in
tandem. In a preferred embodiment, HSA fusions with BNP variants
(e.g., BNP amino acid residues 1-29) may be used for the treatment
of congestive heart failure. In an additional preferred embodiment,
HSA fusions with BNP variants (e.g., BNP amino acid residues 1-29)
may be used in the treatment of post-myocardial infarction. In an
additional embodiment, HSA fusions with BNP variants (e.g., BNP
amino acid residues 1-29) may be used to treat additional
cardiovascular disorders, including, but not limited to,
hypertension, salt-sensitive hypertension, angina pectoris,
peripheral artery disease, hypotension, cardiac volume overload,
cardiac decompensation, cardiac failure, non-hemodynamic CHF, left
ventricular dysfunction, dyspnea, myocardial reperfusion injury, or
left ventricular remodeling. In another embodiment, HSA fusions
with BNP variants (e.g., BNP amino acid residues 1-29) may be used
in the treatment for elevated aldosterone levels, which can lead to
vasoconstriction, impaired cardiac output and/or hypertension. In a
preferred embodiment, HSA fusion with BNP variants (e.g., BNP amino
acid residues 1-29) may be used in the treatment of renal disorders
or diseases, including, but not limited to, diabetic nephropathy;
glomerular hypertrophy, glomerular injury, renal glomerular
disease, acute and/or chronic renal failure. In an additional
embodiment HSA fusions with BNP variants (e.g., BNP amino acid
residues 1-29) may be used to treat stroke or excess fluid in
tissues.
[0080] In related but distinct embodiments, the invention is
directed to natriuretic peptide variants including, but not limited
to BNP amino acid residues 1-29, wherein the peptides are not fused
with HSA. In one embodiment, the BNP variants of the invention have
the sequence of two BNP variants (e.g., BNP amino acid residues
1-29) in tandem. In an additional embodiment, the BNP variants of
the invention have the sequence of three, four, five or more BNP
variants (e.g., BNP amino acid residues 1-29) in tandem. In a
preferred embodiment, the BNP variants (e.g., BNP amino acid
residues 1-29) of the invention may be used for the treatment of
congestive heart failure. In an additional preferred embodiment,
the BNP variants (e.g., BNP amino acid residues 1-29) of the
invention may be used in the treatment of post-myocardial
infarction. In an additional embodiment, the BNP variants (e.g.,
BNP amino acid residues 1-29) of the invention may be used to treat
additional cardiovascular disorders, including, but not limited to,
hypertension, salt-sensitive hypertension, angina pectoris,
peripheral artery disease, hypotension, cardiac volume overload,
cardiac decompensation, cardiac failure, non-hemodynamic CHF, left
ventricular dysfunction, dyspnea, myocardial reperfusion injury, or
left ventricular remodeling. In another embodiment, the BNP
variants (e.g., BNP amino acid residues 1-29) of the invention may
be used in the treatment for elevated aldosterone levels, which can
lead to vasoconstriction, impaired cardiac output and/or
hypertension. In a further preferred embodiment, the BNP variants
(e.g., BNP amino acid residues 1-29) of the invention may be used
in the treatment of renal disorders or diseases, including, but not
limited to, diabetic nephropathy; glomerular hypertrophy,
glomerular injury, renal glomerular disease, acute and/or chronic
renal failure. In an additional embodiment, the BNP variants (e.g.,
BNP amino acid residues 1-29) of the invention may be used to treat
stroke or excess fluid in tissues.
[0081] In a further embodiment, the invention is directed to
natriuretic peptide variants including, but not limited to, BNP
variants (e.g., BNP amino acid residues 1-29), that have been
modified in order to extend half-life, biological activity, and/or
to facilitate purification of the variant. According to this
embodiment, the natriuretic peptide variants (e.g., BNP amino acid
residues 1-29) may be pegylated, methylated, or otherwise
chemically modified or conjugated using techniques known in the
art. Alternatively, methods known in the art may be used to
recombinantly fuse the natriuretic peptide variants of the
invention to other peptide sequences known in the art to extend
half-life, improve biological activity and/or facilitate
purification. For example, natriuretic peptide variants of the
invention may be fused or conjugated to an antibody Fc region, or
portion thereof. The antibody portion fused to a natriuretic
variants (e.g., BNP amino acid residues 1-29) of the 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 natriuretic variants 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 (e.g.,
BNP amino acid residues 1-29) can form dimers through disulfide
bonding between the Fc portions. Higher multimeric forms can be
made by fusing the variants to portions of IgA and IgM. Methods for
fusing or conjugating the variants 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). In an additional
embodiment, the modified BNP variants of the invention have the
sequence of two BNP variants (e.g., BNP amino acid residues 1-29)
in tandem. In an additional embodiment, the modified BNP variants
of the invention have the sequence of three, four, five or more BNP
variants (e.g., BNP amino acid residues 1-29) in tandem. In a
preferred embodiment, the modified BNP variants (e.g., BNP amino
acid residues 1-29) of the invention may be used for the treatment
of congestive heart failure. In a preferred embodiment, the
modified BNP variants (e.g., BNP amino acid residues 1-29) of the
invention may be used in the treatment of post-myocardial
infarction. In an additional embodiment, the modified BNP variants
(e.g., BNP amino acid residues 1-29) of the invention may be used
to treat additional cardiovascular disorders, including, but not
limited to, hypertension, salt-sensitive hypertension, angina
pectoris, peripheral artery disease, hypotension, cardiac volume
overload, cardiac decompensation, cardiac failure, non-hemodynamic
CHF, left ventricular dysfunction, dyspnea, myocardial reperfusion
injury, or left ventricular remodeling. In another embodiment, the
modified BNP variants (e.g., BNP amino acid residues 1-29) of the
invention may be used in the treatment for elevated aldosterone
levels, which can lead to vasoconstriction, impaired cardiac output
and/or hypertension. In a preferred embodiment, the modified BNP
variants (e.g., BNP amino acid residues 1-29) of the invention may
be used in the treatment of renal disorders or diseases, including,
but not limited to, diabetic nephropathy; glomerular hypertrophy,
glomerular injury, renal glomerular disease, acute and/or chronic
renal failure. In an additional embodiment, the modified BNP
variants (e.g., BNP amino acid residues 1-29) of the invention may
be used to treat stroke or excess fluid in tissues.
[0082] In another embodiment, CNP-HSA fusions may be used in the
regulation of endochodral ossification. For example, in a preferred
embodiment, CNP-HSA fusions may be used in the treatment of
skeletal dysplasias, including, but not limited to anchondroplasia,
hypochondroplasia, and thanatophoric dysplasia.
[0083] As used herein, "therapeutic activity" or "activity" may
refer to an activity whose effect is consistent with a desirable
therapeutic outcome in humans, or to desired effects in non-human
mammals or in other species or organisms. Therapeutic activity may
be measured in vivo or in vitro. For example, a desirable effect
may be assayed in cell culture. Such in vitro or cell culture
assays are commonly available for many Therapeutic proteins as
described in the art. Examples of assays include, but are not
limited to those described herein in the Examples section or in the
"Exemplary Activity Assay" column (column 3) of Table 1.
[0084] Therapeutic proteins corresponding to a Therapeutic protein
portion of an albumin fusion protein of the invention, such as cell
surface and secretory proteins, are often modified by the
attachment of one or more oligosaccharide groups. The modification,
referred to as glycosylation, can dramatically affect the physical
properties of proteins and can be important in protein stability,
secretion, and localization. Glycosylation occurs at specific
locations along the polypeptide backbone. There are usually two
major types of glycosylation: glycosylation characterized by
O-linked oligosaccharides, which are attached to serine or
threonine residues; and glycosylation characterized by N-linked
oligosaccharides, which are attached to asparagine residues in an
Asn-X-Ser or Asn-X-Thr sequence, where X can be any amino acid
except proline. N-acetylneuramic acid (also known as silica acid)
is usually the terminal residue of both N-linked and O-linked
oligosaccharides. Variables such as protein structure and cell type
influence the number and nature of the carbohydrate units within
the chains at different glycosylation sites. Glycosylation isomers
are also common at the same site within a given cell type.
[0085] Therapeutic proteins corresponding to a Therapeutic protein
portion of an albumin fusion protein of the invention, as well as
analogs and variants thereof, may be modified so that glycosylation
at one or more sites is altered as a result of manipulation(s) of
their nucleic acid sequence, by the host cell in which they are
expressed, or due to other conditions of their expression. For
example, glycosylation isomers may be produced by abolishing or
introducing glycosylation sites, e.g., by substitution or deletion
of amino acid residues, such as substitution of glutamine for
asparagine, or unglycosylated recombinant proteins may be produced
by expressing the proteins in host cells that will not glycosylate
them, e.g. in E. coli or glycosylation-deficient yeast. These
approaches are described in more detail below and are known in the
art.
[0086] Therapeutic proteins, particularly those disclosed in Table
1, and their nucleic acid and amino acid sequences are well known
in the art and available in public databases such as Chemical
Abstracts Services Databases (e.g., the CAS Registry), GenBank, and
subscription provided databases such as GenSeq (e.g., Derwent).
Exemplary nucleotide sequences of Therapeutic proteins which may be
used to derive a polynucleotide of the invention are shown in
column 7, "SEQ ID NO:X," of Table 2. Sequences shown as SEQ ID NO:X
may be a wild type polynucleotide sequence encoding a given
Therapeutic protein (e.g., either full length or mature), or in
some instances the sequence may be a variant of said wild type
polynucleotide sequence (e.g., a polynucleotide which encodes the
wild type Therapeutic protein, wherein the DNA sequence of said
polynucleotide has been optimized, for example, for expression in a
particular species; or a polynucleotide encoding a variant of the
wild type Therapeutic protein (i.e., a site directed mutant; an
allelic variant)). It is well within the ability of the skilled
artisan to use the sequence shown as SEQ ID NO:X to derive the
construct described in the same row. For example, if SEQ ID NO:X
corresponds to a full length protein, but only a portion of that
protein is used to generate the specific CID, it is within the
skill of the art to rely on molecular biology techniques, such as
PCR, to amplify the specific fragment and clone it into the
appropriate vector.
[0087] Additional Therapeutic proteins corresponding to a
Therapeutic protein portion of an albumin fusion protein of the
invention include, but are not limited to, one or more of the
Therapeutic proteins or peptides disclosed in the "Therapeutic
Protein X" column of Table 1 (column 1), or fragment or variant
thereof.
[0088] Table 1 provides a non-exhaustive list of Therapeutic
proteins that correspond to a Therapeutic protein portion of an
albumin fusion protein of the invention, or an albumin fusion
protein encoded by a polynucleotide of the invention. The first
column, "Therapeutic Protein X," discloses Therapeutic protein
molecules that may be followed by parentheses containing scientific
and brand names of proteins that comprise, or alternatively consist
of, that Therapeutic protein molecule or a fragment or variant
thereof. "Therapeutic protein X" as used herein may refer either to
an individual Therapeutic protein molecule, or to the entire group
of Therapeutic proteins associated with a given Therapeutic protein
molecule disclosed in this column. The "Biological activity" column
(column 2) describes Biological activities associated with the
Therapeutic protein molecule. Column 3, "Exemplary Activity Assay,"
provides references that describe assays which may be used to test
the therapeutic and/or biological activity of a Therapeutic
protein:X or an albumin fusion protein comprising a Therapeutic
protein X (or fragment thereof) portion. Each of the references
cited in the "Exemplary Activity Assay" column are herein
incorporated by reference in their entireties, particularly with
respect to the description of the respective activity assay
described in the reference (see Methods section therein, for
example) for assaying the corresponding biological activity set
forth in the "Biological Activity" column of Table 1. The fourth
column, "Preferred Indication: Y," describes disease, disorders,
and/or conditions that may be treated, prevented, diagnosed, and/or
ameliorated by Therapeutic protein X or an albumin fusion protein
comprising a Therapeutic protein X (or fragment thereof) portion.
The "Construct ID" column (column 5) provides a link to an
exemplary albumin fusion construct disclosed in Table 2 which
encodes an albumin fusion protein comprising, or alternatively
consisting of the referenced Therapeutic Protein X (or fragment
thereof) portion.
TABLE-US-00001 TABLE 1 Therapeutic Protein: X Biological Activity
Exemplary Activity Assay Preferred Indication: Y Construct ID
Therapeutic Protein: Z Interferon alpha Confers a range of
Anti-viral assay: Rubinstein S, Viral infections include Severe
2249, 2343, See Table 2, SEQ ID NO: Z for particular construct.
(Interferon alfa-2b; cellular responses Familletti PC, Pestka S.
Acute Respiratory Syndrome 2366, 2381, Interferon alfa-2a;
including antiviral, (1981) Convenient assay for (SARS) and other
coronavirus 2382, 2410, recombinant; Interferon antiproliferative,
interferons. J. Virol. infections; filoviruses, including but 3165,
3422, alfa-n1; Interferon alfa- antitumor and 37(2): 755-8; Anti-
not limited to Ebola viruses and 3423, 3424, n3; Peginterferon
alpha- immunomodulatory proliferation assay: Gao Y, et Marburg
virus; Arenaviruses, 3476, 3960, 2b; Ribavirin and activities;
stimulate al (1999) Sensitivity of an including but not limited to
4290, 4291, interferon alfa-2b; production of two epstein-barr
virus-positive Pichende virus, Lassa virus, Junin 4292, 4295,
Interferon alfacon-1; enzymes: a protein tumor line, Daudi, to
alpha virus, Machupo virus, Guanarito 4296. interferon consensus;
YM kinase and an interferon correlates with virus; and lymphocytic
643; CIFN; interferon- oligoadenylate expression of a GC-rich viral
choriomeningitis virus (LCMV); alpha consensus; synthetase.
transcript. Mol Cell Biol. Bunyaviruses, including but not
recombinant methionyl 19(11): 7305-13. limited to Punta toro virus,
consensus interferon; Crimean-Congo hemorrhagic fever recombinant
consensus virus, sandfly fever viruses, Rift interferon; CGP 35269;
Valley fever virus, La Crosse virus, RO 253036; RO 258310; and
hantaviruses; Flaviviruses, INTRON A; PEG- including but not
limited to Yellow INTRON; OIF; Fever, Banzi virus, West Nile virus,
OMNIFERON; PEG- Dengue viruses, Japanese OMNIFERON; Encephalitis
virus, Tick-borne VELDONA; PEG- encephalitis, Omsk Hemorrhagic
REBETRON; ROFERON Fever, and Kyasanur Forest Disease A; WELLFERON;
virus; Togaviruses, including but not ALFERON N/LDO; limited to
Venezuelan, eastern, and REBETRON; western equine encephalitis
viruses, ALTEMOL; Ross River virus, and Rubella virus;
VIRAFERONPEG; Orthopox viruses, including but not PEGASYS; limited
to Vaccinia, Cowpox, VIRAFERON; Smallpox, and Monkeypox; VIRAFON;
AMPLIGEN; Herpesviruses; FluA/B; Respiratory INFERGEN; INFAREX;
Sincytial virus (RSV); paraflu; ORAGEN) measles; rhinoviruses;
adenoviruses; Semliki Forest virus; Viral Hemorrhagic fevers;
Rhabdoviruses; Paramyxoviruses, including but not limited to Nipah
virus and Hendra virus; and other viral agents identified by the
U.S. Centers for Disease Control and Prevention as high-priority
disease agents (i.e., Category A, B, and C agents; see, e.g.,
Moran, Emerg. Med. Clin. North. Am. 2002; 20(2): 311-30 and Darling
et al., Emerg. Med. Clin. North Am. 2002; 20(2): 273-309). Atrial
natriuretic peptide ANP is diuretic Renin and aldosterone levels
Hypertension; salt-sensitive 3484, 4174. See Table 2, SEQ ID NO: Z
for particular construct. (ANP; atrial natriuretic (natriuretic),
can be measured using hypertension; congestive heart factor; ANF)
hypotensive, and has methods known in the art, for failure; angina
pectoris, peripheral an inhibitory effect example, in Yamato et
al., artery disease; diabetic nephropathy; on renin and Circ J 2003
May; 67(5): 384-90. stroke; kidney failure; acute and/or
aldosterone secretion. Blood pressure can be chronic renal failure;
acute tubular Involved in measured with a necrosis; acute renal
failure; renal regulation of blood sphygmomanometer or using
disease; renal glomerular disease; pressure and salt and other
methods known in the excess fluid in tissues; hypotension; water
balance/ art, such as in Reddy et al., cardiac volume overload;
cardiac electrolyte Ultrasound Med Biol 2003 decompensation; left
ventricular homeostasis in body Mar; 29(3): 379-85. dysfunction;
dyspnea; treatment for fluids. elevated aldosterone levels, which
can lead to vasoconstriction, impaired cardiac output and/or
hypertension; cardiovascular disease; cardiac failure; myocardial
reperfusion injury; left ventricular remodeling; post-myocardial
infarction. B-type natriuretic peptide Stimulates smooth Inhibition
of angiotensin can Hypertension; salt-sensitive 3618, 3689, See
Table 2, SEQ ID NO: Z for particular construct. (BNP, brain
natriuretic muscle relaxation be determined using assays
hypertension; congestive heart 3690, 3691, peptide) and
vasodilation, known in the art, for example failure; angina
pectoris, peripheral 3692, 3715, natriuresis, and using an in vitro
proliferation artery disease; diabetic nephropathy; 3716, 3723,
suppression of renin- assay with rat cardiac stroke; kidney
failure; acute and/or 3724, 3725, angiotensin and fibroblasts as
described in chronic renal failure; acute tubular 3736, 3741,
endothelin. Naunyn Schmiedebergs Arch necrosis; acute renal
failure; renal 3769, 3778, Pharmacol 1999 disease; renal glomerular
disease; 3783, 3795, May; 359(5): 394-9. excess fluid in tissues;
hypotension; 3796, 3809, Vasodilation can be measured cardiac
volume overload; cardiac 3896, 3897, in animals by measuring the
decompensation; left ventricular 3898, 3899, myogenic responses of
small dysfunction; dyspnea; treatment for 3900, 3956, renal
arteries in an isobaric elevated aldosterone levels, which 3957,
3959, arteriograph system (see Am J can lead to vasoconstriction,
3961, 3962, Physiol Regul Integr Comp impaired cardiac output
and/or 3965, 3966, Physiol 2002 hypertension; cardiovascular 3967,
3968, Aug; 283(2): R349-R355). disease; cardiac failure; myocardial
4005, 4006, Natriuesis is determined by reperfusion injury; left
ventricular 4007, 4062, measuring the amount of remodeling;
post-myocardial 4130, 4160, sodium in the urine. infarction. 4161,
4167, 4168, 4169, 4170, 4171, 4172, 4174. Long-acting natriuretic
Inhibits renal Na.sup.+- Renal Na.sup.+-K.sup.+-ATPase
Hypertension; salt-sensitive 3886, 3887. See Table 2, SEQ ID NO: Z
for particular peptide (LANP; proANP- K.sup.+-ATPase; activity can
be measured hypertension; congestive heart construct. (31-67);
enhances synthesis of using assays known in the art, failure;
angina pectoris, peripheral Also see, Vesely Am J Physiol Renal
prostaglandin E2 that such as in Ku et al., 1987; artery disease;
diabetic nephropathy; Rhysiol 2003; 285: F167-177 which is
incorporated by reference regulates contraction Endocrinology 120:
2166-2173. stroke; kidney failure; acute and/or and relaxation of
Vasodilation can be chronic renal failure; acute tubular smooth
muscle, as measured using assays known necrosis; acute renal
failure; renal well as the dilation in the art (Ashton et al.
disease; renal glomerular disease; and constriction of Pharmacology
2000; excess fluid in tissues; hypotension; blood vessels; 61(2):
101-105. Prostaglandin cardiac volume overload; cardiac inhibits
plasma renin E2 synthesis can be decompensation; left ventricular
activity; causes determined using assays dysfunction; dyspnea;
treatment for diuresis and known in the art, (Cheng et elevated
aldosterone levels, which natriuresis. Involved al., J Endocrinol.
2004 can lead to vasoconstriction, in regulation of blood Aug;
182(2): 249-56). Blood impaired cardiac output and/or pressure and
pressure can be measured hypertension; cardiovascular
salt/water/electrolyte with a sphygmomanometer or disease; cardiac
failure; myocardial balance in body using other methods known in
reperfusion injury; left ventricular fluids; the art, such as in
Reddy et remodeling; post-myocardial renoprotection. al.,
Ultrasound Med Biol infarction. 2003 Mar; 29(3): 379-85. Natriuesis
is determined by measuring the amount of sodium in the urine.
Diuresis is determined by measuring the amount of urine secreted.
Vessel Dialator (VDP; Inhibits renal Na.sup.+- Renal
Na.sup.+-K.sup.+-ATPase Hypertension; salt-sensitive 3888, 3889.
See Table 2, SEQ ID NO: Z for particular construct.
proANP-(79-98)). K.sup.+-ATPase; activity can be measured
hypertension; congestive heart Also see, Vesely Am J Physiol Renal
Rhysiol 2003; enhances synthesis of using assays known in the art,
failure; angina pectoris, peripheral 285: F167-177 which is hereby
incorporated by reference prostaglandin E2 that such as in Ku et
al., 1987; artery disease; diabetic nephropathy; regulates
contraction Endocrinology 120: 2166-2173. stroke; kidney failure;
acute and/or and relaxation of Vasodilation can be chronic renal
failure; acute tubular smooth muscle, as measured using assays
known necrosis; acute renal failure; renal well as the dilation in
the art (Ashton et al. disease; renal glomerular disease; and
constriction of Pharmacology 2000; excess fluid in tissues;
hypotension; blood vessels; 61(2): 101-105. Prostaglandin cardiac
volume overload; cardiac inhibits aldosterone E2 synthesis can be
decompensation; left ventricular secretion; causes determined using
assays dysfunction; dyspnea; treatment for natriuresis in patients
known in the art, (Cheng et elevated aldosterone levels, which with
congestive heart al., J Endocrinol. 2004 can lead to
vasoconstriction, failure; causes Aug; 182(2): 249-56). impaired
cardiac output and/or kaliuresis. Involved Aldosterone levels can
be hypertension; cardiovascular in regulation of blood measured
using methods disease; cardiac failure; myocardial pressure and
known in the art, for example, reperfusion injury; left ventricular
salt/water/electrolyte in Yamato et al., Circ J 2003; remodeling;
post-myocardial balance in body May; 67(5): 384-90. Blood
infarction. fluids; pressure can be measured renoprotection. with a
sphygmomanometer or using other methods known in the art, such as
in Reddy et al., Ultrasound Med Biol 2003 Mar; 29(3): 379-85.
Natriuesis is determined by measuring the amount of sodium in the
urine. Kaliuresis is determined by measuring the amount of
potassium in the urine. Kaliuretic Peptide (KUP; Involved in Blood
pressure can be Hypertension; salt-sensitive 3890, 3891. See Table
2, SEQ ID NO: Z for particular construct. proANP-(99-126)).
regulation of blood measured with a hypertension; congestive heart
Also see, Vesely Am J Physiol Renal Rhysiol 2003; pressure and
sphygmomanometer or using failure; angina pectoris, peripheral 285:
F167-177 which is hereby incorporated by reference
salt/water/electrolyte other methods known in the artery disease;
diabetic nephropathy; balance in body art, such as in Reddy et al.,
stroke; kidney failure; acute and/or fluids. Ultrasound Med Biol
2003 chronic renal failure; acute tubular Mar; 29(3): 379-85.
Natriuesis necrosis; acute renal failure; renal is determined by
measuring disease; renal glomerular disease; the amount of sodium
in the excess fluid in tissues; hypotension; urine. Diuresis is
determined cardiac volume overload; cardiac by measuring the amount
of decompensation; left ventricular urine secreted.. dysfunction;
dyspnea; treatment for elevated aldosterone levels, which can lead
to vasoconstriction, impaired cardiac output and/or
hypertension; cardiovascular disease; cardiac failure; myocardial
reperfusion injury; left ventricular remodeling; post-myocardial
infarction. C-type Natriuretic Peptide Promotes diuresis Natriuesis
is determined by Hypertension; salt-sensitive 3892, 3893. See Table
2, SEQ ID NO: Z for particular construct. (CNP) and natriuresis.
measuring the amount of hypertension; congestive heart Also see,
Vesely Am J Physiol Renal Rhysiol 2003; Involved in sodium in the
urine. Diuresis failure; angina pectoris, peripheral 285: F167-177
which is hereby incorporated by reference regulation of blood is
determined by measuring artery disease; diabetic nephropathy;
pressure and the amount of urine secreted. stroke; kidney failure;
acute and/or salt/water/electrolyte cGMP production in bone can
chronic renal failure; acute tubular balance in body be measured
using assays in necrosis; acute renal failure; renal fluids.
Involved in the art (Yasoda et al., J. Biol. disease; renal
glomerular disease; the regulation of Chem. 1998; 273: 11695-11700.
excess fluid in tissues; hypotension; endochondral cardiac volume
overload; cardiac ossification of bone. decompensation; left
ventricular dysfunction; dyspnea; treatment for elevated
aldosterone levels, which can lead to vasoconstriction, impaired
cardiac output and/or hypertension; cardiovascular disease; cardiac
failure; myocardial reperfusion injury; left ventricular
remodeling; post-myocardial infarction; skeletal dysplasias
including anchondroplasia, hypochondroplasia and thanatophoric
dysplasia. Dendroaspis natriuretic Inhibits Na.sup.+-K.sup.+- Renal
Na.sup.+-K.sup.+-ATPase Hypertension; salt-sensitive 3894, 3895.
See Table 2, SEQ ID NO: Z for particular construct. peptide (DNP)
ATPase; enhances activity can be measured hypertension; congestive
heart Also see, Vesely Am J Physiol Renal Rhysiol 2003; synthesis
of using assays known in the art, failure; angina pectoris,
peripheral 285: F167-177 which is hereby incorporated by reference
prostaglandin E2 that such as in Ku et al., 1987; artery disease;
diabetic nephropathy; regulates contraction Endocrinology 120:
2166-2173. stroke; kidney failure; acute and/or and relaxation of
Vasodilation can be chronic renal failure; acute tubular smooth
muscle, as measured using assays known necrosis; acute renal
failure; renal well as the dilation in the art (Ashton et al.
disease; renal glomerular disease; and constriction of Pharmacology
2000; excess fluid in tissues; hypotension; blood vessels; 61(2):
101-105. Prostaglandin cardiac volume overload; cardiac inhibits
aldosterone E2 synthesis can be decompensation; left ventricular
secretion; causes determined using assays dysfunction; dyspnea;
treatment for diuresis and known in the art, (Cheng et elevated
aldosterone levels, which natriuresis. Involved al., J Endocrinol.
2004 can lead to vasoconstriction, in regulation of blood Aug;
182(2): 249-56). impaired cardiac output and/or pressure and
Aldosterone levels can be hypertension; cardiovascular
salt/water/electrolyte measured using methods disease; cardiac
failure; myocardial balance in body known in the art, for example,
reperfusion injury; left ventricular fluids; in Yamato et al., Circ
J 2003 remodeling; post-myocardial renoprotection. May; 67(5):
384-90. Blood infarction. pressure can be measured with a
sphygmomanometer or using other methods known in the art, such as
in Reddy et al., Ultrasound Med Biol 2003 Mar; 29(3): 379-85.
Natriuesis is determined by measuring the amount of sodium in the
urine. Diuresis is determined by measuring the amount of urine
secreted. Beta defensin-2 (beta Involved in the innate
Antimicrobial activity can be Treatment of fungal, bacterial, or
4173, 4175, See Table 2, SEQ ID NO: Z for particular defensin 4;
SAP1; defense system as an measured using assays known viral
infection; Infection in immune- 4176, 4177, construct. DEFB2;
HBD-2; DEFB- antimicrobial in the art, such as in Bals et
compromised disease states; 4178, 4179, 2; DEFB102; skin- peptide;
kills gram al., J. Clin. Invest. 1998 Sept Inflammation;
Gingivitis; 4180, 4181 antimicrobial peptide 1) negative and gram
102(5): 874-880. Bronchiolitis obliterans syndrome; positive
organisms, Oral squamous cell carcinoma; such as, for example,
Uterine infection; Psoriasis; E. coli, P. aeruginosa, Neonatal
infection; Lung cancer; S. aureus, E. faecalis, Inflammatory bowel
Disease; and Candida sp.; Gastritis; Middle ear infection
stimulates odontoblast differentiation Human chemokine HCC-
Involved in Chemokine activities can be Autoimmune disorders;
Immunity; 1933, 1934, See Table 2, SEQ ID NO: Z for particular 1
(ckBeta-1; CKB-1; inflammation, determined using assays Vascular
and Inflammatory 1947, 1948, construct. HWFBD) allergy, tissue
known in the art: Methods in disorders; HIV; AIDS; infectious 1955,
1998, rejection, viral Molecular Biology, 2000, vol. diseases.
2355, 2412, infection, and tumor 138: Chemokine Protocols. 2449,
2837, biology; enhances Edited by: A. E. I. Proudfoot, 2838, 2839,
proliferation of T. N. C. Wells, and C. A. Power. 2840, 2841, CD34+
myeloid .COPYRGT. Humana Press Inc., 2842, 2843, progenitor cells.
Totowa, NJ 2844, 2845, 2849, 2947, 3066, 3105, 3124, 3125, 3139,
3152, 3153, 3154, 3155, 3156, 3169, 3170, 3202, 3203, 3204, 3205,
3206, 3207, 3272, 3970. Fractalkine (neurotactin; Fractalkine is
Fractalkine activity can be Immune disorders; Leukemia; 4191, 4192,
See Table 2, SEQ ID NO: Z for particular chemokine CX3C) believed
to play a determined using Lymphoma; Bacterial or Yeast 4193, 4194.
construct role in chemotactic Chemotactic leukocyte Infections
leukocyte migration migration assays known in the and neurological
art, for example: J. Immunol. disorders. Methods 33, ((1980));
Nature 1997 Jun 5; 387(6633): 611-7. Oxyntomodulin Stimulates
insulin The effect of oxyntomodulin Most preferred: Hyperglycemia;
3579, 3580, See, Table 2, secretion; stimulates on insulin
secretion can be Obesity; Diabetes; Diabetes 4213, 4215, SEQ ID NO:
Z cAMP production; measured by methods known Insipidus; Diabetes
mellitus; Type 1 4217, 4232, for particular inhibits meal- in the
art, including the MIN6 diabetes; Type 2 diabetes; Insulin 4240,
4253. construct stimulated gastric cell assay described in Ann.
resistance; Insulin deficiency; acid secretion; NY Acad. Sci. 805:
44-51 Hyperlipidemia; Hyperketonemia; regulates gut (1996).
Non-insulin dependent Diabetes motility; inhibits cAMP accumulation
can be Mellitus (NIDDM); Insulin- food intake, measured using
methods dependent Diabetes Mellitus known in the art, including
(IDDM); A Condition Associated the in vitro assay described in With
Diabetes Including, But Not Br J Pharmacol 138(4): 660-70 Limited
To Obesity, Heart Disease, (2003). Hyperglycemia, Infections,
Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X. Killer Toxin; Killer Toxin A
killer toxin (KT) Candidacidal activity can Candidiasis 4227 See,
Table 2, Peptide (KP) produced by the yeast measure in vitro using
assays SEQ ID NO: Z Pichia anomala is a known in the art, such as
for particular glycoprotein capable those disclosed by Magliani et
construct of killing other al., Nat. Biotechnol. 1997;
microorganisms 15: 155-158; or by Polonelli et presenting specific
al, Clin. Diagn. Lab. cell wall receptors Immunol. 1997; 4:
142-146. (KTR) and competing in natural habitats for the same
ecological niche. Killer Toxin Peptide (KP) is a peptide derived
from a recombinant antiidiotypic antibody, which retains killer
toxin microbicidal activity, probably through the interaction with
the beta-glucan killer toxin receptor on target microbial cells.
TIMP-4 (Tissue Inhibitor The proteins encoded TIMP inhibitory
activity can Anti-cancer applications; 4233, 4234, See Table 2, of
Metalloprotease) by this gene family be assayed using assays
Restenosis; Autoimmune Disorders; 4273, 4274 SEQ ID NO: Z are
natural inhibitors known in the art, such as Osteoarthritis for
particular of the matrix those disclosed by Murphy et construct
metalloproteinases, a al., Biochem J 1981 Apr group of peptidases
1; 195(1): 167-70; Suneel et involved in al., J Biol Chem 1995 Jun
degradation of the 16; 270(24): 14313-8. extracellular matrix. PYY
(Peptide YY, Decreases appetite; Appetite and food intake can Most
preferred: Treatment of 3108, 3109, See Table 2, including
PYY.sub.3-36 increases satiety; be can be measured by Obesity;
treatment of Diabetes; 3281, 3117, SEQ ID NO: Z (amino acid
residues 31-64 decreases food methods known in the art suppression
of body weight gain; 3118, 3282, for particular of full length PYY,
intake. (Batterham et al. Nature suppression of appetite. 4215,
4235, construct. amino acid residues 3-36 2002; 418: 650654)
Hyperglycemia; Diabetes; Diabetes 4236, 4262, of mature PYY); also
Insipidus; Diabetes mellitus; Type 1 4267. including PYY(3-36)
diabetes; Type 2 diabetes; Insulin (G9R) (SEQ ID resistance;
Insulin deficiency; NO: 780)) Hyperlipidemia; Hyperketonemia;
Non-insulin dependent Diabetes Mellitus (NIDDM); Insulin- dependent
Diabetes Mellitus (IDDM); A Condition Associated With Diabetes
Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
Other indications for antibodies, antagonists: treatment of weight
loss; treatment of AIDS wasting; appetite stimulant; treatment of
cachexia. Adrenomedullin stimulates Vasodilation can be measured
Treatment of Congestive Heart 3144, 4239, See Table 2,
vasodilation; using assays known in the art Failure; Hypertension;
Myocardial 4260, 4261. SEQ ID NO: Z promotes bone (Ashton et al.
Pharmacology Infarction; Septic Shock; for particular growth. 2000;
61(2): 101-105. The Osteoporosis; Postmenopausal construct
promotion of bone growth osteoporosis; Osteopenia. can be measured
using assays known in the art, such as the osteoblast proliferation
assay (Cornish et al. Am J Physiol 1997 Dec; 273(6 Pt 1): E1113-
20). Ghrelin Stimulates release of Appetite and food intake can
Endocrine; loss
of body weight; loss 4241, 4242, See Table 2, growth hormone be can
be measured by of body weight associated with 4268. SEQ ID NO: Z
from anterior methods known in the art cancer or anorexia nervosa;
loss of for particular pituitary. Stimulates (Batterham et al.
Nature appetite; excessive appetite; body construct. appetite and
reduces 2002; 418: 650654) weight gain; Obesity; Diabetes; fat
burning. Acromegaly; Growth failure; Growth hormone deficiency;
Growth failure and growth retardation Prader-Willi syndrome in
children 2 years or older; Growth deficiencies; Growth failure
associated with chronic renal insufficiency; Postmenopausal
osteoporosis; burns; cachexia; cancer cachexia; dwarfism; metabolic
disorders; obesity; renal failure; Turner's Syndrome, pediatric and
adult; fibromyalgia; fracture treatment; frailty, AIDS wasting
Calcitonin gene-related CGRP is a potent The vasodilatory activity
of Migraine Headaches; Angina 4246, 4247, See Table 2, peptide
(CGRP) vasodilator, and a CGRP can be assayed using Pectoris;
Arrythmias; Heart Failure; 4248, 4249. SEQ ID NO: Z regulator of
the aortic ring vasodilation Hypertension; Postmenopausal for
particular endothelial and assay described in Pharmacol
Osteoporosis; Raynaud's Disease; construct. osteoblast cell Res.
1999 Mar; 39(3): 217-20. Subarachnoid Haemorrhage proliferation.
Endothelial and osteoblast Additional effects of cell proliferation
activities CGRP include can be measured in vitro (Eur reduced
gastric J Pharmacol. 2000 Dec secretion, increased 15; 409(3):
273-8; Proc Natl body temperature, Acad Sci USA 1990 anorexic
effects, and May; 87(9): 3299-303) positive inotropic and
chronotropic effects on the heart Insulin-like growth factor- IGF-I
is a pleiotropic IGF-I activity may be assayed Diabetes mellitus;
Growth disorders; 4251, 4252. See Table 2, 1 (Mecasermin; Somazon;
polypeptide with a in vitro using an serum Frailty; Amyotrophic
lateral SEQ ID NO: Z IGF-1; IGF-1 complex; wide range of actions
withdrawal apoptosis- sclerosis; Osteoarthritis; Kidney for
particular CEP 151; CGP 35126; FK in both central and protection
assay. (J disease & neuropathy; Dwarfism; construct. 780;
Mecar; RHIGF-I; peripheral nervous Endocrinol 2000 Oct; HIV-1
infections; Myocardial Somatomedin-1; sytems. It is involved
167(1): 165-74). Proliferation ischaemia; Osteoporosis; Multiple
Somatomedin-C; in growth and assay using breast carcinoma
sclerosis; Nerve disorders; Burns; SOMATOKINE; development and cell
line MCF-7 (Karey 1988 diabetes; peripheral neuropathies
MYOTROPHIN; IGEF; protects neurons Cancer Res. 48: 4083) DepoIGF-1)
against cell death via the activation of intracellular pathways
implicating phosphatidylinositide 3/Akt kinase. Neuraminidase
(Influenza Neuraminidase is one Neuraminidase activity can be
Vaccine or antigen against Influenza 4254, 4255 See Table 2, A
virus of two glycoproteins assayed in vitro using assays A, strain
H5N1; Avian Flu SEQ ID NO: Z (A/Goose/Guangdong/1/96 on the surface
of the known in the art, such as for particular (H5N1))) Influenza
virus those disclosed by Van construct. which, as an antigen Deusen
et al., Avian Dis. defines the particular 1983 Jul-Sep; 27(3):
745-50; strain of virus. The or by Wetherall et al., J. Clin.
variation of Microbiology 2003 Feb; neuraminidase 41(2): 742-750.
molecules over time permits the virus to evade human immune
responses and therefore necessitates the formulation of a new
vaccine each year. Neuraminidase cleaves the cellular- receptor
sialic acid residues to which the newly formed particles are
attached. This cleavage releases the viruses, which can now invade
new cells. Without neuraminidase, infection would be limited to one
round of replication, rarely enough to cause disease. Neuraminidase
may also facilitate viral invasion of the upper airways, possibly
by cleaving the sialic acid moieties on the mucin that bathes the
airway epithelial cells. Hemagglutinin [Influenza Hemagglutinin
(HA), Complement Fixation (CF) Vaccine or antigen against Influenza
4256, 4257. See Table 2, A virus (A/Hong the major influenza and
Hemagglutination A, strain H5N1; Avian Flu SEQ ID NO: Z
Kong/213/03(HK213:H5N1))] virus surface Inhibition (HI) can be
assayed for particular glycoprotein, has two in vitro using assays
known in construct. functions in the art, such as those infection:
(i) it binds disclosed by Prince et al., the virus to the Clin.
Diagn. Lab. Immunol. cellular receptors, 2003 May; 10(3): 481-482.
sialic acid residues of glycoproteins and glycolipids, and (ii)
following endocytosis it mediates the low pH- induced fusion of
viral and cellular membranes to permit entry of the viral genome
into the cell. All influenza viruses bear two surface
glycoproteins, a hemagglutinin and a neuraminidase, which are the
antigens that define the particular strain of influenza. The
variation of these molecules over time permits the virus to evade
human immune responses and therefore necessitates the formulation
of a new vaccine each year. The hemagglutinin is a sialic acid
receptor- binding molecule and mediates entry of the virus into the
target cell. Butyrylcholinesterase Butyrylcholinesterase BchE
activity assay Detoxification for Cocaine 4258, 4259. See Table 2,
(BchE, Serum accelerates cocaine "Differential inhibition of
Overdose; suxamethonium SEQ ID NO: Z Cholinesterase, metabolism: in
vitro human serum cholinesterase sensitivity; apnea; for particular
pseuocholinesterase E1 and in vivo effects in with fluoride:
recognition of construct. (CHE1)) nonhuman primates two new
phenotypes." Nature and humans. 191: 496-498, 1961. "A rare Carmona
GN, Jufer RA, genetically determined variant Goldberg SR, of
pseudocholinesterase in Gorelick DA, Greig NH, two German families
with Yu QS, Cone EJ, high plasma enzyme activity." Schindler CW.
Europ. J. Biochem. 99: 65-69, Drug Metab Dispos. 1979. "Genetic
analysis of a 2000 Mar; 28(3): 367-71. Japanese patient with An
atypical form of butyrylcholinesterase butyrylcholinesterase
deficiency." Ann. Hum. or the absence of its Genet. 61: 491-496,
1997. activity leads to prolonged apnea following administration of
the muscle relaxant suxamethonium. The widespread expression of
CHE1 in early differentiation suggests development-related
functions for this protein. Endothelin (ET-1; Endothelin is a
potent Endothelin-1-induced CHF, pulmonary hypertension, SEQ ID
NOs: Genbank Accession No. vasoconstrictor. It is vasoconstriction
is mediated hypertension, renal failure, any 781 and 782.
NP_001946) inappropriately by Ca2+ influx through a disease for
which endothelin elevated in non-selective cation channel.
antagonism would be beneficial, as hypertensive diseases It is
mediated by the anti-angiogenic or anti-tumor agent and in heart
failure. endothelin receptors ETA and for treatment of cancer and
macular Potent agonist for the ETB, both of which are G-
degeneration. ETA and ETB protein-coupled receptors. endothelin
receptors. Antagonists can be identified Induces the by their
ability to prevent Ca- production of flux mediated by ET-1
hypoxia-inducible peptide, which can be assayed factor 1.alpha.,
and, thus, in vitro using assays known in the production of art,
such as disclosed in VEGF. Wong-Dusting et al, J. Cardiovasc.
Pharmacol. (1991) 17: S236-S238; Ono et al., Nature (1994) 370:
252-253; Koyama, Y. et al., Neuroscience (2000) 101: 219-227;
Russell, F. D. and Molenaar, P., Trends Pharmacol. Sci. (2000) 21:
353-359; Masaki, T. et al., Eur. J. Pharmacol. (1999) 375: 133-138;
Inoue, A. et al., Proc. Natl. Acad. Sci. USA (1989) 86: 2863-2867;
and Spinella, F. et al., J. Biol. Chem. (2002) 277: 27850-27855.
Mechano Growth Factor A muscle growth Myoblast proliferation and
Wasting disease, cachexia, stroke, SEQ ID NOs: (MGF; IGF-IEc;
Genbank factor that appears differentiation can be assayed MI, CHF,
diseases where 783 and 784. Accession No. P05019) lost with aging.
Can in vitro by assays known in neuroprotection or muscle (skeletal
also act as the art such as disclosed in or smooth) protection or
neuroprotective Dluzniewska et al, FASEB J. regeneration would be
beneficial. agent. Unlike mature (2005) Sep 6; Goldspink G. J
IGF-I, MGF inhibits Musculoskelet Neuronal terminal Interact.
(2004) Jun; 4(2): 143-7; differentiation whilst or Yang SY and
Goldspink G, increasing myoblast FEBS Lett. (2002) Jul 3,
proliferation. 522(1-3): 156-60.
TABLE-US-00002 TABLE 2 Construct Expression SEQ ID SEQ ID Leader
Fusion No. ID Construct Name Description Vector SEQ ID NO: Y NO: X
SEQ ID NO: Z NO: A SEQ ID NO: B Sequence 1 3422
pSAC35:APsp.HSA.IFNa Acid Phosphatase signal peptide pSAC35 192 117
267 342 343 Acid followed by mature HSA and phoshpatase IFNa. 2
3423 pSAC35:INVsp.HSA. Invertase signal peptid followed pSAC35 193
118 268 344 345 Invertase IFNa by mature HSA and IFNa. 3 3424
pSAC35:KTsp.HSA.IFNa Killer Toxin signal peptide pSAC35 194 119 269
346 347 Killer toxin followed by mature HSA and IFNa. 4 2249
pSAC35:IFNa2-HSA Mature IFNa2 fused upstream of pSAC35 195 120 270
348 349 HSA/kex2 also named: mature HSA and downstream of
pSAC23:IFN.alpha.2-HSA HSA/kex2 leader sequence. 5 2343 pSAC35.INV-
Mature Interferon alpha2 fused pSAC35 196 121 271 350 351 invertase
IFNA2.HSA upstream of mature HSA and downstream of invertase signal
peptide. 6 2366 pSAC35.MAF- Mature IFNa2 fused upstream of pSAC35
197 122 272 352 353 MF.alpha.-1 IFNa2.HSA mature HSA and downstream
of yeast mating factor alpha leader sequence. 7 2381 pC4:HSA- Amino
acids C17 to E181 of pC4 198 123 273 354 355 HSA IFNa2(C17-E181)
IFNa2 (fragment shown as amino acids C1 to E165 of SEQ ID NO: 273)
fused downstream of HSA. 8 2382 pC4:IFNa2-HSA IFNa2 fused upstream
of mature pC4 199 124 274 356 357 Native HSA. IFN.alpha.2 leader 9
2410 pSAC35INV:IFNa- Mature IFNa2 fused downstream pSAC35 200 125
275 358 359 invertase HSA of the invertase signal peptide and
upstream of mature HSA. 10 3165 pSAC35:HSA.IFNa HSA fused upstream
of IFN.alpha. and pSAC35 201 126 276 None None HSA/kex2 also named
CID downstream of the HSA/kex2 3165, leader. pSAC35:HSA.INFa 11
3476 pSAC35:G19Rsp.HSA. The Modified HSA/kex2 signal pSAC35 202 127
277 360 361 Modified IFNa sequence followed by mature HSA/Kex2 HSA
followed by INF-alpha. 12 3690 pC4:MPIFSP.BNP/HSA Myeloid
progenitor inhibitory pC4 203 128 278 362 363 MPIF-1 factor-1
(MPIF) signal sequence followed by BNP fused to the N- terminus of
mature HSA. 13 3691 pC4:SPCON.BNP/HSA A consensus signal sequence
pC4 204 129 279 364 365 Consensus followed by BNP fused to the N-
terminus of mature HSA. 14 3715 pSAC35:BNP29/HSA. A single copy of
human BNP pSAC35 205 130 280 366 367 HSA/Kex2 S65 (amino acids
1-29) fused to the N-terminus of HSA (S65-L585), an HSA N-terminal
truncation (delta 1-64). This is downstream of the HSA/Kex2 signal
sequence. 15 3723 pEE12.1:MPIFSP.BNP/ Myeloid progenitor inhibitory
pEE12.1 206 131 281 None None MPIF-1 HSA factor-1 (MPIF) signal
sequence followed by BNP fused to the N- terminus of mature HSA. 16
3724 pEE12.1:SPCON.BNP/ A consensus signal sequence pEE12.1 207 132
282 None None Consensus HSA followed by BNP fused to the N-
terminus of mature HSA. 17 3725 pEE12.1:SPCON2.BNP/ A consensus
signal sequence pEE12.1 208 133 283 None None Consensus HSA
followed by BNP fused to the N- Signal terminus of mature HSA.
Peptide #2 18 3736 pC4:SPCON2.BNP/ A consensus signal sequence pC4
209 134 284 368 369 Consensus HSA followed by BNP fused to the N-
Signal terminus of mature HSA. Peptide #2 19 3769 pC4:BNP(R13G)/HSA
Myeloid progenitor inhibitory pC4 210 135 285 370 371 MPIF-1
factor-1 (MPIF) signal sequence followed by BNP mutant (R13G) fused
to the N-terminus of mature HSA. 20 3778 pC4:SPCON.BNP29/ A single
copy of human BNP (1-29) pC4 211 136 286 372 373 Consensus HSA.S65
fused to the N-terminus of HSA (S65-L585), an HSA N- terminal
truncation (delta 1-64). This is downstream of a consensus signal
sequence. 21 3783 pC4:SPCON.BNP(R13G)/ Consensus signal sequence
pC4 212 137 287 374 375 Consensus HSA followed by BNP mutant (R13G)
fused to the N-terminus of mature HSA. 22 3795 pC4:SPCON.BNP(K14G)/
Consensus signal sequence pC4 213 138 288 376 377 Consensus HSA
followed by BNP mutant (K14G) fused to the N-terminus of mature
HSA. 23 3796 pSAC35:HSA/BNP Followed by mature HSA fused pSAC35 214
139 289 378 379 HSA/Kex2 to the N-terminus of BNP. 24 3809
pSAC35:BNP30(GGG)/ HSA/Kex2 signal sequence pSAC35 215 140 290 380
381 HSA/Kex2 HSA followed by BNP (amino acids 1-30) fused via
tripartite glycines to the N-terminus of mature HSA. 25 3886
pSAC35:HSA/KEX2. HSA/Kex2 signal sequence pSAC35 216 141 291 None
None HSA/Kex2 LANP.HSA followed by LANP fused to the N-terminus of
mature HSA. LANP corresponds to amino acids 26-55 of SeqID No: 291
(hereby referred to as LANP). 26 3887 pSAC35:HSA/KEX2. HSA/Kex2
signal sequence pSAC35 217 142 292 None None HSA/Kex2 HSA.LANP
followed by mature HSA fused to the N-terminus of mature LANP. 27
3888 pSAC35:HSA/KEX2. HSA/Kex2 signal sequence pSAC35 218 143 293
None None HSA/Kex2 VDP.HSA followed by VDP fused to the N- terminus
of mature HSA. VDP corresponds to amino acids 56-92 of SeqID No:
293 (hereby referred to as VDP). 28 3889 pSAC35:HSA/KEX2. HSA/Kex2
signal sequence pSAC35 219 144 294 None None HSA/Kex2 HSA.VDP
followed by mature HSA fused to the N-terminus of mature VDP. 29
3890 pSAC35:HSA/KEX2. HSA/Kex2 signal sequence pSAC35 220 145 295
None None HSA/Kex2 KUP.HSA followed by KUP fused to the N- terminus
of mature HSA. KUP corresponds to amino acids 104-123 of SeqID No:
295 (hereby referred to as KUP). 30 3891 pSAC35:HSA/KEX2. HSA/Kex2
signal sequence pSAC35 221 146 296 None None HSA/Kex2 HSA.KUP
followed by mature HSA fused to the N-terminus of mature KUP. 31
3892 pSAC35:HSA/KEX2. HSA/Kex2 signal sequence pSAC35 222 147 297
None None HSA/Kex2. CNP.HSA followed by CNP fused to the N-
terminus of mature HSA. CNP corresponds to amino acids 105-123 of
SeqID No: 297 (hereby referred to as KUP). 32 3893 pSAC35:HSA/KEX2.
HSA/Kex2 signal sequence pSAC35 223 148 298 None None HSA/Kex2
HSA.CNP followed by mature HSA fused to the N-terminus of mature
CNP. 33 3894 pSAC35:HSA/KEX2. HSA/Kex2 signal sequence pSAC35 224
149 299 None None HSA/Kex2 DNP.HSA followed by DNP fused to the N-
terminus of mature HSA. 34 3895 pSAC35:HSA/KEX2. HSA/Kex2 signal
sequence pSAC35 225 150 300 None None HSA/Kex2 HSA.DNP followed by
mature HSA fused to the N-terminus of mature DNP. 35 3618
pC4:SPCON.BNP1-32 A consensus signal sequence pC4 226 151 301 382
383 Consensus (2x)/HSA followed by two, tandem copies of mature BNP
fused to the N- terminus of mature HSA. 36 3484 pSAC35:ANP/HSA
HSA/kex2 leader followed by pSAC35 227 152 302 384 385 HSA/kex2
atrial natriuretic peptide followed by mature HSA. 37 1933
pSAC35:HCC- Amino acids T20 to N93 of pSAC35 228 153 303 386 387
HSA/kex2 1.T20-N93:HSA HCC-1 fused upstream of mature HSA and
downstream of the HSA/kex2 leader sequence. 38 1934 pSAC35:HCC-
Amino acids T20 to N93 of pSAC35 229 154 304 388 389 HSA/kex2
1C.O.T20-N93:HSA HCC-1 fused upstream of mature HSA and downstream
of the HSA/kex2 leader sequence. DNA sequence is codon optimized
for yeast expression. 39 1947 pSAC35:d8HCC- Amino acids G28 to N93
of pSAC35 230 155 305 390 391 HSA/kex2 1.G28-N93:HSA HCC-1 fused
upstream of mature HSA and downstream of HSA/kex2 leader sequence.
40 1948 pSAC35:d8HCC- Amino acids G28 to N93 of pSAC35 231 156 306
392 393 HSA/kex2 1C.O.G28-N93:HSA HCC-1 fused upstream of mature
HSA and downstream of HSA/kex2 leader sequence. DNA sequence is
codon optimized for yeast expression. 41 1955 pSAC35:t9HCC- Amino
acids G28 to N93 of pSAC35 232 157 307 394 395 HSA/kex2
1.G28-N93:spcHSA HCC-1 fused upstream of a 16 amino acid spacer and
mature HSA and downstream of HSA/kex2 leader sequence. 42 1998
pC4:CKB1.G28-N93. Amino acids G28 to N93 of pC4 233 158 308 396 397
HSA HSA CkBeta1 fused upstream of mature HSA and downstream of the
HSA leader sequence. 43 2355 pSAC35:MATalpha. Amino acids G28 to
N93 of pSAC35 234 159 309 398 399 MF.alpha.-1 d8ckbeta1.G28-N93:
Ckbeta1 fused upstream of HSA mature HSA and downstream of the
yeast mating factor alpha leader sequence. 44 2412
pSAC35:delKEX.d8ckbeta1. Amino acids G28 to N93 of pSAC35 235 160
310 400 401 HSA minus G28-N93: Ckbeta1 fused downstream of the the
KEX HSA HSA signal sequence (with the site KEX site deleted - last
6 amino acids of the leader) and upstream of mature HSA. 45 2449
pSAC35:INV.d8CKB1. Amino acids G28 to N93 of pSAC35 236 161 311 402
403 Invertase G28-N93:HSA Ckbeta1 fused downstream of the invertase
signal peptide and upstream of mature HSA. 46 2837
pSAC35:CKB1.K21-N93: K21-N93 of CKB1 (fragment pScCHSA 237 162 312
404 405 HSA/kex2 HSA shown as K2 to N74 of SEQ ID NO: 1735) fused
downstream of the HSA/kex2 leader and upstream of mature HSA. 47
2838 pSAC35:CKB1.T22-N93: T22-N93 of CKB1 (fragment pScCHSA 238 163
313 406 407 HSA/kex2 HSA shown as T3 to N74 of SEQ ID NO: 1736)
fused downstream of the HSA/kex2 leader and upstream of mature HSA.
48 2839 pSAC35:CKB1.E23-N93: E23-N93 of CKB1 (fragment pScCHSA 239
164 314 408 409 HSA/kex2 HSA shown as E4 to N74 of SEQ ID NO: 1737)
fused downstream of the HSA/kex2 leader and
upstream of mature HSA. 49 2840 pSAC35:CKB1.S24-N93: S24-N93 of
CKB1 (fragment pScCHSA 240 165 315 410 411 HSA/kex2 HSA shown as S5
to N74 of SEQ ID NO: 1738) fused downstream of the HSA/kex2 leader
and upstream of mature HSA. 50 2841 pSAC35:CKB1.S25-N93: S25-N93 of
CKB1 (fragment pScCHSA 241 166 316 412 413 HSA/kex2 HSA shown as S6
to N74 of SEQ ID NO: 1739) fused downstream of the HSA/kex2 leader
and upstream of mature HSA. 51 2842 pSAC35:CKB1.S26-N93: S26-N93 of
CKB1 (fragment pScCHSA 242 167 317 414 415 HSA/kex2 HSA shown as S7
to N74 of SEQ ID NO: 1740) fused downstream of the HSA/kex2 leader
and upstream of mature HSA. 52 2843 pSAC35:CKB1.R27-N93: R27-N93 of
CKB1 (fragment pScCHSA 243 168 318 416 417 HSA/kex2 HSA shown as R8
to N74 of SEQ ID NO: 1741) fused downstream of the HSA/kex2 leader
and upstream of mature HSA. 53 2844 pSAC35:CKB1.P29-N93: P29-N93 of
CKB1 (fragment pScCHSA 244 169 319 418 419 HSA/kex2 HSA shown as
P10 to N74 of SEQ ID NO: 1742) fused downstream of the HSA/kex2
leader and upstream of mature HSA. 54 2845 pSAC35:CKB1.Y30-N93:
Y30-N93 of CKB1 (fragment pScCHSA 245 170 320 420 421 HSA/kex2 HSA
shown as Y11 to N74 of SEQ ID NO: 1743) fused downstream of the
HSA/kex2 leader and upstream of mature HSA. 55 2849
pC4.MPIFsp.CKB1. G28-N93 of CKB1 (fragment pC4 246 171 321 422 423
MPIF G28-N93.HSA shown as G9 to N74 of SEQ ID NO: 1744) fused
downstream of the MPIF signal peptide and upstream of mature HSA.
56 2947 pSAC:CKb- Invertase signal peptide followed pSAC35 247 172
322 424 425 Invertase 1.delta.8(x2).HSA by amino acids G28-N93 of
full length CK.beta.1 (SEQ IDNO: 1769), tandemly repeated, followed
by mature HSA. 57 3066 pSAC35:CKB- Invertase signal peptide
followed pScCHSA 248 173 323 426 427 invertase 1d8.GLP-1(7- by
amino acids G28-N93 of full 36).HSA length CK.beta.1 (SEQ IDNO:
1788), followed by GLP-1(7-36), followed by mature HSA. 58 3105
pSAC35:INV.t9HCC- Invertase signal peptide followed pSAC35 249 174
324 428 429 Invertase 1.G28-N93:spc.HSA by amino acids G28 to N93
of HCC-1 fused upstream of a spacer and mature HSA. 59 3124
pSAC35:INV.CKB1. Invertase signal peptide followed pSAC35 250 175
325 430 431 invertase P29-N93:HSA by amino acids 29 to 93 of full
length ckbeta1 fused to N- terminus of HSA. 60 3125 pSAC35:INV.CKb-
Invertase signal peptide followed pSAC35 251 176 326 432 433
invertase 1.R27-N93:HSA by amino acids 27 to 93 of full length
ckbeta1 fused to N- terminus of HSA. 61 3139 pSAC35:INV.CKB1.
Invertase signal peptide followed pSAC35 252 177 327 434 435
Invertase G28-N93. by amino acids G28-N93 of full DAHK.HSA length
CK.beta.1 (see, e.g, SEQ IDNO: 1788), followed by a 16 amino acid
linker derived from the N-terminus of HSA, followed by mature HSA.
62 3152 pSAC35:INV.CKB1. Invertase signal peptide followed pSAC35
253 178 328 436 437 invertase Met.R27-N93.HSA by a Met, followed by
amino acids R27-N93 of full length CK.beta.1, followed by mature
HSA. 63 3153 pSAC35:INV.CKB1. Invertase signal peptide followed
pSAC35 254 179 329 438 439 Invertase Met.S26-N93.HSA by a Met,
followed by amino acids S26-N93 of full length CK.beta.1, followed
by mature HSA. 64 3154 pSAC35:INV.CKB1. Invertase signal peptide
followed pSAC35 255 180 330 440 441 invertase Met.S25-N93.HSA by a
Met, followed by amino acids S25-N93 of full length CK.beta.1,
followed by mature HSA. 65 3155 pSAC35:INV.CKB1. Invertase signal
peptide followed pSAC35 256 181 331 442 443 invertase
Met.G28-N93.HSA by a Met, followed by amino acids G28-N93 of full
length CK.beta.1, followed by mature HSA. 66 3156 pSAC35:INV.CKB1.
Invertase signal peptide followed pSAC35 257 182 332 444 445
invertase Met.P29-N93.HSA by a Met, followed by amino acids P29-N93
of full length CK.beta.1, followed by mature HSA. 67 3169
pSAC35:KT.CKB1. Killer toxin signal sequence fused pSAC35 258 183
333 None None Killer toxin G28-N93.HSA upstream of amino acids G28
through N93 of CKB1 (fragment shown as amino acids G1 to N66 of SEQ
ID NO: 1822) and mature HSA. 68 3170 pSAC35:KT.HA.CKB1. Killer
toxin signal sequence pSAC35 259 184 334 None None Killer toxin
G28-N93.HSA followed by HA dipeptide and amino acids G28 through
N93 of CKB1 (fragment shown as amino acids G1 to N66 of SEQ ID NO:
1823) and mature HSA. 69 3202 pSAC35:API.d8CKb1/ HSA/kex2 leader
followed by pSAC35 260 185 335 446 447 HSA/kex2 HSA amino acids
"API" followed by d8CKb1 and mature HSA. The sequence of delta 8
for CKB1 is shown in SEQ ID NO: 1833. 70 3203 pSAC35:ASL.d8CKb1/
HSA/kex2 leader followed by pSAC35 261 186 336 448 449 HSA/kex2 HSA
amino acids "ASL" followed by d8CKb1 and mature HSA. 71 3204
pSAC35:SPY.d8CKb1/ HSA/kex2 leader followed by pSAC35 262 187 337
450 451 HSA/kex2 HSA amino acids "SPY" followed by d8CKb1 and
mature HSA. 72 3205 pSAC35:MSPY.d8CKb1/ HSA/kex2 leader followed by
pSAC35 263 188 338 452 453 HSA/kex2 HSA amino acids "MSPY" followed
by d8CKb1 and mature HSA. 73 3206 pSAC35:CPYSC.d8CKb1/ HSA/kex2
leader followed by a pSAC35 264 189 339 454 455 HSA/kex2 HSA five
amino acid linker followed by d8CKb1 and mature HSA. 74 3207
pSAC35:GPY.d8CKb1/ HSA/kex2 leader followed by pSAC35 265 190 340
456 457 HSA/kex2 HSA amino acids "GPY" followed by d8CKb1 and
mature HSA. 75 3272 pSAC35.INV:{D}8CK CKbeta-1 tandem repeat (x2)
pSAC35 266 191 341 458 459 Invertase {b}1(x2)/HSA fusion to the
N-termal HSA. Under the invertase signal peptide. 76 3692
pC4:MPIFSP.cynoBNP/ Myeloid progenitor inhibitory pC4 496 460 532
568 569 MPIF-1 CSA factor-1 (MPIF-1) signal (Mammalian) sequence
followed by cynomolgus monkey BNP fused at the N-terminus of
cynomolgus monkey serum albumin. 77 3716 pC4:ratBNP45/RSA Pre-pro
region of the HSA leader pC4 497 461 533 570 571 HSA sequence
followed by rat BNP(1-45) (Mammalian) fused at the N-terminus of
rat serum albumin. 78 3741 pC4:MPIFSP.ratBNP Myeloid progenitor
inhibitory pC4 498 462 534 572 573 MPIF-1 45/RSA factor-1 (MPIF-1)
signal (Mammalian) sequence followed by rat BNP(1-45) fused at the
N-terminus of rat serum albumin. 79 3956 pSAC35:BNP(K3-H32).
HSA/Kex2 signal sequence pSAC35 499 463 535 574 575 HSA/Kex2 HSA
followed by BNP (amino acids 3-32) fused at the N-terminus of
mature HSA. 80 3957 pSAC35:BNP(K3-R30). HSA/Kex2 signal sequence
pSAC35 500 464 536 576 577 HSA/Kex2 GGG.HSA followed by BNP (amino
acids 3-30) fused via tripartite glycines to the N-terminus of
mature HSA. 81 3959 pSAC35:HSA.BNP HSA/Kex2 signal sequence pSAC35
501 465 537 578 579 HSA/Kex2 (S1-L29) followed by mature HSA fused
to BNP (amino acids 1-29) at its C- terminus. 82 3960
pSAC35:HSA.IFNalpha2a HSA/Kex2 signal sequence pSAC35 502 466 538
580 581 HSA/Kex2 followed by mature HSA fused to interferon alpha
2a at its C- terminus. 83 3961 pSAC35:HSA.(BNP HSA/Kex2 signal
sequence pSAC35 503 467 539 582 583 HSA/Kex2 (S1-L29))x2 followed
by mature HSA fused to two tandem copies of BNP (amino acids 1-29)
at its C- terminus. 84 3962 pcDNA3.1-:BNP(1-32). Pre-pro region of
the HSA signal pCDNA3.1 504 468 540 None None HSA HSA sequence
followed by BNP (amino acids 1-32) fused at the N-terminus of
mature HSA. 85 3965 pSAC35:HSA.BNP HSA/Kex2 signal sequence pSAC35
505 469 541 584 585 HSA/Kex-2 (S1-R30) followed by mature HSA fused
to BNP (amino acids 1-30) at its C- terminus. 86 3966
pSAC35:HSA.BNP HSA/Kex2 signal sequence pSAC35 506 470 542 586 587
HSA/Kex-2 (S1-R31) followed by mature HSA fused to BNP (amino acids
1-31) at its C- terminus. 87 3967 pSAC35:HSAsp.BNP Pre region of
the HSA signal pSAC35 507 471 543 None None HSA (S1-H32).HSA
sequence followed BNP (amino acids 1-32) fused to the N- terminus
of mature HSA. 88 3968 pSAC35:INVsp.BNP Invertase signal peptide
followed 508 472 544 588 589 Invertase (S1-H32).HSA by BNP (amino
acids 1-32) fused to the N-terminus of mature HSA. 89 3970
pSAC35:INVsp.CKB1 Invertase signal peptide followed pSAC35 509 473
545 590 591 Invertase (T20-N93) by mature mutant Ckbeta1 (E34A).HSA
(amino acids 20-93 (E34A)) fused to the N-terminus of mature HSA.
90 4005 pSAC35:BNP(S1-R30). HSA/Kex-2 signal sequence pSAC35 510
474 546 None None HSA/Kex-2 HSA followed by BNP (amino acids 1-30)
fused to the N-terminus of mature HSA. 91 4006 pSAC35:BNP(S1-R30).
HSA/Kex-2 signal sequence pSAC35 511 475 547 None None HSA/Kex-2
HSA(D25-E40). followed by BNP (amino acids 1-30) HSA fused via a 16
amino acid linker derived from HSA (amino acids 25-40) to the
N-terminus of mature HSA. 92 4007 pSAC35:BNP(S1-R30). HSA/Kex-2
signal sequence pSAC35 512 476 548 None None HSA/Kex-2
HSA(D25-Q56). followed by BNP (amino acids 1-30) HSA fused via a 32
amino acid linker derived from HSA (amino acids 25-56) to the
N-terminus of mature HSA. 93 4062 pcDNA3.1:MPIFsp. Myeloid
progenitor inhibitory pCDNA3.1- 513 477 549 None None MPIF-1
HSA.BNP(S1-H32) factor-1 (MPIF-1) signal sequence followed by
mature HSA fused to BNP at its C- terminus. 94 4130 pSAC35:HSA
HSA/Kex-2 signal sequence pSAC35 514 478 550 None None HSA/Kex-2
(C34S)-BNP (1-29) followed by a mutant of mature HSA (C34S) fused
to BNP at its
C-terminus. 95 4160 pSAC35:BNP(S1-R30: HSA/Kex-2 followed by a
mutant pSAC35 515 479 551 None None HSA/Kex-2 P2A).HSA BNP (amino
acids 1-30 (P2A)) fused to the N-terminus of mature HSA. 96 4161
pSAC35:BNP(S1-R30: HSA/Kex-2 followed by a mutant pSAC35 516 480
552 None None HSA/Kex-2 P2L).HSA BNP (amino acids 1-30 (P2L)) fused
to the N-terminus of mature HSA. 97 4167 pSAC35:DSA.dogB Modified
HSA/Kex-2 signal pSAC35 517 481 553 None None Modified NP(1-32)
sequence followed by dog serum HSA/Kex-2 albumin fused to dog BNP
(amino acids 1-32) at its C- terminus. 98 4168 pSAC35:DSA.dogB
Modified HSA/Kex-2 signal pSAC35 518 482 554 None None Modified
NP(1-29) sequence followed by dog serum HSA/Kex-2 albumin fused to
dog BNP (amino acids 1-29) at its C- terminus. 99 4169
pSAC35:KTsp.CSA. Killer toxin signal sequence pSAC35 519 483 555
None None Killer toxin cynoBNP(1-29) followed by cynomolgus monkey
serum fused to cyno BNP (1-29) at its C-terminus. 100 4170
pSAC35:KTsp.RSA.ratBNP Killer toxin signal sequence pSAC35 520 484
556 None None Killer toxin (1-45) followed by rat serum albumin
fused to rat BNP (amino acids 1-45) at its C-terminus. 101 4171
pSAC35:KTsp.RSA.ratBNP Killer toxin signal sequence pSAC35 521 485
557 None None Killer toxin (14-42) followed by rat serum albumin
fused to rat BNP (amino acids 14-42) at its C-terminus. 102 4172
pSAC35:MSA.mouse Invertase signal sequence pSAC35 522 486 558 None
None Invertase BNP(1-45) followed by mouse serum albumin fused to
mouse BNP (amino acids 1-45) at its C- terminus. 103 4173
pc4HSA:HSA.2xBeta Pre-pro region of the HSA signal pC4 523 487 559
592 593 HSA Defensin-2 sequence followed by mature (Mammalian) HSA
fused to two tandem copies of Beta Defensin-2 at its C- terminus.
104 4174 pSAC35:ANP.HSA. HSA/Kex-2 signal sequence pSAC35 524 488
560 594 595 HSA/Kex-2 BNP(S1-L29) followed by mature ANP fused to
the N-terminus of mature HSA and BNP (amino acids 1-29) fused to
the C-terminus of mature HSA. 105 4175 pc4HSA:HSA.beta Pre-pro
region of the HSA signal pC4 525 489 561 596 597 HSA Defensin-2
sequence followed by mature (Mammalian) HSA fused to beta
Defensin-2 at its C-terminus. 106 4176 pc4HSA:HSASp.beta Pre-pro
region of the HSA signal pC4 526 490 562 598 599 HSA Defensin-2.HSA
sequence followed by beta (Mammalian) Defensin-2 fused to the N-
terminus of mature HSA. 107 4177 pc4HSA:HSASp.2xbeta Pre-pro region
of the HSA signal pC4 527 491 563 600 601 HSA Defensin-2.HSA
sequence followed by two tandem (Mammalian) copies of beta
Defensin-2 fused to the N-terminus of mature HSA. 108 4178
pSAC35:KEX2.2xbetaDefensin- HSA/Kex-2 signal sequence pSAC35 528
492 564 None None HSA/Kex-2 2.HSA followed by two tandem copies of
beta Defensin-2 fused to the N- terminus of mature HSA. 109 4179
pSAC35:KEX2.beta HSA/Kex-2 signal sequence pSAC35 529 493 565 None
None HSA/Kex-2 Defensin-2.HSA followed by beta Defensin-2 fused to
the N-terminus of mature HSA. 110 4180 pSAC35:HSA.beta HSA/Kex-2
signal sequence pSAC35 530 494 566 None None HSA/Kex-2 Defensin-2
followed by mature HSA fused to beta defensin-2 at its C-terminus.
111 4181 pSAC35:HSA.2x.betaDefensin-2 HSA/Kex-2 signal sequence
pSAC35 531 495 567 None None HSA/Kex-2 followed by mature HSA fused
to two tandem copies of beta Defensin-2 at its C-terminus. 112 4191
pSAC35:HSA.Fractalkine. HSA/Kex-2 signal sequence pSAC35 641 602
680 None None HSA/Kex-2 Q25-G100 followed by mature HSA fused to
soluble fractalkine (amino acids 25-100) at its C-terminus. 113
4192 pSAC35:Fractalkine. HSA/Kex-2 signal sequence pSAC35 642 603
681 None None HSA/Kex-2 Q25-G100.HSA followed by soluble
fractalkine (amino acids 25-100) fused to the N-terminus of mature
HSA. 114 4193 pSAC35:Fractalkine. HSA/Kex-2 signal sequence pSAC35
643 604 682 None None HSA/Kex-2 Q25-G100 followed by soluble mutant
(R48Q).HSA fractalkine (amino acids 25-100 (R48Q, corresponding to
R71Q of full-length fractalkine)) fused to the N-terminus of mature
HSA. 115 4194 pSAC35:Fractalkine. HSA/Kex-2 signal sequence pSAC35
644 605 683 None None HSA/Kex-2 V29-G100.HSA followed by mature HSA
fused to a soluble dominant negative form of fractalkine (amino
acids 29-100) at its C-terminus. 116 4213 pSAC35:INVsp.OXM.
Invertase signal sequence pSAC35 645 606 684 None None Invertase
HSA followed by full-length oxyntomodulin fused to the N- terminus
of HSA. 117 4215 pSAC35:INVsp.PYY Invertase signal sequence pSAC35
646 607 685 719 720 Invertase 3-36.HSA(D25-E40). followed by PYY
(amino acids 3-36) HSA.OXM fused via a 16 amino acid linker from
HSA (amino acids 25-40) to the N-terminus of mature HSA which is
fused at its C-terminus to full-length oxyntomodulin. 118 4217
pC4:HSA.OXM Pre-pro region of the HSA signal pC4 647 608 686 721
722 HSA sequence followed by mature (Mammalian) HSA fused at its
C-terminus to full-length oxyntomodulin. 119 4227
pSAC35:Invsp.KP.HSA Invertase signal sequence pSAC35 648 609 687
None None Invertase (D25-E40).HSA followed by killer toxin peptide
fused via a 16 amino acid linker from HSA (amino acids 25-40) to
mature HSA. 120 4232 pCDNA3.1+: Pre-pro region of the HSA signal
pCDNA3.1+ 649 610 688 None None HSA HSA.OXM sequence followed by
mature HSA fused at its C-terminus to full length oxyntomodulin.
121 4233 pSAC35:KEX2.TIMP4. HSA/Kex-2 leader sequence pSAC35 650
611 689 723 724 HSA/Kex-2 C30-G157.HSA followed by a C-terminal
truncated form of mature TIMP4 (amino acids 30-157) fused to the
N-terminus of mature HSA. 122 4234 pSAC35:HSA.TIMP. HSA/Kex-2
leader sequence pSAC35 651 612 690 725 726 HSA/Kex-2 C30-G157
followed by mature HSA fused at its C-terminus to a C-terminal
truncated form of mature TIMP4 (amino acids 30-157). 123 4235
pC4:HSA.2x(PYY 3-36) Pre-pro region of the HSA signal pC4 652 613
691 None None HSA sequence followed by mature (Mammalian) HSA fused
to two tandem copies of PYY (amino acids 3-36) at its C-terminus.
124 4236 pC4: A consensus signal sequence pC4 653 614 692 727 None
Consensus SPCON2sp.PYY(3-36). followed by PYY (amino acids 3-36)
(Mammalian) HSA fused to the N-terminus of mature HSA. 125 4239
pSAC35: Modified HSA/Kex-2 leader pSAC35 654 615 693 None None
Modified [HSA/KEX(R19G)]sp. sequence followed by HSA/Kex ADM.HSA
adrenomedullin fused to the N- terminus of mature HSA. 126 4240
pC4: A consensus signal sequence pC4 655 616 694 None None
Consensus SPCON2sp.OXM.HSA followed by oxynotmodulin fused
(Mammalian) to the N-terminus of mature HSA. 127 4241
pC4:HSA.Ghrelin Pre-pro region of the HSA signal pC4 656 617 695
728 729 HSA sequence followed by mature (Mammalian) HSA fused to
ghrelin at its C- terminus 128 4242 pSAC35: Pre-pro region of the
HSA signal pSAC35 657 618 696 730 731 HSA HSA.Ghrelin sequence
followed by mature HSA fused to ghrelin at its C- terminus 129 4246
pSAC35:CGRP(V8-F37). HSA/Kex-2 signal sequence pSAC35 658 619 697
None None HSA/Kex-2 HSA followed by a truncated form of CGRP (amino
acids 8-37) fused to the N-terminus of mature HSA. 130 4247
pSAC35:HSA.CGRP HSA/Kex-2 signal sequence pSAC35 659 620 698 None
None HSA/Kex-2 (V8-F37) followed by mature HSA fused to a truncated
form of CGRP (amino acids 8-37) at its C-terminus. 131 4248
pSAC35:CGRP(L12-F37). HSA/Kex-2 signal sequence pSAC35 660 621 699
None None HSA/Kex-2 HSA followed by a truncated form of CGRP (amino
acids 12-37) fused to the N-terminus of mature HSA. 132 4249
pSAC35:HSA.CGRP HSA/Kex-2 signal sequence pSAC35 661 622 700 None
None HSA/Kex-2 (L12-F37) followed by a mature HSA fused to a
truncated form of CGRP (amino acids 12-37) at its C- terminus. 133
4251 pSAC35:HSA.IGF1 HSA/Kex-2 signal sequence pSAC35 662 623 701
None None HSA/Kex-2 (G49-M153) followed by mature HSA fused to a
mature form of IGF1 (amino acids 49-153) at its C-terminus. 134
4252 pSAC35:IGF1(G49-M153). HSA/Kex-2 signal sequence pSAC35 663
624 702 None None HSA/Kex-2 HSA followed by a mature form of IGF1
(amino acids 49-153) fused to the N-terminus of mature HSA. 135
4253 pC4:SPCON2.OXM. A consensus signal sequence pC4 664 625 703
None None Consensus OXM.HSA followed by two tandem copies of
(Mammalian) oxyntomodulin fused to the N- terminus of mature HSA.
136 4254 pSAC35:NA(S35-K469). HSA/Kex-2 signal sequence pSAC35 665
626 704 None None HSA/Kex-2 HSA followed an N-terminally truncated
form of neuraminidase from Influenza A/Hong Kong/213/03 (HK213;
H5N1) (amino acids 35-469) fused via a Gly-Ser linker
(GGGGSGGGGSGG) to the N- terminus of mature HSA. 137 4255
pC4.SPCON2:NA(S35-K469). A consensus signal sequence pC4 666 627
705 None None Consensus HSA followed by an N-terminally (Mammalian)
truncated form of neuraminidase from Influenza A/Hong Kong/213/03
(HK213; H5N1) (amino acids 35-469) fused via a Gly-Ser
linker(GGGGSGGGGSGG) to the N-terminus of mature HSA. 138 4256
pSAC35:HA(D17-Q531). HSA/Kex-2 signal sequence pSAC35 667 628 706
None None HSA/Kex-2 HSA followed by a truncated form of
hemagglutinin from Influenza A/Hong Kong/213/03 (HK213; H5N1)
(amino acids 17-531) lacking a signal peptide and the C-terminal
hydrophobic domain, fused via a Gly-Ser
linker (GGGGSGGGGSGG) to the N-terminus of mature HSA. 139 4257
pC4:SPCON2.HA(D17-Q531). A consensus signal sequence pC4 668 629
707 None None Consensus HSA followed by a truncated form of
(Mammalian) hemagglutinin from Influenza A/Hong Kong/213/03 (HK213;
H5N1) (amino acids 17-531) lacking a signal peptide and the
C-terminal hydrophobic domain, fused via a Gly-Ser linker
(GGGGSGGGGSGG) to the N-terminus of mature HSA. 140 4258
pSAC35.BChE(E29-V557). HSA/Kex-2 signal sequence pSAC35 669 630 708
None None HSA/Kex-2 HSA followed by butyrylcholinesterase (BChE)
(amino acids 39-529 (A356W, Y360A, N45Q, N483Q, N509Q and N514Q))
fused to the N-terminus of mature HSA. 141 4259 pC4:SPCON2.BChE A
consensus signal sequence pC4 670 631 709 None None Consensus
(E29-V557).HSA followed by butyrylcholinesterase (Mammalian) (BChE)
(amino acids 39-529 (A356W, Y360A, N45Q, N483Q, N509Q and N514Q))
fused to the N-terminus of mature HSA. 142 4260 pC4:HSA.ADM
(Y1-G53) Pre-pro region of the HSA signal pC4 671 632 710 None None
HSA sequence followed by mature (Mammalian) HSA fused at its
C-terminus to adrenomedullin containing an additional glycine
residue at the C-terminus (amino acids 1-53). 143 4261
pSAC35:HSA.ADM HSA/Kex-2 signal sequence pSAC35 672 633 711 None
None HSA/Kex-2 (Y1-G53) followed by mature HSA fused at its
C-terminus to adrenomedullin containing an additional glycine
residue at the C-terminus (amino acids 1-53). 144 4262
pC4:HSA.PYY(I3-G37) Pre-pro region of the HSA signal pC4 673 634
712 None None HSA sequence followed by mature (Mammalian) HSA fused
at its C-terminus to PYY containing an additional glycine residue
at the C-terminus (amino acids 3-37). 145 4267
pSAC35:HSA.PYY(I3-G37) HSA/Kex-2 signal sequence pSAC35 674 635 713
None None HSA/Kex-2 followed by mature HSA fused at its C-terminus
to PYY containing an additional glycine residue at the C-terminus
(amino acids 3-37). 146 4290 pSAC35:HSA(C34S). HSA/Kex-2 signal
sequence pSAC35 675 636 714 None None HSA/Kex-2 INFa followed by a
mutant mature HSA (C34S of mature HSA) fused to mature interferon
alpha at its C-terminus. 147 4291 pSAC35.HSA.INFa HSA/Kex-2 signal
sequence pSAC35 676 637 715 None None HSA/Kex-2 (C586S) followed by
mature HSA fused to a mutant mature interferon alpha (C1S of mature
IFNa) at its C- terminus. 148 4292 pSAC35:HSA.INFa HSA/Kex-2 signal
sequence pSAC35 677 638 716 None None HSA/Kex-2 (C614S) followed by
mature HSA fused to a mutant mature interferon alpha (C29S of
mature IFNa) at its C- terminus. 149 4295 pSAC35:HSA.INFa HSA/Kex-2
signal sequence pSAC35 678 639 717 None None HSA/Kex-2 (C683S)
followed by mature HSA fused to a mutant mature interferon alpha
(C98S of mature IFNa) at its C- terminus. 150 4296 pSAC35:HSA.INFa
HSA/Kex-2 signal sequence pSAC35 679 640 718 None None HSA/Kex-2
(C723S) followed by mature HSA fused to a mutant mature interferon
alpha (C138S of mature IFNa) at its C- terminus. 151 3108
pSAC35HSA.PYY Mature PYY fused downstream pSAC35 744 732 756 None
None HSA/kex2 of mature HSA and the HSA/kex2 leader. 152 3109
pSAC35HSA.PYY3-36 HSA/kex2 leader followed by pSAC35 745 733 757
None None HSA/kex2 mature HSA and then PYY3-36 (SEQ ID NO: 1799).
153 3117 pC4:PYY3-36/HSA HSA leader followed by PYY3-36 pC4 746 734
758 768 769 HSA (SEQ ID NO: 1800) and mature HSA. 154 3118
pSAC35:PYY3-36/ HSA/kex2 leader followed by pSAC35 747 735 759 770
771 HSA/kex2 HSA PYY3-36 (SEQ ID NO: 1801) and mature HSA. 155 3281
pSAC35.PY3-36 PYY3-36 tandem repeat (x2) pSAC35 748 736 760 772 773
HSA/kex2 (x2)/HSA fused upstream of HSA and downstream of the
HSA/kex2 signal peptide. 156 3282 pSAC35:HSA/PYY3-36 PYY3-36 tandem
repeat (x2) pSAC35 749 737 761 774 775 HSA/kex2 (x2) fused
downstream of mature HSA and HSA/kex2 leader. 157 3144
pSAC35:adrenomedullin HSA/kex2 leader followed by pSAC35 750 738
762 776 777 HSA/kex2 (27-52)/HSA amino acids 27-52 of
adrenomedullin followed by mature HSA. 158 3579 pSAC35:HSA/kex2.
HSA/kex2 leader followed by pSAC35 751 739 763 None None HSA/kex2
HSA.oxyntomodulin mature HSA followed by mature oxyntomodulin. 159
3580 pSAC35:HSA/kex2.oxyntomodulin. HSA/kex2 leader followed by
pSAC35 752 740 764 None None HSA/kex2 HSA mature oxyntomodulin
followed by mature HSA. 160 4268 pC4: Consensus signal sequence pC4
753 741 765 778 779 Consensus SPCON2sp.Ghrelin. followed by ghrelin
fused to the (Mammalian) HSA N-terminus of HSA. 161 4273
pSAC35:KEX2.TIMP4. HSA/Kex-2 signal sequence pSAC35 754 742 766
None None HSA/Kex-2 C30-P224.HSA followed by mature TIMP4 (amino
acids 30-224) fused to the N-terminus of mature HSA. 162 4274
pSAC35:HSA.TIMP4. HSA/Kex-2 signal sequence pSAC35 755 743 767 None
None HSA/Kex-2 C30-P224 followed by mature HSA fused to mature
TIMP4 (amino acids 30-224) at its C-terminus.
[0089] Table 2 provides a non-exhaustive list of polynucleotides of
the invention comprising, or alternatively consisting of, nucleic
acid molecules encoding an albumin fusion protein. The first
column, "Fusion No." gives a fusion number to each polynucleotide.
Column 2, "Construct ID" provides a unique numerical identifier for
each polynucleotide of the invention. The Construct IDs may be used
to identify polynucleotides which encode albumin fusion proteins
comprising, or alternatively consisting of, a Therapeutic protein
portion corresponding to a given Therapeutic Protein:X listed in
the corresponding row of Table 1 wherein that Construct ID is
listed in column 5. The "Construct Name" column (column 3) provides
the name of a given albumin fusion construct or polynucleotide.
[0090] The fourth column in Table 2, "Description" provides a
general description of a given albumin fusion construct, and the
fifth column, "Expression Vector" lists the vector into which a
polynucleotide comprising, or alternatively consisting of, a
nucleic acid molecule encoding a given albumin fusion protein was
cloned. Vectors are known in the art, and are available
commercially or described elsewhere. For example, as described in
the Examples, an "expression cassette" comprising, or alternatively
consisting of, one or more of (1) a polynucleotide encoding a given
albumin fusion protein, (2) a leader sequence, (3) a promoter
region, and (4) a transcriptional terminator, may be assembled in a
convenient cloning vector and subsequently be moved into an
alternative vector, such as, for example, an expression vector
including, for example, a yeast expression vector or a mammalian
expression vector. In one embodiment, for expression in S.
cervisiae, an expression cassette comprising, or alternatively
consisting of, a nucleic acid molecule encoding an albumin fusion
protein is cloned into pSAC35. In another embodiment, for
expression in CHO cells, an expression cassette comprising, or
alternatively consisting of, a nucleic acid molecule encoding an
albumin fusion protein is cloned into pC4. In a further embodiment,
a polynucleotide comprising or alternatively consisting of a
nucleic acid molecule encoding the Therapeutic protein portion of
an albumin fusion protein is cloned into pC4:HSA. In a still
further embodiment, for expression in NS0 cells, an expression
cassette comprising, or alternatively consisting of, a nucleic acid
molecule encoding an albumin fusion protein is cloned into pEE12.
Other useful cloning and/or expression vectors will be known to the
skilled artisan and are within the scope of the invention.
[0091] Column 6, "SEQ ID NO:Y," provides the full length amino acid
sequence of the albumin fusion protein of the invention. In most
instances, SEQ ID NO:Y shows the unprocessed form of the albumin
fusion protein encoded--in other words, SEQ ID NO:Y shows the
signal sequence, a HSA portion, and a therapeutic portion all
encoded by the particular construct. Specifically contemplated by
the present invention are all polynucleotides that encode SEQ ID
NO:Y. When these polynucleotides are used to express the encoded
protein from a cell, the cell's natural secretion and processing
steps produces a protein that lacks the signal sequence listed in
columns 4 and/or 11 of Table 2. The specific amino acid sequence of
the listed signal sequence is shown later in the specification or
is well known in the art. Thus, most preferred embodiments of the
present invention include the albumin fusion protein produced by a
cell (which would lack the leader sequence shown in columns 4
and/or 11 of Table 2). Also most preferred are polypeptides
comprising SEQ ID NO:Y without the specific leader sequence listed
in columns 4 and/or 11 of Table 2. Compositions comprising these
two preferred embodiments, including pharmaceutical compositions,
are also preferred. Moreover, it is well within the ability of the
skilled artisan to replace the signal sequence listed in columns 4
and/or 11 of Table 2 with a different signal sequence, such as
those described later in the specification to facilitate secretion
of the processed albumin fusion protein.
[0092] The seventh column, "SEQ ID NO:X," provides the parent
nucleic acid sequence from which a polynucleotide encoding a
Therapeutic protein portion of a given albumin fusion protein may
be derived. In one embodiment, the parent nucleic acid sequence
from which a polynucleotide encoding a Therapeutic protein portion
of an albumin fusion protein may be derived comprises the wild type
gene sequence encoding a Therapeutic protein shown in Table 1. In
an alternative embodiment, the parent nucleic acid sequence from
which a polynucleotide encoding a Therapeutic protein portion of an
albumin fusion protein may be derived comprises a variant or
derivative of a wild type gene sequence encoding a Therapeutic
protein shown in Table 1, such as, for example, a synthetic codon
optimized variant of a wild type gene sequence encoding a
Therapeutic protein.
[0093] The eighth column, "SEQ ID NO:Z," provides a predicted
translation of the parent nucleic acid sequence (SEQ ID NO:X). This
parent sequence can be a full length parent protein used to derive
the particular construct, the mature portion of a parent protein, a
variant or fragment of a wildtype protein, or an artificial
sequence that can be used to create the described construct. One of
skill in the art can use this amino acid sequence shown in SEQ ID
NO:Z to determine which amino acid residues of an albumin fusion
protein encoded by a given construct are provided by the
therapeutic protein. Moreover, it is well within the ability of the
skilled artisan to use the sequence shown as SEQ ID NO:Z to derive
the construct described in the same row. For example, if SEQ ID
NO:Z corresponds to a full length protein, but only a portion of
that protein is used to generate the specific CID, it is within the
skill of the art to rely on molecular biology techniques, such as
PCR, to amplify the specific fragment and clone it into the
appropriate vector.
[0094] Amplification primers provided in columns 9 and 10, "SEQ ID
NO:A" and "SEQ ID NO:B" respectively, are exemplary primers used to
generate a polynucleotide comprising or alternatively consisting of
a nucleic acid molecule encoding the Therapeutic protein portion of
a given albumin fusion protein. In one embodiment of the invention,
oligonucleotide primers having the sequences shown in columns 9
and/or 10 (SEQ ID NOS:A and/or B) are used to PCR amplify a
polynucleotide encoding the Therapeutic protein portion of an
albumin fusion protein using a nucleic acid molecule comprising or
alternatively consisting of the nucleotide sequence provided in
column 7 (SEQ ID NO:X) of the corresponding row as the template
DNA. PCR methods are well-established in the art. Additional useful
primer sequences could readily be envisioned and utilized by those
of ordinary skill in the art.
[0095] In an alternative embodiment, oligonucleotide primers may be
used in overlapping PCR reactions to generate mutations within a
template DNA sequence. PCR methods are known in the art.
[0096] As shown in Table 3, certain albumin fusion constructs
disclosed in this application have been deposited with the
ATCC.RTM..
TABLE-US-00003 TABLE 3 ATCC Deposit No./ Construct ID Construct
Name Date 2249 pSAC35:IFNa2-HSA PTA-3763 also named
pSAC23:IFN.alpha.2-HSA Oct. 4, 2001 2343 pSAC35.INV-IFNA2.HSA
PTA-3940 Dec. 19, 2001 2381 pC4:HSA-IFNa2(C17-E181) PTA-3942 Dec.
19, 2001 2382 pC4:IFNa2-HSA PTA-3939 Dec. 19, 2001 3165
pSAC35:HSA.IFNa PTA-4670 also named CID 3165, Sep. 16, 2002
pSAC35:HSA.INF.alpha.
[0097] It is possible to retrieve a given albumin fusion construct
from the deposit by techniques known in the art and described
elsewhere herein (see, Example 10). The ATCC is located at 10801
University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC
deposits were made pursuant to the terms of the Budapest Treaty on
the international recognition of the deposit of microorganisms for
the purposes of patent procedure.
[0098] In a further embodiment of the invention, an "expression
cassette" comprising, or alternatively consisting of one or more of
(1) a polynucleotide encoding a given albumin fusion protein, (2) a
leader sequence, (3) a promoter region, and (4) a transcriptional
terminator can be moved or "subcloned" from one vector into
another. Fragments to be subcloned may be generated by methods well
known in the art, such as, for example, PCR amplification (e.g.,
using oligonucleotide primers having the sequence shown in SEQ ID
NO:A or B), and/or restriction enzyme digestion.
[0099] In preferred embodiments, the albumin fusion proteins of the
invention are capable of a therapeutic activity and/or biologic
activity corresponding to the therapeutic activity and/or biologic
activity of the Therapeutic protein corresponding to the
Therapeutic protein portion of the albumin fusion protein listed in
the corresponding row of Table 1. In further preferred embodiments,
the therapeutically active protein portions of the albumin fusion
proteins of the invention are fragments or variants of the protein
encoded by the sequence shown in SEQ ID NO:X column of Table 2, and
are capable of the therapeutic activity and/or biologic activity of
the corresponding Therapeutic protein.
[0100] Polypeptide and Polynucleotide Fragments and Variants
[0101] Fragments
[0102] The present invention is further directed to fragments of
the Therapeutic proteins described in Table 1, albumin proteins,
and/or albumin fusion proteins of the invention.
[0103] The present invention is also directed to polynucleotides
encoding fragments of the Therapeutic proteins described in Table
1, albumin proteins, and/or albumin fusion proteins of the
invention.
[0104] Even if deletion of one or more amino acids from the
N-terminus of a protein results in modification or loss of one or
more biological functions of the Therapeutic protein, albumin
protein, and/or albumin fusion protein of the invention, other
Therapeutic activities and/or functional activities (e.g.,
biological activities, ability to multimerize, ability to bind a
ligand) may still be retained. For example, the ability of
polypeptides with N-terminal deletions to induce and/or bind to
antibodies which recognize the complete or mature forms of the
polypeptides generally will be retained when less than the majority
of the residues of the complete polypeptide are removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal
residues of a complete polypeptide retains such immunologic
activities can readily be determined by routine methods described
herein and otherwise known in the art. It is not unlikely that a
mutein with a large number of deleted N-terminal amino acid
residues may retain some biological or immunogenic activities. In
fact, peptides composed of as few as six amino acid residues may
often evoke an immune response.
[0105] Accordingly, fragments of a Therapeutic protein
corresponding to a Therapeutic protein portion of an albumin fusion
protein of the invention, include the full length protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the reference polypeptide
(i.e., a Therapeutic protein referred to in Table 1, or a
Therapeutic protein portion of an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct described in Table 2).
In particular, N-terminal deletions may be described by the general
formula III to q, where q is a whole integer representing the total
number of amino acid residues in a reference polypeptide (e.g., a
Therapeutic protein referred to in Table 1, or a Therapeutic
protein portion of an albumin fusion protein of the invention, or a
Therapeutic protein portion of an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct described in Table 2),
and m is defined as any integer ranging from 2 to q minus 6.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0106] In addition, fragments of serum albumin polypeptides
corresponding to an albumin protein portion of an albumin fusion
protein of the invention, include the full length protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the reference polypeptide
(i.e., serum albumin, or a serum albumin portion of an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 2). In preferred embodiments,
N-terminal deletions may be described by the general formula III to
585, where 585 is a whole integer representing the total number of
amino acid residues in mature human serum albumin (SEQ ID NO: 1),
and m is defined as any integer ranging from 2 to 579.
Polynucleotides encoding these polypeptides are also encompassed by
the invention. In additional embodiments, N-terminal deletions may
be described by the general formula III to 609, where 609 is a
whole integer representing the total number of amino acid residues
in full length human serum albumin (SEQ ID NO:3), and m is defined
as any integer ranging from 2 to 603. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0107] Moreover, fragments of albumin fusion proteins of the
invention, include the full length albumin fusion protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the albumin fusion protein (e.g., an albumin fusion
protein encoded by a polynucleotide or albumin fusion construct
described in Table 2; or an albumin fusion protein having the amino
acid sequence disclosed in column 6 of Table 2). In particular,
N-terminal deletions may be described by the general formula III to
q, where q is a whole integer representing the total number of
amino acid residues in the albumin fusion protein, and m is defined
as any integer ranging from 2 to q minus 6. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0108] Also as mentioned above, even if deletion of one or more
amino acids from the N-terminus or C-terminus of a reference
polypeptide (e.g., a Therapeutic protein; serum albumin protein; or
albumin fusion protein of the invention) results in modification or
loss of one or more biological functions of the protein, other
functional activities (e.g., biological activities, ability to
multimerize, ability to bind a ligand) and/or Therapeutic
activities may still be retained. For example the ability of
polypeptides with C-terminal deletions to induce and/or bind to
antibodies which recognize the complete or mature forms of the
polypeptide generally will be retained when less than the majority
of the residues of the complete or mature polypeptide are removed
from the C-terminus. Whether a particular polypeptide lacking the
N-terminal and/or C-terminal residues of a reference polypeptide
retains Therapeutic activity can readily be determined by routine
methods described herein and/or otherwise known in the art.
[0109] The present invention further provides polypeptides having
one or more residues deleted from the carboxy terminus of the amino
acid sequence of a Therapeutic protein corresponding to a
Therapeutic protein portion of an albumin fusion protein of the
invention (e.g., a Therapeutic protein referred to in Table 1, or a
Therapeutic protein portion of an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct described in Table 2).
In particular, C-terminal deletions may be described by the general
formula I to n, where n is any whole integer ranging from 6 to q
minus 1, and where q is a whole integer representing the total
number of amino acid residues in a reference polypeptide (e.g., a
Therapeutic protein referred to in Table 1, or a Therapeutic
protein portion of an albumin fusion protein encoded by a
polynucleotide or albumin fusion construct described in Table 2).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0110] In addition, the present invention provides polypeptides
having one or more residues deleted from the carboxy terminus of
the amino acid sequence of an albumin protein corresponding to an
albumin protein portion of an albumin fusion protein of the
invention (e.g., serum albumin or an albumin protein portion of an
albumin fusion protein encoded by a polynucleotide or albumin
fusion construct described in Table 2). In particular, C-terminal
deletions may be described by the general formula I to n, where n
is any whole integer ranging from 6 to 584, where 584 is the whole
integer representing the total number of amino acid residues in
mature human serum albumin (SEQ ID NO:1) minus 1. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
In particular, C-terminal deletions may be described by the general
formula I to n, where n is any whole integer ranging from 6 to 608,
where 608 is the whole integer representing the total number of
amino acid residues in serum albumin (SEQ ID NO:3) minus 1.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0111] Moreover, the present invention provides polypeptides having
one or more residues deleted from the carboxy terminus of an
albumin fusion protein of the invention. In particular, C-terminal
deletions may be described by the general formula I to n, where n
is any whole integer ranging from 6 to q minus 1, and where q is a
whole integer representing the total number of amino acid residues
in an albumin fusion protein of the invention. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0112] In addition, any of the above described N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
reference polypeptide. The invention also provides polypeptides
having one or more amino acids deleted from both the amino and the
carboxyl termini, which may be described generally as having
residues m to n of a reference polypeptide (e.g., a Therapeutic
protein referred to in Table 1, or a Therapeutic protein portion of
an albumin fusion protein of the invention, or a Therapeutic
protein portion encoded by a polynucleotide or albumin fusion
construct described in Table 2, or serum albumin (e.g., SEQ ID
NO:1), or an albumin protein portion of an albumin fusion protein
of the invention, or an albumin protein portion encoded by a
polynucleotide or albumin fusion construct described in Table 2, or
an albumin fusion protein, or an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct of the invention)
where n and m are integers as described above. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0113] The present application is also directed to proteins
containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%
or 99% identical to a reference polypeptide sequence (e.g., a
Therapeutic protein referred to in Table 1, or a Therapeutic
protein portion of an albumin fusion protein of the invention, or a
Therapeutic protein portion encoded by a polynucleotide or albumin
fusion construct described in Table 2, or serum albumin (e.g., SEQ
ID NO:1), or an albumin protein portion of an albumin fusion
protein of the invention, or an albumin protein portion encoded by
a polynucleotide or albumin fusion construct described in Table 2,
or an albumin fusion protein, or an albumin fusion protein encoded
by a polynucleotide or albumin fusion construct of the invention)
set forth herein, or fragments thereof. In preferred embodiments,
the application is directed to proteins comprising polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
reference polypeptides having the amino acid sequence of N- and
C-terminal deletions as described above. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0114] Preferred polypeptide fragments of the invention are
fragments comprising, or alternatively, consisting of, an amino
acid sequence that displays a Therapeutic activity and/or
functional activity (e.g. biological activity) of the polypeptide
sequence of the Therapeutic protein or serum albumin protein of
which the amino acid sequence is a fragment.
[0115] Other preferred polypeptide fragments are biologically
active fragments. Biologically active fragments are those
exhibiting activity similar, but not necessarily identical, to an
activity of the polypeptide of the present invention. The
biological activity of the fragments may include an improved
desired activity, or a decreased undesirable activity.
[0116] Variants
[0117] "Variant" refers to a polynucleotide or nucleic acid
differing from a reference nucleic acid or polypeptide, but
retaining essential properties thereof. Generally, variants are
overall closely similar, and, in many regions, identical to the
reference nucleic acid or polypeptide.
[0118] As used herein, "variant", refers to a Therapeutic protein
portion of an albumin fusion protein of the invention, albumin
portion of an albumin fusion protein of the invention, or albumin
fusion protein of the invention differing in sequence from a
Therapeutic protein (e.g. see "therapeutic" column of Table 1),
albumin protein, and/or albumin fusion protein, respectively, but
retaining at least one functional and/or therapeutic property
thereof as described elsewhere herein or otherwise known in the
art. Generally, variants are overall very similar, and, in many
regions, identical to the amino acid sequence of the Therapeutic
protein corresponding to a Therapeutic protein portion of an
albumin fusion protein, albumin protein corresponding to an albumin
protein portion of an albumin fusion protein, and/or albumin fusion
protein. Nucleic acids encoding these variants are also encompassed
by the invention.
[0119] The present invention is also directed to proteins which
comprise, or alternatively consist of, an amino acid sequence which
is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%,
identical to, for example, the amino acid sequence of a Therapeutic
protein corresponding to a Therapeutic protein portion of an
albumin fusion protein of the invention (e.g., the amino acid
sequence of a Therapeutic protein:X disclosed in Table 1; or the
amino acid sequence of a Therapeutic protein portion of an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 1 and 2, or fragments or variants
thereof), albumin proteins corresponding to an albumin protein
portion of an albumin fusion protein of the invention (e.g., the
amino acid sequence of an albumin protein portion of an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 1 and 2; the amino acid sequence shown
in SEQ ID NO: 1; or fragments or variants thereof), and/or albumin
fusion proteins. Fragments of these polypeptides are also provided
(e.g., those fragments described herein). Further polypeptides
encompassed by the invention are polypeptides encoded by
polynucleotides which hybridize to the complement of a nucleic acid
molecule encoding an albumin fusion protein of the invention under
stringent hybridization conditions (e.g., hybridization to filter
bound DNA in 6.times. Sodium chloride/Sodium citrate (SSC) at about
45 degrees Celsius, followed by one or more washes in
0.2.times.SSC, 0.1% SDS at about 50-65 degrees Celsius), under
highly stringent conditions (e.g., hybridization to filter bound
DNA in 6.times. sodium chloride/Sodium citrate (SSC) at about 45
degrees Celsius, followed by one or more washes in 0.1.times.SSC,
0.2% SDS at about 68 degrees Celsius), 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
protocol in Molecular Biology, Green publishing associates, Inc.,
and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and
2.10.3). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0120] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence, it is
intended that the amino acid sequence of the subject polypeptide is
identical to the query sequence except that the subject polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the query amino acid sequence. In other words, to
obtain a polypeptide having an amino acid sequence at least 95%
identical to a query amino acid sequence, up to 5% of the amino
acid residues in the subject sequence may be inserted, deleted, or
substituted with another amino acid. These alterations of the
reference sequence may occur at the amino- or carboxy-terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0121] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequence of an albumin fusion protein of
the invention or a fragment thereof (such as a Therapeutic protein
portion of the albumin fusion protein or an albumin portion of the
albumin fusion protein), can be determined conventionally using
known computer programs. A preferred method for determining the
best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a
global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp.
App. Biosci. 6:237-245 (1990)). In a sequence alignment the query
and subject sequences are either both nucleotide sequences or both
amino acid sequences. The result of said global sequence alignment
is expressed as percent identity. Preferred parameters used in a
FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty-1, Joining Penalty=20, Randomization Group Length=0, Cutoff
Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size
Penalty=0.05, Window Size=500 or the length of the subject amino
acid sequence, whichever is shorter.
[0122] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0123] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0124] The variant will usually have at least 75% (preferably at
least about 80%, 90%, 95% or 99%) sequence identity with a length
of normal HA or Therapeutic protein which is the same length as the
variant. Homology or identity at the nucleotide or amino acid
sequence level is determined by BLAST (Basic Local Alignment Search
Tool) analysis using the algorithm employed by the programs blastp,
blastn, blastx, tblastn and tblastx (Karlin et al., Proc. Natl.
Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, J. Mol. Evol. 36:
290-300 (1993), fully incorporated by reference) which are tailored
for sequence similarity searching.
[0125] The approach used by the BLAST program is to first consider
similar segments between a query sequence and a database sequence,
then to evaluate the statistical significance of all matches that
are identified and finally to summarize only those matches which
satisfy a preselected threshold of significance. For a discussion
of basic issues in similarity searching of sequence databases, see
Altschul et al., (Nature Genetics 6: 119-129 (1994)) which is fully
incorporated by reference. The search parameters for histogram,
descriptions, alignments, expect (i.e., the statistical
significance threshold for reporting matches against database
sequences), cutoff, matrix and filter are at the default settings.
The default scoring matrix used by blastp, blastx, tblastn, and
tblastx is the BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad.
Sci. USA 89: 10915-10919 (1992), fully incorporated by reference).
For blastn, the scoring matrix is set by the ratios of M (i.e., the
reward score for a pair of matching residues) to N (i.e., the
penalty score for mismatching residues), wherein the default values
for M and N are 5 and -4, respectively. Four blastn parameters may
be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap
extension penalty); wink=1 (generates word hits at every
wink.sup.th position along the query); and gapw=16 (sets the window
width within which gapped alignments are generated). The equivalent
Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32. A
Bestfit comparison between sequences, available in the GCG package
version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and
LEN=3 (gap extension penalty) and the equivalent settings in
protein comparisons are GAP=8 and LEN=2.
[0126] The polynucleotide variants of the invention may contain
alterations in the coding regions, non-coding regions, or both.
Especially preferred are polynucleotide variants containing
alterations which produce silent substitutions, additions, or
deletions, but do not alter the properties or activities of the
encoded polypeptide. Nucleotide variants produced by silent
substitutions due to the degeneracy of the genetic code are
preferred. Moreover, polypeptide variants in which less than 50,
less than 40, less than 30, less than 20, less than 10, or 5-50,
5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or
added in any combination are also preferred. Polynucleotide
variants can be produced for a variety of reasons, e.g., to
optimize codon expression for a particular host (change codons in
the human mRNA to those preferred by a bacterial host, such as,
yeast or E. coli).
[0127] In a preferred embodiment, a polynucleotide of the invention
which encodes the albumin portion of an albumin fusion protein is
optimized for expression in yeast or mammalian cells. In a further
preferred embodiment, a polynucleotide of the invention which
encodes the Therapeutic protein portion of an albumin fusion
protein is optimized for expression in yeast or mammalian cells. In
a still further preferred embodiment, a polynucleotide encoding an
albumin fusion protein of the invention is optimized for expression
in yeast or mammalian cells.
[0128] In an alternative embodiment, a codon optimized
polynucleotide which encodes a Therapeutic protein portion of an
albumin fusion protein does not hybridize to the wild type
polynucleotide encoding the Therapeutic protein under stringent
hybridization conditions as described herein. In a further
embodiment, a codon optimized polynucleotide which encodes an
albumin portion of an albumin fusion protein does not hybridize to
the wild type polynucleotide encoding the albumin protein under
stringent hybridization conditions as described herein. In another
embodiment, a codon optimized polynucleotide which encodes an
albumin fusion protein does not hybridize to the wild type
polynucleotide encoding the Therapeutic protein portion or the
albumin protein portion under stringent hybridization conditions as
described herein.
[0129] In an additional embodiment, a polynucleotide which encodes
a Therapeutic protein portion of an albumin fusion protein does not
comprise, or alternatively consist of, the naturally occurring
sequence of that Therapeutic protein. In a further embodiment, a
polynucleotide which encodes an albumin protein portion of an
albumin fusion protein does not comprise, or alternatively consist
of, the naturally occurring sequence of albumin protein. In an
alternative embodiment, a polynucleotide which encodes an albumin
fusion protein does not comprise, or alternatively consist of, the
naturally occurring sequence of a Therapeutic protein portion or
the albumin protein portion.
[0130] Naturally occurring variants are called "allelic variants,"
and refer to one of several alternate forms of a gene occupying a
given locus on a chromosome of an organism. (Genes II, Lewin, B.,
ed., John Wiley & Sons, New York (1985)). These allelic
variants can vary at either the polynucleotide and/or polypeptide
level and are included in the present invention. Alternatively,
non-naturally occurring variants may be produced by mutagenesis
techniques or by direct synthesis.
[0131] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the polypeptides of the present invention. For
instance, one or more amino acids can be deleted from the
N-terminus or C-terminus of the polypeptide of the present
invention without substantial loss of biological function. As an
example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported
variant KGF proteins having heparin binding activity even after
deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly,
Interferon gamma exhibited up to ten times higher activity after
deleting 8-10 amino acid residues from the carboxy terminus of this
protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
[0132] Moreover, ample evidence demonstrates that variants often
retain a biological activity similar to that of the naturally
occurring protein. For example, Gayle and coworkers (J. Biol. Chem.
268:22105-22111 (1993)) conducted extensive mutational analysis of
human cytokine IL-1a. They used random mutagenesis to generate over
3,500 individual IL-1a mutants that averaged 2.5 amino acid changes
per variant over the entire length of the molecule. Multiple
mutations were examined at every possible amino acid position. The
investigators found that "[m]ost of the molecule could be altered
with little effect on either [binding or biological activity]." In
fact, only 23 unique amino acid sequences, out of more than 3,500
nucleotide sequences examined, produced a protein that
significantly differed in activity from wild-type.
[0133] Furthermore, even if deleting one or more amino acids from
the N-terminus or C-terminus of a polypeptide results in
modification or loss of one or more biological functions, other
biological activities may still be retained. For example, the
ability of a deletion variant to induce and/or to bind antibodies
which recognize the secreted form will likely be retained when less
than the majority of the residues of the secreted form are removed
from the N-terminus or C-terminus. Whether a particular polypeptide
lacking N- or C-terminal residues of a protein retains such
immunogenic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0134] Thus, the invention further includes polypeptide variants
which have a functional activity (e.g., biological activity and/or
therapeutic activity). In one embodiment, the invention provides
variants of albumin fusion proteins that have a functional activity
(e.g., biological activity and/or therapeutic activity) that
corresponds to one or more biological and/or therapeutic activities
of the Therapeutic protein corresponding to the Therapeutic protein
portion of the albumin fusion protein. In another embodiment, the
invention provides variants of albumin fusion proteins that have a
functional activity (e.g., biological activity and/or therapeutic
activity) that corresponds to one or more biological and/or
therapeutic activities of the Therapeutic protein corresponding to
the Therapeutic protein portion of the albumin fusion protein. Such
variants include deletions, insertions, inversions, repeats, and
substitutions selected according to general rules known in the art
so as have little effect on activity. Polynucleotides encoding such
variants are also encompassed by the invention.
[0135] In preferred embodiments, the variants of the invention have
conservative substitutions. By "conservative substitutions" is
intended swaps within groups such as replacement of the aliphatic
or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of
the hydroxyl residues Ser and Thr; replacement of the acidic
residues Asp and Glu; replacement of the amide residues Asn and
Gln, replacement of the basic residues Lys, Arg, and His;
replacement of the aromatic residues Phe, Tyr, and Trp, and
replacement of the small-sized amino acids Ala, Ser, Thr, Met, and
Gly.
[0136] Guidance concerning how to make phenotypically silent amino
acid substitutions is provided, for example, in Bowie et al.,
"Deciphereing the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that there are two main strategies for studying
the tolerance of an amino acid sequence to change.
[0137] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, conserved
amino acids can be identified. These conserved amino acids are
likely important for protein function. In contrast, the amino acid
positions where substitutions have been tolerated by natural
selection indicates that these positions are not critical for
protein function. Thus, positions tolerating amino acid
substitution could be modified while still maintaining biological
activity of the protein.
[0138] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example, site
directed mutagenesis or alanine-scanning mutagenesis (introduction
of single alanine mutations at every residue in the molecule) can
be used. See Cunningham and Wells, Science 244:1081-1085 (1989).
The resulting mutant molecules can then be tested for biological
activity.
[0139] As the authors state, these two strategies have revealed
that proteins are surprisingly tolerant of amino acid
substitutions. The authors further indicate which amino acid
changes are likely to be permissive at certain amino acid positions
in the protein. For example, most buried (within the tertiary
structure of the protein) amino acid residues require nonpolar side
chains, whereas few features of surface side chains are generally
conserved. Moreover, tolerated conservative amino acid
substitutions involve replacement of the aliphatic or hydrophobic
amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl
residues Ser and Thr; replacement of the acidic residues Asp and
Glu; replacement of the amide residues Asn and Gln, replacement of
the basic residues Lys, Arg, and His; replacement of the aromatic
residues Phe, Tyr, and Trp, and replacement of the small-sized
amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino
acid substitution, variants of the present invention include (i)
polypeptides containing substitutions of one or more of the
non-conserved amino acid residues, where the substituted amino acid
residues may or may not be one encoded by the genetic code, or (ii)
polypeptides containing substitutions of one or more of the amino
acid residues having a substituent group, or (iii) polypeptides
which have been fused with or chemically conjugated to another
compound, such as a compound to increase the stability and/or
solubility of the polypeptide (for example, polyethylene glycol),
(iv) polypeptide containing additional amino acids, such as, for
example, an IgG Fc fusion region peptide. Such variant polypeptides
are deemed to be within the scope of those skilled in the art from
the teachings herein.
[0140] For example, polypeptide variants containing amino acid
substitutions of charged amino acids with other charged or neutral
amino acids may produce proteins with improved characteristics,
such as less aggregation. Aggregation of pharmaceutical
formulations both reduces activity and increases clearance due to
the aggregate's immunogenic activity. See Pinckard et al., Clin.
Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:
838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier
Systems 10:307-377 (1993).
[0141] In specific embodiments, the polypeptides of the invention
comprise, or alternatively, consist of, fragments or variants of
the amino acid sequence of an albumin fusion protein, the amino
acid sequence of a Therapeutic protein and/or human serum albumin,
wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50
or 50-150, amino acid residue additions, substitutions, and/or
deletions when compared to the reference amino acid sequence. In
preferred embodiments, the amino acid substitutions are
conservative. Nucleic acids encoding these polypeptides are also
encompassed by the invention.
[0142] The polypeptide of the present invention can be composed of
amino acids joined to each other by peptide bonds or modified
peptide bonds, i.e., peptide isosteres, and may contain amino acids
other than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as post-translational
processing, or by chemical modification techniques which are well
known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous
research literature. Modifications can occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990);
Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[0143] Functional Activity
[0144] "A polypeptide having functional activity" refers to a
polypeptide capable of displaying one or more known functional
activities associated with the full-length, pro-protein, and/or
mature form of a Therapeutic protein. Such functional activities
include, but are not limited to, biological activity, antigenicity
[ability to bind (or compete with a polypeptide for binding) to an
anti-polypeptide antibody], immunogenicity (ability to generate
antibody which binds to a specific polypeptide of the invention),
ability to form multimers with polypeptides of the invention, and
ability to bind to a receptor or ligand for a polypeptide.
[0145] "A polypeptide having biological activity" refers to a
polypeptide exhibiting activity similar to, but not necessarily
identical to, an activity of a Therapeutic protein of the present
invention, including mature forms, as measured in a particular
biological assay, with or without dose dependency. In the case
where dose dependency does exist, it need not be identical to that
of the polypeptide, but rather substantially similar to the
dose-dependence in a given activity as compared to the polypeptide
of the present invention (i.e., the candidate polypeptide will
exhibit greater activity or not more than about 25-fold less and,
preferably, not more than about tenfold less activity, and most
preferably, not more than about three-fold less activity relative
to the polypeptide of the present invention).
[0146] In preferred embodiments, an albumin fusion protein of the
invention has at least one biological and/or therapeutic activity
associated with the Therapeutic protein portion (or fragment or
variant thereof) when it is not fused to albumin.
[0147] In additional preferred embodiments, the albumin fusion
protein of the invention has an increased plasma stability compared
to the Therapeutic protein portion (or fragment or variant thereof)
in an unfused state. Plasma stability of the albumin fusion protein
of the invention or of the unfused Therapeutic protein portion (or
fragment or variant thereof) can be assayed using or routinely
modifying assays known in the art.
[0148] The albumin fusion proteins of the invention can be assayed
for functional activity (e.g., biological activity) using or
routinely modifying assays known in the art, as well as assays
described herein. Additionally, one of skill in the art may
routinely assay fragments of a Therapeutic protein corresponding to
a Therapeutic protein portion of an albumin fusion protein, for
activity using assays referenced in its corresponding row of Table
1 (e.g., in column 3 of Table 1). Further, one of skill in the art
may routinely assay fragments of an albumin protein corresponding
to an albumin protein portion of an albumin fusion protein, for
activity using assays known in the art and/or as described in the
Examples section below.
[0149] For example, in one embodiment where one is assaying for the
ability of an albumin fusion protein to bind or compete with a
Therapeutic protein for binding to an anti-Therapeutic polypeptide
antibody and/or anti-albumin antibody, various immunoassays known
in the art can be used, including but not limited to, competitive
and non-competitive assay systems using techniques such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ
immunoassays (using colloidal gold, enzyme or radioisotope labels,
for example), western blots, precipitation reactions, agglutination
assays (e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody is detected
by detecting binding of a secondary antibody or reagent to the
primary antibody. In a further embodiment, the secondary antibody
is labeled. Many means are known in the art for detecting binding
in an immunoassay and are within the scope of the present
invention.
[0150] In a preferred embodiment, where a binding partner (e.g., a
receptor or a ligand) of a Therapeutic protein is identified,
binding to that binding partner by an albumin fusion protein which
comprises that Therapeutic protein as the Therapeutic protein
portion of the fusion can be assayed, e.g., by means well-known in
the art, such as, for example, reducing and non-reducing gel
chromatography, protein affinity chromatography, and affinity
blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123
(1995). In another embodiment, the ability of physiological
correlates of an albumin fusion protein to bind to a substrate(s)
of the Therapeutic polypeptide corresponding to the Therapeutic
protein portion of the fusion can be routinely assayed using
techniques known in the art.
[0151] In an alternative embodiment, where the ability of an
albumin fusion protein to multimerize is being evaluated,
association with other components of the multimer can be assayed,
e.g., by means well-known in the art, such as, for example,
reducing and non-reducing gel chromatography, protein affinity
chromatography, and affinity blotting. See generally, Phizicky et
al., supra.
[0152] In preferred embodiments, an albumin fusion protein
comprising all or a portion of an antibody that binds a Therapeutic
protein, has at least one biological and/or therapeutic activity
(e.g., to specifically bind a polypeptide or epitope) associated
with the antibody that binds a Therapeutic protein (or fragment or
variant thereof) when it is not fused to albumin. In other
preferred embodiments, the biological activity and/or therapeutic
activity of an albumin fusion protein comprising all or a portion
of an antibody that binds a Therapeutic protein is the inhibition
(i.e., antagonism) or activation (i.e., agonism) of one or more of
the biological activities and/or therapeutic activities associated
with the polypeptide that is specifically bound by antibody that
binds a Therapeutic protein.
[0153] Albumin fusion proteins comprising at least a fragment or
variant of an antibody that binds a Therapeutic protein may be
characterized in a variety of ways. In particular, albumin fusion
proteins comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein may be assayed for the ability to
specifically bind to the same antigens specifically bound by the
antibody that binds a Therapeutic protein corresponding to the
Therapeutic protein portion of the albumin fusion protein using
techniques described herein or routinely modifying techniques known
in the art.
[0154] Assays for the ability of the albumin fusion proteins (e.g.,
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein) to (specifically) bind a specific protein or
epitope 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., U.S. 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). Albumin fusion
proteins comprising at least a fragment or variant of a Therapeutic
antibody may also be assayed for their specificity and affinity for
a specific protein or epitope using or routinely modifying
techniques described herein or otherwise known in the art.
[0155] The albumin fusion proteins comprising at least a fragment
or variant of an antibody that binds a Therapeutic protein may be
assayed for cross-reactivity with other antigens (e.g., molecules
that have sequence/structure conservation with the molecule(s)
specifically bound by the antibody that binds a Therapeutic protein
(or fragment or variant thereof) corresponding to the Therapeutic
protein portion of the albumin fusion protein of the invention) by
any method known in the art.
[0156] Immunoassays which can be used to analyze (immunospecific)
binding and cross-reactivity 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, and
protein A immunoassays, to name but a few. Such assays are routine
and well known in the art (see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, which is incorporated by reference herein in
its entirety). Exemplary immunoassays are described briefly below
(but are not intended by way of limitation).
[0157] 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 albumin fusion protein of
the invention (e.g., comprising at least a fragment or variant of
an antibody that binds a Therapeutic protein) to the cell lysate,
incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees
C., adding sepharose beads coupled to an anti-albumin antibody, for
example, 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. The ability of the
albumin fusion protein 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 albumin fusion protein to
an antigen and decrease the background (e.g., pre-clearing the cell
lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York at 10.16.1.
[0158] 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),
applying the albumin fusion protein of the invention (diluted in
blocking buffer) to the membrane, washing the membrane in washing
buffer, applying a secondary antibody (which recognizes the albumin
fusion protein, e.g., an anti-human serum albumin antibody)
conjugated to an enzymatic substrate (e.g., horseradish peroxidase
or alkaline phosphatase) or radioactive molecule (e.g., .sup.32P or
.sup.125I) diluted in blocking buffer, washing the membrane in wash
buffer, and detecting the presence of the antigen. One of skill in
the art would be knowledgeable as to the parameters that can be
modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0159] ELISAs comprise preparing antigen, coating the well of a
96-well microtiter plate with the antigen, washing away antigen
that did not bind the wells, adding the albumin fusion protein
(e.g., comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein) of the invention conjugated to a
detectable compound such as an enzymatic substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) to the wells and
incubating for a period of time, washing away unbound or
non-specifically bound albumin fusion proteins, and detecting the
presence of the albumin fusion proteins specifically bound to the
antigen coating the well. In ELISAs the albumin fusion protein does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes albumin fusion protein)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the albumin
fusion protein may be coated to the well. In this case, the
detectable molecule could be the antigen conjugated to a detectable
compound such as an enzymatic substrate (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., Ausubel et al, eds, 1994, Current Protocols in Molecular
Biology, Vol. 1, John Wiley & Sons, Inc., New York at
11.2.1.
[0160] The binding affinity of an albumin fusion protein to a
protein, antigen, or epitope and the off-rate of an albumin fusion
protein-protein/antigen/epitope 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 albumin fusion
protein of the invention in the presence of increasing amounts of
unlabeled antigen, and the detection of the antibody bound to the
labeled antigen. The affinity of the albumin fusion protein for a
specific protein, antigen, or epitope and the binding off-rates can
be determined from the data by Scatchard plot analysis. Competition
with a second protein that binds the same protein, antigen or
epitope as the albumin fusion protein, can also be determined using
radioimmunoassays. In this case, the protein, antigen or epitope is
incubated with an albumin fusion protein conjugated to a labeled
compound (e.g., .sup.3H or .sup.125I) in the presence of increasing
amounts of an unlabeled second protein that binds the same protein,
antigen, or epitope as the albumin fusion protein of the
invention.
[0161] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of albumin fusion
proteins of the invention to a protein, antigen or epitope. BIAcore
kinetic analysis comprises analyzing the binding and dissociation
of albumin fusion proteins, or specific polypeptides, antigens or
epitopes from chips with immobilized specific polypeptides,
antigens or epitopes or albumin fusion proteins, respectively, on
their surface.
[0162] Antibodies that bind a Therapeutic protein corresponding to
the Therapeutic protein portion of an albumin fusion protein may
also be described or specified in terms of their binding affinity
for a given protein or antigen, preferably the antigen which they
specifically bind. Preferred binding affinities include those with
a dissociation constant or Kd less than 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. More 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 or 10.sup.-8 M. Even more preferred
binding affinities include those with a dissociation constant or Kd
less than 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, .sup.10-12 M, 5.times.10.sup.-13 M, 5.times.10.sup.-14 M,
5.times.10.sup.-15 M, or 10.sup.-15 M. In preferred embodiments,
albumin fusion proteins comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, has an affinity
for a given protein or epitope similar to that of the corresponding
antibody (not fused to albumin) that binds a Therapeutic protein,
taking into account the valency of the albumin fusion protein
(comprising at least a fragment or variant of an antibody that
binds a Therapeutic protein) and the valency of the corresponding
antibody. In addition, assays described herein (see Examples and
Table 1) and otherwise known in the art may routinely be applied to
measure the ability of albumin fusion proteins and fragments,
variants and derivatives thereof to elicit biological activity
and/or Therapeutic activity (either in vitro or in vivo) related to
either the Therapeutic protein portion and/or albumin portion of
the albumin fusion protein. Other methods will be known to the
skilled artisan and are within the scope of the invention.
[0163] Albumin
[0164] As described above, an albumin fusion protein of the
invention comprises at least a fragment or variant of a Therapeutic
protein and at least a fragment or variant of human serum albumin,
which are associated with one another, preferably by genetic
fusion.
[0165] An additional embodiment comprises at least a fragment or
variant of a Therapeutic protein and at least a fragment or variant
of human serum albumin, which are linked to one another by chemical
conjugation.
[0166] The terms, human serum albumin (HSA) and human albumin (HA)
are used interchangeably herein. The terms, "albumin and "serum
albumin" are broader, and encompass human serum albumin (and
fragments and variants thereof) as well as albumin from other
species (and fragments and variants thereof).
[0167] As used herein, "albumin" refers collectively to albumin
protein or amino acid sequence, or an albumin fragment or variant,
having one or more functional activities (e.g., biological
activities) of albumin. In particular, "albumin" refers to human
albumin or fragments thereof (see for example, EP 201 239, EP 322
094 WO 97/24445, WO95/23857) especially the mature form of human
albumin as shown in FIG. 1 and SEQ ID NO: 1, or albumin from other
vertebrates or fragments thereof, or analogs or variants of these
molecules or fragments thereof.
[0168] In preferred embodiments, the human serum albumin protein
used in the albumin fusion proteins of the invention contains one
or both of the following sets of point mutations with reference to
SEQ ID NO: 1: Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and
Arg-410 to Ala; or Arg-410 to A, Lys-413 to Gln, and Lys-414 to Gln
(see, e.g., International Publication No. WO95/23857, hereby
incorporated in its entirety by reference herein). In even more
preferred embodiments, albumin fusion proteins of the invention
that contain one or both of above-described sets of point mutations
have improved stability/resistance to yeast Yap3p proteolytic
cleavage, allowing increased production of recombinant albumin
fusion proteins expressed in yeast host cells.
[0169] As used herein, a portion of albumin sufficient to prolong
the therapeutic activity or plasma stability or shelf-life of the
Therapeutic protein refers to a portion of albumin sufficient in
length or structure to stabilize or prolong the therapeutic
activity or plasma stability of the protein so that the shelf life
or plasma stability of the Therapeutic protein portion of the
albumin fusion protein is prolonged or extended compared to the
shelf-life or plasma stability in the non-fusion state. The albumin
portion of the albumin fusion proteins may comprise the full length
of the HA sequence as described above, or may include one or more
fragments thereof that are capable of stabilizing or prolonging the
therapeutic activity. Such fragments may be of 10 or more amino
acids in length or may include about 15, 20, 25, 30, 50, or more
contiguous amino acids from the HA sequence or may include part or
all of specific domains of HA. For instance, one or more fragments
of HA spanning the first two immunoglobulin-like domains may be
used. In a preferred embodiment, the HA fragment is the mature form
of HA.
[0170] The albumin portion of the albumin fusion proteins of the
invention may be a variant of normal HA. The Therapeutic protein
portion of the albumin fusion proteins of the invention may also be
variants of the Therapeutic proteins as described herein. The term
"variants" includes insertions, deletions and substitutions, either
conservative or non conservative, where such changes do not
substantially alter one or more of the oncotic, useful
ligand-binding and non-immunogenic properties of albumin, or the
active site, or active domain which confers the therapeutic
activities of the Therapeutic proteins.
[0171] In particular, the albumin fusion proteins of the invention
may include naturally occurring polymorphic variants of human
albumin and fragments of human albumin, for example those fragments
disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419).
The albumin may be derived from any vertebrate, especially any
mammal, for example human, cow, sheep, or pig. Non-mammalian
albumins include, but are not limited to, hen and salmon. The
albumin portion of the albumin fusion protein may be from a
different animal than the Therapeutic protein portion.
[0172] Generally speaking, an HA fragment or variant will be at
least 100 amino acids long, preferably at least 150 amino acids
long. The HA variant may consist of or alternatively comprise at
least one whole domain of HA, for example domains 1 (amino acids
1-194 of SEQ ID NO: 1), domain 2 (amino acids 195-387 of SEQ ID
NO:1), domain 3 (amino acids 388-585 of SEQ ID NO:1), domains 1 and
2 (1-387 of SEQ ID NO:1), domains 2 and 3 (195-585 of SEQ ID NO:1)
or domains 1 and 3 (amino acids 1-194 of SEQ ID NO:1 and amino
acids 388-585 of SEQ ID NO:1). Each domain is itself made up of two
homologous subdomains namely 1-105, 120-194, 195-291, 316-387,
388-491 and 512-585, with flexible inter-subdomain linker regions
comprising residues Lys106 to Glu119, Glu292 to Val315 and Glu492
to Ala511.
[0173] Preferably, the albumin portion of an albumin fusion protein
of the invention comprises at least one subdomain or domain of HA
or conservative modifications thereof. If the fusion is based on
subdomains, some or all of the adjacent linker is preferably used
to link to the Therapeutic protein moiety.
[0174] Antibodies that Specifically Bind Therapeutic Proteins are
also Therapeutic Proteins
[0175] The present invention also encompasses albumin fusion
proteins that comprise at least a fragment or variant of an
antibody that specifically binds a Therapeutic protein disclosed in
Table 1. It is specifically contemplated that the term "Therapeutic
protein" encompasses antibodies that bind a Therapeutic protein
(e.g., as Described in column I of Table 1) and fragments and
variants thereof. Thus an albumin fusion protein of the invention
may contain at least a fragment or variant of a Therapeutic
protein, and/or at least a fragment or variant of an antibody that
binds a Therapeutic protein.
[0176] Antibody Structure and Background
[0177] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa and lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
See generally, Fundamental Immunology Chapters 3-5 (Paul, W., ed.,
4th ed. Raven Press, N.Y. (1998)) (incorporated by reference in its
entirety for all purposes). The variable regions of each
light/heavy chain pair form the antibody binding site.
[0178] Thus, an intact IgG antibody has two binding sites. Except
in bifunctional or bispecific antibodies, the two binding sites are
the same.
[0179] The chains all exhibit the same general structure of
relatively conserved framework regions (FR) joined by three
hypervariable regions, also called complementarity determining
regions or CDRs. The CDR regions, in general, are the portions of
the antibody which make contact with the antigen and determine its
specificity. The CDRs from the heavy and the light chains of each
pair are aligned by the framework regions, enabling binding to a
specific epitope. From N-terminal to C-terminal, both light and
heavy chains variable regions comprise the domains FR1, CDR1, FR2,
CDR2, FR3, CDR3 and FR4. The variable regions are connected to the
heavy or light chain constant region. The assignment of amino acids
to each domain is in accordance with the definitions of Kabat
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature
342:878-883 (1989).
[0180] As used herein, "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that specifically binds an antigen (e.g., a molecule containing one
or more CDR regions of an antibody). Antibodies that may correspond
to a Therapeutic protein portion of an albumin fusion protein
include, but are not limited to, monoclonal, multispecific, human,
humanized or chimeric antibodies, single chain antibodies (e.g.,
single chain Fvs), Fab fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies specific to
antibodies of the invention), and epitope-binding fragments of any
of the above (e.g., VH domains, VL domains, or one or more CDR
regions).
[0181] Antibodies that Bind Therapeutic Proteins
[0182] The present invention encompasses albumin fusion proteins
that comprise at least a fragment or variant of an antibody that
binds a Therapeutic Protein (e.g., as disclosed in Table 1) or
fragment or variant thereof.
[0183] Antibodies that bind a Therapeutic protein (or fragment or
variant thereof) may be from any animal origin, including birds and
mammals. Preferably, the antibodies are human, murine (e.g., mouse
and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or
chicken antibodies. Most preferably, the antibodies are human
antibodies. As used herein, "human" antibodies include antibodies
having the amino acid sequence of a human immunoglobulin and
include antibodies isolated from human immunoglobulin libraries and
xenomice or other organisms that have been genetically engineered
to produce human antibodies.
[0184] The antibody molecules that bind to a Therapeutic protein
and that may correspond to a Therapeutic protein portion of an
albumin fusion protein of the invention can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,
IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In
preferred embodiments, the antibody molecules that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein portion of an albumin fusion protein are IgG1. In other
preferred embodiments, the immunoglobulin molecules that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein portion of an albumin fusion protein are IgG2. In other
preferred embodiments, the immunoglobulin molecules that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein portion of an albumin fusion protein are IgG4.
[0185] Most preferably the antibodies that bind to a Therapeutic
protein and that may correspond to a Therapeutic protein portion of
an albumin fusion protein 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.
[0186] The antibodies that bind to a Therapeutic protein and that
may correspond to a Therapeutic protein portion of an albumin
fusion protein may be monospecific, bispecific, trispecific or of
greater multispecificity. Multispecific antibodies may be specific
for different epitopes of a Therapeutic protein or may be specific
for both a Therapeutic protein 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).
[0187] Antibodies that bind a Therapeutic protein (or fragment or
variant thereof) may be bispecific or bifunctional which means that
the antibody is an artificial hybrid antibody having two different
heavy/light chain pairs and two different binding sites. Bispecific
antibodies can be produced by a variety of methods including fusion
of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai
& Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et
al. J. Immunol. 148:1547 1553 (1992). In addition, bispecific
antibodies may be formed as "diabodies" (Holliger et al.
"`Diabodies`: small bivalent and bispecific antibody fragments"
PNAS USA 90:6444-6448 (1993)) or "Janusins" (Traunecker et al.
"Bispecific single chain molecules (Janusins) target cytotoxic
lymphocytes on HIV infected cells" EMBO J. 10:3655-3659 (1991) and
Traunecker et al. "Janusin: new molecular design for bispecific
reagents" Int J Cancer Suppl 7:51-52 (1992)).
[0188] The present invention also provides albumin fusion proteins
that comprise, fragments or variants (including derivatives) of an
antibody described herein or known elsewhere in the art. Standard
techniques known to those of skill in the art can be used to
introduce mutations in the nucleotide sequence encoding a molecule
of the invention, including, for example, site-directed mutagenesis
and PCR-mediated mutagenesis which result in amino acid
substitutions. Preferably, the variants (including derivatives)
encode less than 50 amino acid substitutions, less than 40 amino
acid substitutions, less than 30 amino acid substitutions, less
than 25 amino acid substitutions, less than 20 amino acid
substitutions, less than 15 amino acid substitutions, less than 10
amino acid substitutions, less than 5 amino acid substitutions,
less than 4 amino acid substitutions, less than 3 amino acid
substitutions, or less than 2 amino acid substitutions relative to
the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1,
VLCDR2, or VLCDR3. In specific embodiments, the variants encode
substitutions of VHCDR3. In a preferred embodiment, the variants
have conservative amino acid substitutions at one or more predicted
non-essential amino acid residues.
[0189] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein may be described or specified in terms of the epitope(s) or
portion(s) of a Therapeutic protein which they recognize or
specifically bind. Antibodies which specifically bind a Therapeutic
protein or a specific epitope of a Therapeutic protein may also be
excluded. Therefore, the present invention encompasses antibodies
that specifically bind Therapeutic proteins, and allows for the
exclusion of the same. In preferred embodiments, albumin fusion
proteins comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein, binds the same epitopes as the
unfused fragment or variant of that antibody itself.
[0190] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein may also be described or specified in terms of their
cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of a Therapeutic protein 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% sequence identity
(as calculated using methods known in the art and described herein)
to a Therapeutic protein are also included in the present
invention. In specific embodiments, antibodies that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein portion of an albumin fusion protein cross-react with
murine, rat and/or rabbit homologs of human proteins and the
corresponding epitopes thereof. Antibodies that do not bind
polypeptides with less than 95%, less than 90%, less than 85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than 55%, and less than 50% sequence identity (as
calculated using methods known in the art and described herein) to
a Therapeutic protein are also included in the present invention.
In a specific embodiment, the above-described cross-reactivity is
with respect to any single specific antigenic or immunogenic
polypeptide, or combination(s) of 2, 3, 4, 5, or more of the
specific antigenic and/or immunogenic polypeptides disclosed
herein. In preferred embodiments, albumin fusion proteins
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein, has similar or substantially identical cross
reactivity characteristics compared to the fragment or variant of
that particular antibody itself.
[0191] Further included in the present invention are antibodies
which bind polypeptides encoded by polynucleotides which hybridize
to a polynucleotide encoding a Therapeutic protein under stringent
hybridization conditions (as described herein). Antibodies that
bind to a Therapeutic protein and that may correspond to a
Therapeutic protein portion of an albumin fusion protein of the
invention may also be described or specified in terms of their
binding affinity to a polypeptide of the invention. Preferred
binding affinities include those with a dissociation constant or Kd
less than 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. More 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 or
10.sup.-8 M. Even more preferred binding affinities include those
with a dissociation constant or Kd less than 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, .sup.10-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 1.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, or 10.sup.-15 M. In preferred
embodiments, albumin fusion proteins comprising at least a fragment
or variant of an antibody that binds a Therapeutic protein, has an
affinity for a given protein or epitope similar to that of the
corresponding antibody (not fused to albumin) that binds a
Therapeutic protein, taking into account the valency of the albumin
fusion protein (comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein) and the valency of the
corresponding antibody.
[0192] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of a Therapeutic
protein as determined by any method known in the art for
determining competitive binding, for example, the immunoassays
described herein. In preferred embodiments, the antibody
competitively inhibits binding to the epitope by at least 95%, at
least 90%, at least 85%, at least 80%, at least 75%, at least 70%,
at least 60%, or at least 50%. In preferred embodiments, albumin
fusion proteins comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein, competitively inhibits
binding of a second antibody to an epitope of a Therapeutic
protein. In other preferred embodiments, albumin fusion proteins
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein, competitively inhibits binding of a second
antibody to an epitope of a Therapeutic protein 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%.
[0193] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein of the invention may act as agonists or antagonists of the
Therapeutic protein. For example, the present invention includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. The
invention features both receptor-specific antibodies and
ligand-specific antibodies. The invention also features
receptor-specific antibodies which do not prevent ligand binding
but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody. In preferred embodiments, albumin fusion
proteins comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein, has similar or substantially
similar characteristics with regard to preventing ligand binding
and/or preventing receptor activation compared to an un-fused
fragment or variant of the antibody that binds the Therapeutic
protein.
[0194] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the Therapeutic
proteins (e.g. as disclosed in Table 1). The above antibody
agonists can be made using methods known in the art. See, e.g., PCT
publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al.,
Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214
(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et
al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine
9(4):233-241 (1997); Carlson et al., J. Biol. Chem.
272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762
(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et
al., Cytokine 8(1):14-20 (1996) (which are all incorporated by
reference herein in their entireties). In preferred embodiments,
albumin fusion proteins comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, have similar or
substantially identical agonist or antagonist properties as an
un-fused fragment or variant of the antibody that binds the
Therapeutic protein.
[0195] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein of the invention may be used, for example, to purify,
detect, and target Therapeutic proteins, including both in in vitro
and in vivo diagnostic and therapeutic methods. For example, the
antibodies have utility in immunoassays for qualitatively and
quantitatively measuring levels of the Therapeutic protein in
biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); incorporated by reference herein in its entirety. Likewise,
albumin fusion proteins comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, may be used, for
example, to purify, detect, and target Therapeutic proteins,
including both in vitro and in vivo diagnostic and therapeutic
methods.
[0196] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein include derivatives that are modified, i.e., by the
covalent attachment of any type of molecule to the antibody. 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. Albumin fusion proteins of the invention may also be
modified as described above.
[0197] Methods of Producing Antibodies that Bind Therapeutic
Proteins
[0198] The antibodies that bind to a Therapeutic protein and that
may correspond to a Therapeutic protein portion of an albumin
fusion protein of the 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 Therapeutic protein may 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.
[0199] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0200] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with a Therapeutic
protein or fragment or variant thereof, an albumin fusion protein,
or a cell expressing such a Therapeutic protein or fragment or
variant thereof or albumin fusion protein. Once an immune response
is detected, e.g., antibodies specific for the antigen are detected
in the mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC. Hybridomas are selected and cloned by
limited dilution. The hybridoma clones are then assayed by methods
known in the art for cells that secrete antibodies capable of
binding a polypeptide of the invention. Ascites fluid, which
generally contains high levels of antibodies, can be generated by
immunizing mice with positive hybridoma clones.
[0201] 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 wherein, preferably, the hybridoma is generated by fusing
splenocytes isolated from a mouse immunized with an antigen of the
invention with myeloma cells and then screening the hybridomas
resulting from the fusion for hybridoma clones that secrete an
antibody able to bind a polypeptide of the invention.
[0202] Another well known method for producing both polyclonal and
monoclonal human B cell lines is transformation using Epstein Barr
Virus (EBV). Protocols for generating EBV-transformed B cell lines
are commonly known in the art, such as, for example, the protocol
outlined in Chapter 7.22 of Current Protocols in Immunology,
Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is
hereby incorporated in its entirety by reference. The source of B
cells for transformation is commonly human peripheral blood, but B
cells for transformation may also be derived from other sources
including, but not limited to, lymph nodes, tonsil, spleen, tumor
tissue, and infected tissues. Tissues are generally made into
single cell suspensions prior to EBV transformation. Additionally,
steps may be taken to either physically remove or inactivate T
cells (e.g., by treatment with cyclosporin A) in B cell-containing
samples, because T cells from individuals seropositive for anti-EBV
antibodies can suppress B cell immortalization by EBV.
[0203] In general, the sample containing human B cells is
innoculated with EBV, and cultured for 3-4 weeks. A typical source
of EBV is the culture supernatant of the B95-8 cell line (ATCC
#VR-1492). Physical signs of EBV transformation can generally be
seen towards the end of the 3-4 week culture period. By
phase-contrast microscopy, transformed cells may appear large,
clear, hairy and tend to aggregate in tight clusters of cells.
Initially, EBV lines are generally polyclonal. However, over
prolonged periods of cell cultures, EBV lines may become monoclonal
or polyclonal as a result of the selective outgrowth of particular
B cell clones. Alternatively, polyclonal EBV transformed lines may
be subcloned (e.g., by limiting dilution culture) or fused with a
suitable fusion partner and plated at limiting dilution to obtain
monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g.,
SP2/0, X63-Ag8.653), heteromyeloma cell lines (human.times.mouse;
e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM
1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention
also provides a method of generating polyclonal or monoclonal human
antibodies against polypeptides of the invention or fragments
thereof, comprising EBV-transformation of human B cells.
[0204] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0205] For example, antibodies that bind to a Therapeutic protein
can also be generated using various phage display methods known in
the art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make antibodies that bind to a Therapeutic protein
include those disclosed in Brinkman et al., J. Immunol. Methods
182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186
(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in
Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134;
PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.
5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;
5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
[0206] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0207] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816,397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework 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., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[0208] 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.
[0209] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which
are incorporated by reference herein in their entirety. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.) and
Genpharm (San Jose, Calif.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0210] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0211] Polynucleotides Encoding Antibodies
[0212] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody and fragments thereof. The
invention also encompasses polynucleotides that hybridize under
stringent or alternatively, under lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a Therapeutic
protein, and more preferably, an antibody that binds to a
polypeptide having the amino acid sequence of a "Therapeutic
protein:X" as disclosed in the "SEQ ID NO:Z" column of Table 2.
[0213] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0214] 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) by PCR amplification using synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning
using an oligonucleotide probe specific for the particular gene
sequence to identify, e.g., a cDNA clone from a cDNA library that
encodes the antibody. Amplified nucleic acids generated by PCR may
then be cloned into replicable cloning vectors using any method
well known in the art (See Example 65).
[0215] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0216] 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 know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0217] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0218] 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)).
[0219] Recombinant Expression of Antibodies
[0220] 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), of the
invention has been obtained, the vector for the production of the
antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art. Thus, methods for preparing
a protein by expressing a polynucleotide containing an antibody
encoding nucleotide sequence are described herein. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0221] 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 of the invention, 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.
[0222] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0223] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0224] 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).
[0225] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0226] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0227] 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.
[0228] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt- cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers
resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods commonly known in the art of recombinant DNA technology may
be routinely applied to select the desired recombinant clone, and
such methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0229] 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)).
[0230] Vectors which use glutamine synthase (GS) or DHFR as the
selectable markers can be amplified in the presence of the drugs
methionine sulphoximine or methotrexate, respectively. An advantage
of glutamine synthase based vectors are the availability of cell
lines (e.g., the murine myeloma cell line, NS0) which are glutamine
synthase negative. Glutamine synthase expression systems can also
function in glutamine synthase expressing cells (e.g. Chinese
Hamster Ovary (CHO) cells) by providing additional inhibitor to
prevent the functioning of the endogenous gene. A glutamine
synthase expression system and components thereof are detailed in
PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404;
and WO91/06657 which are incorporated in their entireties by
reference herein. Additionally, glutamine synthase expression
vectors that may be used according to the present invention are
commercially available from suppliers, including, for example Lonza
Biologics, Inc. (Portsmouth, N.H.). Expression and production of
monoclonal antibodies using a GS expression system in murine
myeloma cells is described in Bebbington et al., Bio/technology
10:169 (1992) and in Biblia and Robinson Biotechnol. Prog. 11:1
(1995) which are incorporated in their entireties by reference
herein.
[0231] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0232] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies that bind to a Therapeutic protein and
that may correspond to a Therapeutic protein portion of an albumin
fusion protein of the invention or fragments thereof can be fused
to heterologous polypeptide sequences described herein or otherwise
known in the art, to facilitate purification.
[0233] Modifications of Antibodies
[0234] Antibodies that bind a Therapeutic protein or fragments or
variants 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
hemagglutinin tag (also called 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.
[0235] 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 125I, 131I, 111In or 99Tc. Other examples of
detectable substances have been described elsewhere herein.
[0236] Further, an antibody of the invention 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, 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).
[0237] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, alpha-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), 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.
[0238] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0239] Techniques for conjugating such therapeutic moiety to
antibodies are well known. See, for example, Arnon et al.,
"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0240] 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.
[0241] 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.
[0242] Antibody-Albumin Fusion
[0243] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein of the invention include, but are not limited to,
antibodies that bind a Therapeutic protein disclosed in the
"Therapeutic Protein X" column of Table 1, or a fragment or variant
thereof.
[0244] In specific embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VH
domain. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, one, two or
three VH CDRs. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VH
CDR1. In other embodiments, the fragment or variant of an antibody
that immunospecifically binds a Therapeutic protein and that
corresponds to a Therapeutic protein portion of an albumin fusion
protein comprises, or alternatively consists of, the VH CDR2. In
other embodiments, the fragment or variant of an antibody that
immunospecifically binds a Therapeutic protein and that corresponds
to a Therapeutic protein portion of an albumin fusion protein
comprises, or alternatively consists of, the VH CDR3.
[0245] In specific embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VL
domain. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, one, two or
three VL CDRs. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VL
CDR1. In other embodiments, the fragment or variant of an antibody
that immunospecifically binds a Therapeutic protein and that
corresponds to a Therapeutic protein portion of an albumin fusion
protein comprises, or alternatively consists of, the VL CDR2. In
other embodiments, the fragment or variant of an antibody that
immunospecifically binds a Therapeutic protein and that corresponds
to a Therapeutic protein portion of an albumin fusion protein
comprises, or alternatively consists of, the VL CDR3.
[0246] In other embodiments, the fragment or variant of an antibody
that immunospecifically binds a Therapeutic protein and that
corresponds to a Therapeutic protein portion of an albumin fusion
protein comprises, or alternatively consists of, one, two, three,
four, five, or six VH and/or VL CDRs.
[0247] In preferred embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, an scFv
comprising the VH domain of the Therapeutic antibody, linked to the
VL domain of the therapeutic antibody by a peptide linker such as
(Gly.sub.4Ser).sub.3 (SEQ ID NO:4).
[0248] Immunophenotyping
[0249] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein (or fragment or variant
thereof) may be utilized for immunophenotyping of cell lines and
biological samples. Therapeutic proteins of the present invention
may be useful as cell-specific markers, or more specifically as
cellular markers that are differentially expressed at various
stages of differentiation and/or maturation of particular cell
types. Monoclonal antibodies (or albumin fusion proteins comprising
at least a fragment or variant of an antibody that binds a
Therapeutic protein) directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies (or albumin fusion proteins
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein) to screen for cellular populations
expressing the marker(s), and include magnetic separation using
antibody-coated magnetic beads, "panning" with antibody attached to
a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S.
Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49
(1999)).
[0250] 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.
[0251] Characterizing Antibodies that Bind a Therapeutic Protein
and Albumin Fusion Proteins Comprising a Fragment or Variant of an
Antibody that Binds a Therapeutic Protein
[0252] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein (or fragment or variant
thereof) may be characterized in a variety of ways. In particular,
Albumin fusion proteins of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
may be assayed for the ability to specifically bind to the same
antigens specifically bound by the antibody that binds a
Therapeutic protein corresponding to the antibody that binds a
Therapeutic protein portion of the albumin fusion protein using
techniques described herein or routinely modifying techniques known
in the art.
[0253] Assays for the ability of the antibodies of the invention or
albumin fusion proteins of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
(or fragment or variant thereof) to (specifically) bind a specific
protein or epitope 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., U.S. 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). The antibodies
of the invention or albumin fusion proteins of the invention
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein (or fragment or variant thereof) may also be
assayed for their specificity and affinity for a specific protein
or epitope using or routinely modifying techniques described herein
or otherwise known in the art.
[0254] The albumin fusion proteins of the invention comprising at
least a fragment or variant of an antibody that binds a Therapeutic
protein may be assayed for cross-reactivity with other antigens
(e.g., molecules that have sequence/structure conservation with the
molecule(s) specifically bound by the antibody that binds a
Therapeutic protein (or fragment or variant thereof) corresponding
to the Therapeutic protein portion of the albumin fusion protein of
the invention) by any method known in the art.
[0255] Immunoassays which can be used to analyze (immunospecific)
binding and cross-reactivity 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, and
protein A immunoassays, to name but a few. Such assays are routine
and well known in the art (see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, which is incorporated by reference herein in
its entirety). Exemplary immunoassays are described briefly below
(but are not intended by way of limitation).
[0256] 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 an antibody of the invention or
albumin fusion protein of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
(or fragment or variant thereof) 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 (or beads coated with an
appropriate anti-idiotypic antibody or anti-albumin antibody in the
case when an albumin fusion protein comprising at least a fragment
or variant of a Therapeutic antibody) 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. The ability of the antibody or albumin fusion protein of
the invention 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 or albumin fusion protein to
an antigen and decrease the background (e.g., pre-clearing the cell
lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York at 10.16.1.
[0257] 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),
applying the antibody or albumin fusion protein of the invention
(diluted in blocking buffer) to the membrane, washing the membrane
in washing buffer, applying a secondary antibody (which recognizes
the albumin fusion protein, e.g., an anti-human serum albumin
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
.sup.32P or .sup.125I) diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0258] ELISAs comprise preparing antigen, coating the well of a
96-well microtiter plate with the antigen, washing away antigen
that did not bind the wells, adding the antibody or albumin fusion
protein (comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein) of the invention conjugated to a
detectable compound such as an enzymatic substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) to the wells and
incubating for a period of time, washing away unbound or
non-specifically bound albumin fusion proteins, and detecting the
presence of the antibody or albumin fusion proteins specifically
bound to the antigen coating the well. In ELISAs the antibody or
albumin fusion protein does not have to be conjugated to a
detectable compound; instead, a second antibody (which recognizes
the antibody or albumin fusion protein, respectively) conjugated to
a detectable compound may be added to the well. Further, instead of
coating the well with the antigen, antibody or the albumin fusion
protein may be coated to the well. In this case, the detectable
molecule could be the antigen conjugated to a detectable compound
such as an enzymatic substrate (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., Ausubel
et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1,
John Wiley & Sons, Inc., New York at 11.2.1.
[0259] The binding affinity of an albumin fusion protein to a
protein, antigen, or epitope and the off-rate of an antibody- or
albumin fusion protein-protein/antigen/epitope 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 or albumin fusion protein of the invention 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 or albumin fusion protein of the invention for a specific
protein, antigen, or epitope and the binding off-rates can be
determined from the data by Scatchard plot analysis. Competition
with a second protein that binds the same protein, antigen or
epitope as the antibody or albumin fusion protein, can also be
determined using radioimmunoassays. In this case, the protein,
antigen or epitope is incubated with an antibody or albumin fusion
protein of the invention conjugated to a labeled compound (e.g.,
.sup.3H or .sup.125I) in the presence of increasing amounts of an
unlabeled second protein that binds the same protein, antigen, or
epitope as the albumin fusion protein of the invention.
[0260] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of antibody or albumin
fusion proteins of the invention to a protein, antigen or epitope.
BIAcore kinetic analysis comprises analyzing the binding and
dissociation of antibodies, albumin fusion proteins, or specific
polypeptides, antigens or epitopes from chips with immobilized
specific polypeptides, antigens or epitopes, antibodies or albumin
fusion proteins, respectively, on their surface.
[0261] Therapeutic Uses
[0262] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
or albumin fusion proteins of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein), nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein), albumin fusion
proteins of the invention comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, and nucleic acids
encoding such albumin fusion proteins. The antibodies of the
invention or albumin fusion proteins of the invention comprising at
least a fragment or variant of an antibody that binds a Therapeutic
protein can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a Therapeutic protein, 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 expression and/or activity of a
Therapeutic protein includes, but is not limited to, alleviating
symptoms associated with those diseases, disorders or conditions.
antibodies of the invention or albumin fusion proteins of the
invention comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0263] In a specific and preferred embodiment, the present
invention is directed to antibody-based therapies which involve
administering antibodies of the invention or albumin fusion
proteins of the invention comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein to an animal,
preferably a mammal, and most preferably a human, patient for
treating one or more diseases, disorders, or conditions, including
but not limited to: neural disorders, immune system disorders,
muscular disorders, reproductive disorders, gastrointestinal
disorders, pulmonary disorders, cardiovascular disorders, renal
disorders, proliferative disorders, and/or cancerous diseases and
conditions, and/or as described elsewhere herein. Therapeutic
compounds of the invention include, but are not limited to,
antibodies of the invention (e.g., antibodies directed to the full
length protein expressed on the cell surface of a mammalian cell;
antibodies directed to an epitope of a Therapeutic protein and
nucleic acids encoding antibodies of the invention (including
fragments, analogs and derivatives thereof and anti-idiotypic
antibodies as described herein). The antibodies of the invention
can be used to treat, inhibit or prevent diseases, disorders or
conditions associated with aberrant expression and/or activity of a
Therapeutic protein, 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 expression and/or activity of a
Therapeutic protein includes, but is not limited to, alleviating
symptoms associated with those diseases, disorders or conditions.
Antibodies of the invention or albumin fusion proteins of the
invention comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0264] A summary of the ways in which the antibodies of the
invention or albumin fusion proteins of the invention comprising at
least a fragment or variant of an antibody that binds a Therapeutic
protein may be used therapeutically includes binding Therapeutic
proteins locally or systemically in the body or by direct
cytotoxicity of the antibody, e.g. as mediated by complement (CDC)
or by effector cells (ADCC). Some of these approaches are described
in more detail below. Armed with the teachings provided herein, one
of ordinary skill in the art will know how to use the antibodies of
the invention or albumin fusion proteins of the invention
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein for diagnostic, monitoring or therapeutic
purposes without undue experimentation.
[0265] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein 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.
[0266] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein may be administered alone
or in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). 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.
[0267] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against Therapeutic
proteins, fragments or regions thereof, (or the albumin fusion
protein correlate of such an antibody) for both immunoassays
directed to and therapy of disorders related to polynucleotides or
polypeptides, including fragments thereof, of the present
invention. Such antibodies, fragments, or regions, will preferably
have an affinity for polynucleotides or polypeptides of the
invention, including fragments thereof. Preferred binding
affinities include dissociation constants or Kd's less than
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. More 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 or 10.sup.-8
M. Even more preferred binding affinities include those with a
dissociation constant or Kd less than 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.
[0268] Gene Therapy
[0269] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies that bind therapeutic proteins or albumin
fusion proteins comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein are administered to
treat, inhibit or prevent a disease or disorder associated with
aberrant expression and/or activity of a Therapeutic protein, 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.
[0270] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described in more detail elsewhere in this application.
[0271] Demonstration of Therapeutic or Prophylactic Activity
[0272] 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.
[0273] Therapeutic/Prophylactic Administration and Composition
[0274] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention. 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.
[0275] 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.
[0276] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0277] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0278] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[0279] 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 Pres., 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, 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)).
[0280] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0281] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0282] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0283] 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.
[0284] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0285] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a Therapeutic protein 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.
[0286] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0287] Diagnosis and Imaging
[0288] Labeled antibodies and derivatives and analogs thereof that
bind a Therapeutic protein (or fragment or variant thereof)
(including albumin fusion proteins comprising at least a fragment
or variant of an antibody that binds a Therapeutic protein), can be
used for diagnostic purposes to detect, diagnose, or monitor
diseases, disorders, and/or conditions associated with the aberrant
expression and/or activity of Therapeutic protein. The invention
provides for the detection of aberrant expression of a Therapeutic
protein, comprising (a) assaying the expression of the Therapeutic
protein in cells or body fluid of an individual using one or more
antibodies specific to the polypeptide interest and (b) comparing
the level of gene expression with a standard gene expression level,
whereby an increase or decrease in the assayed Therapeutic protein
expression level compared to the standard expression level is
indicative of aberrant expression.
[0289] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the Therapeutic
protein in cells or body fluid of an individual using one or more
antibodies specific to the Therapeutic protein or albumin fusion
proteins comprising at least a fragment of variant of an antibody
specific to a Therapeutic protein, and (b) comparing the level of
gene expression with a standard gene expression level, whereby an
increase or decrease in the assayed Therapeutic protein gene
expression level compared to the standard expression level is
indicative of a particular disorder. With respect to cancer, the
presence of a relatively high amount of transcript in biopsied
tissue from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0290] Antibodies of the invention or albumin fusion proteins
comprising at least a fragment of variant of an antibody specific
to a Therapeutic protein can be used to assay protein levels in a
biological sample using classical immunohistological methods known
to those of skill in the art (e.g., see Jalkanen et al., J. Cell.
Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for
detecting protein gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA). Suitable antibody assay labels are known in the art and
include enzyme labels, such as, glucose oxidase; radioisotopes,
such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium
(3H), indium (112In), and technetium (99Tc); luminescent labels,
such as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0291] One facet of the invention is the detection and diagnosis of
a disease or disorder associated with aberrant expression of a
Therapeutic protein in an animal, preferably a mammal and most
preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the Therapeutic protein 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 therapeutic protein. 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.
[0292] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99mTc. The labeled antibody, antibody fragment, or
albumin fusion protein comprising at least a fragment or variant of
an antibody that binds a Therapeutic protein will then
preferentially accumulate at the location of cells which contain
the specific Therapeutic protein. In vivo tumor imaging is
described in S. W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor
Imaging The Radiochemical Detection of Cancer, S. W. Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
[0293] 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.
[0294] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0295] 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.
[0296] 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). Antibodies that specifically detect the albumin fusion
protein but not albumin or the therapeutic protein alone are a
preferred embodiment. These can be used to detect the albumin
fusion protein as described throughout the specification.
[0297] Kits
[0298] 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 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).
[0299] 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.
[0300] 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.
[0301] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0302] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, Mo.).
[0303] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0304] 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.
[0305] Albumin Fusion Proteins
[0306] The present invention relates generally to albumin fusion
proteins and methods of treating, preventing, or ameliorating
diseases or disorders. As used herein, "albumin fusion protein"
refers to a protein formed by the fusion of at least one molecule
of albumin (or a fragment or variant thereof) to at least one
molecule of a Therapeutic protein (or fragment or variant thereof).
An albumin fusion protein of the invention comprises at least a
fragment or variant of a Therapeutic protein and at least a
fragment or variant of human serum albumin, which are associated
with one another, preferably by genetic fusion (i.e., the albumin
fusion protein is generated by translation of a nucleic acid in
which a polynucleotide encoding all or a portion of a Therapeutic
protein is joined in-frame with a polynucleotide encoding all or a
portion of albumin) or to one another. The Therapeutic protein and
albumin protein, once part of the albumin fusion protein, may each
be referred to as a "portion", "region" or "moiety" of the albumin
fusion protein.
[0307] In a preferred embodiment, the invention provides an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 1 or Table 2. Polynucleotides encoding
these albumin fusion proteins are also encompassed by the
invention.
[0308] Preferred albumin fusion proteins of the invention, include,
but are not limited to, albumin fusion proteins encoded by a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of a Therapeutic
protein (or fragment or variant thereof); a nucleic acid molecule
comprising, or alternatively consisting of, a polynucleotide
encoding at least one molecule of albumin (or a fragment or variant
thereof) joined in frame to at least one polynucleotide encoding at
least one molecule of a Therapeutic protein (or fragment or variant
thereof) generated as described in Table 1, Table 2 or in the
Examples; or a nucleic acid molecule comprising, or alternatively
consisting of, a polynucleotide encoding at least one molecule of
albumin (or a fragment or variant thereof) joined in frame to at
least one polynucleotide encoding at least one molecule of a
Therapeutic protein (or fragment or variant thereof), further
comprising, for example, one or more of the following elements: (1)
a functional self-replicating vector (including but not limited to,
a shuttle vector, an expression vector, an integration vector,
and/or a replication system), (2) a region for initiation of
transcription (e.g., a promoter region, such as for example, a
regulatable or inducible promoter, a constitutive promoter), (3) a
region for termination of transcription, (4) a leader sequence, and
(5) a selectable marker.
[0309] In one embodiment, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, a Therapeutic
protein (e.g., as described in Table 1) and a serum albumin
protein. In other embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
biologically active and/or therapeutically active fragment of a
Therapeutic protein and a serum albumin protein. In other
embodiments, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, a biologically active
and/or therapeutically active variant of a Therapeutic protein and
a serum albumin protein. In preferred embodiments, the serum
albumin protein component of the albumin fusion protein is the
mature portion of serum albumin.
[0310] In further embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
Therapeutic protein, and a biologically active and/or
therapeutically active fragment of serum albumin. In further
embodiments, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, a Therapeutic protein
and a biologically active and/or therapeutically active variant of
serum albumin. In preferred embodiments, the Therapeutic protein
portion of the albumin fusion protein is the mature portion of the
Therapeutic protein.
[0311] In further embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
biologically active and/or therapeutically active fragment or
variant of a Therapeutic protein and a biologically active and/or
therapeutically active fragment or variant of serum albumin. In
preferred embodiments, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, the mature
portion of a Therapeutic protein and the mature portion of serum
albumin.
[0312] Preferably, the albumin fusion protein comprises HA as the
N-terminal portion, and a Therapeutic protein as the C-terminal
portion. Alternatively, an albumin fusion protein comprising HA as
the C-terminal portion, and a Therapeutic protein as the N-terminal
portion may also be used.
[0313] In other embodiments, the albumin fusion protein has a
Therapeutic protein fused to both the N-terminus and the C-terminus
of albumin. In a preferred embodiment, the Therapeutic proteins
fused at the N- and C-termini are the same Therapeutic proteins. In
an alternative preferred embodiment, the Therapeutic proteins fused
at the N- and C-termini are different Therapeutic proteins. In
another preferred embodiment, the Therapeutic proteins fused at the
N- and C-termini are different Therapeutic proteins which may be
used to treat or prevent the same or a related disease, disorder,
or condition (e.g. as listed in the "Preferred Indication Y" column
of Table 1). In another preferred embodiment, the Therapeutic
proteins fused at the N- and C-termini are different Therapeutic
proteins which may be used to treat, ameliorate, or prevent
diseases or disorders (e.g. as listed in the "Preferred Indication
Y" column of Table 1) which are known in the art to commonly occur
in patients simultaneously, concurrently, or consecutively, or
which commonly occur in patients in association with one
another.
[0314] Albumin fusion proteins of the invention encompass proteins
containing one, two, three, four, or more molecules of a given
Therapeutic protein X or variant thereof fused to the N- or
C-terminus of an albumin fusion protein of the invention, and/or to
the N- and/or C-terminus of albumin or variant thereof. Molecules
of a given Therapeutic protein X or variants thereof may be in any
number of orientations, including, but not limited to, a `head to
head` orientation (e.g., wherein the N-terminus of one molecule of
a Therapeutic protein X is fused to the N-terminus of another
molecule of the Therapeutic protein X), or a `head to tail`
orientation (e.g., wherein the C-terminus of one molecule of a
Therapeutic protein X is fused to the N-terminus of another
molecule of Therapeutic protein X).
[0315] In one embodiment, one, two, three, or more tandemly
oriented Therapeutic protein X polypeptides (or fragments or
variants thereof) are fused to the N- or C-terminus of an albumin
fusion protein of the invention, and/or to the N- and/or C-terminus
of albumin or variant thereof.
[0316] Albumin fusion proteins of the invention further encompass
proteins containing one, two, three, four, or more molecules of a
given Therapeutic protein X or variant thereof fused to the N- or
C-terminus of an albumin fusion protein of the invention, and/or to
the N- and/or C-terminus of albumin or variant thereof, wherein the
molecules are joined through peptide linkers. Examples include
those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby
incorporated by reference). Albumin fusion proteins comprising
multiple Therapeutic protein X polypeptides separated by peptide
linkers may be produced using conventional recombinant DNA
technology. Linkers are particularly important when fusing a small
peptide to the large HSA molecule. The peptide itself can be a
linker by fusing tandem copies of the peptide or other known
linkers can be used. Constructs that incorporate linkers are
described in Table 2 or are apparent when examining SEQ ID
NO:Y.
[0317] Further, albumin fusion proteins of the invention may also
be produced by fusing a Therapeutic protein X or variants thereof
to the N-terminal and/or C-terminal of albumin or variants thereof
in such a way as to allow the formation of intramolecular and/or
intermolecular multimeric forms. In one embodiment of the
invention, albumin fusion proteins may be in monomeric or
multimeric forms (i.e., dimers, trimers, tetramers and higher
multimers). In a further embodiment of the invention, the
Therapeutic protein portion of an albumin fusion protein may be in
monomeric form or multimeric form (i.e., dimers, trimers, tetramers
and higher multimers). In a specific embodiment, the Therapeutic
protein portion of an albumin fusion protein is in multimeric form
(i.e., dimers, trimers, tetramers and higher multimers), and the
albumin protein portion is in monomeric form.
[0318] In addition to albumin fusion protein in which the albumin
portion is fused N-terminal and/or C-terminal of the Therapeutic
protein portion, albumin fusion proteins of the invention may also
be produced by inserting the Therapeutic protein or peptide of
interest (e.g., a Therapeutic protein X as disclosed in Table 1, or
an antibody that binds a Therapeutic protein or a fragment or
variant thereof) into an internal region 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. The loops, as determined from
the crystal structure of HA (PDB identifiers 1AO6, 1BJ5, 1BKE,
1BM0, 1E7E to 1E7I 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.
[0319] Loops in human albumin structure into which peptides or
polypeptides may be inserted to generate albumin fusion proteins of
the invention 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, peptides or
polypeptides are inserted into the Val54-Asn61, Gln170-Ala176,
and/or Lys560-Thr566 loops of mature human albumin (SEQ ID
NO:1).
[0320] Peptides to be inserted may be derived from either phage
display or synthetic peptide libraries screened for specific
biological activity or from the active portions of a molecule with
the desired function. Additionally, random peptide libraries may be
generated within particular loops or by insertions of randomized
peptides into particular loops of the HA molecule and in which all
possible combinations of amino acids are represented.
[0321] Such library(s) could be generated on HA or domain fragments
of HA by one of the following methods:
[0322] randomized mutation of amino acids within one or more
peptide loops of HA or HA domain fragments. Either one, more or all
the residues within a loop could be mutated in this manner;
[0323] replacement of, or insertion into one or more loops of HA or
HA domain fragments (i.e., internal fusion) of a randomized
peptide(s) of length X.sub.n (where X is an amino acid and n is the
number of residues;
[0324] N-, C- or N- and C-terminal peptide/protein fusions in
addition to (a) and/or (b).
[0325] The HA or HA domain fragment may also be made
multifunctional by grafting the peptides derived from different
screens of different loops against different targets into the same
HA or HA domain fragment.
[0326] In preferred embodiments, peptides inserted into a loop of
human serum albumin are peptide fragments or peptide variants of
the Therapeutic proteins disclosed in Table 1. More particularly,
the invention encompasses albumin fusion proteins which comprise
peptide fragments or peptide variants at least 7 at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 20, at least 25, at least 30, at
least 35, or at least 40 amino acids in length inserted into a loop
of human serum albumin. The invention also encompasses albumin
fusion proteins which comprise peptide fragments or peptide
variants at least 7 at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 35, or at least 40 amino
acids fused to the N-terminus of human serum albumin. The invention
also encompasses albumin fusion proteins which comprise peptide
fragments or peptide variants at least 7 at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 20, at least 25, at least 30, at least 35, or at
least 40 amino acids fused to the C-terminus of human serum
albumin. For example, short peptides described in Table 1 and 2
(e.g., Therapeutic Y) can be inserted into the albumin loops.
[0327] Generally, the albumin fusion proteins of the invention may
have one HA-derived region and one Therapeutic protein-derived
region. Multiple regions of each protein, however, may be used to
make an albumin fusion protein of the invention. Similarly, more
than one Therapeutic protein may be used to make an albumin fusion
protein of the invention. For instance, a Therapeutic protein may
be fused to both the N- and C-terminal ends of the HA. In such a
configuration, the Therapeutic protein portions may be the same or
different Therapeutic protein molecules. The structure of
bifunctional albumin fusion proteins may be represented as: X--HA-Y
or Y--HA-X.
[0328] For example, an anti-BLyS.TM. scFv-HA-IFN.alpha.-2b fusion
may be prepared to modulate the immune response to IFN.alpha.-2b by
anti-BLyS.TM. scFv. An alternative is making a bi (or even multi)
functional dose of HA-fusions e.g. HA-IFN.alpha.-2b fusion mixed
with HA-anti-BLyS.TM. scFv fusion or other HA-fusions in various
ratio's depending on function, half-life etc.
[0329] Bi- or multi-functional albumin fusion proteins may also be
prepared to target the Therapeutic protein portion of a fusion to a
target organ or cell type via protein or peptide at the opposite
terminus of HA.
[0330] As an alternative to the fusion of known therapeutic
molecules, the peptides could be obtained by screening libraries
constructed as fusions to the N-, C- or N- and C-termini of HA, or
domain fragment of HA, of typically 6, 8, 12, 20 or 25 or X.sub.n
(where X is an amino acid (aa) and n equals the number of residues)
randomized amino acids, and in which all possible combinations of
amino acids were represented. A particular advantage of this
approach is that the peptides may be selected in situ on the HA
molecule and the properties of the peptide would therefore be as
selected for rather than, potentially, modified as might be the
case for a peptide derived by any other method then being attached
to HA.
[0331] Additionally, the albumin fusion proteins of the invention
may include a linker peptide between the fused portions to provide
greater physical separation between the moieties and thus maximize
the accessibility of the Therapeutic protein portion, for instance,
for binding to its cognate receptor. The linker peptide may consist
of amino acids such that it is flexible or more rigid.
[0332] The linker sequence may be cleavable by a protease or
chemically to yield the growth hormone related moiety. Preferably,
the protease is one which is produced naturally by the host, for
example the S. cerevisiae protease kex2 or equivalent
proteases.
[0333] Therefore, as described above, the albumin fusion proteins
of the invention may have the following formula R1-L-R2; R2-L-R1;
or R1-L-R2-L-R1, wherein R1 is at least one Therapeutic protein,
peptide or polypeptide sequence, and not necessarily the same
Therapeutic protein, L is a linker and R2 is a serum albumin
sequence.
[0334] In preferred embodiments, albumin fusion proteins of the
invention comprising a Therapeutic protein have a higher plasma
stability compared to the plasma stability of the same Therapeutic
protein when not fused to albumin. Plasma stability typically
refers to the time period between when the Therapeutic protein is
administered in vivo and carried into the bloodstream and when the
therapeutic protein is degraded and cleared from the bloodstream,
into an organ, such as the kidney or liver, that ultimately clears
the Therapeutic protein from the body. Plasma stability is
calculated in terms of the half-life of the Therapeutic protein in
the bloodstream. The half-life of the Therapeutic protein in the
bloodstream can be readily determined by common assays known in the
art.
[0335] In preferred embodiments, Albumin fusion proteins of the
invention comprising a Therapeutic protein have extended shelf life
compared to the shelf life the same Therapeutic protein when not
fused to albumin. Shelf-life typically refers to the time period
over which the therapeutic activity of a Therapeutic protein in
solution or in some other storage formulation, is stable without
undue loss of therapeutic activity. Many of the Therapeutic
proteins are highly labile in their unfused state. As described
below, the typical shelf-life of these Therapeutic proteins is
markedly prolonged upon incorporation into the albumin fusion
protein of the invention.
[0336] Albumin fusion proteins of the invention with "prolonged" or
"extended" shelf-life exhibit greater therapeutic activity relative
to a standard that has been subjected to the same storage and
handling conditions. The standard may be the unfused full-length
Therapeutic protein. When the Therapeutic protein portion of the
albumin fusion protein is an analog, a variant, or is otherwise
altered or does not include the complete sequence for that protein,
the prolongation of therapeutic activity may alternatively be
compared to the unfused equivalent of that analog, variant, altered
peptide or incomplete sequence. As an example, an albumin fusion
protein of the invention may retain greater than about 100% of the
therapeutic activity, or greater than about 105%, 110%, 120%, 130%,
150% or 200% of the therapeutic activity of a standard when
subjected to the same storage and handling conditions as the
standard when compared at a given time point.
[0337] Shelf-life may also be assessed in terms of therapeutic
activity remaining after storage, normalized to therapeutic
activity when storage began. Albumin fusion proteins of the
invention with prolonged or extended shelf-life as exhibited by
prolonged or extended therapeutic activity may retain greater than
about 50% of the therapeutic activity, about 60%, 70%, 80%, or 90%
or more of the therapeutic activity of the equivalent unfused
Therapeutic protein when subjected to the same conditions.
[0338] Expression of Fusion Proteins
[0339] The albumin fusion proteins of the invention may be produced
as recombinant molecules by secretion from yeast, a microorganism
such as a bacterium, or a human or animal cell line. Preferably,
the polypeptide is secreted from the host cells.
[0340] A particular embodiment of the invention comprises a DNA
construct encoding a signal sequence effective for directing
secretion in yeast, particularly a yeast-derived signal sequence
(especially one which is homologous to the yeast host), and the
fused molecule of the first aspect of the invention, there being no
yeast-derived pro sequence between the signal and the mature
polypeptide.
[0341] The Saccharomyces cerevisiae invertase signal is a preferred
example of a yeast-derived signal sequence.
[0342] Conjugates of the kind prepared by Poznansky et al., (FEBS
Lett. 239:18 (1988)), in which separately-prepared polypeptides are
joined by chemical cross-linking, are not contemplated.
[0343] The present invention also includes a cell, preferably a
yeast cell transformed to express an albumin fusion protein of the
invention. In addition to the transformed host cells themselves,
the present invention also contemplates a culture of those cells,
preferably a monoclonal (clonally homogeneous) culture, or a
culture derived from a monoclonal culture, in a nutrient medium. If
the polypeptide is secreted, the medium will contain the
polypeptide, with the cells, or without the cells if they have been
filtered or centrifuged away. Many expression systems are known and
may be used, including bacteria (for example E. coli and Bacillus
subtilis), yeasts (for example Saccharomyces cerevisiae,
Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for
example Aspergillus), plant cells, animal cells and insect
cells.
[0344] Preferred yeast strains to be used in the production of
albumin fusion proteins are D88, DXY1 and BXP10. D88 [leu2-3,
leu2-122, can1, pra1, ubc4] is a derivative of parent strain
AH22his.sup.+ (also known as DB1; see, e.g., Sleep et al.
Biotechnology 8:42-46 (1990)). The strain contains a leu2 mutation
which allows for auxotropic selection of 2 micron-based plasmids
that contain the LEU2 gene. D88 also exhibits a derepression of
PRB1 in glucose excess. The PRB1 promoter is normally controlled by
two checkpoints that monitor glucose levels and growth stage. The
promoter is activated in wild type yeast upon glucose depletion and
entry into stationary phase. Strain D88 exhibits the repression by
glucose but maintains the induction upon entry into stationary
phase. The PRA1 gene encodes a yeast vacuolar protease, YscA
endoprotease A, that is localized in the ER. The UBC4 gene is in
the ubiquitination pathway and is involved in targeting short lived
and abnormal proteins for ubiquitin dependant degradation.
Isolation of this ubc4 mutation was found to increase the copy
number of an expression plasmid in the cell and cause an increased
level of expression of a desired protein expressed from the plasmid
(see, e.g., International Publication No. WO99/00504, hereby
incorporated in its entirety by reference herein).
[0345] DXY1, a derivative of D88, has the following genotype:
[leu2-3, leu2-122, can1, pra1, ubc4, ura3::yap3]. In addition to
the mutations isolated in D88, this strain also has a knockout of
the YAP3 protease. This protease causes cleavage of mostly di-basic
residues (RR, RK, KR, KK) but can also promote cleavage at single
basic residues in proteins. Isolation of this yap3 mutation
resulted in higher levels of full length HSA production (see, e.g.,
U.S. Pat. No. 5,965,386 and Kerry-Williams et al., Yeast 14:161-169
(1998), hereby incorporated in their entireties by reference
herein).
[0346] BXP10 has the following genotype: leu2-3, leu2-122, can1,
pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3. In
addition to the mutations isolated in DXY1, this strain also has a
knockout of the PMT1 gene and the HSP150 gene. The PMT1 gene is a
member of the evolutionarily conserved family of
dolichyl-phosphate-D-mannose protein O-mannosyltransferases (Pmts).
The transmembrane topology of Pmt1p suggests that it is an integral
membrane protein of the endoplasmic reticulum with a role in
O-linked glycosylation. This mutation serves to reduce/eliminate
O-linked glycosylation of HSA fusions (see, e.g., International
Publication No. WO00/44772, hereby incorporated in its entirety by
reference herein). Studies revealed that the Hsp150 protein is
inefficiently separated from rHA by ion exchange chromatography.
The mutation in the HSP150 gene removes a potential contaminant
that has proven difficult to remove by standard purification
techniques. See, e.g., U.S. Pat. No. 5,783,423, hereby incorporated
in its entirety by reference herein.
[0347] The desired protein is produced in conventional ways, for
example from a coding sequence inserted in the host chromosome or
on a free plasmid. The yeasts are transformed with a coding
sequence for the desired protein in any of the usual ways, for
example electroporation. Methods for transformation of yeast by
electroporation are disclosed in Becker & Guarente (1990)
Methods Enzymol. 194, 182.
[0348] Successfully transformed cells, i.e., cells that contain a
DNA construct of the present invention, can be identified by well
known techniques. For example, cells resulting from the
introduction of an expression construct can be grown to produce the
desired polypeptide. Cells can be harvested and lysed and their DNA
content examined for the presence of the DNA using a method such as
that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent
et al. (1985) Biotech. 3, 208. Alternatively, the presence of the
protein in the supernatant can be detected using antibodies.
[0349] Useful yeast plasmid vectors include pRS403-406 and
pRS413-416 and are generally available from Stratagene Cloning
Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404,
pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and
incorporate the yeast selectable markers HIS3, 7RP1, LEU2 and URA3.
Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
[0350] Preferred vectors for making albumin fusion proteins for
expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA
which are described in detail in Example 1. FIG. 2 shows a map of
the pPPC0005 plasmid that can be used as the base vector into which
polynucleotides encoding Therapeutic proteins may be cloned to form
HA-fusions. It contains a PRB1 S. cerevisiae promoter (PRB1p), a
Fusion leader sequence (FL), DNA encoding HA (rHA) and an ADH1 S.
cerevisiae terminator sequence. The sequence of the fusion leader
sequence consists of the first 19 amino acids of the signal peptide
of human serum albumin (SEQ ID NO:3) and the last five amino acids
of the mating factor alpha 1 promoter (SLDKR, see EP-A-387 319
which is hereby incorporated by reference in its entirety).
[0351] The plasmids, pPPC0005, pScCHSA, pScNHSA, and pC4:HSA were
deposited on Apr. 11, 2001 at the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209 and given
accession numbers ATCC PTA-3278, PTA-3276, PTA-3279, and PTA-3277,
respectively. Another vector useful for expressing an albumin
fusion protein in yeast the pSAC35 vector which is described in
Sleep et al., BioTechnology 8:42 (1990) which is hereby
incorporated by reference in its entirety.
[0352] A yeast promoter that can be used to express the albumin
fusion protein is the MET25 promoter. See, for example, Dominik
Mumburg, Rolf Muller and Martin Funk. Nucleic Acids Research, 1994,
Vol. 22, No. 25, pp. 5767-5768. The Met25 promoter is 383 bases
long (bases -382 to -1) and the genes expressed by this promoter
are also known as Met15, Met17, and YLR303W. A preferred embodiment
uses the sequence below, where, at the 5' end of the sequence
below, the Not 1 site used in the cloning is underlined and at the
3' end, the ATG start codon is underlined:
GCGGCCGCCGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTTGCTTTTTCTCTTGAGGTCACATGA-
TCGCAAAATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGT-
TAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAA-
TTGAACACGCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCC-
TTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAATACAGGGTCGTCAGATACATAGATACAA-
TTCTATTACCCCCATCCATACAATG (SEQ ID NO:5)
[0353] Additional promoters that can be used to express the albumin
fusion protein in yeast include the following:
[0354] a) the cbh1 promoter:
TABLE-US-00004 (SEQ ID NO:113)
TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCAT
CTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAA
AGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGG
AGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTC
CGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGT
AGCGATGGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGAC
GGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCAC
CTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCG
TAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGA
GAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCT
GTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCA
TGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGG
GAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAG
CATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCT
AAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAAC
GTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGC
CCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGAT
CCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGG
TAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGG
AAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGA
GTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGT
CACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGG
CAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGG
CCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGA
TCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAA
ATGTTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCAA
GAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGC
AAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCA
TGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACAC
CATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC
[0355] b) the cysD promoter from Aspergillus nidulans:
TABLE-US-00005 (SEQ ID NO:114)
AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAG
CCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCCTATTTGG
ACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGCAATTA
CGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACG
CGATCACGCCATTGTGGCCGCCGGCGTTGTGCTGCTGCTATTCCCCGCAT
ATAAACAACCCCTCCACCAGTTCGTTGGGCTTTGCGAATGCTGTACTCTA
TTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAA
AGATATCAAAGCCATC
[0356] c) a modified cbh1 promoter having the sequence:
TABLE-US-00006 (SEQ ID NO:115)
TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCAT
CTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAA
AGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGG
AGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTC
CGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGT
AGCGATGGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGAC
GGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCAC
CTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCG
TAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGA
GAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCT
GTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCA
TGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGG
GAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAG
CATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCT
AAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAAC
GTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGC
CCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGAT
CCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGG
TAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGG
AAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGA
GTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGT
CACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGG
CAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGG
CCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGA
TCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAA
ATGTTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCTG
CAGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAA
AAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTAT
TGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGCTTAGCCAAG
AACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCA
AAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCAT
GCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACC
ATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC
[0357] d) a cysD promoter from Aspergillus nidulans having the
sequence:
TABLE-US-00007 (SEQ ID NO:116)
AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAG
CCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCCTATTTGG
ACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGCAATTA
CGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACG
CGATCACGCCATTGTGGCCGCCGGCGCTGCAGCCTCTTATCGAGAAAGAA
ATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACC
CAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCG
TCACACAACAAGGTCCTACGCCGGCGTTGTGCTGCTGCTATTCCCCGCAT
ATAAACAACCCCTCCACCAGTTCGTTGGGCTTTGCGAATGCTGTACTCTA
TTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAA
AGATATCAAAGCCATC
[0358] A variety of methods have been developed to operably link
DNA to vectors via complementary cohesive termini. For instance,
complementary homopolymer tracts can be added to the DNA segment to
be inserted to the vector DNA. The vector and DNA segment are then
joined by hydrogen bonding between the complementary homopolymeric
tails to form recombinant DNA molecules.
[0359] Synthetic linkers containing one or more restriction sites
provide an alternative method of joining the DNA segment to
vectors. The DNA segment, generated by endonuclease restriction
digestion, is treated with bacteriophage T4 DNA polymerase or E.
coli DNA polymerase I, enzymes that remove protruding,
gamma-single-stranded termini with their 3' 5'-exonucleolytic
activities, and fill in recessed 3'-ends with their polymerizing
activities.
[0360] The combination of these activities therefore generates
blunt-ended DNA segments. The blunt-ended segments are then
incubated with a large molar excess of linker molecules in the
presence of an enzyme that is able to catalyze the ligation of
blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
Thus, the products of the reaction are DNA segments carrying
polymeric linker sequences at their ends. These DNA segments are
then cleaved with the appropriate restriction enzyme and ligated to
an expression vector that has been cleaved with an enzyme that
produces termini compatible with those of the DNA segment.
[0361] Synthetic linkers containing a variety of restriction
endonuclease sites are commercially available from a number of
sources including International Biotechnologies Inc, New Haven,
Conn., USA.
[0362] A desirable way to modify the DNA in accordance with the
invention, if, for example, HA variants are to be prepared, is to
use the polymerase chain reaction as disclosed by Saiki et al.
(1988) Science 239, 487-491. In this method the DNA to be
enzymatically amplified is flanked by two specific oligonucleotide
primers which themselves become incorporated into the amplified
DNA. The specific primers may contain restriction endonuclease
recognition sites which can be used for cloning into expression
vectors using methods known in the art.
[0363] Exemplary genera of yeast contemplated to be useful in the
practice of the present invention as hosts for expressing the
albumin fusion proteins are Pichia (Hansenula), Saccharomyces,
Kluyveromyces, Candida, Torulopsis, Torulaspora,
Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces,
Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,
Sporidiobolus, Endomycopsis, and the like. Preferred genera are
those selected from the group consisting of Saccharomyces,
Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora.
Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and
S. rouxii.
[0364] Examples of Kluyveromyces spp. are K. fragilis, K. lactis
and K. marxianus. A suitable Torulaspora species is T. delbrueckii.
Examples of Pichia (Hansenula) spp. are P. angusta (formerly H.
polymorpha), P. anomala (formerly H. anomala) and P. pastoris.
Methods for the transformation of S. cerevisiae are taught
generally in EP 251 744, EP 258 067 and WO 90/01063, all of which
are incorporated herein by reference.
[0365] Preferred exemplary species of Saccharomyces include S.
cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces
rouxii. Preferred exemplary species of Kluyveromyces include K.
fragilis and K. lactis. Preferred exemplary species of Hansenula
include H. polymorpha (now Pichia angusta), H. anomala (now Pichia
anomala), and Pichia capsulata. Additional preferred exemplary
species of Pichia include P. pastoris. Preferred exemplary species
of Aspergillus include A. niger and A. nidulans. Preferred
exemplary species of Yarrowia include Y. lipolytica. Many preferred
yeast species are available from the ATCC. For example, the
following preferred yeast species are available from the ATCC and
are useful in the expression of albumin fusion proteins:
Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3
mutant (ATCC Accession No. 4022731); Saccharomyces cerevisiae
Hansen, teleomorph strain BY4743 hsp150 mutant (ATCC Accession No.
4021266); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743
pmt1 mutant (ATCC Accession No. 4023792); Saccharomyces cerevisiae
Hansen, teleomorph (ATCC Accession Nos. 20626; 44773; 44774; and
62995); Saccharomyces diastaticus Andrews et Gilliland ex van der
Walt, teleomorph (ATCC Accession No. 62987); Kluyveromyces lactis
(Dombrowski) van der Walt, teleomorph (ATCC Accession No. 76492);
Pichia angusta (Teunisson et al.) Kurtzman, teleomorph deposited as
Hansenula polymorpha de Morais et Maia, teleomorph (ATCC Accession
No. 26012); Aspergillus niger van Tieghem, anamorph (ATCC Accession
No. 9029); Aspergillus niger van Tieghem, anamorph (ATCC Accession
No. 16404); Aspergillus nidulans (Eidam) Winter, anamorph (ATCC
Accession No. 48756); and Yarrowia lipolytica (Wickerham et al.)
van der Walt et von Arx, teleomorph (ATCC Accession No.
201847).
[0366] Suitable promoters for S. cerevisiae include those
associated with the PGKI gene, GAL1 or GAL10 genes, CYCI, PHO5,
TRPI, ADHI, ADH2, the genes for glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, triose phosphate isomerase, phosphoglucose
isomerase, glucokinase, alpha-mating factor pheromone, [a mating
factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDI
promoter, and hybrid promoters involving hybrids of parts of 5'
regulatory regions with parts of 5' regulatory regions of other
promoters or with upstream activation sites (e.g. the promoter of
EP-A-258 067).
[0367] Convenient regulatable promoters for use in
Schizosaccharomyces pombe are the thiamine-repressible promoter
from the nmt gene as described by Maundrell (1990) J. Biol. Chem.
265, 10857-10864 and the glucose repressible jbp1 gene promoter as
described by Hoffman & Winston (1990) Genetics 124,
807-816.
[0368] Methods of transforming Pichia for expression of foreign
genes are taught in, for example, Cregg et al. (1993), and various
Phillips patents (e.g. U.S. Pat. No. 4,857,467, incorporated herein
by reference), and Pichia expression kits are commercially
available from Invitrogen BV, Leek, Netherlands, and Invitrogen
Corp., San Diego, Calif. Suitable promoters include AOXI and AOX2.
Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465 include
information on Hansenula vectors and transformation, suitable
promoters being MOX1 and FMD1; whilst EP 361 991, Fleer et al.
(1991) and other-publications from Rhone-Poulenc Rorer teach how to
express foreign proteins in Kluyveromyces spp., a suitable promoter
being PGKI.
[0369] The transcription termination signal is preferably the 3'
flanking sequence of a eukaryotic gene which contains proper
signals for transcription termination and polyadenylation. Suitable
3' flanking sequences may, for example, be those of the gene
naturally linked to the expression control sequence used, i.e. may
correspond to the promoter. Alternatively, they may be different in
which case the termination signal of the S. cerevisiae ADHI gene is
preferred.
[0370] The desired albumin fusion protein may be initially
expressed with a secretion leader sequence, which may be any leader
effective in the yeast chosen. Leaders useful in yeast include any
of the following: [0371] a) the MPIF-1 signal sequence (e.g., amino
acids 1-21 of GenBank Accession number AAB51134)
MKVSVAALSCLMLVTALGSQA (SEQ ID NO:6) [0372] b) the stanniocalcin
signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7) [0373] c) the
pre-pro region of the HSA signal sequence (e.g.,
MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:8) [0374] d) the pre region of
the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYS, SEQ ID NO:9) or
variants thereof, such as, for example, MKWVSFISLLFLFSSAYS, (SEQ ID
NO:10) [0375] e) the invertase signal sequence (e.g.,
MLLQAFLFLLAGFAAKISA, SEQ ID NO:11) [0376] f) the yeast mating
factor alpha signal sequence (e.g.,
MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIAS-
IAAKEEGVSLEKR, SEQ ID NO:12 or
MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIAS-
IAAKEEGVSLDKR, SEQ ID NO:12) [0377] g) K lactis killer toxin leader
sequence [0378] h) a hybrid signal sequence (e.g.,
MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:13) [0379] i) an
HSA/MF.alpha.-1 hybrid signal sequence (also known as HSA/kex2)
(e.g., MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:14) [0380] j) a K.
lactis killer/MF.alpha.-1 fusion leader sequence (e.g.,
MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15) [0381] k) the Immunoglobulin
Ig signal sequence (e.g., MGWSCIILFLVATATGVHS, SEQ ID NO:16) [0382]
l) the Fibulin B precursor signal sequence (e.g.,
MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:17) [0383] m) the
clusterin precursor signal sequence (e.g., MMKTLLLFVGLLLTWESGQVLG,
SEQ ID NO:18) [0384] n) the insulin-like growth factor-binding
protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID
NO:19) [0385] o) variants of the pre-pro-region of the HSA signal
sequence such as, for example, [0386] MKWVSFISLLFLFSSAYSRGVFRR (SEQ
ID NO:20), [0387] MKWVTFISLLFLFAGVLG (SEQ ID NO:21), [0388]
MKWVTFISLLFLFSGVLG (SEQ ID NO:22), [0389] MKWVTFISLLFLFGGVLG (SEQ
ID NO:23), [0390] Modified HSA leader HSA #64--MKWVTFISLLFLFAGVSG
(SEQ ID NO:24); [0391] Modified HSA leader HSA
#66--MKWVTFISLLFLFGGVSG (SEQ ID NO:25); [0392] Modified HSA (A14)
leader--MKWVTFISLLFLFAGVSG (SEQ ID NO:26); [0393] Modified HSA
(S14) leader (also known as modified HSA #65)--MKWVTFISLLFLFSGVSG
(SEQ ID NO:27), [0394] Modified HSA (G14)
leader--MKWVTFISLLFLFGGVSG (SEQ ID NO:28), or [0395]
MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:29) [0396] p) a consensus signal
sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:30) [0397] q) acid
phosphatase (PH05) leader (e.g., MFKSVVYSILAASLANA SEQ ID NO:31)
[0398] r) the pre-sequence of MFoz-1 [0399] s) the pre-sequence of
0 glucanase (BGL2) [0400] t) killer toxin leader [0401] u) the
presequence of killer toxin [0402] v) k. lactis killer toxin prepro
(29 amino acids; 16 amino acids of pre and 13 amino acids of pro)
MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:32) [0403] w) S.
diastaticus glucoamylase II secretion leader sequence [0404] x) S.
carlsbergensis .beta.-galactosidase (MEL1) secretion leader
sequence [0405] y) Candida glucoamylase leader sequence [0406] z)
The hybrid leaders disclosed in EP-A-387 319 (herein incorporated
by reference) [0407] aa) the gp67 signal sequence (in conjunction
with baculoviral expression systems) (e.g., amino acids 1-19 of
GenBank Accession Number AAA72759) or [0408] bb) the natural leader
of the therapeutic protein X; [0409] cc) S. cerevisiae invertase
(SUC2) leader, as disclosed in JP 62-096086 (granted as 911036516,
herein incorporate by reference); or [0410] dd)
Inulinase--MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:33). [0411] ee) A
modified TA57 propeptide leader variant
#1--MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR
(SEQ ID NO:34) [0412] ff) A modified TA57 propeptide leader variant
#2--MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAEEGEPK-
R (SEQ ID NO:35) [0413] gg) A consensus signal
peptide--MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO:111) [0414] hh) A
modified HSA/kex2 signal sequence--MKWVSFISLLFLFSSAYSGSLDKR (SEQ ID
NO:112) [0415] ii) A consensus signal peptide
#2--MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:105)
[0416] Additional Methods of Recombinant and Synthetic Production
of Albumin Fusion Proteins
[0417] The present invention also relates to vectors containing a
polynucleotide encoding an albumin fusion protein of the present
invention, host cells, and the production of albumin fusion
proteins by synthetic and recombinant techniques. The vector may
be, for example, a phage, plasmid, viral, or retroviral vector.
Retroviral vectors may be replication competent or replication
defective. In the latter case, viral propagation generally will
occur only in complementing host cells.
[0418] The polynucleotides encoding albumin fusion proteins of the
invention may be joined to a vector containing a selectable marker
for propagation in a host. Generally, a plasmid vector is
introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0419] The polynucleotide insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination, and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0420] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418, glutamine synthase, or neomycin resistance for
eukaryotic cell culture, and tetracycline, kanamycin or ampicillin
resistance genes for culturing in E. coli and other bacteria.
Representative examples of appropriate hosts include, but are not
limited to, bacterial cells, such as E. coli, Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast cells
(e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession
No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9
cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma
cells; and plant cells. Appropriate culture mediums and conditions
for the above-described host cells are known in the art.
[0421] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors,
Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and
pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Preferred expression vectors for use in
yeast systems include, but are not limited to pYES2, pYD1,
pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5,
pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from
Invitrogen, Carlbad, Calif.). Other suitable vectors will be
readily apparent to the skilled artisan.
[0422] In one embodiment, polynucleotides encoding an albumin
fusion protein of the invention may be fused to signal sequences
which will direct the localization of a protein of the invention to
particular compartments of a prokaryotic or eukaryotic cell and/or
direct the secretion of a protein of the invention from a
prokaryotic or eukaryotic cell. For example, in E. coli, one may
wish to direct the expression of the protein to the periplasmic
space. Examples of signal sequences or proteins (or fragments
thereof) to which the albumin fusion proteins of the invention may
be fused in order to direct the expression of the polypeptide to
the periplasmic space of bacteria include, but are not limited to,
the pelB signal sequence, the maltose binding protein (MBP) signal
sequence, MBP, the ompA signal sequence, the signal sequence of the
periplasmic E. coli heat-labile enterotoxin B-subunit, and the
signal sequence of alkaline phosphatase. Several vectors are
commercially available for the construction of fusion proteins
which will direct the localization of a protein, such as the pMAL
series of vectors (particularly the pMAL-p series) available from
New England Biolabs. In a specific embodiment, polynucleotides
albumin fusion proteins of the invention may be fused to the pelB
pectate lyase signal sequence to increase the efficiency of
expression and purification of such polypeptides in Gram-negative
bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents
of which are herein incorporated by reference in their
entireties.
[0423] Examples of signal peptides that may be fused to an albumin
fusion protein of the invention in order to direct its secretion in
mammalian cells include, but are not limited to: [0424] a) the
MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession
number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO:6) [0425] b) the
stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7)
[0426] c) the pre-pro region of the HSA signal sequence (e.g.,
MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:8) [0427] d) the pre region of
the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYS, SEQ ID NO:9) or
variants thereof, such as, for example, MKWVSFISLLFLFSSAYS, (SEQ ID
NO:10) [0428] e) the invertase signal sequence (e.g.,
MLLQAFLFLLAGFAAKISA, SEQ ID NO:11) [0429] f) the yeast mating
factor alpha signal sequence (e.g.,
MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIAS-
IAAKEEGVSLEKR, SEQ ID NO:12 or
MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIAS-
IAAKEEGVSLDKR, SEQ ID NO:12) [0430] g) K. lactis killer toxin
leader sequence [0431] h) a hybrid signal sequence (e.g.,
MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:13) [0432] i) an
HSA/MF.alpha.-1 hybrid signal sequence (also known as HSA/kex2)
(e.g., MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:14) [0433] j) a K.
lactis killer/MF.alpha.-1 fusion leader sequence (e.g.,
MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15) [0434] k) the Immunoglobulin
Ig signal sequence (e.g., MGWSCIILFLVATATGVHS, SEQ ID NO:16) [0435]
l) the Fibulin B precursor signal sequence (e.g.,
MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:17) [0436] m) the
clusterin precursor signal sequence (e.g., MMKTLLLFVGLLLTWESGQVLG,
SEQ ID NO:18) [0437] n) the insulin-like growth factor-binding
protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID
NO:19) [0438] o) variants of the pre-pro-region of the HSA signal
sequence such as, for example, [0439] MKWVSFISLLFLFSSAYSRGVFRR (SEQ
ID NO:20), [0440] MKWVTFISLLFLFAGVLG (SEQ ID NO:21), [0441]
MKWVTFISLLFLFSGVLG (SEQ ID NO:22), [0442] MKWVTFISLLFLFGGVLG (SEQ
ID NO:23), [0443] Modified HSA leader HSA #64--MKWVTFISLLFLFAGVSG
(SEQ ID NO:24); [0444] Modified HSA leader HSA
#66--MKWVTFISLLFLFGGVSG (SEQ ID NO:25); [0445] Modified HSA (A 14)
leader--MKWVTFISLLFLFAGVSG (SEQ ID NO:26); [0446] Modified HSA
(S14) leader (also known as modified HSA #65)--MKWVTFISLLFLFSGVSG
(SEQ ID NO:27), [0447] Modified HSA (G14)
leader--MKWVTFISLLFLFGGVSG (SEQ ID NO:28), or [0448]
MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:29) [0449] p) a consensus signal
sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:30) [0450] q) acid
phosphatase (PH05) leader (e.g., MFKSVVYSILAASLANA SEQ ID NO:31)
[0451] r) the pre-sequence of MFoz-1 [0452] s) the pre-sequence of
0 glucanase (BGL2) [0453] t) killer toxin leader [0454] u) the
presequence of killer toxin [0455] v) k. lactis killer toxin prepro
(29 amino acids; 16 amino acids of pre and 13 amino acids of pro)
MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:32) [0456] w) S.
diastaticus glucoamylase II secretion leader sequence [0457] x) S.
carlsbergensis .beta.-galactosidase (MEL1) secretion leader
sequence [0458] y) Candida glucoamylase leader sequence [0459] z)
The hybrid leaders disclosed in EP-A-387 319 (herein incorporated
by reference) [0460] aa) the gp67 signal sequence (in conjunction
with baculoviral expression systems) (e.g., amino acids 1-19 of
GenBank Accession Number AAA72759) or [0461] bb) the natural leader
of the therapeutic protein X; cc) S. cerevisiae invertase (SUC2)
leader, as disclosed in JP 62-096086 (granted as 911036516, herein
incorporate by reference); or [0462] dd)
Inulinase--MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:33). [0463] ee) A
modified TA57 propeptide leader variant
#1--MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR
(SEQ ID NO:34) [0464] ff) A modified TA57 propeptide leader variant
#2--MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAEEGEPK-
R (SEQ ID NO:35) [0465] gg) A consensus signal
peptide--MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO:111) [0466] jj) A
modified HSA/kex2 signal sequence--MKWVSFISLLFLFSSAYSGSLDKR (SEQ ID
NO:112) [0467] kk) A consensus signal peptide
#2--MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:105)
[0468] In a preferred embodiment, the modified HSA/kex2 signal
sequence (SEQ ID NO:112) is fused to the amino terminus of an
albumin fusion protein, including fusion proteins comprising
albumin and a therapeutic protein as described herein, as well as
albumin fusion proteins disclosed in WO93/15199; WO97/24445;
WO03/60071; WO03/59934; and PCT/US04/01369, each of which are
incorporated herein by reference in their entireties. The modified
HSA/kex2 signal sequence is based on the HSA/kex2 signal sequence
(SEQ ID NO:14) disclosed, e.g., in Sleep et al., Biotechnology
1990, vol. 8, pp. 42-46; and U.S. Pat. No. 5,302,697, both of which
are incorporated herein by reference in their entireties. The
modified HSA/kex2 leader sequence disclosed herein contains a
non-conservative amino acid substitution (Arg to Gly) at residue 19
of the parent signal peptide. The modified HSA/kex2 signal peptide
has been found to produce unexpectedly better expression yield
and/or better cleavage efficiency of albumin fusion proteins when
expressed in yeast than the unmodified HSA/kex2 signal sequence.
Variants of the modified HSA/kex2 signal peptide are also
encompassed by the invention. In particular the Gly residue at
position 19 of SEQ ID NO:112 may be substituted with a Pro residue.
Other conservative substitution variants of the modified HSA/kex2
signal sequence are also contemplated. Nucleic acids encoding the
modified HSA/kex2 signal sequence of SEQ ID NO:112, as well as
conservative substitution variants thereof, are also encompassed by
the invention.
[0469] Vectors which use glutamine synthase (GS) or DHFR as the
selectable markers can be amplified in the presence of the drugs
methionine sulphoximine or methotrexate, respectively. An advantage
of glutamine synthase based vectors are the availability of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine
synthase negative. Glutamine synthase expression systems can also
function in glutamine synthase expressing cells (e.g., Chinese
Hamster Ovary (CHO) cells) by providing additional inhibitor to
prevent the functioning of the endogenous gene. A glutamine
synthase expression system and components thereof are detailed in
PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404;
and WO91/06657, which are hereby incorporated in their entireties
by reference herein. Additionally, glutamine synthase expression
vectors can be obtained from Lonza Biologics, Inc. (Portsmouth,
N.H.). Expression and production of monoclonal antibodies using a
GS expression system in murine myeloma cells is described in
Bebbington et al., Bio/technology 10:169 (1992) and in Biblia and
Robinson Biotechnol. Prog. 11:1 (1995) which are herein
incorporated by reference.
[0470] The present invention also relates to host cells containing
the above-described vector constructs described herein, and
additionally encompasses host cells containing nucleotide sequences
of the invention that are operably associated with one or more
heterologous control regions (e.g., promoter and/or enhancer) using
techniques known of in the art. The host cell can be a higher
eukaryotic cell, such as a mammalian cell (e.g., a human derived
cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell can be a prokaryotic cell, such as a bacterial cell. A
host strain may be chosen which modulates the expression of the
inserted gene sequences, or modifies and processes the gene product
in the specific fashion desired. Expression from certain promoters
can be elevated in the presence of certain inducers; thus
expression of the genetically engineered polypeptide may be
controlled. Furthermore, different host cells have characteristics
and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
phosphorylation, cleavage) of proteins. Appropriate cell lines can
be chosen to ensure the desired modifications and processing of the
foreign protein expressed.
[0471] Introduction of the nucleic acids and nucleic acid
constructs of the invention into the host cell can be effected by
calcium phosphate transfection, DEAE-dextran mediated transfection,
cationic lipid-mediated transfection, electroporation,
transduction, infection, or other methods. Such methods are
described in many standard laboratory manuals, such as Davis et
al., Basic Methods In Molecular Biology (1986). It is specifically
contemplated that the polypeptides of the present invention may in
fact be expressed by a host cell lacking a recombinant vector.
[0472] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., the coding
sequence corresponding to a Therapeutic protein may be replaced
with an albumin fusion protein corresponding to the Therapeutic
protein), and/or to include genetic material (e.g., heterologous
polynucleotide sequences such as for example, an albumin fusion
protein of the invention corresponding to the Therapeutic protein
may be included). The genetic material operably associated with the
endogenous polynucleotide may activate, alter, and/or amplify
endogenous polynucleotides.
[0473] In addition, techniques known in the art may be used to
operably associate heterologous polynucleotides (e.g.,
polynucleotides encoding an albumin protein, or a fragment or
variant thereof) and/or heterologous control regions (e.g.,
promoter and/or enhancer) with endogenous polynucleotide sequences
encoding a Therapeutic protein via homologous recombination (see,
e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International
Publication Number WO 96/29411; International Publication Number WO
94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989), the
disclosures of each of which are incorporated by reference in their
entireties).
[0474] Albumin fusion proteins of the invention can be recovered
and purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, hydrophobic charge interaction chromatography and
lectin chromatography. Most preferably, high performance liquid
chromatography ("HPLC") is employed for purification.
[0475] In preferred embodiments the albumin fusion proteins of the
invention are purified using Anion Exchange Chromatography
including, but not limited to, chromatography on Q-sepharose, DEAE
sepharose, poros HQ, poros DEAE, Toyopearl Q, Toyopearl QAE,
Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE
columns.
[0476] In specific embodiments the albumin fusion proteins of the
invention are purified using Cation Exchange Chromatography
including, but not limited to, SP-sepharose, CM sepharose, poros
HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM,
Fractogel S and CM columns and their equivalents and
comparables.
[0477] In specific embodiments the albumin fusion proteins of the
invention are purified using Hydrophobic Interaction Chromatography
including, but not limited to, Phenyl, Butyl, Methyl, Octyl,
Hexyl-sepharose, poros Phenyl, Butyl, Methyl, Octyl, Hexyl,
Toyopearl Phenyl, Butyl, Methyl, Octyl, Hexyl Resource/Source
Phenyl, Butyl, Methyl, Octyl, Hexyl, Fractogel Phenyl, Butyl,
Methyl, Octyl, Hexyl columns and their equivalents and
comparables.
[0478] In specific embodiments the albumin fusion proteins of the
invention are purified using Size Exclusion Chromatography
including, but not limited to, sepharose S100, S200, S300, superdex
resin columns and their equivalents and comparables.
[0479] In specific embodiments the albumin fusion proteins of the
invention are purified using Affinity Chromatography including, but
not limited to, Mimetic Dye affinity, peptide affinity and antibody
affinity columns that are selective for either the HSA or the
"fusion target" molecules.
[0480] In preferred embodiments albumin fusion proteins of the
invention are purified using one or more Chromatography methods
listed above. In other preferred embodiments, albumin fusion
proteins of the invention are purified using one or more of the
following Chromatography columns, Q sepharose FF column, SP
Sepharose FF column, Q Sepharose High Performance Column, Blue
Sepharose FF column, Blue Column, Phenyl Sepharose FF column, DEAE
Sepharose FF, or Methyl Column.
[0481] Additionally, albumin fusion proteins of the invention may
be purified using the process described in PCT International
Publication WO 00/44772 which is herein incorporated by reference
in its entirety. One of skill in the art could easily modify the
process described therein for use in the purification of albumin
fusion proteins of the invention.
[0482] Albumin fusion proteins of the present invention may be
recovered from: 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, albumin fusion proteins of the
invention may also include an initial modified methionine residue,
in some cases as a result of host-mediated processes. Thus, it is
well known in the art that the N-terminal methionine encoded by the
translation initiation codon generally is removed with high
efficiency from any protein after translation in all eukaryotic
cells. While the N-terminal methionine on most proteins also is
efficiently removed in most prokaryotes, for some proteins, this
prokaryotic removal process is inefficient, depending on the nature
of the amino acid to which the N-terminal methionine is covalently
linked.
[0483] In one embodiment, the yeast Pichia pastoris is used to
express albumin fusion proteins of the invention in a eukaryotic
system. Pichia pastoris is a methylotrophic yeast which can
metabolize methanol as its sole carbon source. A main step in the
methanol metabolization pathway is the oxidation of methanol to
formaldehyde using O.sub.2. This reaction is catalyzed by the
enzyme alcohol oxidase. In order to metabolize methanol as its sole
carbon source, Pichia pastoris must generate high levels of alcohol
oxidase due, in part, to the relatively low affinity of alcohol
oxidase for O.sub.2. Consequently, in a growth medium depending on
methanol as a main carbon source, the promoter region of one of the
two alcohol oxidase genes (AOX1) is highly active. In the presence
of methanol, alcohol oxidase produced from the AOX1 gene comprises
up to approximately 30% of the total soluble protein in Pichia
pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21
(1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F.,
et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous
coding sequence, such as, for example, a polynucleotide of the
present invention, under the transcriptional regulation of all or
part of the AOX1 regulatory sequence is expressed at exceptionally
high levels in Pichia yeast grown in the presence of methanol.
[0484] In one example, the plasmid vector pPIC9K is used to express
DNA encoding an albumin fusion protein of the invention, as set
forth herein, in a Pichea yeast system essentially as described in
"Pichia Protocols: Methods in Molecular Biology," D. R. Higgins and
J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This
expression vector allows expression and secretion of a polypeptide
of the invention by virtue of the strong AOX1 promoter linked to
the Pichia pastoris alkaline phosphatase (PHO) secretory signal
peptide (i.e., leader) located upstream of a multiple cloning
site.
[0485] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815,
as one skilled in the art would readily appreciate, as long as the
proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG as required.
[0486] In another embodiment, high-level expression of a
heterologous coding sequence, such as, for example, a
polynucleotide encoding an albumin fusion protein of the present
invention, may be achieved by cloning the heterologous
polynucleotide of the invention into an expression vector such as,
for example, pGAPZ or pGAPZalpha, and growing the yeast culture in
the absence of methanol.
[0487] In addition, albumin fusion proteins of the invention can be
chemically synthesized using techniques known in the art (e.g., see
Creighton, 1983, Proteins: Structures and Molecular Principles,
W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature,
310:105-111 (1984)). For example, a polypeptide corresponding to a
fragment of a polypeptide can be synthesized by use of a peptide
synthesizer. Furthermore, if desired, nonclassical amino acids or
chemical amino acid analogs can be introduced as a substitution or
addition into the polypeptide sequence. Non-classical amino acids
include, but are not limited to, to the D-isomers of the common
amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid,
4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,
6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino
propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine, citrulline, homocitrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids,
and amino acid analogs in general. Furthermore, the amino acid can
be D (dextrorotary) or L (levorotary).
[0488] The invention encompasses albumin fusion proteins of the
present invention which are differentially modified during or after
translation, e.g., by glycosylation, acetylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to an antibody molecule or other
cellular ligand, etc. Any of numerous chemical modifications may be
carried out by known techniques, including but not limited, to
specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, V8 protease, NaBH.sub.4; acetylation,
formylation, oxidation, reduction; metabolic synthesis in the
presence of tunicamycin; etc.
[0489] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The albumin fusion
proteins may also be modified with a detectable label, such as an
enzymatic, fluorescent, isotopic or affinity label to allow for
detection and isolation of the protein.
[0490] 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 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.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.104La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47SC,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, and .sup.97Ru.
[0491] In specific embodiments, albumin fusion proteins of the
present invention or fragments or variants thereof are attached to
macrocyclic chelators that associate with radiometal ions,
including but not limited to, .sup.177Lu, .sup.90Y, .sup.166Ho, and
.sup.153Sm, to polypeptides. In a preferred embodiment, the
radiometal ion associated with the macrocyclic chelators is
.sup.111In. In another preferred embodiment, the radiometal ion
associated with the macrocyclic chelator is .sup.90Y. In specific
embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, DOTA is attached to an
antibody of the invention or fragment thereof via 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.
[0492] As mentioned, the albumin fusion proteins of the invention
may be modified by either natural processes, such as
post-translational processing, or by chemical modification
techniques which are well known in the art. It will be appreciated
that the same type of modification may be present in the same or
varying degrees at several sites in a given polypeptide.
Polypeptides of the invention may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990);
Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[0493] Albumin fusion proteins of the invention and antibodies that
bind a Therapeutic protein or fragments or variants thereof 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.
[0494] Further, an albumin fusion protein of the invention 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, 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).
[0495] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, alpha-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), 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. Techniques for conjugating such therapeutic
moiety to proteins (e.g., albumin fusion proteins) are well known
in the art.
[0496] Albumin fusion proteins may also be attached to solid
supports, which are particularly useful for immunoassays or
purification of polypeptides that are bound by, that bind to, or
associate with albumin fusion proteins of the invention. Such solid
supports include, but are not limited to, glass, cellulose,
polyacrylamide, nylon, polystyrene, polyvinyl chloride or
polypropylene.
[0497] Albumin fusion proteins, 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.
[0498] In embodiments where the albumin fusion protein of the
invention comprises only the VH domain of an antibody that binds a
Therapeutic protein, it may be necessary and/or desirable to
coexpress the fusion protein with the VL domain of the same
antibody that binds a Therapeutic protein, such that the VH-albumin
fusion protein and VL protein will associate (either covalently or
non-covalently) post-translationally.
[0499] In embodiments where the albumin fusion protein of the
invention comprises only the VL domain of an antibody that binds a
Therapeutic protein, it may be necessary and/or desirable to
coexpress the fusion protein with the VH domain of the same
antibody that binds a Therapeutic protein, such that the VL-albumin
fusion protein and VH protein will associate (either covalently or
non-covalently) post-translationally.
[0500] Some Therapeutic antibodies are bispecific antibodies,
meaning the antibody that binds a Therapeutic protein is an
artificial hybrid antibody having two different heavy/light chain
pairs and two different binding sites. In order to create an
albumin fusion protein corresponding to that Therapeutic protein,
it is possible to create an albumin fusion protein which has an
scFv fragment fused to both the N- and C-terminus of the albumin
protein moiety. More particularly, the scFv fused to the N-terminus
of albumin would correspond to one of the heavy/light (VH/VL) pairs
of the original antibody that binds a Therapeutic protein and the
scFv fused to the C-terminus of albumin would correspond to the
other heavy/light (VH/VL) pair of the original antibody that binds
a Therapeutic protein.
[0501] Also provided by the invention are chemically modified
derivatives of the albumin fusion proteins of the invention which
may provide additional advantages such as increased solubility,
stability and 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 albumin fusion proteins 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.
[0502] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about lkDa 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,
45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000,
85,000, 90,000, 95,000, or 100,000 kDa.
[0503] 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.
[0504] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, such as, for example, the method disclosed in EP 0 401 384
(coupling PEG to G-CSF), herein incorporated by reference; 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 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 lysine residues and the N-terminal amino
acid residues; those having a free carboxyl group may include
aspartic acid residues glutamic acid residues and the C-terminal
amino acid residue. Sulfhydryl groups may also be used as a
reactive group for attaching the polyethylene glycol molecules.
Preferred for therapeutic purposes is attachment at an amino group,
such as attachment at the N-terminus or lysine group.
[0505] As suggested above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to proteins via covalent
bonds to lysine, histidine, aspartic acid, glutamic acid, or
cysteine residues. One or more reaction chemistries may be employed
to attach polyethylene glycol to specific amino acid residues
(e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the protein or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the protein.
[0506] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation
reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. Selective proteins
chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group containing polymer is achieved.
[0507] As indicated above, pegylation of the albumin fusion
proteins of the invention may be accomplished by any number of
means. For example, polyethylene glycol may be attached to the
albumin fusion 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.
[0508] One system for attaching polyethylene glycol directly to
amino acid residues of proteins without an intervening linker
employs tresylated MPEG, which is produced by the modification of
monmethoxy polyethylene glycol (MPEG) using tresylchloride
(ClSO.sub.2CH.sub.2CF.sub.3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine
groups of the protein. Thus, the invention includes
protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule
having a 2,2,2-trifluoreothane sulphonyl group.
[0509] Polyethylene glycol can also be attached to proteins using a
number of different intervening linkers. For example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by
reference, discloses urethane linkers for connecting polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein
the polyethylene glycol is attached to the protein by a linker can
also be produced by reaction of proteins with compounds such as
MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,
MPEG-p-nitrophenolcarbonate, and various MPEG-succinate
derivatives. A number of additional polyethylene glycol derivatives
and reaction chemistries for attaching polyethylene glycol to
proteins are described in International Publication No. WO
98/32466, the entire disclosure of which is incorporated herein by
reference. Pegylated protein products produced using the reaction
chemistries set out herein are included within the scope of the
invention.
[0510] The number of polyethylene glycol moieties attached to each
albumin fusion protein of the invention (i.e., the degree of
substitution) may also vary. For example, the pegylated proteins of
the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
Similarly, the average degree of substitution within ranges such as
1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14,
13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol
moieties per protein molecule. Methods for determining the degree
of substitution are discussed, for example, in Delgado et al.,
Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[0511] The polypeptides of the invention can be recovered and
purified from chemical synthesis and recombinant cell cultures by
standard 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. Well known techniques for refolding
protein may be employed to regenerate active conformation when the
polypeptide is denatured during isolation and/or purification.
[0512] The presence and quantity of albumin fusion proteins of the
invention may be determined using ELISA, a well known immunoassay
known in the art. In one ELISA protocol that would be useful for
detecting/quantifying albumin fusion proteins of the invention,
comprises the steps of coating an ELISA plate with an anti-human
serum albumin antibody, blocking the plate to prevent non-specific
binding, washing the ELISA plate, adding a solution containing the
albumin fusion protein of the invention (at one or more different
concentrations), adding a secondary anti-Therapeutic protein
specific antibody coupled to a detectable label (as described
herein or otherwise known in the art), and detecting the presence
of the secondary antibody. In an alternate version of this
protocol, the ELISA plate might be coated with the anti-Therapeutic
protein specific antibody and the labeled secondary reagent might
be the anti-human albumin specific antibody.
[0513] Uses of the Polynucleotides
[0514] Each of the polynucleotides identified herein can be used in
numerous ways as reagents. The following description should be
considered exemplary and utilizes known techniques.
[0515] The polynucleotides of the present invention are useful to
produce the albumin fusion proteins of the invention. As described
in more detail below, polynucleotides of the invention (encoding
albumin fusion proteins) may be used in recombinant DNA methods
useful in genetic engineering to make cells, cell lines, or tissues
that express the albumin fusion protein encoded by the
polynucleotides encoding albumin fusion proteins of the
invention.
[0516] Polynucleotides of the present invention are also useful in
gene therapy. One goal of gene therapy is to insert a normal gene
into an organism having a defective gene, in an effort to correct
the genetic defect. The polynucleotides disclosed in the present
invention offer a means of targeting such genetic defects in a
highly accurate manner. Another goal is to insert a new gene that
was not present in the host genome, thereby producing a new trait
in the host cell. Additional non-limiting examples of gene therapy
methods encompassed by the present invention are more thoroughly
described elsewhere herein (see, e.g., the sections labeled "Gene
Therapy", and Examples 61 and 62).
[0517] Uses of the Polypeptides
[0518] Each of the polypeptides identified herein can be used in
numerous ways. The following description should be considered
exemplary and utilizes known techniques.
[0519] Albumin fusion proteins of the invention are useful to
provide immunological probes for differential identification of the
tissue(s) (e.g., immunohistochemistry assays such as, for example,
ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem.
29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistry
assays).
[0520] Albumin fusion proteins can be used to assay levels of
polypeptides in a biological sample using classical
immunohistological methods known to those of skill in the art
(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other
methods useful for detecting protein gene expression include
immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the radioimmunoassay (RIA). Suitable assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.201Ti), gallium (.sup.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, 188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru; luminescent
labels, such as luminol; and fluorescent labels, such as
fluorescein and rhodamine, and biotin.
[0521] Albumin fusion proteins of the invention can also be
detected in vivo by imaging. Labels or markers for in vivo imaging
of protein include those detectable by X-radiography, nuclear
magnetic resonance (NMR) or electron spin relaxation (ESR). For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the albumin fusion protein by
labeling of nutrients given to a cell line expressing the albumin
fusion protein of the invention.
[0522] An albumin fusion protein which has been labeled with an
appropriate detectable imaging moiety, such as a radioisotope (for
example, .sup.311I, .sup.112In, .sup.99mTc, (.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.103
Pd), molybdenum (.sup.99Mo), xenon (.sup.133 Xe), 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), a
radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined
for immune system disorder. It will be understood in the art that
the size of the subject and the imaging system used will determine
the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the
quantity of radioactivity injected will normally range from about 5
to 20 millicuries of .sup.99mTc. The labeled albumin fusion protein
will then preferentially accumulate at locations in the body (e.g.,
organs, cells, extracellular spaces or matrices) where one or more
receptors, ligands or substrates (corresponding to that of the
Therapeutic protein used to make the albumin fusion protein of the
invention) are located. Alternatively, in the case where the
albumin fusion protein comprises at least a fragment or variant of
a Therapeutic antibody, the labeled albumin fusion protein will
then preferentially accumulate at the locations in the body (e.g.,
organs, cells, extracellular spaces or matrices) where the
polypeptides/epitopes corresponding to those bound by the
Therapeutic antibody (used to make the albumin fusion protein of
the invention) are located. 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)). The protocols described
therein could easily be modified by one of skill in the art for use
with the albumin fusion proteins of the invention.
[0523] In one embodiment, the invention provides a method for the
specific delivery of albumin fusion proteins of the invention to
cells by administering albumin fusion proteins of the invention
(e.g., polypeptides encoded by polynucleotides encoding albumin
fusion proteins of the invention and/or antibodies) that are
associated with heterologous polypeptides or nucleic acids. In one
example, the invention provides a method for delivering a
Therapeutic protein into the targeted cell. In another example, the
invention provides a method for delivering a single stranded
nucleic acid (e.g., antisense or ribozymes) or double stranded
nucleic acid (e.g., DNA that can integrate into the cell's genome
or replicate episomally and that can be transcribed) into the
targeted cell.
[0524] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering albumin fusion proteins of the invention in
association with toxins or cytotoxic prodrugs.
[0525] By "toxin" is meant one or more compounds that bind and
activate endogenous cytotoxic effector systems, radioisotopes,
holotoxins, modified toxins, catalytic subunits of toxins, or any
molecules or enzymes not normally present in or on the surface of a
cell that under defined conditions cause the cell's death. Toxins
that may be used according to the methods of the invention include,
but are not limited to, radioisotopes known in the art, compounds
such as, for example, antibodies (or complement fixing containing
portions thereof) that bind an inherent or induced endogenous
cytotoxic effector system, thymidine kinase, endonuclease, RNAse,
alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria
toxin, saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic
or cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, .sup.213Bi, or other
radioisotopes such as, for example, .sup.103Pd, .sup.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; luminescent
labels, such as luminol; and fluorescent labels, such as
fluorescein and rhodamine, and biotin. In a specific embodiment,
the invention provides a method for the specific destruction of
cells (e.g., the destruction of tumor cells) by administering
polypeptides of the invention or antibodies of the invention in
association with the radioisotope .sup.90Y. In another specific
embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by
administering polypeptides of the invention or antibodies of the
invention in association with the radioisotope .sup.111In. In a
further specific embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering polypeptides of the invention or antibodies
of the invention in association with the radioisotope
.sup.131I.
[0526] Techniques known in the art may be applied to lable
polypeptides of the invention. Such techniques include, but are not
limited to, the use of bifunctional conjugating agents (see e.g.,
U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;
5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274;119;
4,994,560; and 5,808,003; the contents of each of which are hereby
incorporated by reference in its entirety).
[0527] The albumin fusion proteins of the present invention are
useful for diagnosis, treatment, prevention and/or prognosis of
various disorders in mammals, preferably humans. Such disorders
include, but are not limited to, those described herein under the
section heading "Biological Activities," below.
[0528] Thus, the invention provides a diagnostic method of a
disorder, which involves (a) assaying the expression level of a
certain polypeptide in cells or body fluid of an individual using
an albumin fusion protein of the invention; and (b) comparing the
assayed polypeptide expression level with a standard polypeptide
expression level, whereby an increase or decrease in the assayed
polypeptide expression level compared to the standard expression
level is indicative of a disorder. With respect to cancer, the
presence of a relatively high amount of transcript in biopsied
tissue from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0529] Moreover, albumin fusion proteins of the present invention
can be used to treat or prevent diseases or conditions such as, for
example, neural disorders, immune system disorders, muscular
disorders, reproductive disorders, gastrointestinal disorders,
pulmonary disorders, cardiovascular disorders, renal disorders,
proliferative disorders, and/or cancerous diseases and conditions.
For example, patients can be administered a polypeptide of the
present invention in an effort to replace absent or decreased
levels of the polypeptide (e.g., insulin), to supplement absent or
decreased levels of a different polypeptide (e.g., hemoglobin S for
hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the
activity of a polypeptide (e.g., an oncogene or tumor supressor),
to activate the activity of a polypeptide (e.g., by binding to a
receptor), to reduce the activity of a membrane bound receptor by
competing with it for free ligand (e.g., soluble TNF receptors used
in reducing inflammation), or to bring about a desired response
(e.g., blood vessel growth inhibition, enhancement of the immune
response to proliferative cells or tissues).
[0530] In particular, albumin fusion proteins comprising of at
least a fragment or variant of a Therapeutic antibody can also be
used to treat disease (as described supra, and elsewhere herein).
For example, administration of an albumin fusion protein comprising
of at least a fragment or variant of a Therapeutic antibody can
bind, and/or neutralize the polypeptide to which the Therapeutic
antibody used to make the albumin fusion protein specifically
binds, and/or reduce overproduction of the polypeptide to which the
Therapeutic antibody used to make the albumin fusion protein
specifically binds. Similarly, administration of an albumin fusion
protein comprising of at least a fragment or variant of a
Therapeutic antibody can activate the polypeptide to which the
Therapeutic antibody used to make the albumin fusion protein
specifically binds, by binding to the polypeptide bound to a
membrane (receptor).
[0531] At the very least, the albumin fusion proteins of the
invention of the present invention can be used as molecular weight
markers on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the art.
Albumin fusion proteins of the invention can also be used to raise
antibodies, which in turn may be used to measure protein expression
of the Therapeutic protein, albumin protein, and/or the albumin
fusion protein of the invention from a recombinant cell, as a way
of assessing transformation of the host cell, or in a biological
sample. Moreover, the albumin fusion proteins of the present
invention can be used to test the biological activities described
herein.
[0532] Diagnostic Assays
[0533] The compounds of the present invention are useful for
diagnosis, treatment, prevention and/or prognosis of various
disorders in mammals, preferably humans. Such disorders include,
but are not limited to, those described for each Therapeutic
protein in the corresponding row of Table 1 and herein under the
section headings "Immune Activity," "Blood Related Disorders,"
"Hyperproliferative Disorders," "Renal Disorders," "Cardiovascular
Disorders," "Respiratory Disorders," "Anti-Angiogenesis Activity,"
"Diseases at the Cellular Level," "Wound Healing and Epithelial
Cell Proliferation," "Neural Activity and Neurological Diseases,"
"Endocrine Disorders," "Reproductive System Disorders," "Infectious
Disease," "Regeneration," and/or "Gastrointestinal Disorders,"
infra.
[0534] For a number of disorders, substantially altered (increased
or decreased) levels of gene expression can be detected in tissues,
cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to a "standard" gene expression level, that is,
the expression level in tissues or bodily fluids from an individual
not having the disorder. Thus, the invention provides a diagnostic
method useful during diagnosis of a disorder, which involves
measuring the expression level of the gene encoding a polypeptide
in tissues, cells or body fluid from an individual and comparing
the measured gene expression level with a standard gene expression
level, whereby an increase or decrease in the gene expression
level(s) compared to the standard is indicative of a disorder.
These diagnostic assays may be performed in vivo or in vitro, such
as, for example, on blood samples, biopsy tissue or autopsy
tissue.
[0535] The present invention is also useful as a prognostic
indicator, whereby patients exhibiting enhanced or depressed gene
expression will experience a worse clinical outcome.
[0536] By "assaying the expression level of the gene encoding a
polypeptide" is intended qualitatively or quantitatively measuring
or estimating the level of a particular polypeptide (e.g. a
polypeptide corresponding to a Therapeutic protein disclosed in
Table 1) or the level of the mRNA encoding the polypeptide of the
invention in a first biological sample either directly (e.g., by
determining or estimating absolute protein level or mRNA level) or
relatively (e.g., by comparing to the polypeptide level or mRNA
level in a second biological sample). Preferably, the polypeptide
expression level or mRNA level in the first biological sample is
measured or estimated and compared to a standard polypeptide level
or mRNA level, the standard being taken from a second biological
sample obtained from an individual not having the disorder or being
determined by averaging levels from a population of individuals not
having the disorder. As will be appreciated in the art, once a
standard polypeptide level or mRNA level is known, it can be used
repeatedly as a standard for comparison.
[0537] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source containing polypeptides of the invention (including portions
thereof) or mRNA. As indicated, biological samples include body
fluids (such as sera, plasma, urine, synovial fluid and spinal
fluid) and tissue sources found to express the full length or
fragments thereof of a polypeptide or mRNA. 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.
[0538] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels
of mRNA encoding the polypeptides of the invention are then assayed
using any appropriate method. These include Northern blot analysis,
S1 nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0539] The present invention also relates to diagnostic assays such
as quantitative and diagnostic assays for detecting levels of
polypeptides that bind to, are bound by, or associate with albumin
fusion proteins of the invention, in a biological sample (e.g.,
cells and tissues), including determination of normal and abnormal
levels of polypeptides. Thus, for instance, a diagnostic assay in
accordance with the invention for detecting abnormal expression of
polypeptides that bind to, are bound by, or associate with albumin
fusion proteins compared to normal control tissue samples may be
used to detect the presence of tumors. Assay techniques that can be
used to determine levels of a polypeptide that bind to, are bound
by, or associate with albumin fusion proteins of the present
invention in a sample derived from a host are well-known to those
of skill in the art. Such assay methods include radioimmunoassays,
competitive-binding assays, Western Blot analysis and ELISA assays.
Assaying polypeptide levels in a biological sample can occur using
any art-known method.
[0540] Assaying polypeptide levels in a biological sample can occur
using a variety of techniques. For example, polypeptide expression
in tissues can be studied with classical immunohistological methods
(Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,
et al., J. Cell. Biol. 105:3087-3096 (1987)). Other methods useful
for detecting polypeptide 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, and
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0541] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the gene of
interest (such as, for example, cancer). The protein isolation
methods employed herein may, for example, be such as those
described in Harlow and Lane (Harlow, E. and Lane, D., 1988,
"Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.), which is incorporated herein by
reference in its entirety. The isolated cells can be derived from
cell culture or from a patient. The analysis of cells taken from
culture may be a necessary step in the assessment of cells that
could be used as part of a cell-based gene therapy technique or,
alternatively, to test the effect of compounds on the expression of
the gene.
[0542] For example, albumin fusion proteins may be used to
quantitatively or qualitatively detect the presence of polypeptides
that bind to, are bound by, or associate with albumin fusion
proteins of the present invention. This can be accomplished, for
example, by immunofluorescence techniques employing a fluorescently
labeled albumin fusion protein coupled with light microscopic, flow
cytometric, or fluorimetric detection.
[0543] In a preferred embodiment, albumin fusion proteins
comprising at least a fragment or variant of an antibody that
specifically binds at least a Therapeutic protein disclosed herein
(e.g., the Therapeutic proteins disclosed in Table 1) or otherwise
known in the art may be used to quantitatively or qualitatively
detect the presence of gene products or conserved variants or
peptide fragments thereof. This can be accomplished, for example,
by immunofluorescence techniques employing a fluorescently labeled
antibody coupled with light microscopic, flow cytometric, or
fluorimetric detection.
[0544] The albumin fusion proteins of the present invention may,
additionally, be employed histologically, as in immunofluorescence,
immunoelectron microscopy or non-immunological assays, for in situ
detection of polypeptides that bind to, are bound by, or associate
with an albumin fusion protein of the present invention. In situ
detection may be accomplished by removing a histological specimen
from a patient, and applying thereto a labeled antibody or
polypeptide of the present invention. The albumin fusion proteins
are preferably applied by overlaying the labeled albumin fusion
proteins onto a biological sample. Through the use of such a
procedure, it is possible to determine not only the presence of the
polypeptides that bind to, are bound by, or associate with albumin
fusion proteins, but also its distribution in the examined tissue.
Using the present invention, those of ordinary skill will readily
perceive that any of a wide variety of histological methods (such
as staining procedures) can be modified in order to achieve such in
situ detection.
[0545] Immunoassays and non-immunoassays that detect polypeptides
that bind to, are bound by, or associate with albumin fusion
proteins will typically comprise incubating a sample, such as a
biological fluid, a tissue extract, freshly harvested cells, or
lysates of cells which have been incubated in cell culture, in the
presence of a detectably labeled antibody capable of binding gene
products or conserved variants or peptide fragments thereof, and
detecting the bound antibody by any of a number of techniques
well-known in the art.
[0546] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled albumin fusion protein of the invention. The
solid phase support may then be washed with the buffer a second
time to remove unbound antibody or polypeptide. Optionally the
antibody is subsequently labeled. The amount of bound label on
solid support may then be detected by conventional means.
[0547] By "solid phase support or carrier" is intended any support
capable of binding a polypeptide (e.g., an albumin fusion protein,
or polypeptide that binds, is bound by, or associates with an
albumin fusion protein of the invention.) Well-known supports or
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides, gabbros, and magnetite. The nature of the carrier
can be either soluble to some extent or insoluble for the purposes
of the present invention. The support material may have virtually
any possible structural configuration so long as the coupled
molecule is capable of binding to a polypeptide. Thus, the support
configuration may be spherical, as in a bead, or cylindrical, as in
the inside surface of a test tube, or the external surface of a
rod. Alternatively, the surface may be flat such as a sheet, test
strip, etc. Preferred supports include polystyrene beads. Those
skilled in the art will know many other suitable carriers for
binding antibody or antigen, or will be able to ascertain the same
by use of routine experimentation.
[0548] The binding activity of a given lot of albumin fusion
protein may be determined according to well known methods. Those
skilled in the art will be able to determine operative and optimal
assay conditions for each determination by employing routine
experimentation.
[0549] In addition to assaying polypeptide levels in a biological
sample obtained from an individual, polypeptide can also be
detected in vivo by imaging. For example, in one embodiment of the
invention, albumin fusion proteins of the invention are used to
image diseased or neoplastic cells.
[0550] Labels or markers for in vivo imaging of albumin fusion
proteins of the invention include those detectable by
X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography,
suitable labels include radioisotopes such as barium or cesium,
which emit detectable radiation but are not overtly harmful to the
subject. Suitable markers for NMR and ESR include those with a
detectable characteristic spin, such as deuterium, which may be
incorporated into the albumin fusion protein by labeling of
nutrients of a cell line (or bacterial or yeast strain)
engineered.
[0551] Additionally, albumin fusion proteins of the invention whose
presence can be detected, can be administered. For example, albumin
fusion proteins of the invention labeled with a radio-opaque or
other appropriate compound can be administered and visualized in
vivo, as discussed, above for labeled antibodies. Further, such
polypeptides can be utilized for in vitro diagnostic
procedures.
[0552] A polypeptide-specific antibody or antibody fragment which
has been labeled with an appropriate detectable imaging moiety,
such as a radioisotope (for example, .sup.131I, .sup.112In,
.sup.99mTc), a radio-opaque substance, or a material detectable by
nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the mammal
to be examined for a disorder. It will be understood in the art
that the size of the subject and the imaging system used will
determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of .sup.99mTc. The labeled
albumin fusion protein will then preferentially accumulate at the
locations in the body which contain a polypeptide or other
substance that binds to, is bound by or associates with an albumin
fusion protein of the present invention. 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)).
[0553] One of the ways in which an albumin fusion protein of the
present invention can be detectably labeled is by linking the same
to a reporter enzyme and using the linked product in an enzyme
immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent
Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological
Associates Quarterly Publication, Walkersville, Md.); Voller et
al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth.
Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme
Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al.,
(eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter
enzyme which is bound to the antibody will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual means.
Reporter enzymes which can be used to detectably label the antibody
include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. Additionally, the detection can be
accomplished by colorimetric methods which employ a chromogenic
substrate for the reporter enzyme. Detection may also be
accomplished by visual comparison of the extent of enzymatic
reaction of a substrate in comparison with similarly prepared
standards.
[0554] Albumin fusion proteins may also be radiolabelled and used
in any of a variety of other immunoassays. For example, by
radioactively labeling the albumin fusion proteins, it is possible
to the use the albumin fusion proteins in a radioimmunoassay (RIA)
(see, for example, Weintraub, B., Principles of Radioimmunoassays,
Seventh Training Course on Radioligand Assay Techniques, The
Endocrine Society, March, 1986, which is incorporated by reference
herein). The radioactive isotope can be detected by means
including, but not limited to, a gamma counter, a scintillation
counter, or autoradiography.
[0555] Additionally, chelator molecules, are known in the art and
can be used to label the Albumin fusion proteins. Chelator
molecules may be attached Albumin fusion proteins of the invention
to facilitate labeling said protein with metal ions including
radionuclides or fluorescent labels. For example, see Subramanian,
R. and Meares, C. F., "Bifunctional Chelating Agents for
Radiometal-labeled monoclonal Antibodies," in Cancer Imaging with
Radiolabeled Antibodies (D. M. Goldenberg, Ed.) Kluwer Academic
Publications, Boston; Saji, H., "Targeted delivery of radiolabeled
imaging and therapeutic agents: bifunctional radiopharmaceuticals."
Crit. Rev. Ther. Drug Carrier Syst. 16:209-244 (1999); Srivastava
S. C. and Mease R. C., "Progress in research on ligands, nuclides
and techniques for labeling monoclonal antibodies." Int. J. Rad.
Appl. Instrum. B 18:589-603 (1991); and Liu, S, and Edwards, D. S.,
"Bifunctional chelators for therapeutic lanthanide
radiopharmaceuticals." Bioconjug. Chem. 12:7-34 (2001). Any
chelator which can be covalently bound to said Albumin fusion
proteins may be used according to the present invention. The
chelator may further comprise a linker moiety that connects the
chelating moiety to the Albumin fusion protein.
[0556] In one embodiment, the Albumin fusion protein of the
invention are attached to an acyclic chelator such as diethylene
triamine-N,N,N',N'',N''-pentaacetic acid (DPTA), analogues of DPTA,
and derivatives of DPTA. As non-limiting examples, the chelator may
be 2-(p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic
acid (1B4M-DPTA, also known as MX-DTPA),
2-methyl-6-(rho-nitrobenzyl)-1,4,7-triazaheptane-N,N,N',N'',N''-pentaacet-
ic acid (nitro-1B4M-DTPA or nitro-MX-DTPA);
2-(p-isothiocyanatobenzyl)-cyclohexyldiethylenetriaminepentaacetic
acid (CHX-DTPA), or
N-[2-amino-3-(rho-nitrophenyl)propyl]-trans-cyclohexane-1,2-diamine-N,N',-
N''-pentaacetic acid (nitro-CHX-A-DTPA).
[0557] In another embodiment, the Albumin fusion protein of the
invention are attached to an acyclic terpyridine chelator such as
6,6''-bis[[N,N,N'',N''-tetra(carboxymethyl)amino]methyl]-4'-(3-amino-4-me-
thoxyphenyl)-2,2':6',2''-terpyridine (TMT-amine).
[0558] In specific embodiments, the macrocyclic chelator which is
attached to the Albumin fusion protein of the invention is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to the
Albumin fusion protein of the invention via a linker molecule.
Examples of linker molecules useful for conjugating DOTA to a
polypeptide are commonly known in the art--see, for example,
DeNardo et al., Clin. Cancer Res. 4(10):2483-90, 1998; Peterson et
al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al.,
Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated
by reference in their entirety. In addition, U.S. Pat. Nos.
5,652,361 and 5,756,065, which disclose chelating agents that may
be conjugated to antibodies, and methods for making and using them,
are hereby incorporated by reference in their entireties. Though
U.S. Pat. Nos. 5,652,361 and 5,756,065 focus on conjugating
chelating agents to antibodies, one skilled in the art could
readily adapt the method disclosed therein in order to conjugate
chelating agents to other polypeptides.
[0559] Bifunctional chelators based on macrocyclic ligands in which
conjugation is via an activated arm, or functional group, attached
to the carbon backbone of the ligand can be employed as described
by M. Moi et al., J. Amer. Chem. Soc. 49:2639 (1989)
(2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic
acid); S. V. Deshpande et al., J. Nucl. Med. 31:473 (1990); G.
Ruser et al., Bioconj. Chem. 1:345 (1990); C. J. Broan et al., J.
C. S. Chem. Comm. 23:1739 (1990); and C. J. Anderson et al., J.
Nucl. Med. 36:850 (1995).
[0560] In one embodiment, a macrocyclic chelator, such as
polyazamacrocyclic chelators, optionally containing one or more
carboxy, amino, hydroxamate, phosphonate, or phosphate groups, are
attached to the Albumin fusion protein of the invention. In another
embodiment, the chelator is a chelator selected from the group
consisting of DOTA, analogues of DOTA, and derivatives of DOTA.
[0561] In one embodiment, suitable chelator molecules that may be
attached to the Albumin fusion protein of the invention include
DOXA (1-oxa-4,7,10-triazacyclododecanetriacetic acid), NOTA
(1,4,7-triazacyclononanetriacetic acid), TETA
(1,4,8,11-tetraazacyclotetradecanetetraacetic acid), and THT
(4'-(3-amino-4-methoxy-phenyl)-6,6''-bis(N',N'-dicarboxymethyl-N-methylhy-
dra zino)-2,2':6',2''-terpyridine), and analogs and derivatives
thereof. See, e.g., Ohmono et al., J. Med. Chem. 35: 157-162
(1992); Kung et al., J. Nucl. Med. 25: 326-332 (1984); Jurisson et
al., Chem. Rev. 93:1137-1156 (1993); and U.S. Pat. No. 5,367,080.
Other suitable chelators include chelating agents disclosed in U.S.
Pat. Nos. 4,647,447; 4,687,659; 4,885,363; EP-A-71564; WO89/00557;
and EP-A-232751.
[0562] In another embodiment, suitable macrocyclic carboxylic acid
chelators which can be used in the present invention include
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA); 1,4,8,12-tetraazacyclopentadecane-N,N',N'',N'''-tetraacetic
acid (15N4); 1,4,7-triazacyclononane-N,N',N''-triacetic acid (9N3);
1,5,9-triazacyclododecane-N,N',N''-triacetic acid (12N3); and
6-bromoacetamido-benzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-t-
etraacetic acid (BAT).
[0563] A preferred chelator that can be attached to the Albumin
Fusion protein of the invention is
.quadrature.-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclodod-
ecane-1,4,7,10-tetraacetic acid, which is also known as
MeO-DOTA-NCS. A salt or ester of
.quadrature.-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclodod-
ecane-1,4,7,10-tetraacetic acid may also be used.
[0564] Albumin fusion proteins of the invention to which chelators
such as those described are covalently attached may be labeled (via
the coordination site of the chelator) with radionuclides that are
suitable for therapeutic, diagnostic, or both therapeutic and
diagnostic purposes. Examples of appropriate metals include Ag, At,
Au, Bi, Cu, Ga, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc,
Tl, Y, and Yb. Examples of the radionuclide used for diagnostic
purposes are Fe, Gd, .sup.111n, .sup.67Ga, or .sup.68Ga. In another
embodiment, the radionuclide used for diagnostic purposes is
.sup.111In, or .sup.67Ga. Examples of the radionuclide used for
therapeutic purposes are .sup.166Ho, .sup.165Dy, .sup.90Y,
.sup.115mIn, .sup.52Fe, or .sup.72Ga. In one embodiment, the
radionuclide used for diagnostic purposes is .sup.166Ho or
.sup.90Y. Examples of the radionuclides used for both therapeutic
and diagnostic purposes include .sup.153Sm, .sup.177Lu, .sup.159Gd,
.sup.175Yb, or .sup.47Sc. In one embodiment, the radionuclide is
.sup.153Sm, .sup.177Lu, .sup.175Yb, or .sup.159Gd.
[0565] Preferred metal radionuclides include .sup.90Y, .sup.99mTc,
.sup.111In, .sup.47Sc, .sup.67Ga, .sup.51Cr, .sup.77mSn, .sup.67Cu,
.sup.167Tm, .sup.97Ru, .sup.188Re, .sup.177Lu, .sup.199Au,
.sup.47Sc, .sup.67Ga .sup.51Cr, .sup.177mSn, .sup.67Cu, .sup.167Tm,
.sup.95Ru, .sup.188Re, .sup.177Lu, .sup.199Au, .sup.203Pb and
.sup.141Ce.
[0566] In a particular embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with a metal ion selected from the group consisting of .sup.90Y,
.sup.111In, .sup.177Lu, .sup.166Ho, .sup.215Bi, and .sup.255Ac.
[0567] Moreover, .gamma.-emitting radionuclides, such as
.sup.99mTc, .sup.111In, .sup.67Ga, and .sup.169Yb have been
approved or under investigation for diagnostic imaging, while
.beta.-emitters, such as .sup.67Cu, .sup.111Ag, .sup.186Re, and
.sup.90Y are useful for the applications in tumor therapy. Also
other useful radionuclides include .gamma.-emitters, such as
.sup.99mTc, .sup.111In, .sup.67Ga, and .sup.169Yb, and
.beta.-emitters, such as .sup.67Cu, .sup.111Ag, .sup.186Re,
.sup.188Re and .sup.90Y, as well as other radionuclides of interest
such as .sup.211At, .sup.212Bi, .sup.177Lu, .sup.86Rb, .sup.105Rh,
.sup.153Sm, .sup.198Au, .sup.149Pm, .sup.85Sr, .sup.142Pr,
.sup.214Pb, .sup.109Pd, .sup.166Ho, .sup.208Tl, and .sup.44Sc.
Albumin fusion proteins of the invention to which chelators are
covalently attached may be labeled with the radionuclides described
above.
[0568] In another embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with paramagnetic metal ions including ions of transition and
lanthanide metal, such as metals having atomic numbers of 21-29,
42, 43, 44, or 57-71, in particular ions of Cr, V, Mn, Fe, Co, Ni,
Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.
The paramagnetic metals used in compositions for magnetic resonance
imaging include the elements having atomic numbers of 22 to 29, 42,
44 and 58-70.
[0569] In another embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with fluorescent metal ions including lanthanides, in particular
La, Ce, Pr, Nd, Pm, Sm, Eu (e.g., .sup.152Eu), Gd, Tb, Dy, Ho, Er,
Tm, Yb, and Lu.
[0570] In another embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with heavy metal-containing reporters may include atoms of Mo, Bi,
Si, and W.
[0571] It is also possible to label the albumin fusion proteins
with a fluorescent compound. When the fluorescently labeled
antibody is exposed to light of the proper wave length, its
presence can then be detected due to fluorescence. Among the most
commonly used fluorescent labeling compounds are fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, ophthaldehyde and fluorescamine.
[0572] The albumin fusion protein can also be detectably labeled
using fluorescence emitting metals such as .sup.152Eu, or others of
the lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0573] The albumin fusion proteins can also can be detectably
labeled by coupling it to a chemiluminescent compound. The presence
of the chemiluminescent-tagged albumin fusion protein is then
determined by detecting the presence of luminescence that arises
during the course of a chemical reaction. Examples of particularly
useful chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester.
[0574] Likewise, a bioluminescent compound may be used to label
albumin fusion proteins of the present invention. Bioluminescence
is a type of chemiluminescence found in biological systems in,
which a catalytic protein increases the efficiency of the
chemiluminescent reaction. The presence of a bioluminescent protein
is determined by detecting the presence of luminescence. Important
bioluminescent compounds for purposes of labeling are luciferin,
luciferase and aequorin.
[0575] Transgenic Organisms
[0576] Transgenic organisms that express the albumin fusion
proteins of the invention are also included in the invention.
Transgenic organisms are genetically modified organisms into which
recombinant, exogenous or cloned genetic material has been
transferred. Such genetic material is often referred to as a
transgene. The nucleic acid sequence of the transgene may include
one or more transcriptional regulatory sequences and other nucleic
acid sequences such as introns, that may be necessary for optimal
expression and secretion of the encoded protein. The transgene may
be designed to direct the expression of the encoded protein in a
manner that facilitates its recovery from the organism or from a
product produced by the organism, e.g. from the milk, blood, urine,
eggs, hair or seeds of the organism. The transgene may consist of
nucleic acid sequences derived from the genome of the same species
or of a different species than the species of the target animal.
The transgene may be integrated either at a locus of a genome where
that particular nucleic acid sequence is not otherwise normally
found or at the normal locus for the transgene.
[0577] The term "germ cell line transgenic organism" refers to a
transgenic organism in which the genetic alteration or genetic
information was introduced into a germ line cell, thereby
conferring the ability of the transgenic organism to transfer the
genetic information to offspring. If such offspring in fact possess
some or all of that alteration or genetic information, then they
too are transgenic organisms. The alteration or genetic information
may be foreign to the species of organism to which the recipient
belongs, foreign only to the particular individual recipient, or
may be genetic information already possessed by the recipient. In
the last case, the altered or introduced gene may be expressed
differently than the native gene.
[0578] A transgenic organism may be a transgenic animal or a
transgenic plant. Transgenic animals can be produced by a variety
of different methods including transfection, electroporation,
microinjection, gene targeting in embryonic stem cells and
recombinant viral and retroviral infection (see, e.g., U.S. Pat.
No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins et al. (1993)
Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol. 6(2)
99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods
in Molecular Biology No. 62, Humana Press (1997)). The method of
introduction of nucleic acid fragments into recombination competent
mammalian cells can be by any method which favors co-transformation
of multiple nucleic acid molecules. Detailed procedures for
producing transgenic animals are readily available to one skilled
in the art, including the disclosures in U.S. Pat. No. 5,489,743
and U.S. Pat. No. 5,602,307.
[0579] A number of recombinant or transgenic mice have been
produced, including those which express an activated oncogene
sequence (U.S. Pat. No. 4,736,866); express simian SV40 T-antigen
(U.S. Pat. No. 5,728,915); lack the expression of interferon
regulatory factor 1 (IRF-1) (U.S. Pat. No. 5,731,490); exhibit
dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at
least one human gene which participates in blood pressure control
(U.S. Pat. No. 5,731,489); display greater similarity to the
conditions existing in naturally occurring Alzheimer's disease
(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate
cellular adhesion (U.S. Pat. No. 5,602,307); possess a bovine
growth hormone gene (Clutter et al. (1996) Genetics
143(4):1753-1760); or, are capable of generating a fully human
antibody response (McCarthy (1997) The Lancet 349(9049):405).
[0580] While mice and rats remain the animals of choice for most
transgenic experimentation, in some instances it is preferable or
even necessary to use alternative animal species. Transgenic
procedures have been successfully utilized in a variety of
non-murine animals, including sheep, goats, pigs, dogs, cats,
monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see,
e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515-526; Houdebine
(1995) Reprod. Nutr. Dev. 35(6):609-617; Petters (1994) Reprod.
Fertil. Dev. 6(5):643-645; Schnieke et al. (1997) Science
278(5346):2130-2133; and Amoah (1997) J. Animal Science
75(2):578-585).
[0581] To direct the secretion of the transgene-encoded protein of
the invention into the milk of transgenic mammals, it may be put
under the control of a promoter that is preferentially activated in
mammary epithelial cells. Promoters that control the genes encoding
milk proteins are preferred, for example the promoter for casein,
beta lactoglobulin, whey acid protein, or lactalbumin (see, e.g.,
DiTullio (1992) BioTechnology 10:74-77; Clark et al. (1989)
BioTechnology 7:487-492; Gorton et al. (1987) BioTechnology
5:1183-1187; and Soulier et al. (1992) FEBS Letts. 297:13). The
transgenic mammals of choice would produce large volumes of milk
and have long lactating periods, for example goats, cows, camels or
sheep.
[0582] An albumin fusion protein of the invention can also be
expressed in a transgenic plant, e.g. a plant in which the DNA
transgene is inserted into the nuclear or plastidic genome. Plant
transformation procedures used to introduce foreign nucleic acids
into plant cells or protoplasts are known in the art. See, in
general, Methods in Enzymology Vol. 153 ("Recombinant DNA Part D")
1987, Wu and Grossman Eds., Academic Press and European Patent
Application EP 693554. Methods for generation of genetically
engineered plants are further described in U.S. Pat. No. 5,283,184,
U.S. Pat. No. 5,482,852, and European Patent Application EP 693
554, all of which are hereby incorporated by reference.
[0583] Pharmaceutical or Therapeutic Compositions
[0584] The albumin fusion proteins of the invention or formulations
thereof may be administered by any conventional method including
parenteral (e.g. subcutaneous or intramuscular) injection or
intravenous infusion. The treatment may consist of a single dose or
a plurality of doses over a period of time.
[0585] While it is possible for an albumin fusion protein of the
invention to be administered alone, it is preferable to present it
as a pharmaceutical formulation, together with one or more
acceptable carriers. The carrier(s) must be "acceptable" in the
sense of being compatible with the albumin fusion protein and not
deleterious to the recipients thereof. Typically, the carriers will
be water or saline which will be sterile and pyrogen free. Albumin
fusion proteins of the invention are particularly well suited to
formulation in aqueous carriers such as sterile pyrogen free water,
saline or other isotonic solutions because of their extended
shelf-life in solution. For instance, pharmaceutical compositions
of the invention may be formulated well in advance in aqueous form,
for instance, weeks or months or longer time periods before being
dispensed.
[0586] For example, formulations containing the albumin fusion
protein may be prepared taking into account the extended shelf-life
of the albumin fusion protein in aqueous formulations. As discussed
above, the shelf-life of many of these Therapeutic proteins are
markedly increased or prolonged after fusion to HA.
[0587] In instances where aerosol administration is appropriate,
the albumin fusion proteins of the invention can be formulated as
aerosols using standard procedures. The term "aerosol" includes any
gas-borne suspended phase of an albumin fusion protein of the
instant invention which is capable of being inhaled into the
bronchioles or nasal passages. Specifically, aerosol includes a
gas-borne suspension of droplets of an albumin fusion protein of
the instant invention, as may be produced in a metered dose inhaler
or nebulizer, or in a mist sprayer. Aerosol also includes a dry
powder composition of a compound of the instant invention suspended
in air or other carrier gas, which may be delivered by insufflation
from an inhaler device, for example. See Ganderton & Jones,
Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda
(1990) Critical Reviews in Therapeutic Drug Carrier Systems
6:273-313; and Raeburn et al., (1992) Pharmacol. Toxicol. Methods
27:143-159.
[0588] The formulations of the invention are also typically
non-immunogenic, in part, because of the use of the components of
the albumin fusion protein being derived from the proper species.
For instance, for human use, both the Therapeutic protein and
albumin portions of the albumin fusion protein will typically be
human. In some cases, wherein either component is non
human-derived, that component may be humanized by substitution of
key amino acids so that specific epitopes appear to the human
immune system to be human in nature rather than foreign.
[0589] The formulations may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. Such methods include the step of bringing into
association the albumin fusion protein with the carrier that
constitutes one or more accessory ingredients. In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0590] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation appropriate for the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example sealed
ampules, vials or syringes, and may be stored in a freeze-dried
(lyophilised) condition requiring only the addition of the sterile
liquid carrier, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders. Dosage formulations may contain the
Therapeutic protein portion at a lower molar concentration or lower
dosage compared to the non-fused standard formulation for the
Therapeutic protein given the extended serum half-life exhibited by
many of the albumin fusion proteins of the invention.
[0591] As an example, when an albumin fusion protein of the
invention comprises one of the proteins listed in the "Therapeutic
Protein:X" column of Table 1 as one or more of the Therapeutic
protein regions, the dosage form can be calculated on the basis of
the potency of the albumin fusion protein relative to the potency
of the therapeutic protein alone, while taking into account the
prolonged serum half-life and shelf-life of the albumin fusion
proteins compared to that of native therapeutic protein. For
example, if the therapeutic protein is typically administered at
0.3 to 30.0 IU/kg/week, or 0.9 to 12.0 IU/kg/week, given in three
or seven divided doses for a year or more. In an albumin fusion
protein consisting of full length HA fused to a therapeutic
protein, an equivalent dose in terms of units would represent a
greater weight of agent but the dosage frequency can be reduced,
for example to twice a week, once a week or less.
[0592] Formulations or compositions of the invention may be
packaged together with, or included in a kit with, instructions or
a package insert referring to the extended shelf-life of the
albumin fusion protein component. For instance, such instructions
or package inserts may address recommended storage conditions, such
as time, temperature and light, taking into account the extended or
prolonged shelf-life of the albumin fusion proteins of the
invention. Such instructions or package inserts may also address
the particular advantages of the albumin fusion proteins of the
inventions, such as the ease of storage for formulations that may
require use in the field, outside of controlled hospital, clinic or
office conditions. As described above, formulations of the
invention may be in aqueous form and may be stored under less than
ideal circumstances without significant loss of therapeutic
activity.
[0593] Albumin fusion proteins of the invention can also be
included in nutraceuticals. For instance, certain albumin fusion
proteins of the invention may be administered in natural products,
including milk or milk product obtained from a transgenic mammal
which expresses albumin fusion protein. Such compositions can also
include plant or plant products obtained from a transgenic plant
which expresses the albumin fusion protein. The albumin fusion
protein can also be provided in powder or tablet form, with or
without other known additives, carriers, fillers and diluents.
Nutraceuticals are described in Scott Hegenhart, Food Product
Design, December 1993.
[0594] The invention also provides methods of treatment and/or
prevention of diseases or disorders (such as, for example, any one
or more of the diseases or disorders disclosed herein) by
administration to a subject of an effective amount of an albumin
fusion protein of the invention or a polynucleotide encoding an
albumin fusion protein of the invention ("albumin fusion
polynucleotide") in a pharmaceutically acceptable carrier.
[0595] The albumin fusion protein and/or polynucleotide will be
formulated and dosed in a fashion consistent with good medical
practice, taking into account the clinical condition of the
individual patient (especially the side effects of treatment with
the albumin fusion protein and/or polynucleotide alone), the site
of delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amount" for purposes herein is thus determined by such
considerations.
[0596] As a general proposition, the total pharmaceutically
effective amount of the albumin fusion protein administered
parenterally per dose will be in the range of about 1 ug/kg/day to
10 mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. More preferably, this
dose is at least 0.01 mg/kg/day, and most preferably for humans
between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, the albumin fusion protein is typically administered
at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either
by 1-4 injections per day or by continuous subcutaneous infusions,
for example, using a mini-pump. An intravenous bag solution may
also be employed. The length of treatment needed to observe changes
and the interval following treatment for responses to occur appears
to vary depending on the desired effect.
[0597] As noted above, the albumin fusion protein of the invention
has a higher plasma stability compared to the Therapeutic protein
portion (or fragment or variant thereof) alone. This increase in
plasma stability should be taken into account when determining the
effective amount of the albumin fusion protein to be administered
per dose and the dosing administration schedule. In particular,
higher plasma stability may allow the albumin fusion protein to be
administered at a lower dose at the same frequency of
administrations, or alternatively, may allow the albumin fusion
protein to be administered in fewer dosings. Preferably, the higher
stability allows the albumin fusion protein of the invention to be
administered less often in fewer dosings. More preferably, the
albumin fusion protein can be administered once every two weeks.
Still more preferably, the albumin fusion protein can be
administered once every three, four, five, or more weeks depending
on the pharmacokinetics of the albumin fusion protein. For example,
as discussed above, the pharmacokinetics of an IFN-alpha-HSA fusion
protein supports a dosing regimen of once every 2-4 weeks or more,
and even dosing at intervals of 4 weeks or more than every 4
weeks.
[0598] The effective amount of the albumin fusion protein to be
administered per dose can also be denoted as the total formulated
albumin fusion protein concentration given per dose. In one
embodiment, the total formulated albumin fusion protein
concentration administered to a patient per dose is in the range of
about 10 ug/dose to about 2000 ug/dose. More preferably, the total
concentration is in the range of about 100 ug/dose to about 1000
ug/dose, or alternatively, about 1000 ug/dose to about 1200 ug/dose
or about 900 ug/dose to about 1800 ug/dose.
[0599] In a specific embodiment, an IFN-alpha-HSA fusion protein of
the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is dosed in a total formulated concentration of about 90
ug/dose to about 2000 ug/dose. In more preferred embodiments, an
IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is dosed in a total
formulated concentration of about 900 ug/dose to about 2000
ug/dose, about 900 ug/dose to about 1200 ug/dose, about 900 ug/dose
to about 1800 ug/dose and most preferably in a total formulated
concentration of about 1200 ug/dose to about 1800 ug/dose. In
additional preferred embodiments, an IFN-alpha-HSA fusion protein
of the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381,
2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292,
4295, or 4296) is dosed in a total formulated concentration of 600
ug/dose, 720 ug/dose, 800 ug/dose, 900 ug/dose, 1000 ug/dose, 1200
ug/dose, 1500 ug/dose, 1800 ug/dose, or 2000 ug/dose. In additional
embodiments, the total formulated dose of an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered either alone or in combination
with an antiviral compound, such as ribavirin. In additionally
preferred embodiments, the total formulated dose of an
IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered in
combination with one, two, three, or more antiviral compounds,
including, but not limited to, ribavirin and optionally another
antiviral compound.
[0600] In an additional embodiment, the total formulated
concentration of an IFN-alpha-HSA fusion proteins of the invention
(e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165,
3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) is
administered to treat a patient infected with HCV. In a specific
embodiment, the IFN-alpha-HSA fusion proteins of the invention
(e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165,
3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) are
administered to a Treatment naive patient with HCV either alone or
in combination with an effective amount of an antiviral compound,
such as ribavirin, in a total formulated concentration of about 90
ug/dose to about 2000 ug/dose. In more preferred embodiments, the
IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered to a
Treatment naive patient with HCV either alone or in combination
with an effective amount of antiviral compound, such as ribavirin,
in a total formulated concentration of about 900 ug/dose to about
2000 ug/dose, about 900 ug/dose to about 1200 ug/dose, about 900
ug/dose to about 1800 ug/dose and most preferably in a total
formulated concentration of about 1200 ug/dose to about 1800
ug/dose. In additional preferred embodiments, an IFN-alpha-HSA
fusion protein of the invention (e.g., produced by CIDs 2249, 2343,
2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290,
4291, 4292, 4295, or 4296) is administered to a Treatment naive
patient with HCV either alone or in combination with an effective
amount of antiviral compound, such as ribavirin, in a total
formulated concentration of 600 ug/dose, 720 ug/dose, 800 ug/dose,
900 ug/dose, 1000 ug/dose, 1200 ug/dose, 1500 ug/dose, 1800
ug/dose, or 2000 ug/dose.
[0601] In an additional embodiment, the total formulated
concentration of an IFN-alpha-HSA fusion proteins of the invention
are administered to a Treatment naive patient with HCV in
combination with an effective amount of one or more antiviral
compounds, including, for example, ribavirin, in a total formulated
concentration of about 90 ug/dose to about 2000 ug/dose. In
additional preferred embodiments, the IFN-alpha-HSA fusion protein
of the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381,
2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292,
4295, or 4296) is administered to a Treatment naive patient with
HCV in combination with one, two, three, or more antiviral
compounds, including, for example, ribavirin and optionally another
antiviral compound, in a total formulated concentration of about
900 ug/dose to about 2000 ug/dose, about 900 ug/dose to about 1200
ug/dose, about 900 ug/dose to about 1800 ug/dose and most
preferably in a total formulated concentration of about 1200
ug/dose to about 1800 ug/dose. In additional preferred embodiments,
an IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered to a
Treatment naive patient with HCV in combination with an effective
amount of one, two, three, or more antiviral compounds, including,
for example, ribavirin, in a total formulated concentration of 600
ug/dose, 720 ug/dose, 800 ug/dose, 900 ug/dose, 1000 ug/dose, 1200
ug/dose, 1500 ug/dose, 1800 ug/dose, or 2000 ug/dose.
[0602] In an additional embodiment, an IFN-alpha-HSA fusion protein
of the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381,
2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292,
4295, or 4296) is administered to a Treatment experienced patient
with HCV either alone or in combination with an effective amount of
antiviral compound, such as ribavirin, in a total formulated
concentration of about 90 ug/dose to about 2000 ug/dose. In more
preferred embodiments, an IFN-alpha-HSA fusion protein of the
invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is administered to a Treatment experienced patient with
HCV either alone or in combination with an effective amount of
antiviral compound, such as ribavirin, in a total formulated
concentration of about 900 ug/dose to about 2000 ug/dose, about 900
ug/dose to about 1200 ug/dose, about 900 ug/dose to about 1800
ug/dose and most preferably in a total formulated concentration of
about 1200 ug/dose to about 1800 ug/dose. In additional preferred
embodiments, an IFN-alpha-HSA fusion proteins of the invention
(e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165,
3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) is
administered to a Treatment experienced patient with HCV either
alone or in combination with an effective amount of antiviral
compound, such as ribavirin, in a total formulated concentration of
600 ug/dose, 720 ug/dose, 800 ug/dose, 900 ug/dose, 1000 ug/dose,
1200 ug/dose, 1500 ug/dose, 1800 ug/dose, or 2000 ug/dose.
[0603] In an additional embodiment, an IFN-alpha-HSA fusion protein
of the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381,
2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292,
4295, or 4296) is administered to a Treatment experienced patient
with HCV in combination with one, two, three, or more antiviral
compounds, including, for example, ribavirin and optionally another
antiviral compound, in a total formulated concentration of about 90
ug/dose to about 2000 ug/dose. In more preferred embodiments, an
IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered to a
Treatment experienced patient with HCV in combination with one,
two, three, or more antiviral compounds, including, for example,
ribavirin and optionally another antiviral compound, in a total
formulated concentration of about 900 ug/dose to about 2000
ug/dose, about 900 ug/dose to about 1200 ug/dose, about 900 ug/dose
to about 1800 ug/dose and most preferably in a total formulated
concentration of about 1200 ug/dose to about 1800 ug/dose. In
additional preferred embodiments, an IFN-alpha-HSA fusion proteins
of the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381,
2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292,
4295, or 4296) is administered to a Treatment experienced patient
with HCV in combination with an effective amount of one, two,
three, or more antiviral compounds, including, for example,
ribavirin and optionally another antiviral compound, in a total
formulated concentration of 600 ug/dose, 720 ug/dose, 800 ug/dose,
900 ug/dose, 1000 ug/dose, 1200 ug/dose, 1500 ug/dose, 1800
ug/dose, or 2000 ug/dose.
[0604] The total formulated concentration of the albumin fusion
protein and the dosing interval in which the dosing interval at
which the albumin fusion protein will administered will vary
depending on the desired effect and the particular therapeutic
protein adminstered. In one embodiment, the total formulated
albumin fusion protein concentration administered to a patient per
dose is in the range of about 10 ug/dose to about 2000 ug/dose once
a week, once every two weeks, once every three weeks, once every
four weeks or more. More preferably, the total concentration is in
the range of about 100 ug/dose to about 1000 ug/dose once a week,
once every two weeks, once every three weeks, once every four weeks
or more, or alternatively, about 1000 ug/dose to about 1200 ug/dose
or about 900 ug/dose to about 1800 ug/dose once a week, once every
two weeks, once every three weeks, once every four weeks or
more.
[0605] In a specific embodiment, an IFN-alpha-HSA fusion protein of
the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is administered at a total formulated concentration of
about 90 ug/dose to about 2000 ug/dose once every two, three, four,
or five weeks. In more preferred embodiments, an IFN-alpha-HSA
fusion protein of the invention (e.g., produced by CIDs 2249, 2343,
2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290,
4291, 4292, 4295, or 4296) is dosed in a total formulated
concentration of about 900 ug/dose to about 2000 ug/dose once every
one, two, three, four or five weeks; about 900 ug/dose to about
1200 ug/dose once every one, two, three, four or five weeks; about
900 ug/dose to about 1800 ug/dose once every one, two, three, four
or five weeks; and most preferably in a total formulated
concentration of about 1200 ug/dose to 1800 ug/dose once every one,
two, three, four or five weeks. In additional embodiments, an
IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered at a
total formulated concentration of about 600 ug/dose once every one,
two, three, four or five weeks; 800 ug/dose once every one, two,
three, four or five weeks, 900 ug/dose once every one, two, three,
four or five weeks; 1000 ug/dose once every one, two, three, four
or five weeks; 1200 ug/dose once every one, two, three, four or
five weeks; 1500 ug/dose once every one, two, three, four or five
weeks; 1600 ug/dose once every one, two, three, four or five weeks;
1800 ug/dose once every one, two, three, four or five weeks; or
2000 ug/dose once every one, two, three, four or five weeks. In
more preferred embodiments, the IFN-alpha-HSA fusion protein of the
invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is administered at a total formulated concentration of 900
ug/dose once every two weeks, and more preferably at a total
concentration of 1200 ug/dose once every two weeks, 1200 ug/dose
once every four weeks, or 1800 ug/dose once every four weeks. In
additional embodiments, the total formulated dose of an
IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered either
alone or in combination with an antiviral compound, such as
ribavirin. In additional preferred embodiments, the total
formulated dose of an IFN-alpha-HSA fusion protein of the invention
(e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165,
3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) is
administered in combination with one, two, three, or more antiviral
compounds, including, for example, ribavirin and optionally another
antiviral compound.
[0606] In specific embodiments, an IFN-alpha-HSA fusion protein of
the invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is administered to a Treatment naive HCV patient at a
total formulated concentration of about 90 ug/dose to about 2000
ug/dose once every two, three, four, or five weeks either alone or
in combination with an antiviral compound, such as ribavirin. In
more preferred embodiments, an IFN-alpha-HSA fusion protein of the
invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is administered to a Treatment naive HCV patient in a
total formulated concentration of about 900 ug/dose to about 2000
ug/dose once every one, two, three, four or five weeks; about 900
ug/dose to about 1200 ug/dose once every one, two, three, four or
five weeks; about 900 ug/dose to about 1800 ug/dose once every one,
two, three, four or five weeks; and most preferably in a total
formulated concentration of about 1200 ug/dose to about 1800
ug/dose once every one, two, three, four or five weeks either alone
or in combination with an antiviral compound, including, such as
ribavirin. In additional embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment naive HCV
patient at a total formulated concentration of about 600 ug/dose
once every one, two, three, four or five weeks; 800 ug/dose once
every one, two, three, four or five weeks, 900 ug/dose once every
one, two, three, four or five weeks; 1000 ug/dose once every one,
two, three, four or five weeks; 1200 ug/dose once every one, two,
three, four or five weeks; 1500 ug/dose once every one, two, three,
four or five weeks; 1600 ug/dose once every one, two, three, four
or five weeks; 1800 ug/dose once every one, two, three, four or
five weeks; or 2000 ug/dose once every one, two, three, four or
five weeks either alone or in combination with an antiviral
compound, such as ribavirin.
[0607] In preferred specific embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment naive HCV
patient at a total formulated concentration of about 90 ug/dose to
about 2000 ug/dose once every two, three, four, or five weeks in
combination with one, two, three, or more antiviral compounds,
including, for example, ribavirin and optionally another antiviral
compound. In more preferred embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment naive HCV
patient in a total formulated concentration of about 900 ug/dose to
about 2000 ug/dose once every one, two, three, four or five weeks;
about 900 ug/dose to about 1200 ug/dose once every one, two, three,
four or five weeks; about 900 ug/dose to about 1800 ug/dose once
every one, two, three, four or five weeks; and most preferably in a
total formulated concentration of about 1200 ug/dose to about 1800
ug/dose once every one, two, three, four or five weeks in
combination with one or more antiviral compounds, including, for
example, ribavirin in combination with one, two, three, or more
antiviral compounds, including, for example, ribavirin and
optionally another antiviral compound. In additional embodiments,
an IFN-alpha-HSA fusion protein of the invention (e.g., produced by
CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered to a
Treatment naive HCV patient at a total formulated concentration of
about 600 ug/dose once every one, two, three, four or five weeks;
800 ug/dose once every one, two, three, four or five weeks, 900
ug/dose once every one, two, three, four or five weeks; 1000
ug/dose once every one, two, three, four or five weeks; 1200
ug/dose once every one, two, three, four or five weeks; 1500
ug/dose once every one, two, three, four or five weeks; 1600
ug/dose once every one, two, three, four or five weeks; 1800
ug/dose once every one, two, three, four or five weeks; or 2000
ug/dose once every one, two, three, four or five weeks in
combination with one, two, three, or more antiviral compounds,
including, for example, ribavirin and optionally another antiviral
compound.
[0608] In more preferred embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment naive HCV
patient at a total formulated concentration of 900 ug/dose once
every two weeks, and more preferably at a total concentration of
1200 ug/dose once every two weeks, 1200 ug/dose once every four
weeks, or 1800 ug/dose once every four weeks, either alone or in
combination with an antiviral compound, such as ribavirin. In most
preferred embodiments, an IFN-alpha-HSA fusion protein of the
invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is administered to a Treatment naive HCV patient at a
total formulated concentration of 900 ug/dose once every two weeks,
and more preferably at a total concentration of 1200 ug/dose once
every two weeks, 1200 ug/dose once every four weeks, or 1800
ug/dose once every four weeks, in combination with one, two, three,
or more antiviral compounds, including, for example, ribavirin and
optionally another antiviral compound.
[0609] In additional specific embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment experienced HCV
patient at a total formulated concentration of about 90 ug/dose to
about 2000 ug/dose once every two, three, four, or five weeks
either alone or in combination with an antiviral compound, such as
ribavirin. In more preferred embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment experienced HCV
patient in a total formulated concentration of about 900 ug/dose to
about 2000 ug/dose once every one, two, three, four or five weeks;
about 900 ug/dose to about 1200 ug/dose once every one, two, three,
four or five weeks; about 900 ug/dose to about 1800 ug/dose once
every one, two, three, four or five weeks; and most preferably in a
total formulated concentration of about 1200 ug/dose to about 1800
ug/dose once every one, three, four or five weeks, or most
preferably every two weeks either alone or in combination with an
antiviral compound, such as ribavirin. In additional embodiments,
an IFN-alpha-HSA fusion proteins of the invention (e.g., produced
by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered to a
Treatment experienced HCV patient at a total formulated
concentration of about 600 ug/dose once every one, two, three, four
or five weeks; 800 ug/dose once every one, two, three, four or five
weeks, 900 ug/dose once every one, two, three, four or five weeks;
1000 ug/dose once every one, two, three, four or five weeks; 1200
ug/dose once every one, two, three, four or five weeks; 1500
ug/dose once every one, two, three, four or five weeks; 1600
ug/dose once every one, two, three, four or five weeks; 1800
ug/dose once every one, two, three, four or five weeks; or 2000
ug/dose once every one, two, three, four or five weeks either alone
or in combination with an antiviral compound, such as
ribavirin.
[0610] In more specific embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment experienced HCV
patient at a total formulated concentration of about 90 ug/dose to
about 2000 ug/dose once every two, three, four, or five weeks in
combination with one, two, three, or more antiviral compounds,
including, for example, ribavirin and optionally another antiviral
compound. In more preferred embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment experienced HCV
patient in a total formulated concentration of about 900 ug/dose to
about 2000 ug/dose once every one, two, three, four or five weeks;
about 900 ug/dose to about 1200 ug/dose once every one, two, three,
four or five weeks; about 900 ug/dose to about 1800 ug/dose once
every one, two, three, four or five weeks; and most preferably in a
total formulated concentration of about 1200 ug/dose to about 1800
ug/dose once every one, three, four or five weeks, or most
preferably every two weeks in combination with one, two, three, or
more antiviral compounds, including, for example, ribavirin and
optionally another antiviral compound. In additional embodiments,
an IFN-alpha-HSA fusion proteins of the invention (e.g., produced
by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424,
3476, 3960, 4290, 4291, 4292, 4295, or 4296) is administered to a
Treatment experienced HCV patient at a total formulated
concentration of about 600 ug/dose once every one, two, three, four
or five weeks; 800 ug/dose once every one, two, three, four or five
weeks, 900 ug/dose once every one, two, three, four or five weeks;
1000 ug/dose once every one, two, three, four or five weeks; 1200
ug/dose once every one, two, three, four or five weeks; 1500
ug/dose once every one, two, three, four or five weeks; 1600
ug/dose once every one, two, three, four or five weeks; 1800
ug/dose once every one, two, three, four or five weeks; or 2000
ug/dose once every one, two, three, four or five weeks in
combination with one, two, three, or more antiviral compounds,
including, for example, ribavirin and optionally another antiviral
compound.
[0611] In more preferred embodiments, an IFN-alpha-HSA fusion
protein of the invention (e.g., produced by CIDs 2249, 2343, 2366,
2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291,
4292, 4295, or 4296) is administered to a Treatment experienced HCV
patient at a total formulated concentration of 900 ug/dose once
every two weeks, and more preferably at a total concentration of
1200 ug/dose once every two weeks, 1200 ug/dose once every four
weeks, or 1800 ug/dose once every four weeks, either alone or in
combination with an antiviral compound, such as ribavirin. In most
preferred embodiments, an IFN-alpha-HSA fusion protein of the
invention (e.g., produced by CIDs 2249, 2343, 2366, 2381, 2382,
2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295,
or 4296) is administered to a Treatment experienced HCV patient at
a total formulated concentration of 900 ug/dose once every two
weeks, and more preferably at a total concentration of 1200 ug/dose
once every two weeks, 1200 ug/dose once every four weeks, or 1800
ug/dose once every four weeks, in combination with one, two, three,
or more antiviral compounds, including, for example, ribavirin and
optionally another antiviral compound.
[0612] Albumin fusion proteins and/or polynucleotides can be are
administered orally, rectally, parenterally, intracisternally,
intravaginally, intraperitoneally, topically (as by powders,
ointments, gels, drops or transdermal patch), bucally, or as an
oral or nasal spray. "Pharmaceutically acceptable carrier" refers
to a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any. The term
"parenteral" as used herein refers to modes of administration which
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion.
[0613] Albumin fusion proteins and/or polynucleotides of the
invention are also suitably administered by sustained-release
systems. Examples of sustained-release albumin fusion proteins
and/or polynucleotides are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. The term "parenteral" as used herein refers
to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion. Additional examples of
sustained-release albumin fusion proteins and/or polynucleotides
include suitable polymeric materials (such as, for example,
semi-permeable polymer matrices in the form of shaped articles,
e.g., films, or mirocapsules), suitable hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, and sparingly soluble derivatives (such as, for example, a
sparingly soluble salt).
[0614] Sustained-release matrices include polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556
(1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0615] Sustained-release albumin fusion proteins and/or
polynucleotides also include liposomally entrapped albumin fusion
proteins and/or polynucleotides of the invention (see generally,
Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in
the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)).
Liposomes containing the albumin fusion protein and/or
polynucleotide are prepared by methods known per se: DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985);
Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP
52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.
Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324. Ordinarily, the liposomes are of the small (about 200-800
Angstroms) unilamellar type in which the lipid content is greater
than about 30 mol. percent cholesterol, the selected proportion
being adjusted for the optimal Therapeutic.
[0616] In yet an additional embodiment, the albumin fusion proteins
and/or polynucleotides of the invention are delivered by way of a
pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201
(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.
Engl. J. Med. 321:574 (1989)).
[0617] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0618] For parenteral administration, in one embodiment, the
albumin fusion protein and/or polynucleotide is formulated
generally by mixing it at the desired degree of purity, in a unit
dosage injectable form (solution, suspension, or emulsion), with a
pharmaceutically acceptable carrier, i.e., one that is non-toxic to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation. For example,
the formulation preferably does not include oxidizing agents and
other compounds that are known to be deleterious to the
Therapeutic.
[0619] Generally, the formulations are prepared by contacting the
albumin fusion protein and/or polynucleotide uniformly and
intimately with liquid carriers or finely divided solid carriers or
both. Then, if necessary, the product is shaped into the desired
formulation. Preferably the carrier is a parenteral carrier, more
preferably a solution that is isotonic with the blood of the
recipient. Examples of such carrier vehicles include water, saline,
Ringer's solution, and dextrose solution. Non-aqueous vehicles such
as fixed oils and ethyl oleate are also useful herein, as well as
liposomes.
[0620] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates (including, for example,
Tween-20), poloxamers, or PEG.
[0621] The albumin fusion protein is typically formulated in such
vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml,
preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of
polypeptide salts.
[0622] Any pharmaceutical used for therapeutic administration can
be sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes). Albumin
fusion proteins and/or polynucleotides generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0623] Albumin fusion proteins and/or polynucleotides ordinarily
will be stored in unit or multi-dose containers, for example,
sealed ampoules or vials, as an aqueous solution or as a
lyophilized formulation for reconstitution. As an example of a
lyophilized formulation, 10-ml vials are filled with 5 ml of
sterile-filtered 1% (w/v) aqueous albumin fusion protein and/or
polynucleotide solution, and the resulting mixture is lyophilized.
The infusion solution is prepared by reconstituting the lyophilized
albumin fusion protein and/or polynucleotide using bacteriostatic
Water-for-Injection.
[0624] In a specific and preferred embodiment, the Albumin fusion
protein formulations comprises 0.01 M sodium phosphate, 0.15 mM
sodium chloride, 0.16 micromole sodium octanoate/milligram of
fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. In
another specific and preferred embodiment, the Albumin fusion
protein formulations consists 0.01 M sodium phosphate, 0.15 mM
sodium chloride, 0.16 micromole sodium octanoate/milligram of
fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2.
The pH and buffer are chosen to match physiological conditions and
the salt is added as a tonicifier. Sodium octanoate has been chosen
due to its reported ability to increase the thermal stability of
the protein in solution. Finally, polysorbate has been added as a
generic surfactant, which lowers the surface tension of the
solution and lowers non-specific adsorption of the albumin fusion
protein to the container closure system.
[0625] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the albumin fusion proteins and/or polynucleotides
of the invention. 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 addition, the albumin fusion
proteins and/or polynucleotides may be employed in conjunction with
other therapeutic compounds.
[0626] The albumin fusion proteins and/or polynucleotides of the
invention may be administered alone or in combination with
adjuvants. Adjuvants that may be administered with the albumin
fusion proteins and/or polynucleotides of the invention include,
but are not limited to, alum, alum plus deoxycholate (ImmunoAg),
MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g.,
THERACYS.RTM.), MPL and nonviable preparations of Corynebacterium
parvum. In a specific embodiment, albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with alum. In another specific embodiment, albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with QS-21. Further adjuvants that may be administered
with the albumin fusion proteins and/or polynucleotides of the
invention include, but are not limited to, Monophosphoryl lipid
immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum
salts, MF-59, and Virosomal adjuvant technology. Vaccines that may
be administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
vaccines directed toward protection against MMR (measles, mumps,
rubella), polio, varicella, tetanus/diptheria, hepatitis A,
hepatitis B, Haemophilus influenzae B, whooping cough, pneumonia,
influenza, Lyme's Disease, rotavirus, cholera, yellow fever,
Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and
pertussis. 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.
[0627] The albumin fusion proteins and/or polynucleotides of the
invention may be administered alone or in combination with other
therapeutic agents. Albumin fusion protein and/or polynucleotide
agents that may be administered in combination with the albumin
fusion proteins and/or polynucleotides of the invention, include
but not limited to, chemotherapeutic agents, antibiotics, steroidal
and non-steroidal anti-inflammatories, conventional
immunotherapeutic agents, and/or therapeutic treatments described
below. 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.
[0628] In one embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with an anticoagulant. Anticoagulants that may be administered with
the compositions of the invention include, but are not limited to,
heparin, low molecular weight heparin, warfarin sodium (e.g.,
COUMADIN.RTM.), dicumarol, 4-hydroxycoumarin, anisindione (e.g.,
MIRADON.TM.), acenocoumarol (e.g., nicoumalone, SINTHROME.TM.),
indan-1,3-dione, phenprocoumon (e.g., MARCUMAR.TM.), ethyl
biscoumacetate (e.g., TROMEXAN.TM.), and aspirin. In a specific
embodiment, compositions of the invention are administered in
combination with heparin and/or warfarin. In another specific
embodiment, compositions of the invention are administered in
combination with warfarin. In another specific embodiment,
compositions of the invention are administered in combination with
warfarin and aspirin. In another specific embodiment, compositions
of the invention are administered in combination with heparin. In
another specific embodiment, compositions of the invention are
administered in combination with heparin and aspirin.
[0629] In another embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with thrombolytic drugs. Thrombolytic drugs that may be
administered with the compositions of the invention include, but
are not limited to, plasminogen, lys-plasminogen,
alpha2-antiplasmin, streptokinae (e.g., KABIKINASE.TM.),
antiresplace (e.g., EMINASE.TM.), tissue plasminogen activator
(t-PA, altevase, ACTIVASE.TM.), urokinase (e.g., ABBOKINASE.TM.),
sauruplase, (Prourokinase, single chain urokinase), and
aminocaproic acid (e.g., AMICAR.TM.). In a specific embodiment,
compositions of the invention are administered in combination with
tissue plasminogen activator and aspirin.
[0630] In another embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with antiplatelet drugs. Antiplatelet drugs that may be
administered with the compositions of the invention include, but
are not limited to, aspirin, dipyridamole (e.g., PERSANTWE.TM.),
and ticlopidine (e.g., TICLID.TM.).
[0631] In specific embodiments, the use of anti-coagulants,
thrombolytic and/or antiplatelet drugs in combination with albumin
fusion proteins and/or polynucleotides of the invention is
contemplated for the prevention, diagnosis, and/or treatment of
thrombosis, arterial thrombosis, venous thrombosis,
thromboembolism, pulmonary embolism, atherosclerosis, myocardial
infarction, transient ischemic attack, unstable angina. In specific
embodiments, the use of anticoagulants, thrombolytic drugs and/or
antiplatelet drugs in combination with albumin fusion proteins
and/or polynucleotides of the invention is contemplated for the
prevention of occlusion of saphenous grafts, for reducing the risk
of periprocedural thrombosis as might accompany angioplasty
procedures, for reducing the risk of stroke in patients with atrial
fibrillation including nonrheumatic atrial fibrillation, for
reducing the risk of embolism associated with mechanical heart
valves and or mitral valves disease. Other uses for the
therapeutics of the invention, alone or in combination with
antiplatelet, anticoagulant, and/or thrombolytic drugs, include,
but are not limited to, the prevention of occlusions in
extracorporeal devices (e.g., intravascular canulas, vascular
access shunts in hemodialysis patients, hemodialysis machines, and
cardiopulmonary bypass machines).
[0632] In certain embodiments, albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with antiretroviral agents, nucleoside/nucleotide reverse
transcriptase inhibitors (NRTIs), non-nucleoside reverse
transcriptase inhibitors (NNRTIs), and/or protease inhibitors
(PIs). NRTIs that may be administered in combination with the
albumin fusion proteins and/or polynucleotides of the invention,
include, but are not limited to, RETROVIR.TM. (zidovudine/AZT),
VIDEX.TM. (didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). NNRTIs that may be administered in
combination with the albumin fusion proteins and/or polynucleotides
of the invention, include, but are not limited to, VIRAMUNE.TM.
(nevirapine), RESCRIPTOR.TM. (delavirdine), and SUSTIVA.TM.
(efavirenz). Protease inhibitors that may be administered in
combination with the albumin fusion proteins and/or polynucleotides
of the invention, include, but are not limited to, CRIXIVAN.TM.
(indinavir), NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and
VIRACEPT.TM. (nelfinavir). In a specific embodiment, antiretroviral
agents, nucleoside reverse transcriptase inhibitors, non-nucleoside
reverse transcriptase inhibitors, and/or protease inhibitors may be
used in any combination with albumin fusion proteins and/or
polynucleotides of the invention to treat AIDS and/or to prevent or
treat HIV infection.
[0633] Additional NRTIs include LODENOSINE.TM. (F-ddA; an
acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL.TM.
(emtricitabine/FTC; structurally related to lamivudine (3TC) but
with 3- to 10-fold greater activity in vitro; Triangle/Abbott);
dOTC (BCH-10652, also structurally related to lamivudine but
retains activity against a substantial proportion of
lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused
approval for anti-HIV therapy by FDA; Gilead Sciences);
PREVEON.RTM. (Adefovir Dipivoxil, the active prodrug of adefovir;
its active form is PMEA-pp); TENOFOVIR.TM. (bis-POC PMPA, a PMPA
prodrug; Gilead); DAPD/DXG (active metabolite of DAPD;
Triangle/Abbott); D-D4FC (related to 3TC, with activity against
AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN.TM.
(abacavir/159U89; Glaxo Wellcome Inc.); CS-87
(3'azido-2',3'-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl
(SATE)-bearing prodrug forms of .beta.-L-FD4C and .beta.-L-FddC (WO
98/17281).
[0634] Additional NNRTIs include COACTINON.TM. (Emivirine/MKC-442,
potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE.TM.
(AG-1549/S-1153, a next generation NNRTI with activity against
viruses containing the K103N mutation; Agouron); PNU-142721 (has
20- to 50-fold greater activity than its predecessor delavirdine
and is active against K103N mutants; Pharmacia & Upjohn);
DPC-961 and DPC-963 (second-generation derivatives of efavirenz,
designed to be active against viruses with the K103N mutation;
DuPont); GW-420867X (has 25-fold greater activity than HBY097 and
is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A
(naturally occurring agent from the latex tree; active against
viruses containing either or both the Y181C and K103N mutations);
and Propolis (WO 99/49830).
[0635] Additional protease inhibitors include LOPINAVIR.TM.
(ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide;
Bristol-Myres Squibb); TIPRANAVIR.TM. (PNU-140690, a non-peptic
dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic
dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide;
Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck);
DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a
peptidomimetic with in vitro activity against protease
inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate
prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755
(Ciba); and AGENERASE.TM. (amprenavir; Glaxo Wellcome Inc.).
[0636] Additional antiretroviral agents include fusion
inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include
T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane
protein ectodomain which binds to gp41 in its resting state and
prevents transformation to the fusogenic state; Trimeris) and
T-1249 (a second-generation fusion inhibitor; Trimeris).
[0637] Additional antiretroviral agents include fusion
inhibitors/chemokine receptor antagonists. Fusion
inhibitors/chemokine receptor antagonists include CXCR4 antagonists
such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C
(a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and
the T22 analogs T134 and T140; CCR5 antagonists such as RANTES
(9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4
antagonists such as NSC 651016 (a distamycin analog). Also included
are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists
such as RANTES, SDF-1, MIP-1.alpha., MIP-1.beta., etc., may also
inhibit fusion.
[0638] Additional antiretroviral agents include integrase
inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA)
acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid);
quinalizarin (QLC) and related anthraquinones; ZINTEVIR.TM. (AR
177, an oligonucleotide that probably acts at cell surface rather
than being a true integrase inhibitor; Arondex); and naphthols such
as those disclosed in WO 98/50347.
[0639] Additional antiretroviral agents include hydroxyurea-like
compounds such as BCX-34 (a purine nucleoside phosphorylase
inhibitor; Biocryst); ribonucleotide reductase inhibitors such as
DIDOX.TM. (Molecules for Health); inosine monophosphate
dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and
mycopholic acids such as CellCept (mycophenolate mofetil;
Roche).
[0640] Additional antiretroviral agents include inhibitors of viral
integrase, inhibitors of viral genome nuclear translocation such as
arylene bis(methylketone) compounds; inhibitors of HIV entry such
as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble
complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100;
nucleocapsid zinc finger inhibitors such as dithiane compounds;
targets of HIV Tat and Rev; and pharmacoenhancers such as
ABT-378.
[0641] Other antiretroviral therapies and adjunct therapies include
cytokines and lymphokines such as MIP-1.alpha., MIP-1.beta.,
SDF-1.alpha., IL-2, PROLEUKIN.TM. (aldesleukin/L2-7001; Chiron),
IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-alpha2a,
IFN-alpha2b, or IFN-beta; antagonists of TNFs, NF.kappa.B, GM-CSF,
M-CSF, and IL-10; agents that modulate immune activation such as
cyclosporin and prednisone; vaccines such as Remune.TM. (HIV
Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments,
bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN
rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL
protein, branched synthetic peptide derived from discontinuous
gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef,
and Tat vaccines; gene-based therapies such as genetic suppressor
elements (GSEs; WO 98/54366), and intrakines (genetically modified
CC chemokines targetted to the ER to block surface expression of
newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen
et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the
anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8,
PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and
RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b,
48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies,
anti-TNF-.alpha. antibodies, and monoclonal antibody 33A; aryl
hydrocarbon (AH) receptor agonists and antagonists such as TCDD,
3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and
.alpha.-naphthoflavone (WO 98/30213); and antioxidants such as
7-L-glutamyl-L-cysteine ethyl ester (.gamma.-GCE; WO 99/56764).
[0642] In a further embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with one or more antiviral agent. Antiviral agents that may be
administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
acyclovir, ribavirin, ribavirin analog, amantadine, remantidine,
maxamine, or thymalfasin. Specifically, interferon albumin fusion
protein can be administered in combination with any of these
agents. Moreover, interferon alpha albumin fusion protein can also
be administered with any of these agents, and preferably,
interferon alpha 2a or 2b albumin fusion protein can be
administered with any of these agents. Furthermore, interferon beta
albumin fusion protein can also be administered with any of these
agents. Additionally, any of the IFN hybrids albumin fusion
proteins can be administered in combination with any of these
agents.
[0643] In a most preferred embodiment, an interferon albumin fusion
protein of the invention is administered in combination with
ribavirin or a ribavirin analog. In a preferred embodiment, the
ribavirin or ribavirin analogs that may be administered in
combination with an interferon albumin fusion protein include but
are not limited to COPEGUS.RTM. (Hoffman-La Roche, Nutley, N.J.),
REBETOL.RTM. (Schereing Corp., Kenilworth, N.J.), VIRAZOLE.RTM.
(Valeant, Costa Mesa, Calif.), RIBAVIN.TM. (Lupin, Baltimore, Md.),
RIBAZID.TM. (Epla, Kirachi, Pakistan), tribavirin, VIRAMIDINE.TM.
(Valeant, Costa Mesa, Calif.), and RIBASPHERE.TM. (Three Rivers
Pharmaceuticals, Cranberry Township, Pa.). In a further preferred
embodiment, interferon alpha albumin fusion protein is administered
in combination with ribavirin or ribavirin analog. In a further
preferred embodiment, interferon alpha 2a albumin fusion protein is
administered in combination with ribavirin or ribavirin analog. In
a further preferred embodiment, interferon alpha 2b albumin fusion
protein is administered in combination with ribavirin or ribavirin
analog. In a further preferred embodiment, interferon beta albumin
fusion protein is administered in combination with ribavirin or
ribavirin analog. In a further preferred embodiment, hybrid
interferon albumin fusion protein is administered in combination
with ribavirin or ribavirin analog.
[0644] In a further embodiment, the albumin fusion proteins and/or
polynucleotides of the invention may be administered alone or in
combination with one or more antiviral agents for the treatment of
viral infection. In a preferred embodiment, an interferon-albumin
fusion protein of the invention may be administered in combination
with one or more antiviral agents. In an additional preferred
embodiment, the viral infection results from infection with a
hepatitis virus. In a most preferred embodiment, the hepatitis
virus is hepatitis C virus (HCV). Antiviral agents that may be
administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
small-molecule inhibitors of viral enzymes, small-molecule
inhibitors of RNA polymerase, nucleic acid based antiviral agents,
antisense oligonucleotide inhibitors, thiazolides, novel
immunomodulatory agents, and interferon enhancers. Anti-viral
enzyme inhibitors that may be administered in combination with the
albumin fusion proteins and/or polynucleotides of the invention
include, but are not limited to, VX-950 (protease inhibitor,
Vertex, Cambridge, Mass.), VX-497 (merimepodib, oral IMPDH
inhibitor, Vertex, Cambridge, Mass.), BILB 1941 (protease
inhibitor, Boehringer Ingelheim, Germany), SCH7 (protease
inhibitor, Schereing Corp., Kenilworth, N.J.), MX-3253 (glucosidase
inhibitor, Migenix, Vancouver, BC), IDN-6556 (caspase inhibitor,
Pfizer, New York, N.Y.), UT231B (glucosidase inhibitor, United
Therapeutics, Silver Spring, Md.), R1626 (viral protease inhibitor,
F. Hoffman-La Roche, Switzerland), ITMN-B (ITMN-191, protease
inhibitor, InterMune, Brisbane, Calif.), Celgosivir (MBI-3253,
.alpha.-glucosidase inhibitor, Migenix, Inc., Vancouver, B.C.), SCH
503034 (protease inhibitor, Schereing Corp., Kenilworth, N.J.), ACH
806 (GS9132, oral protease inhibitor, Achillion, New Haven,
Conn./Gilead Sciences, Foster City, Calif.). Anti-viral polymerase
inhibitors that may be administered in combination with the albumin
fusion proteins and/or polynucleotides of the invention may be
nucleoside analogs or non-nucleoside inhibitors (NNIs). In a
preferred embodiment, the anti-viral polymerase inhibitors inhibit
HCV RNA polymerase. In one embodiment, the anti-viral polymerase
inhibitors may be nucleoside analogs including, but not limited to,
NM283 (oral prodrug of 23'-C-methyl-cytidine, Idenix, Cambridge,
Mass.), and 2'-C-methyl nucleosides. In another embodiment, the
anti-viral polymerase inhibitors may be non-nucleoside inhibitors,
including, but not limited to, JTK-103, JTK-003, and JTK-109 (Japan
Tabacco, Tokyo, Japan), R803 (Rigel, South San Francisco, Calif.),
HCV-371, HCV-086, and HCV-796 (ViroPharm, Exton, Pa./Wyeth,
Madison, N.J.), and XTL-2125 (BC2125, XTLbio, New York, N.Y.).
Anti-viral nucleic acid based agents that may be administered in
combination with the albumin fusion proteins and/or polynucleotides
of the invention include, but are not limited to, antisense
oligonucleotides, ribozymes, and siRNAs or short hairpin RNAs
(shRNA). Anti-viral antisense oligonucleotide inhibitor agents that
may be administered in combination with the albumin fusion proteins
and/or polynucleotides of the invention include, but are not
limited to, NEUGENE.RTM. AVI-4065 (AVI Biopharma, Portland, Oreg.).
In another embodiment, a thiazolide may be administered in
combination with the albumin fusion proteins and/or polynucleotides
of the invention. In a preferred embodiment, thiazolides that may
be administered in combination with the albumin fusion proteins
and/or polynucleotides of the invention include, but are not
limited to ALINIA.RTM. (nitazoxanide, Romark Laboratories, L.C.,
Tampa, Fla.). Anti-viral immunomodulatory agents that may be
administered in combination with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
ZADXIN.RTM. (thymosin alpha 1, thymalfasin, SciClone
Pharmaceuticals Int'l, Hong Kong) and toll-like receptor (TLR)
agonists, including, but not limited to, ANA245 (TLR-7 agonist,
Anadys Pharmaceuticals, San Diego, Calif.), ANA975 (oral prodrug of
ANA245, Anadys Pharmaceuticals, San Diego, Calif.), and CPG-10101
(ACTILON.TM., TLR-9 agonist, Coley Pharmaceutical Group, Wellesley,
Mass.). Interferon enhancers that may be administered in
combination with the albumin fusion proteins and/or polynucleotides
of the invention include, but are not limited to EMZ702 (Transition
Therapeutics, Toronto, Ontario). Moreover, anti-viral antibodies
that may be administered in combination with the albumin fusion
proteins and/or polynucleotides of the invention include, but are
not limited to Tarvacin (humanized monoclonal antibody that targets
phosphatidylserine on the surface of tumor endothelial cells,
Peregrine Pharmaceuticals, Inc., Tustin, Calif.).
[0645] In a preferred embodiment the albumin fusion protein that
may be administered alone or in combination with one or more of the
antiviral agents encompassed by the invention is an
inteferon-albumin fusion protein. In additional embodiment, the
interferon portion of the interferon-albumin fusion protein is an
interferon alpha. Non-limiting examples of interferon alpha
encompassed by the invention include, but are not limited to, the
interferon alpha proteins disclosed in the Therapeutic protein
column of Table 1. In particular embodiments, the interferon alpha
portion consists or alternatively comprises interferon alpha-2a,
interferon alpha-2b, interferon alpha-2c, consensus interferon,
interferon alfacon-1, interferon alpha-n1, interferon alpha-n3, any
commercially available form of interferon alpha, such as, for
example, INTRON.RTM. A (Schereing Corp., Kenilworth, N.J.),
ROFERON.RTM. A (Hoffman-La Roche, Nutley, N.J.), Berofor alpha
inteferon (Boehringer Ingelheim Pharmaceutical, Inc., Ridgefied,
Conn.), OMNIFERON.TM. (Viragen, Inc., Plantation, Fla.),
MULTIFERON.TM. (Viragen, Inc., Plantation, Fla.) WELLFERON.RTM.
(GlaxoSmithKline, London, Great Britian), INFERGEN.RTM. (Amgen,
Inc., Thousands Oaks, Calif.), SUMIFERON.RTM. (Sumitomo, Japan),
BELEROFON.RTM. (Nautilus Biotech, France), MAXY-ALPHA.TM. (Maxygen,
Redwood City, Calif./Hoffman-La Roche, Nutley, N.J.), or any
purified interferon alpha product or a fragment thereof. In further
embodiments, the interferon alpha portion of the IFN-alpha-HSA
fusion protein consists or alternatively comprises interferon alpha
modified or formulated for extended or controlled release. For
example, the interferon alpha portion consists, or alternatively
comprises commercially available extended release or controlled
release interferon alpha, including, but not limited to
interferon-alpha-XL (Flamel Technologies, France) and LOCTERON.TM.
(BioLex Therapeutics/OctoPlus, Pittsboro, N.C.). In additional
embodiments, the interferon alpha portion of the IFN-alpha-HSA
fusion protein may be modified by the attachment of chemical
moieties. For example, the inteferon alpha portion may be modified
by pegylation. Accordingly, in additional embodiments, the
interferon alpha portion of the IFN-alpha-HSA fusion protein
consists or alternatively comprises pegylated forms of interferon
alpha-2a,2b, or consensus interferon and include, but are not
limited to, a commercially available pegylated interferon alpha,
such as, for example, PEG-INTRON.RTM. (Schereing Corp., Kenilworth,
N.J.), PEGASYS.RTM. (Hoffman-La Roche, Nutley, N.J.),
PEG-OMNIFERON.TM. (Viragen, Inc., Plantation, Fla.) or a fragment
thereof. In an additional preferred embodiment the interferon
portion of the albumin fusion protein is interferon alpha 2a or 2b
interferon, interferon albumin fusion protein can be administered
in combination with any of these agents. Moreover, in another
embodiment, the interferon portion of the interferon-albumin fusion
protein is an interferon beta or an interferon hybrids. In a
further embodiment, the unfused interferon portion of the
inteferon-albumin fusion protein may be used alone or in
combination with one or more of the antiviral agents encompassed by
the invention.
[0646] In other embodiments, albumin fusion proteins and/or
polynucleotides of the invention may be administered in combination
with anti-opportunistic infection agents. Anti-opportunistic agents
that may be administered in combination with the albumin fusion
proteins and/or polynucleotides of the invention, include, but are
not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLE.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
albumin fusion proteins and/or polynucleotides of the invention are
used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE.TM.,
DAPSONE.TM., PENTAMIDINE.TM., and/or ATOVAQUONE.TM. to
prophylactically treat or prevent an opportunistic Pneumocystis
carinii pneumonia infection. In another specific embodiment,
albumin fusion proteins and/or polynucleotides of the invention are
used in any combination with ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., and/or ETHAMBUTOL.TM. to prophylactically treat
or prevent an opportunistic Mycobacterium avium complex infection.
In another specific embodiment, albumin fusion proteins and/or
polynucleotides of the invention are used in any combination with
RIFABUTIN.TM., CLARITHROMYCIN.TM., and/or AZITHROMYCIN.TM. to
prophylactically treat or prevent an opportunistic Mycobacterium
tuberculosis infection. In another specific embodiment, albumin
fusion proteins and/or polynucleotides of the invention are used in
any combination with GANCICLOVIR.TM., FOSCARNET.TM., and/or
CIDOFOVIR.TM. to prophylactically treat or prevent an opportunistic
cytomegalovirus infection. In another specific embodiment, albumin
fusion proteins and/or polynucleotides of the invention are used in
any combination with FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or
KETOCONAZOLE.TM. to prophylactically treat or prevent an
opportunistic fungal infection. In another specific embodiment,
albumin fusion proteins and/or polynucleotides of the invention are
used in any combination with ACYCLOVIR.TM. and/or FAMCICOLVIR.TM.
to prophylactically treat or prevent an opportunistic herpes
simplex virus type I and/or type II infection. In another specific
embodiment, albumin fusion proteins and/or polynucleotides of the
invention are used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat or prevent an
opportunistic Toxoplasma gondii infection. In another specific
embodiment, albumin fusion proteins and/or polynucleotides of the
invention are used in any combination with LEUCOVORIN.TM. and/or
NEUPOGEN.TM. to prophylactically treat or prevent an opportunistic
bacterial infection.
[0647] In a further embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with an antibiotic agent. Antibiotic agents that may be
administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
amoxicillin, beta-lactamases, aminoglycosides, beta-lactam
(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,
cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones,
macrolides, metronidazole, penicillins, quinolones, rapamycin,
rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamethoxazole, and vancomycin.
[0648] In other embodiments, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with immunestimulants. Immunostimulants that may be administered in
combination with the albumin fusion proteins and/or polynucleotides
of the invention include, but are not limited to, levamisole (e.g.,
ERGAMISOL.TM.), isoprinosine (e.g. INOSIPLEX.TM.), interferons
(e.g. interferon alpha), and interleukins (e.g., IL-2).
[0649] In other embodiments, albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with immunosuppressive agents. Immunosuppressive agents that may be
administered in combination with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
steroids, cyclosporine, cyclosporine analogs, cyclophosphamide
methylprednisone, prednisone, azathioprine, FK-506,
15-deoxyspergualin, and other immunosuppressive agents that act by
suppressing the function of responding T cells. Other
immunosuppressive agents that may be administered in combination
with the albumin fusion proteins and/or polynucleotides of the
invention include, but are not limited to, prednisolone,
methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide,
mizoribine (BREDININ.TM.), brequinar, deoxyspergualin, and
azaspirane (SKF 105685), ORTHOCLONE OKT.RTM. 3 (muromonab-CD3),
SANDIMMUNE.TM., NEORAL.TM., SANGDYA.TM. (cyclosporine),
PROGRAF.RTM. (FK506, tacrolimus), CELLCEPT.RTM. (mycophenolate
motefil, of which the active metabolite is mycophenolic acid),
IMURAN.TM. (azathioprine), glucocorticosteroids, adrenocortical
steroids such as DELTASONE.TM. (prednisone) and HYDELTRASOL.TM.
(prednisolone), FOLEX.TM. and MEXATE.TM. (methotrxate),
OXSORALEN-ULTRA.TM. (methoxsalen) and RAPAMUNE.TM. (sirolimus). In
a specific embodiment, immunosuppressants may be used to prevent
rejection of organ or bone marrow transplantation.
[0650] In an additional embodiment, albumin fusion proteins and/or
polynucleotides of the invention are administered alone or in
combination with one or more intravenous immune globulin
preparations. Intravenous immune globulin preparations that may be
administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but not limited to,
GAMMAR.TM., IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM.,
ATGAM.TM. (antithymocyte glubulin), and GAMIMUNE.TM.. In a specific
embodiment, albumin fusion proteins and/or polynucleotides of the
invention are administered in combination with intravenous immune
globulin preparations in transplantation therapy (e.g., bone marrow
transplant).
[0651] In another embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered alone or as part
of a combination therapy, either in vivo to patients or in vitro to
cells, for the treatment of cancer. In a specific embodiment, the
albumin fusion proteins, particularly IL-2-albumin fusions, are
administered repeatedly during passive immunotherapy for cancer,
such as adoptive cell transfer therapy for metastatic melanoma as
described in Dudley et al. (Science Express, 19 Sep. 2002., at
www.scienceexpress.org, hereby incorporated by reference in its
entirety).
[0652] In certain embodiments, the albumin fusion proteins and/or
polynucleotides of the invention are administered alone or in
combination with an anti-inflammatory agent. Anti-inflammatory
agents that may be administered with the albumin fusion proteins
and/or polynucleotides of the invention include, but are not
limited to, corticosteroids (e.g. betamethasone, budesonide,
cortisone, dexamethasone, hydrocortisone, methylprednisolone,
prednisolone, prednisone, and triamcinolone), nonsteroidal
anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac,
fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin,
ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone,
naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac,
tenoxicam, tiaprofenic acid, and tolmetin.), as well as
antihistamines, 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.
[0653] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-angiogenic agent. Anti-angiogenic agents that may be
administered with the compositions of the invention include, but
are not limited to, Angiostatin (Entremed, Rockville, Md.),
Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol),
Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor
of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1,
Plasminogen Activator Inhibitor-2, and various forms of the lighter
"d group" transition metals.
[0654] 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.
[0655] 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.
[0656] 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.
[0657] 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.
[0658] Additional anti-angiogenic factors that may also be utilized
within the context of the present invention include Thalidomide,
(Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and
J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v
beta 3 antagonist (C. Storgard et al., J Clin. Invest. 103:47-54
(1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI)
(National Cancer Institute, Bethesda, Md.); Conbretastatin A-4
(CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin
Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap
Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London,
UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251
(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;
Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide
(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88;
Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen
(Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine);
and 5-Fluorouracil.
[0659] Anti-angiogenic agents that may be administer in combination
with the compounds of the invention may work through a variety of
mechanisms including, but not limited to, inhibiting proteolysis of
the extracellular matrix, blocking the function of endothelial
cell-extracellular matrix adhesion molecules, by antagonizing the
function of angiogenesis inducers such as growth factors, and
inhibiting integrin receptors expressed on proliferating
endothelial cells. Examples of anti-angiogenic inhibitors that
interfere with extracellular matrix proteolysis and which may be
administered in combination with the compositions of the invention
include, but are not limited to, AG-3340 (Agouron, La Jolla,
Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291
(Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East
Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and
Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic
inhibitors that act by blocking the function of endothelial
cell-extracellular matrix adhesion molecules and which may be
administered in combination with the compositons of the invention
include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt,
Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune,
Gaithersburg, Md.). Examples of anti-angiogenic agents that act by
directly antagonizing or inhibiting angiogenesis inducers and which
may be administered in combination with the compositons of the
invention include, but are not limited to, Angiozyme (Ribozyme,
Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco,
Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101
(Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn,
Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic
agents act to indirectly inhibit angiogenesis. Examples of indirect
inhibitors of angiogenesis which may be administered in combination
with the compositions of the invention include, but are not limited
to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12
(Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown
University, Washington, D.C.).
[0660] In particular embodiments, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
an autoimmune disease, such as for example, an autoimmune disease
described herein.
[0661] In a particular embodiment, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
arthritis. In a more particular embodiment, the use of compositions
of the invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
rheumatoid arthritis.
[0662] In another embodiment, the polynucleotides encoding a
polypeptide of the present invention are administered in
combination with an angiogenic protein, or polynucleotides encoding
an angiogenic protein. Examples of angiogenic proteins that may be
administered with the compositions of the invention include, but
are not limited to, acidic and basic fibroblast growth factors,
VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta,
platelet-derived endothelial cell growth factor, platelet-derived
growth factor, tumor necrosis factor alpha, hepatocyte growth
factor, insulin-like growth factor, colony stimulating factor,
macrophage colony stimulating factor, granulocyte/macrophage colony
stimulating factor, and nitric oxide synthase.
[0663] In additional embodiments, compositions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the albumin
fusion proteins and/or polynucleotides of the invention include,
but are not limited to alkylating agents such as nitrogen mustards
(for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide
Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil),
ethylenimines and methylmelamines (for example, Hexamethylmelamine
and Thiotepa), alkyl sulfonates (for example, Busulfan),
nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU),
Semustine (methyl-CCNU), and Streptozocin (streptozotocin)),
triazenes (for example, Dacarbazine (DTIC;
dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for
example, Methotrexate (amethopterin)), pyrimidine analogs (for
example, Fluorouacil (5-fluorouracil; 5-FU), Floxuridine
(fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)),
purine analogs and related inhibitors (for example, Mercaptopurine
(6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and
Pentostatin (2'-deoxycoformycin)), vinca alkaloids (for example,
Vinblastine (VLB, vinblastine sulfate)) and Vincristine
(vincristine sulfate)), epipodophyllotoxins (for example, Etoposide
and Teniposide), antibiotics (for example, Dactinomycin
(actinomycin D), Daunorubicin (daunomycin; rubidomycin),
Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin
(mitomycin C), enzymes (for example, L-Asparaginase), biological
response modifiers (for example, Interferon-alpha and
interferon-alpha-2b), platinum coordination compounds (for example,
Cisplatin (cis-DDP) and Carboplatin), anthracenedione
(Mitoxantrone), substituted ureas (for example, Hydroxyurea),
methylhydrazine derivatives (for example, Procarbazine
(N-methylhydrazine; M1H), adrenocorticosteroids (for example,
Prednisone), progestins (for example, Hydroxyprogesterone caproate,
Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol
acetate), estrogens (for example, Diethylstilbestrol (DES),
Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol),
antiestrogens (for example, Tamoxifen), androgens (Testosterone
proprionate, and Fluoxymesterone), antiandrogens (for example,
Flutamide), gonadotropin-releasing horomone analogs (for example,
Leuprolide), other hormones and hormone analogs (for example,
methyltestosterone, estramustine, estramustine phosphate sodium,
chlorotrianisene, and testolactone), and others (for example,
dicarbazine, glutamic acid, and mitotane).
[0664] In one embodiment, the compositions of the invention are
administered in combination with one or more of the following
drugs: infliximab (also known as Remicade.TM. Centocor, Inc.),
Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava.TM.
from Hoechst Marion Roussel), Kineret.TM. (an IL-1 Receptor
antagonist also known as Anakinra from Amgen, Inc.)
[0665] In a specific embodiment, compositions of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or combination of one or
more of the components of CHOP. In one embodiment, the compositions
of the invention are administered in combination with
anti-CD.sub.20 antibodies, human monoclonal anti-CD.sub.20
antibodies. In another embodiment, the compositions of the
invention are administered in combination with anti-CD.sub.20
antibodies and CHOP, or anti-CD.sub.20 antibodies and any
combination of one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. In a specific embodiment,
compositions of the invention are administered in combination with
Rituximab. In a further embodiment, compositions of the invention
are administered with Rituximab and CHOP, or Rituximab and any
combination of one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. In a specific embodiment,
compositions of the invention are administered in combination with
tositumomab. In a further embodiment, compositions of the invention
are administered with tositumomab and CHOP, or tositumomab and any
combination of one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. The anti-CD.sub.20 antibodies
may optionally be associated with radioisotopes, toxins or
cytotoxic prodrugs.
[0666] In another specific embodiment, the compositions of the
invention are administered in combination Zevalin.TM.. In a further
embodiment, compositions of the invention are administered with
Zevalin.TM. and CHOP, or Zevalin.TM. and any combination of one or
more of the components of CHOP, particularly cyclophosphamide
and/or prednisone. Zevalin.TM. may be associated with one or more
radisotopes. Particularly preferred isotopes are .sup.90Y and
.sup.111In.
[0667] In an additional embodiment, the albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with cytokines. Cytokines that may be administered with
the albumin fusion proteins and/or polynucleotides of the invention
include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha.
In another embodiment, albumin fusion proteins and/or
polynucleotides of the invention may be administered with any
interleukin, including, but not limited to, IL-1alpha, IL-1beta,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and
IL-21.
[0668] In one embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with members of the TNF family. TNF, TNF-related or TNF-like
molecules that may be administered with the albumin fusion proteins
and/or polynucleotides of the invention include, but are not
limited to, soluble forms of TNF-alpha, lymphotoxin-alpha
(LT-alpha, also known as TNF-beta), LT-beta (found in complex
heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,
4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO
96/14328), AIM-I (International Publication No. WO 97/33899),
endokine-alpha (International Publication No. WO 98/07880), OPG,
and neutrokine-alpha (International Publication No. WO 98/18921,
OX40, and nerve growth factor (NGF), and soluble forms of Fas,
CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/33904), DR4
(International Publication No. WO 98/32856), TR5 (International
Publication No. WO 98/30693), TRANK, TR9 (International Publication
No. WO 98/56892), TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153.
[0669] In an additional embodiment, the albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with angiogenic proteins. Angiogenic proteins that may
be administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
Glioma Derived Growth Factor (GDGF), as disclosed in European
Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A),
as disclosed in European Patent Number EP-682110; Platelet Derived
Growth Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (PlGF), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular
Endothelial Growth Factor B-186 (VEGF-B 186), as disclosed in
International Publication Number WO 96/26736; Vascular Endothelial
Growth Factor-D (VEGF-D), as disclosed in International Publication
Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D),
as disclosed in International Publication Number WO 98/07832; and
Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in
German Patent Number DE19639601. The above mentioned references are
herein incorporated by reference in their entireties.
[0670] In an additional embodiment, the albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with Fibroblast Growth Factors. Fibroblast Growth
Factors that may be administered with the albumin fusion proteins
and/or polynucleotides of the invention include, but are not
limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8,
FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0671] In an additional embodiment, the albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with hematopoietic growth factors. Hematopoietic growth
factors that may be administered with the albumin fusion proteins
and/or polynucleotides of the invention include, but are not
limited to, granulocyte macrophage colony stimulating factor
(GM-CSF) (sargramostim, LEUKINE.TM., PROKINE.TM.), granulocyte
colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN.TM.),
macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin
(epoetin alfa, EPOGEN.TM., PROCRIT.TM.), stem cell factor (SCF,
c-kit ligand, steel factor), megakaryocyte colony stimulating
factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins,
especially any one or more of IL-1 through IL-12, interferon-gamma,
or thrombopoietin.
[0672] In certain embodiments, albumin fusion proteins and/or
polynucleotides of the present invention are administered in
combination with adrenergic blockers, such as, for example,
acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol,
metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol,
sotalol, and timolol.
[0673] In another embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with an antiarrhythmic drug (e.g., adenosine, amidoarone,
bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide,
esmolol, flecamide, lidocaine, mexiletine, moricizine, phenyloin,
procainamide, N-acetyl procainamide, propafenone, propranolol,
quinidine, sotalol, tocamide, and verapamil).
[0674] In another embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with diuretic agents, such as carbonic anhydrase-inhibiting agents
(e.g., acetazolamide, dichlorphenamide, and methazolamide), osmotic
diuretics (e.g., glycerin, isosorbide, mannitol, and urea),
diuretics that inhibit Na.sup.+-K.sup.+-2Cl.sup.- symport (e.g.,
furosemide, bumetanide, azosemide, piretanide, tripamide,
ethacrynic acid, muzolimine, and torsemide), thiazide and
thiazide-like diuretics (e.g., bendroflumethiazide, benzthiazide,
chlorothiazide, hydrochlorothiazide, hydroflumethiazide,
methyclothiazide, polythiazide, trichormethiazide, chlorthalidone,
indapamide, metolazone, and quinethazone), potassium sparing
diuretics (e.g., amiloride and triamterene), and mineralcorticoid
receptor antagonists (e.g., spironolactone, canrenone, and
potassium canrenoate).
[0675] In one embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with treatments for endocrine and/or hormone imbalance disorders.
Treatments for endocrine and/or hormone imbalance disorders
include, but are not limited to, .sup.127I, radioactive isotopes of
iodine such as .sup.131I and .sup.123I; recombinant growth hormone,
such as HUMATROPE.TM. (recombinant somatropin); growth hormone
analogs such as PROTROPIN.TM. (somatrem); dopamine agonists such as
PARLODEL.TM. (bromocriptine); somatostatin analogs such as
SANDOSTATIN.TM. (octreotide); gonadotropin preparations such as
PREGNYL.TM., A.P.L..TM. and PROFASI.TM. (chorionic gonadotropin
(CG)), PERGONAL.TM. (menotropins), and METRODIN.TM. (urofollitropin
(uFSH)); synthetic human gonadotropin releasing hormone
preparations such as FACTREL.TM. and LUTREPULSE.TM. (gonadorelin
hydrochloride); synthetic gonadotropin agonists such as LUPRON.TM.
(leuprolide acetate), SUPPRELIN.TM. (histrelin acetate),
SYNAREL.TM. (nafarelin acetate), and ZOLADEX.TM. (goserelin
acetate); synthetic preparations of thyrotropin-releasing hormone
such as RELEFACT TRH.TM. and THYPINONE.TM. (protirelin);
recombinant human TSH such as THYROGEN.TM.; synthetic preparations
of the sodium salts of the natural isomers of thyroid hormones such
as L-T.sub.4.TM., SYNTHROID.TM. and LEVOTHROID.TM. (levothyroxine
sodium), L-T.sub.3.TM., CYTOMEL.TM. and TRIOSTAT.TM. (liothyroine
sodium), and THYROLAR.TM. (liotrix); antithyroid compounds such as
6-n-propylthiouracil (propylthiouracil),
1-methyl-2-mercaptoimidazole and TAPAZOLE.TM. (methimazole),
NEO-MERCAZOLE.TM. (carbimazole); beta-adrenergic receptor
antagonists such as propranolol and esmolol; Ca channel blockers;
dexamethasone and iodinated radiological contrast agents such as
TELEPAQUE.TM. (iopanoic acid) and ORAGRAFIN.TM. (sodium
ipodate).
[0676] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to, estrogens or congugated
estrogens such as ESTRACE.TM. (estradiol), ESTWYL.TM. (ethinyl
estradiol), PREMARIN.TM., ESTRATAB.TM., ORTHO-EST.TM., OGEN.TM. and
estropipate (estrone), ESTROVIS.TM. (quinestrol), ESTRADERM.TM.
(estradiol), DELESTROGEN.TM. and VALERGEN.TM. (estradiol valerate),
DEPO-ESTRADIOL CYPIONATE.TM. and ESTROJECT LA.TM. (estradiol
cypionate); antiestrogens such as NOLVADEX.TM. (tamoxifen),
SEROPHENE.TM. and CLOMID.TM. (clomiphene); progestins such as
DURALUTIN.TM. (hydroxyprogesterone caproate), MPA.TM. and
DEPO-PROVERA.TM. (medroxyprogesterone acetate), PROVERA.TM. and
CYCRIN.TM. (MPA), MEGACE.TM. (megestrol acetate), NORLUTIN.TM.
(norethindrone), and NORLUTATE.TM. and AYGESTIN.TM. (norethindrone
acetate); progesterone implants such as NORPLANT SYSTEM.TM.
(subdermal implants of norgestrel); antiprogestins such as RU
486.TM. (mifepristone); hormonal contraceptives such as ENOVID.TM.
(norethynodrel plus mestranol), PROGESTASERT.TM. (intrauterine
device that releases progesterone), LOESTRIN.TM., BREVICON.TM.,
MODICON.TM., GENORA.TM., NELONA.TM., NORINYL.TM., OVACON-35.TM. and
OVACON-50.TM. (ethinyl estradiol/norethindrone), LEVLEN.TM.,
NORDETTE.TM., TR1-LEVLEN.TM. and TRIPHASIL-21.TM. (ethinyl
estradiol/levonorgestrel) LO/OVRAL.TM. and OVRAL.TM. (ethinyl
estradiol/norgestrel), DEMULEN.TM. (ethinyl estradiol/ethynodiol
diacetate), NORINYL.TM., ORTHO-NOVUM.TM., NORETHIN.TM., GENORA.TM.,
and NELOVA.TM. (norethindrone/mestranol), DESOGEN.TM. and
ORTHO-CEPT.TM. (ethinyl estradiol/desogestrel), ORTHO-CYCLEN.TM.
and ORTHO-TRICYCLEN.TM. (ethinyl estradiol/norgestimate),
MICRONOR.TM. and NOR-QD.TM. (norethindrone), and OVRETTE.TM.
(norgestrel).
[0677] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to, testosterone esters such
as methenolone acetate and testosterone undecanoate; parenteral and
oral androgens such as TESTOJECT-50.TM. (testosterone), TESTEX.TM.
(testosterone propionate), DELATESTRYL.TM. (testosterone
enanthate), DEPO-TESTOSTERONE.TM. (testosterone cypionate),
DANOCRINE.TM. (danazol), HALOTESTIN.TM. (fluoxymesterone), ORETON
METHYL.TM., TESTRED.TM. and VIRILON.TM. (methyltestosterone), and
OXANDRIN.TM. (oxandrolone); testosterone transdermal systems such
as TESTODERM.TM.; androgen receptor antagonist and
5-alpha-reductase inhibitors such as ANDROCUR.TM. (cyproterone
acetate), EULEXIN.TM. (flutamide), and PROSCAR.TM. (finasteride);
adrenocorticotropic hormone preparations such as CORTROSYN.TM.
(cosyntropin); adrenocortical steroids and their synthetic analogs
such as ACLOVATE.TM. (alclometasone dipropionate), CYCLOCORT.TM.
(amcinonide), BECLOVENT.TM. and VANCERIL.TM. (beclomethasone
dipropionate), CELESTONE.TM. (betamethasone), BENISONE.TM. and
UTICORT.TM. (betamethasone benzoate), DIPROSONE.TM. (betamethasone
dipropionate), CELESTONE PHOSPHATE.TM. (betamethasone sodium
phosphate), CELESTONE SOLUSPAN.TM. (betamethasone sodium phosphate
and acetate), BETA-VAL.TM. and VALISONE.TM. (betamethasone
valerate), TEMOVATE.TM. (clobetasol propionate), CLODERM.TM.
(clocortolone pivalate), CORTEF.TM. and HYDROCORTONE.TM. (cortisol
(hydrocortisone)), HYDROCORTONE ACETATE.TM. (cortisol
(hydrocortisone) acetate), LOCOID.TM. (cortisol (hydrocortisone)
butyrate), HYDROCORTONE PHOSPHATE.TM. (cortisol (hydrocortisone)
sodium phosphate), A-HYDROCORT.TM. and SOLU CORTEF.TM. (cortisol
(hydrocortisone) sodium succinate), WESTCORT.TM. (cortisol
(hydrocortisone) valerate), CORTISONE ACETATE.TM. (cortisone
acetate), DESOWEN.TM. and TRIDESILON.TM. (desonide), TOPICORT.TM.
(desoximetasone), DECADRON.TM. (dexamethasone), DECADRON LA.TM.
(dexamethasone acetate), DECADRON PHOSPHATE.TM. and HEXADROL
PHOSPHATE.TM. (dexamethasone sodium phosphate), FLORONE.TM. and
MAXIFLOR.TM. (diflorasone diacetate), FLORINEF ACETATE.TM.
(fludrocortisone acetate), AEROBID.TM. and NASALIDE.TM.
(flunisolide), FLUONID.TM. and SYNALAR.TM. (fluocinolone
acetonide), LIDEX.TM. (fluocinonide), FLUOR-OP.TM. and FML.TM.
(fluorometholone), CORDRAN.TM. (flurandrenolide), HALOG.TM.
(halcinonide), HMS LIZUIFILM.TM. (medrysone), MEDROL.TM.
(methylprednisolone), DEPO-MEDROL.TM. and MEDROL ACETATE.TM.
(methylprednisone acetate), A-METHAPRED.TM. and SOLUMEDROL.TM.
(methylprednisolone sodium succinate), ELOCON.TM. (mometasone
furoate), HALDRONE.TM. (paramethasone acetate), DELTA-CORTEF.TM.
(prednisolone), ECONOPRED.TM. (prednisolone acetate),
HYDELTRASOL.TM. (prednisolone sodium phosphate), HYDELTRA-T.B.A.TM.
(prednisolone tebutate), DELTASONE.TM. (prednisone), ARISTOCORT.TM.
and KENACORT.TM. (triamcinolone), KENALOG.TM. (triamcinolone
acetonide), ARISTOCORT.TM. and KENACORT DIACETATE.TM.
(triamcinolone diacetate), and ARISTOSPAN.TM. (triamcinolone
hexacetonide); inhibitors of biosynthesis and action of
adrenocortical steroids such as CYTADREN.TM. (aminoglutethimide),
NIZORAL.TM. (ketoconazole), MODRASTANE.TM. (trilostane), and
METOPIRONE.TM. (metyrapone); bovine, porcine or human insulin or
mixtures thereof; insulin analogs; recombinant human insulin such
as HUMULIN.TM. and NOVOLIN.TM.; oral hypoglycemic agents such as
ORAMIDE.TM. and ORINASE.TM. (tolbutamide), DIABINESE.TM.
(chlorpropamide), TOLAMIDE.TM. and TOLINASE.TM. (tolazamide),
DYMELOR.TM. (acetohexamide), glibenclamide, MICRONASE.TM.,
DIBETA.TM. and GLYNASE.TM. (glyburide), GLUCOTROL.TM. (glipizide),
and DIAMICRON.TM. (gliclazide), GLUCOPHAGE.TM. (metformin),
ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine
or porcine glucagon; somatostatins such as SANDOSTATIN.TM.
(octreotide); and diazoxides such as PROGLYCEM.TM. (diazoxide).
[0678] In one embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with treatments for uterine motility disorders. Treatments for
uterine motility disorders include, but are not limited to,
estrogen drugs such as conjugated estrogens (e.g., PREMARIN.RTM.
and ESTRATAB.RTM.), estradiols (e.g., CLIMARA.RTM. and ALORA.RTM.),
estropipate, and chlorotrianisene; progestin drugs (e.g., AMEN.RTM.
(medroxyprogesterone), MICRONOR.RTM. (norethidrone acetate),
PROMETRIUM.RTM. progesterone, and megestrol acetate); and
estrogen/progesterone combination therapies such as, for example,
conjugated estrogens/medroxyprogesterone (e.g., PREMPRO.TM. and
PREMPHASE.RTM.) and norethindrone acetate/ethinyl estsradiol (e.g.,
FEMHRT.TM.).
[0679] In an additional embodiment, the albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with drugs effective in treating iron deficiency and
hypochromic anemias, including but not limited to, ferrous sulfate
(iron sulfate, FEOSOL.TM.), ferrous fumarate (e.g., FEOSTAT.TM.),
ferrous gluconate (e.g., FERGON.TM.), polysaccharide-iron complex
(e.g., NIFEREX.TM.), iron dextran injection (e.g., INFED.TM.),
cupric sulfate, pyroxidine, riboflavin, Vitamin B.sub.12,
cyancobalamin injection (e.g., REDISOL.TM., RUBRAMIN PC.TM.),
hydroxocobalamin, folic acid (e.g., FOLVITE.TM.), leucovorin
(folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN
(Calcium salt of leucovorin), transferrin or ferritin.
[0680] In certain embodiments, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with agents used to treat psychiatric disorders. Psychiatric drugs
that may be administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
antipsychotic agents (e.g., chlorpromazine, chlorprothixene,
clozapine, fluphenazine, haloperidol, loxapine, mesoridazine,
molindone, olanzapine, perphenazine, pimozide, quetiapine,
risperidone, thioridazine, thiothixene, trifluoperazine, and
triflupromazine), antimanic agents (e.g., carbamazepine, divalproex
sodium, lithium carbonate, and lithium citrate), antidepressants
(e.g., amitriptyline, amoxapine, bupropion, citalopram,
clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine,
imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone,
nortriptyline, paroxetine, phenelzine, protriptyline, sertraline,
tranylcypromine, trazodone, trimipramine, and venlafaxine),
antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide,
clorazepate, diazepam, halazepam, lorazepam, oxazepam, and
prazepam), and stimulants (e.g., d-amphetamine, methylphenidate,
and pemoline).
[0681] In other embodiments, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with agents used to treat neurological disorders. Neurological
agents that may be administered with the albumin fusion proteins
and/or polynucleotides of the invention include, but are not
limited to, antiepileptic agents (e.g., carbamazepine, clonazepam,
ethosuximide, phenobarbital, phenyloin, primidone, valproic acid,
divalproex sodium, felbamate, gabapentin, lamotrigine,
levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide,
diazepam, lorazepam, and clonazepam), antiparkinsonian agents
(e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine,
pergolide, ropinirole, pramipexole, benztropine; biperiden;
ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS
therapeutics (e.g. riluzole).
[0682] In another embodiment, albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with vasodilating agents and/or calcium channel blocking agents.
Vasodilating agents that may be administered with the albumin
fusion proteins and/or polynucleotides of the invention include,
but are not limited to, Angiotensin Converting Enzyme (ACE)
inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril,
cilazapril, enalapril, enalaprilat, fosinopril, lisinopril,
moexipril, perindopril, quinapril, ramipril, spirapril,
trandolapril, and nylidrin), and nitrates (e.g., isosorbide
dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of
calcium channel blocking agents that may be administered in
combination with the albumin fusion proteins and/or polynucleotides
of the invention include, but are not limited to amlodipine,
bepridil, diltiazem, felodipine, flunarizine, isradipine,
nicardipine, nifedipine, nimodipine, and verapamil.
[0683] In certain embodiments, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with treatments for gastrointestinal disorders. Treatments for
gastrointestinal disorders that may be administered with the
albumin fusion protein and/or polynucleotide of the invention
include, but are not limited to, H.sub.2 histamine receptor
antagonists (e.g., TAGAMET.TM. (cimetidine), ZANTAC.TM.
(ranitidine), PEPCID.TM. (famotidine), and AXID.TM. (nizatidine));
inhibitors of H.sup.+, K.sup.+ ATPase (e.g., PREVACID.TM.
(lansoprazole) and PRILOSEC.TM. (omeprazole)); Bismuth compounds
(e.g., PEPTO-BISMOL.TM. (bismuth subsalicylate) and DE-NOL.TM.
(bismuth subcitrate)); various antacids; sucralfate; prostaglandin
analogs (e.g. CYTOTEC.TM. (misoprostol)); muscarinic cholinergic
antagonists; laxatives (e.g., surfactant laxatives, stimulant
laxatives, saline and osmotic laxatives); antidiarrheal agents
(e.g., LOMOTIL.TM. (diphenoxylate), MOTOFEN.TM. (diphenoxin), and
IMODIUM.TM. (loperamide hydrochloride)), synthetic analogs of
somatostatin such as SANDOSTATIN.TM. (octreotide), antiemetic
agents (e.g., ZOFRAN.TM. (ondansetron), KYTRIL.TM. (granisetron
hydrochloride), tropisetron, dolasetron, metoclopramide,
chlorpromazine, perphenazine, prochlorperazine, promethazine,
thiethylperazine, triflupromazine, domperidone, haloperidol,
droperidol, trimethobenzamide, dexamethasone, methylprednisolone,
dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide,
trimethobenzamide and chlorpromazine); bile salts; chenodeoxycholic
acid; ursodeoxycholic acid; and pancreatic enzyme preparations such
as pancreatin and pancrelipase.
[0684] In additional embodiments, the albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with other therapeutic or prophylactic regimens, such
as, for example, radiation therapy.
[0685] 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 comprising albumin
fusion proteins of the invention. Optionally associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0686] Gene Therapy
[0687] Constructs encoding albumin fusion proteins of the invention
can be used as a part of a gene therapy protocol to deliver
therapeutically effective doses of the albumin fusion protein. A
preferred approach for in vivo introduction of nucleic acid into a
cell is by use of a viral vector containing nucleic acid, encoding
an albumin fusion protein of the invention. Infection of cells with
a viral vector has the advantage that a large proportion of the
targeted cells can receive the nucleic acid. Additionally,
molecules encoded within the viral vector, e.g., by a cDNA
contained in the viral vector, are expressed efficiently in cells
which have taken up viral vector nucleic acid.
[0688] Retrovirus vectors and adeno-associated virus vectors can be
used as a recombinant gene delivery system for the transfer of
exogenous nucleic acid molecules encoding albumin fusion proteins
in vivo. These vectors provide efficient delivery of nucleic acids
into cells, and the transferred nucleic acids are stably integrated
into the chromosomal DNA of the host. The development of
specialized cell lines (termed "packaging cells") which produce
only replication-defective retroviruses has increased the utility
of retroviruses for gene therapy, and defective retroviruses are
characterized for use in gene transfer for gene therapy purposes
(for a review see Miller, A. D. (1990) Blood 76:271). A replication
defective retrovirus can be packaged into virions which can be used
to infect a target cell through the use of a helper virus by
standard techniques. Protocols for producing recombinant
retroviruses and for infecting cells in vitro or in vivo with such
viruses can be found in Current Protocols in Molecular Biology,
Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989),
Sections 9.10-9.14 and other standard laboratory manuals.
[0689] Another viral gene delivery system useful in the present
invention uses adenovirus-derived vectors. The genome of an
adenovirus can be manipulated such that it encodes and expresses a
gene product of interest but is inactivated in terms of its ability
to replicate in a normal lytic viral life cycle. See, for example,
Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al.,
Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155
(1992). Suitable adenoviral vectors derived from the adenovirus
strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2,
Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant
adenoviruses can be advantageous in certain circumstances in that
they are not capable of infecting nondividing cells and can be used
to infect a wide variety of cell types, including epithelial cells
(Rosenfeld et al., (1992) cited supra). Furthermore, the virus
particle is relatively stable and amenable to purification and
concentration, and as above, can be modified so as to affect the
spectrum of infectivity. Additionally, introduced adenoviral DNA
(and foreign DNA contained therein) is not integrated into the
genome of a host cell but remains episomal, thereby avoiding
potential problems that can occur as a result of insertional
mutagenesis in situations where introduced DNA becomes integrated
into the host genome (e.g., retroviral DNA). Moreover, the carrying
capacity of the adenoviral genome for foreign DNA is large (up to 8
kilobases) relative to other gene delivery vectors (Berkner et al.,
cited supra; Haj-Ahmand et al., J. Virol. 57:267 (1986)).
[0690] In another embodiment, non-viral gene delivery systems of
the present invention rely on endocytic pathways for the uptake of
the subject nucleotide molecule by the targeted cell. Exemplary
gene delivery systems of this type include liposomal derived
systems, poly-lysine conjugates, and artificial viral envelopes. In
a representative embodiment, a nucleic acid molecule encoding an
albumin fusion protein of the invention can be entrapped in
liposomes bearing positive charges on their surface (e.g.,
lipofectins) and (optionally) which are tagged with antibodies
against cell surface antigens of the target tissue (Mizuno et al.
(1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309;
Japanese patent application 1047381; and European patent
publication EP-A-43075).
[0691] Gene delivery systems for a gene encoding an albumin fusion
protein of the invention can be introduced into a patient by any of
a number of methods. For instance, a pharmaceutical preparation of
the gene delivery system can be introduced systemically, e.g. by
intravenous injection, and specific transduction of the protein in
the target cells occurs predominantly from specificity of
transfection provided by the gene delivery vehicle, cell-type or
tissue-type expression due to the transcriptional regulatory
sequences controlling expression of the receptor gene, or a
combination thereof. In other embodiments, initial delivery of the
recombinant gene is more limited with introduction into the animal
being quite localized. For example, the gene delivery vehicle can
be introduced by catheter (see U.S. Pat. No. 5,328,470) or by
Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3 054-3 05
7). The pharmaceutical preparation of the gene therapy construct
can consist essentially of the gene delivery system in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Where the albumin fusion
protein can be produced intact from recombinant cells, e.g.
retroviral vectors, the pharmaceutical preparation can comprise one
or more cells which produce the albumin fusion protein.
[0692] Additional Gene Therapy Methods
[0693] Also encompassed by the invention are gene therapy methods
for treating or preventing disorders, diseases and conditions. The
gene therapy methods relate to the introduction of nucleic acid
(DNA, RNA and antisense DNA or RNA) sequences into an animal to
achieve expression of an albumin fusion protein of the invention.
This method requires a polynucleotide which codes for an albumin
fusion protein of the present invention operatively linked to a
promoter and any other genetic elements necessary for the
expression of the fusion protein by the target tissue. Such gene
therapy and delivery techniques are known in the art, see, for
example, WO90/11092, which is herein incorporated by reference.
[0694] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a polynucleotide encoding an albumin fusion protein of
the present invention ex vivo, with the engineered cells then being
provided to a patient to be treated with the fusion protein of the
present invention. Such methods are well-known in the art. For
example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85:
207-216 (1993); Ferrantini, M. et al., Cancer Research 53:
1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:
4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229
(1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);
Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996);
Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and
Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which
are herein incorporated by reference. In one embodiment, the cells
which are engineered are arterial cells. The arterial cells may be
reintroduced into the patient through direct injection to the
artery, the tissues surrounding the artery, or through catheter
injection.
[0695] As discussed in more detail below, the polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
[0696] In one embodiment, polynucleotides encoding the albumin
fusion proteins of the present invention is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers
to sequences that are free from any delivery vehicle that acts to
assist, promote or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, lipofectin or
precipitating agents and the like. However, polynucleotides
encoding the albumin fusion proteins of the present invention can
also be delivered in liposome formulations and lipofectin
formulations and the like can be prepared by methods well known to
those skilled in the art. Such methods are described, for example,
in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are
herein incorporated by reference.
[0697] The polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44,
pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2
available from Invitrogen. Other suitable vectors will be readily
apparent to the skilled artisan.
[0698] Any strong promoter known to those skilled in the art can be
used for driving the expression of the polynucleotide sequence.
Suitable promoters include adenoviral promoters, such as the
adenoviral major late promoter; or heterologous promoters, such as
the cytomegalovirus (CMV) promoter; the respiratory syncytial virus
(RSV) promoter; inducible promoters, such as the MMT promoter, the
metallothionein promoter; heat shock promoters; the albumin
promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter for the gene corresponding to the Therapeutic protein
portion of the albumin fusion proteins of the invention.
[0699] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0700] The polynucleotide construct can be delivered to the
interstitial space of tissues within the an animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular, fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[0701] For the naked nucleic acid sequence injection, an effective
dosage amount of DNA or RNA will be in the range of from about 0.05
mg/kg body weight to about 50 mg/kg body weight. Preferably the
dosage will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[0702] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
DNA constructs can be delivered to arteries during angioplasty by
the catheter used in the procedure.
[0703] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[0704] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
lipofectin, precipitating agents, etc. Such methods of delivery are
known in the art.
[0705] In certain embodiments, the polynucleotide constructs are
complexed in a liposome preparation. Liposomal preparations for use
in the instant invention include cationic (positively charged),
anionic (negatively charged) and neutral preparations. However,
cationic liposomes are particularly preferred because a tight
charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner et al.,
Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein
incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad.
Sci. USA (1989) 86:6077-6081, which is herein incorporated by
reference); and purified transcription factors (Debs et al., J.
Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by
reference), in functional form.
[0706] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner
et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is
herein incorporated by reference). Other commercially available
liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0707] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication No. WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[0708] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0709] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15 degrees celcius. Alternatively, negatively
charged vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[0710] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983),
101:512-527, which is herein incorporated by reference. For
example, MLVs containing nucleic acid can be prepared by depositing
a thin film of phospholipid on the walls of a glass tube and
subsequently hydrating with a solution of the material to be
encapsulated. SUVs are prepared by extended sonication of MLVs to
produce a homogeneous population of unilamellar liposomes. The
material to be entrapped is added to a suspension of preformed MLVs
and then sonicated. When using liposomes containing cationic
lipids, the dried lipid film is resuspended in an appropriate
solution such as sterile water or an isotonic buffer solution such
as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are
mixed directly with the DNA. The liposome and DNA form a very
stable complex due to binding of the positively charged liposomes
to the cationic DNA. SUVs find use with small nucleic acid
fragments. LUVs are prepared by a number of methods, well known in
the art. Commonly used methods include Ca.sup.2+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and
Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al.,
Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc.
Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H.
and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979));
and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl.
Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science
215:166 (1982)), which are herein incorporated by reference.
[0711] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[0712] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication no. WO 94/9469 provide methods for
delivering DNA-cationic lipid complexes to mammals.
[0713] In certain embodiments, cells are engineered, ex vivo or in
vivo, using a retroviral particle containing RNA which comprises a
sequence encoding an albumin fusion protein of the present
invention. Retroviruses from which the retroviral plasmid vectors
may be derived include, but are not limited to, Moloney Murine
Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey
Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus,
human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and
mammary tumor virus.
[0714] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0715] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding an albumin
fusion protein of the present invention. Such retroviral vector
particles then may be employed, to transduce eukaryotic cells,
either in vitro or in vivo. The transduced eukaryotic cells will
express a fusion protin of the present invention.
[0716] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with polynucleotide contained in an adenovirus vector.
Adenovirus can be manipulated such that it encodes and expresses
fusion protein of the present invention, and at the same time is
inactivated in terms of its ability to replicate in a normal lytic
viral life cycle. Adenovirus expression is achieved without
integration of the viral DNA into the host cell chromosome, thereby
alleviating concerns about insertional mutagenesis. Furthermore,
adenoviruses have been used as live enteric vaccines for many years
with an excellent safety profile (Schwartz et al. Am. Rev. Respir.
Dis. 109:233-238 (1974)). Finally, adenovirus mediated gene
transfer has been demonstrated in a number of instances including
transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton
rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld
et al., (1992) Cell 68:143-155). Furthermore, extensive studies to
attempt to establish adenovirus as a causative agent in human
cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.
Acad. Sci. USA 76:6606).
[0717] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155
(1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature
365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2
is useful and can be grown in human 293 cells. These cells contain
the E1 region of adenovirus and constitutively express E1a and E1b,
which complement the defective adenoviruses by providing the
products of the genes deleted from the vector. In addition to Ad2,
other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also
useful in the present invention.
[0718] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, but cannot replicate in most
cells. Replication deficient adenoviruses may be deleted in one or
more of all or a portion of the following genes: E1a, E1b, E3, E4,
E2a, or L1 through L5.
[0719] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, N., Curr. Topics in
Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0720] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The
polynucleotide construct is inserted into the AAV vector using
standard cloning methods, such as those found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
(1989). The recombinant AAV vector is then transfected into
packaging cells which are infected with a helper virus, using any
standard technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
polynucleotide construct. These viral particles are then used to
transduce eukaryotic cells, either ex vivo or in vivo. The
transduced cells will contain the polynucleotide construct
integrated into its genome, and will express a fusion protein of
the invention.
[0721] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding a polypeptide of the present invention)
via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670,
issued Jun. 24, 1997; International Publication No. WO 96/29411,
published Sep. 26, 1996; International Publication No. WO 94/12650,
published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438
(1989), which are herein encorporated by reference. This method
involves the activation of a gene which is present in the target
cells, but which is not normally expressed in the cells, or is
expressed at a lower level than desired.
[0722] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the desired endogenous polynucleotide sequence
so the promoter will be operably linked to the endogenous sequence
upon homologous recombination.
[0723] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0724] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0725] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous sequence
is placed under the control of the promoter. The promoter then
drives the expression of the endogenous sequence.
[0726] The polynucleotide encoding an albumin fusion protein of the
present invention may contain a secretory signal sequence that
facilitates secretion of the protein. Typically, the signal
sequence is positioned in the coding region of the polynucleotide
to be expressed towards or at the 5' end of the coding region. The
signal sequence may be homologous or heterologous to the
polynucleotide of interest and may be homologous or heterologous to
the cells to be transfected. Additionally, the signal sequence may
be chemically synthesized using methods known in the art.
[0727] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill) pharmaceutical formulations, and decanting or
topical applications during surgery. For example, direct injection
of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers
(Kaneda et al., Science 243:375 (1989)).
[0728] A preferred method of local administration is by direct
injection. Preferably, an albumin fusion protein of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0729] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0730] Therapeutic compositions useful in systemic administration,
include fusion proteins of the present invention complexed to a
targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site. In specific embodiments, suitable delivery
vehicles for use with systemic administration comprise liposomes
comprising albumin fusion proteins of the invention for targeting
the vehicle to a particular site.
[0731] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[0732] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
[0733] Albumin fusion proteins of the present invention can be
administered to any animal, preferably to mammals and birds.
Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle, horses and pigs, with humans being particularly
preferred.
[0734] Biological Activities
[0735] Albumin fusion proteins and/or polynucleotides encoding
albumin fusion proteins of the present invention, can be used in
assays to test for one or more biological activities. If an albumin
fusion protein and/or polynucleotide exhibits an activity in a
particular assay, it is likely that the Therapeutic protein
corresponding to the fusion protein may be involved in the diseases
associated with the biological activity. Thus, the fusion protein
could be used to treat the associated disease.
[0736] In preferred embodiments, the present invention encompasses
a method of treating a disease or disorder listed in the "Preferred
Indication Y" column of Table 1 comprising administering to a
patient in which such treatment, prevention or amelioration is
desired an albumin fusion protein of the invention that comprises a
Therapeutic protein portion corresponding to a Therapeutic protein
disclosed in the "Therapeutic Protein X" column of Table 1 (in the
same row as the disease or disorder to be treated is listed in the
"Preferred Indication Y" column of Table 1) in an amount effective
to treat, prevent or ameliorate the disease or disorder.
[0737] In a further preferred embodiment, the present invention
encompasses a method of treating a disease or disorder listed for a
particular Therapeutic protein in the "Preferred Indication:Y"
column of Table 1 comprising administering to a patient in which
such treatment, prevention or amelioration is desired an albumin
fusion protein of the invention that comprises a Therapeutic
protein portion corresponding to the Therapeutic protein for which
the indications in the Examples are related in an amount effective
to treat, prevent or ameliorate the disease or disorder.
[0738] Specifically contemplated by the present invention are
albumin fusion proteins produced by a cell when encoded by the
polynucleotides that encode SEQ ID NO:Y. When these polynucleotides
are used to express the encoded protein from a cell, the cell's
natural secretion and processing steps produces a protein that
lacks the signal sequence explicitly listed in columns 4 and/or 11
of Table 2. The specific amino acid sequence of the listed signal
sequence is shown in the specification or is well known in the art.
Thus, most preferred embodiments of the present invention include
the albumin fusion protein produced by a cell (which would lack the
leader sequence shown in columns 4 and/or 11 of Table 2). Also most
preferred are polypeptides comprising SEQ ID NO:Y without the
specific leader sequence listed in columns 4 and/or 11 of Table 2.
Compositions comprising these two preferred embodiments, including
pharmaceutical compositions, are also preferred. These albumin
fusion proteins are specifically contemplated to treat, prevent, or
ameliorate a disease or disorder listed for a particular
Therapeutic protein in the "Preferred Indication:Y" column of Table
1.
[0739] In preferred embodiments, fusion proteins of the present
invention may be used in the diagnosis, prognosis, prevention
and/or treatment of diseases and/or disorders relating to diseases
and disorders of the endocrine system (see, for example, "Endocrine
Disorders" section below), the nervous system (see, for example,
"Neurological Disorders" section below), the immune system (see,
for example, "Immune Activity" section below), respiratory system
(see, for example, "Respiratory Disorders" section below),
cardiovascular system (see, for example, "Cardiovascular Disorders"
section below), reproductive system (see, for example,
"Reproductive System Disorders" section below) digestive system
(see, for example, "Gastrointestinal Disorders" section below),
diseases and/or disorders relating to cell proliferation (see, for
example, "Hyperproliferative Disorders" section below), and/or
diseases or disorders relating to the blood (see, for example,
"Blood-Related Disorders" section below).
[0740] In certain embodiments, an albumin fusion protein of the
present invention may be used to diagnose and/or prognose diseases
and/or disorders associated with the tissue(s) in which the gene
corresponding to the Therapeutic protein portion of the fusion
protein of the invention is expressed.
[0741] Thus, fusion proteins of the invention and polynucleotides
encoding albumin fusion proteins of the invention are useful in the
diagnosis, detection and/or treatment of diseases and/or disorders
associated with activities that include, but are not limited to,
prohormone activation, neurotransmitter activity, cellular
signaling, cellular proliferation, cellular differentiation, and
cell migration.
[0742] More generally, fusion proteins of the invention and
polynucleotides encoding albumin fusion proteins of the invention
may be useful for the diagnosis, prognosis, prevention and/or
treatment of diseases and/or disorders associated with the
following systems.
[0743] Immune Activity
[0744] Albumin fusion proteins of the invention and polynucleotides
encoding albumin fusion proteins of the invention may be useful in
treating, preventing, diagnosing and/or prognosing diseases,
disorders, and/or conditions of the immune system, by, for example,
activating or inhibiting the proliferation, differentiation, or
mobilization (chemotaxis) of immune cells. Immune cells develop
through a process called hematopoiesis, producing myeloid
(platelets, red blood cells, neutrophils, and macrophages) and
lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
The etiology of these immune diseases, disorders, and/or conditions
may be genetic, somatic, such as cancer and some autoimmune
diseases, acquired (e.g., by chemotherapy or toxins), or
infectious. Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used as a marker or detector of a particular immune system
disease or disorder.
[0745] In another embodiment, a fusion protein of the invention
and/or polynucleotide encoding an albumin fusion protein of the
invention, may be used to treat diseases and disorders of the
immune system and/or to inhibit or enhance an immune response
generated by cells associated with the tissue(s) in which the
polypeptide of the invention is expressed.
[0746] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, diagnosing, and/or
prognosing immunodeficiencies, including both congenital and
acquired immunodeficiencies. Examples of B cell immunodeficiencies
in which immunoglobulin levels B cell function and/or B cell
numbers are decreased include: X-linked agammaglobulinemia
(Bruton's disease), X-linked infantile agammaglobulinemia, X-linked
immunodeficiency with hyper IgM, non X-linked immunodeficiency with
hyper IgM, X-linked lymphoproliferative syndrome (XLP),
agammaglobulinemia including congenital and acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
unspecified hypogammaglobulinemia, recessive agammaglobulinemia
(Swiss type), Selective IgM deficiency, selective IgA deficiency,
selective IgG subclass deficiencies, IgG subclass deficiency (with
or without IgA deficiency), Ig deficiency with increased IgM, IgG
and IgA deficiency with increased IgM, antibody deficiency with
normal or elevated Igs, Ig heavy chain deletions, kappa chain
deficiency, B cell lymphoproliferative disorder (BLPD), common
variable immunodeficiency (CVID), common variable immunodeficiency
(CVI) (acquired), and transient hypogammaglobulinemia of
infancy.
[0747] In specific embodiments, ataxia-telangiectasia or conditions
associated with ataxia-telangiectasia are treated, prevented,
diagnosed, and/or prognosing using the, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention.
[0748] Examples of congenital immunodeficiencies in which T cell
and/or B cell function and/or number is decreased include, but are
not limited to: DiGeorge anomaly, severe combined
immunodeficiencies (SCID) (including, but not limited to, X-linked
SCID, autosomal recessive SCID, adenosine deaminase deficiency,
purine nucleoside phosphorylase (PNP) deficiency, Class II MHC
deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome,
and ataxia telangiectasia), thymic hypoplasia, third and fourth
pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous
candidiasis, natural killer cell deficiency (NK), idiopathic CD4+
T-lymphocytopenia, immunodeficiency with predominant T cell defect
(unspecified), and unspecified immunodeficiency of cell mediated
immunity.
[0749] In specific embodiments, DiGeorge anomaly or conditions
associated with DiGeorge anomaly are treated, prevented, diagnosed,
and/or prognosed using fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the
invention.
[0750] Other immunodeficiencies that may be treated, prevented,
diagnosed, and/or prognosed using fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, include, but are not limited to, chronic granulomatous
disease, Chediak-Higashi syndrome, myeloperoxidase deficiency,
leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked
lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency,
complement component deficiencies (including C1, C2, C3, C4, C5,
C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic
alymphoplasia-aplasia, immunodeficiency with thymoma, severe
congenital leukopenia, dysplasia with immunodeficiency, neonatal
neutropenia, short limbed dwarfism, and Nezelof syndrome-combined
immunodeficiency with Igs.
[0751] In a preferred embodiment, the immunodeficiencies and/or
conditions associated with the immunodeficiencies recited above are
treated, prevented, diagnosed and/or prognosed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention.
[0752] In a preferred embodiment fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention could be used as an agent to boost immunoresponsiveness
among immunodeficient individuals. In specific embodiments, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention could be used as an agent to boost
immunoresponsiveness among B cell and/or T cell immunodeficient
individuals.
[0753] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, diagnosing and/or prognosing
autoimmune disorders. 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 fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
that can inhibit an immune response, particularly the
proliferation, differentiation, or chemotaxis of T-cells, may be an
effective therapy in preventing autoimmune disorders.
[0754] Autoimmune diseases or disorders that may be treated,
prevented, diagnosed and/or prognosed by fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to, one or more of
the following: systemic lupus erythematosus, rheumatoid arthritis,
ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis,
Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic
anemia, thrombocytopenia, autoimmune thrombocytopenia purpura,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, purpura (e.g., Henloch-Scoenlein purpura),
autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris,
myasthenia gravis, Grave's disease (hyperthyroidism), and
insulin-resistant diabetes mellitus.
[0755] Additional disorders that are likely to have an autoimmune
component that may be treated, prevented, and/or diagnosed with the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention include, but are
not limited to, type II collagen-induced arthritis,
antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,
myocarditis, relapsing polychondritis, rheumatic heart disease,
neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's
Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation,
autism, Guillain-Barre Syndrome, insulin dependent diabetes
mellitus, and autoimmune inflammatory eye disorders.
[0756] Additional disorders that are likely to have an autoimmune
component that may be treated, prevented, diagnosed and/or
prognosed with the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to, scleroderma with anti-collagen
antibodies (often characterized, e.g., by nucleolar and other
nuclear antibodies), mixed connective tissue disease (often
characterized, e.g., by antibodies to extractable nuclear antigens
(e.g., ribonucleoprotein)), polymyositis (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),
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 mellitus (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).
[0757] Additional disorders that may have an autoimmune component
that may be treated, prevented, diagnosed and/or prognosed with the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention include, but are
not limited to, chronic active hepatitis (often characterized,
e.g., by smooth muscle antibodies), primary biliary cirrhosis
(often characterized, e.g., by mitochondria 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), and
many other inflammatory, granulomatous, degenerative, and atrophic
disorders.
[0758] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, diagnosed and/or
prognosed using for example, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention. In a specific preferred embodiment, rheumatoid arthritis
is treated, prevented, and/or diagnosed using fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention.
[0759] In another specific preferred embodiment, systemic lupus
erythematosus is treated, prevented, and/or diagnosed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention. In another specific preferred
embodiment, idiopathic thrombocytopenia purpura is treated,
prevented, and/or diagnosed using fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention.
[0760] In another specific preferred embodiment IgA nephropathy is
treated, prevented, and/or diagnosed using fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention.
[0761] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, diagnosed and/or
prognosed using fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the
invention.
[0762] In preferred embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used as a immunosuppressive agent(s).
[0763] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, prognosing, and/or
diagnosing diseases, disorders, and/or conditions of hematopoietic
cells. Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
could be used to increase differentiation and proliferation of
hematopoietic cells, including the pluripotent stem cells, in an
effort to treat or prevent those diseases, disorders, and/or
conditions associated with a decrease in certain (or many) types
hematopoietic cells, including but not limited to, leukopenia,
neutropenia, anemia, and thrombocytopenia. Alternatively, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention could be used to increase
differentiation and proliferation of hematopoietic cells, including
the pluripotent stem cells, in an effort to treat or prevent those
diseases, disorders, and/or conditions associated with an increase
in certain (or many) types of hematopoietic cells, including but
not limited to, histiocytosis.
[0764] Allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated, prevented, diagnosed and/or prognosed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention. Moreover, these molecules can be
used to treat, prevent, prognose, and/or diagnose anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[0765] Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose and/or prognose
IgE-mediated allergic reactions. Such allergic reactions include,
but are not limited to, asthma, rhinitis, and eczema. In specific
embodiments, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to modulate IgE concentrations in vitro or in vivo.
[0766] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
have uses in the diagnosis, prognosis, prevention, and/or treatment
of inflammatory conditions. For example, since fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may inhibit the activation, proliferation
and/or differentiation of cells involved in an inflammatory
response, these molecules can be used to prevent and/or treat
chronic and acute inflammatory conditions. Such inflammatory
conditions include, but are not limited to, for example,
inflammation associated with infection (e.g., septic shock, sepsis,
or systemic inflammatory response syndrome), ischemia-reperfusion
injury, endotoxin lethality, complement-mediated hyperacute
rejection, nephritis, cytokine or chemokine induced lung injury,
inflammatory bowel disease, Crohn's disease, over production of
cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma
and allergy); gastrointestinal disorders (e.g., inflammatory bowel
disease); cancers (e.g., gastric, ovarian, lung, bladder, liver,
and breast); CNS disorders (e.g., multiple sclerosis; ischemic
brain injury and/or stroke, traumatic brain injury,
neurodegenerative disorders (e.g., Parkinson's disease and
Alzheimer's disease); AIDS-related dementia; and prion disease);
cardiovascular disorders (e.g., atherosclerosis, myocarditis,
cardiovascular disease, and cardiopulmonary bypass complications);
as well as many additional diseases, conditions, and disorders that
are characterized by inflammation (e.g., hepatitis, rheumatoid
arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis,
renal ischemia-reperfusion injury, Grave's disease, systemic lupus
erythematosus, diabetes mellitus, and allogenic transplant
rejection).
[0767] Because inflammation is a fundamental defense mechanism,
inflammatory disorders can effect virtually any tissue of the body.
Accordingly, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
have uses in the treatment of tissue-specific inflammatory
disorders, including, but not limited to, adrenalitis, alveolitis,
angiocholecystitis, appendicitis, balanitis, blepharitis,
bronchitis, bursitis, carditis, cellulitis, cervicitis,
cholecystitis, chorditis, cochlitis, colitis, conjunctivitis,
cystitis, dermatitis, diverticulitis, encephalitis, endocarditis,
esophagitis, eustachitis, fibrositis, folliculitis, gastritis,
gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis,
labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis,
meningitis, metritis, mucitis, myocarditis, myosititis, myringitis,
nephritis, neuritis, orchitis, osteochondritis, otitis,
pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis,
poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis,
salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis,
spondylitis, steatitis, stomatitis, synovitis, syringitis,
tendonitis, tonsillitis, urethritis, and vaginitis.
[0768] In specific embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, are useful to diagnose, prognose, prevent, and/or treat
organ transplant rejections and graft-versus-host disease. 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.
Polypeptides, antibodies, or polynucleotides of the invention,
and/or agonists or antagonists thereof, that inhibit an immune
response, particularly the activation, proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD. In specific
embodiments, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
that inhibit an immune response, particularly the activation,
proliferation, differentiation, or chemotaxis of T-cells, may be an
effective therapy in preventing experimental allergic and
hyperacute xenograft rejection.
[0769] In other embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, are useful to diagnose, prognose, prevent, and/or treat
immune complex diseases, including, but not limited to, serum
sickness, post streptococcal glomerulonephritis, polyarteritis
nodosa, and immune complex-induced vasculitis.
[0770] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used to treat, detect, and/or prevent infectious agents. For
example, by increasing the immune response, particularly increasing
the proliferation activation and/or differentiation of B and/or T
cells, infectious diseases may be treated, detected, and/or
prevented. The immune response may be increased by either enhancing
an existing immune response, or by initiating a new immune
response. Alternatively, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also directly inhibit the infectious agent (refer to section of
application listing infectious agents, etc), without necessarily
eliciting an immune response.
[0771] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used as a vaccine adjuvant that enhances
immune responsiveness to an antigen. In a specific embodiment,
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention are used as an
adjuvant to enhance tumor-specific immune responses.
[0772] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an adjuvant to enhance
anti-viral immune responses. Anti-viral immune responses that may
be enhanced using the compositions of the invention as an adjuvant,
include virus and virus associated diseases or symptoms described
herein or otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a virus, disease, or symptom selected from the
group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis
(e.g., hepatitis B). In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a virus, disease, or symptom selected from the
group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue,
rotavirus, Japanese B encephalitis, influenza A and B,
parainfluenza, measles, cytomegalovirus, rabies, Junin,
Chikungunya, Rift Valley Fever, herpes simplex, and yellow
fever.
[0773] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an adjuvant to enhance
anti-bacterial or anti-fungal immune responses. Anti-bacterial or
anti-fungal immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include bacteria or
fungus and bacteria or fungus associated diseases or symptoms
described herein or otherwise known in the art. In specific
embodiments, the compositions of the invention are used as an
adjuvant to enhance an immune response to a bacteria or fungus,
disease, or symptom selected from the group consisting of: tetanus,
Diphtheria, botulism, and meningitis type B.
[0774] In another specific embodiment, the compositions of the
invention are used as an adjuvant to enhance an immune response to
a bacteria or fungus, disease, or symptom selected from the group
consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella
typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus
pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic
Escherichia coli, Enterohemorrhagic E. coli, and Borrelia
burgdorferi.
[0775] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an adjuvant to enhance
anti-parasitic immune responses. Anti-parasitic immune responses
that may be enhanced using the compositions of the invention as an
adjuvant, include parasite and parasite associated diseases or
symptoms described herein or otherwise known in the art. In
specific embodiments, the compositions of the invention are used as
an adjuvant to enhance an immune response to a parasite. In another
specific embodiment, the compositions of the invention are used as
an adjuvant to enhance an immune response to Plasmodium (malaria)
or Leishmania.
[0776] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may also be employed to treat infectious
diseases including silicosis, sarcoidosis, and idiopathic pulmonary
fibrosis; for example, by preventing the recruitment and activation
of mononuclear phagocytes.
[0777] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an antigen for the generation
of antibodies to inhibit or enhance immune mediated responses
against polypeptides of the invention.
[0778] In one embodiment, albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are administered 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 and immunoglobulin class
switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an
immune response.
[0779] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a stimulator of B cell
responsiveness to pathogens.
[0780] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an activator of T cells.
[0781] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent that elevates the
immune status of an individual prior to their receipt of
immunosuppressive therapies.
[0782] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to induce higher
affinity antibodies.
[0783] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to increase serum
immunoglobulin concentrations.
[0784] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to accelerate
recovery of immunocompromised individuals.
[0785] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to boost
immunoresponsiveness among aged populations and/or neonates.
[0786] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an immune system enhancer
prior to, during, or after bone marrow transplant and/or other
transplants (e.g., allogeneic or xenogeneic organ transplantation).
With respect to transplantation, compositions of the invention may
be administered prior to, concomitant with, and/or after
transplantation. In a specific embodiment, compositions of the
invention are administered after transplantation, prior to the
beginning of recovery of T-cell populations. In another specific
embodiment, compositions of the invention are first administered
after transplantation after the beginning of recovery of T cell
populations, but prior to full recovery of B cell populations.
[0787] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to boost
immunoresponsiveness among individuals having an acquired loss of B
cell function. Conditions resulting in an acquired loss of B cell
function that may be ameliorated or treated by administering the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, include, but are
not limited to, HIV Infection, AIDS, bone marrow transplant, and B
cell chronic lymphocytic leukemia (CLL).
[0788] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to boost
immunoresponsiveness among individuals having a temporary immune
deficiency. Conditions resulting in a temporary immune deficiency
that may be ameliorated or treated by administering the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, 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, and recovery from
surgery.
[0789] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a regulator of antigen
presentation by monocytes, dendritic cells, and/or B-cells. In one
embodiment, albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
enhance antigen presentation or antagonize antigen presentation in
vitro or in vivo. Moreover, in related embodiments, this
enhancement or antagonism of antigen presentation may be useful as
an anti-tumor treatment or to modulate the immune system.
[0790] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used 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.
[0791] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used 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.
[0792] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a stimulator of B cell
production in pathologies such as AIDS, chronic lymphocyte disorder
and/or Common Variable Immunodificiency.
[0793] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for generation
and/or regeneration of lymphoid tissues following surgery, trauma
or genetic defect. In another specific embodiment, albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention are used in the pretreatment of
bone marrow samples prior to transplant.
[0794] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a gene-based therapy for
genetically inherited disorders resulting in
immuno-incompetence/immunodeficiency such as observed among SCID
patients.
[0795] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of activating
monocytes/macrophages to defend against parasitic diseases that
effect monocytes such as Leishmania.
[0796] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of regulating
secreted cytokines that are elicited by polypeptides of the
invention.
[0797] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used in one or more of the applications
described herein, as they may apply to veterinary medicine.
[0798] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of blocking various
aspects of immune responses to foreign agents or self. Examples of
diseases or conditions in which blocking of certain aspects of
immune responses may be desired include autoimmune disorders such
as lupus, and arthritis, as well as immunoresponsiveness to skin
allergies, inflammation, bowel disease, injury and
diseases/disorders associated with pathogens.
[0799] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for preventing the
B cell proliferation and Ig secretion associated with autoimmune
diseases such as idiopathic thrombocytopenic purpura, systemic
lupus erythematosus and multiple sclerosis.
[0800] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used as a inhibitor of
B and/or T cell migration in endothelial cells. This activity
disrupts tissue architecture or cognate responses and is useful,
for example in disrupting immune responses, and blocking
sepsis.
[0801] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for chronic
hypergammaglobulinemia evident in such diseases as monoclonal
gammopathy of undetermined significance (MGUS), Waldenstrom's
disease, related idiopathic monoclonal gammopathies, and
plasmacytomas.
[0802] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be employed for instance to inhibit
polypeptide chemotaxis and activation of macrophages and their
precursors, and of neutrophils, basophils, B lymphocytes and some
T-cell subsets, e.g., activated and CD8 cytotoxic T cells and
natural killer cells, in certain autoimmune and chronic
inflammatory and infective diseases. Examples of autoimmune
diseases are described herein and include multiple sclerosis, and
insulin-dependent diabetes.
[0803] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also be employed to treat idiopathic hyper-eosinophilic
syndrome by, for example, preventing eosinophil production and
migration.
[0804] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to enhance or inhibit complement
mediated cell lysis.
[0805] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to enhance or inhibit antibody
dependent cellular cytotoxicity.
[0806] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may also be employed for treating
atherosclerosis, for example, by preventing monocyte infiltration
in the artery wall.
[0807] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be employed to treat adult
respiratory distress syndrome (ARDS).
[0808] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be useful for stimulating wound and
tissue repair, stimulating angiogenesis, and/or stimulating the
repair of vascular or lymphatic diseases or disorders.
Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to stimulate the regeneration of mucosal surfaces.
[0809] In a specific embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to diagnose, prognose, treat, and/or
prevent a disorder characterized by primary or acquired
immunodeficiency, deficient serum immunoglobulin production,
recurrent infections, and/or immune system dysfunction. Moreover,
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention 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 pneumocystis carnii. Other
diseases and disorders that may be prevented, diagnosed, prognosed,
and/or treated with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to, HIV infection, HTLV-BLV infection,
lymphopenia, phagocyte bactericidal dysfunction anemia,
thrombocytopenia, and hemoglobinuria.
[0810] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to treat, and/or diagnose an individual
having common variable immunodeficiency disease ("CVID"; also known
as "acquired agammaglobulinemia" and "acquired
hypogammaglobulinemia") or a subset of this disease.
[0811] In a specific embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used to diagnose, prognose, prevent, and/or
treat cancers or neoplasms including immune cell or immune
tissue-related cancers or neoplasms. Examples of cancers or
neoplasms that may be prevented, diagnosed, or treated by fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention include, but are not limited to,
acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's
disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL)
Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma,
Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and
disorders described in the section entitled "Hyperproliferative
Disorders" elsewhere herein.
[0812] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for decreasing
cellular proliferation of Large B-cell Lymphomas.
[0813] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of decreasing the
involvement of B cells and Ig associated with Chronic Myelogenous
Leukemia.
[0814] In specific embodiments, the compositions of the invention
are used 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.
[0815] Blood-Related Disorders
[0816] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to modulate hemostatic (the stopping of bleeding) or
thrombolytic (clot dissolving) activity. For example, by increasing
hemostatic or thrombolytic activity, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention could be used to treat or prevent blood
coagulation diseases, disorders, and/or conditions (e.g.,
afibrinogenemia, factor deficiencies, hemophilia), blood platelet
diseases, disorders, and/or conditions (e.g., thrombocytopenia), or
wounds resulting from trauma, surgery, or other causes.
Alternatively, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
that can decrease hemostatic or thrombolytic activity could be used
to inhibit or dissolve clotting. These molecules could be important
in the treatment or prevention of heart attacks (infarction),
strokes, or scarring.
[0817] In specific embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used to prevent, diagnose, prognose, and/or
treat thrombosis, arterial thrombosis, venous thrombosis,
thromboembolism, pulmonary embolism, atherosclerosis, myocardial
infarction, transient ischemic attack, unstable angina. In specific
embodiments, the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used for the prevention of occlusion of saphenous grafts,
for reducing the risk of periprocedural thrombosis as might
accompany angioplasty procedures, for reducing the risk of stroke
in patients with atrial fibrillation including nonrheumatic atrial
fibrillation, for reducing the risk of embolism associated with
mechanical heart valves and or mitral valves disease. Other uses
for the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
include, but are not limited to, the prevention of occlusions in
extrcorporeal devices (e.g., intravascular canulas, vascular access
shunts in hemodialysis patients, hemodialysis machines, and
cardiopulmonary bypass machines).
[0818] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, may be used to prevent, diagnose, prognose,
and/or treat diseases and disorders of the blood and/or blood
forming organs associated with the tissue(s) in which the
polypeptide of the invention is expressed.
[0819] The fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may be used to
modulate hematopoietic activity (the formation of blood cells). For
example, the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to increase the quantity of all or subsets of blood
cells, such as, for example, erythrocytes, lymphocytes (B or T
cells), myeloid cells (e.g., basophils, eosinophils, neutrophils,
mast cells, macrophages) and platelets. The ability to decrease the
quantity of blood cells or subsets of blood cells may be useful in
the prevention, detection, diagnosis and/or treatment of anemias
and leukopenias described below. Alternatively, the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be used to decrease the
quantity of all or subsets of blood cells, such as, for example,
erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g.,
basophils, eosinophils, neutrophils, mast cells, macrophages) and
platelets. The ability to decrease the quantity of blood cells or
subsets of blood cells may be useful in the prevention, detection,
diagnosis and/or treatment of leukocytoses, such as, for example
eosinophilia.
[0820] The fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may be used to
prevent, treat, or diagnose blood dyscrasia.
[0821] Anemias are conditions in which the number of red blood
cells or amount of hemoglobin (the protein that carries oxygen) in
them is below normal. Anemia may be caused by excessive bleeding,
decreased red blood cell production, or increased red blood cell
destruction (hemolysis). The albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in treating, preventing, and/or
diagnosing anemias. Anemias that may be treated prevented or
diagnosed by the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include iron deficiency anemia, hypochromic anemia, microcytic
anemia, chlorosis, hereditary siderob; astic anemia, idiopathic
acquired sideroblastic anemia, red cell aplasia, megaloblastic
anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic
acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g.,
autoimmune helolytic anemia, microangiopathic hemolytic anemia, and
paroxysmal nocturnal hemoglobinuria). The albumin fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be useful in treating, preventing,
and/or diagnosing anemias associated with diseases including but
not limited to, anemias associated with systemic lupus
erythematosus, cancers, lymphomas, chronic renal disease, and
enlarged spleens. The albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention may be useful in treating, preventing, and/or diagnosing
anemias arising from drug treatments such as anemias associated
with methyldopa, dapsone, and/or sulfadrugs. Additionally, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be useful in treating,
preventing, and/or diagnosing anemias associated with abnormal red
blood cell architecture including but not limited to, hereditary
spherocytosis, hereditary elliptocytosis, glucose-6-phosphate
dehydrogenase deficiency, and sickle cell anemia.
[0822] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, and/or diagnosing hemoglobin
abnormalities, (e.g., those associated with sickle cell anemia,
hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E
disease). Additionally, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating thalassemias, including, but not
limited to, major and minor forms of alpha-thalassemia and
beta-thalassemia.
[0823] In another embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating bleeding disorders including, but not
limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic
purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's
disease, hereditary platelet disorders (e.g., storage pool disease
such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane
A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome),
hemolytic-uremic syndrome, hemophelias such as hemophelia A or
Factor VII deficiency and Christmas disease or Factor IX
deficiency, Hereditary Hemorhhagic Telangiectsia, also known as
Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein
purpura) and disseminated intravascular coagulation.
[0824] The effect of the albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention on the clotting time of blood may be monitored using any
of the clotting tests known in the art including, but not limited
to, whole blood partial thromboplastin time (PTT), the activated
partial thromboplastin time (aPTT), the activated clotting time
(ACT), the recalcified activated clotting time, or the Lee-White
Clotting time.
[0825] Several diseases and a variety of drugs can cause platelet
dysfunction. Thus, in a specific embodiment, the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be useful in diagnosing,
prognosing, preventing, and/or treating acquired platelet
dysfunction such as platelet dysfunction accompanying kidney
failure, leukemia, multiple myeloma, cirrhosis of the liver, and
systemic lupus erythematosus as well as platelet dysfunction
associated with drug treatments, including treatment with aspirin,
ticlopidine, nonsteroidal anti-inflammatory drugs (used for
arthritis, pain, and sprains), and penicillin in high doses.
[0826] In another embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating diseases and disorders characterized by
or associated with increased or decreased numbers of white blood
cells. Leukopenia occurs when the number of white blood cells
decreases below normal. Leukopenias include, but are not limited
to, neutropenia and lymphocytopenia. An increase in the number of
white blood cells compared to normal is known as leukocytosis. The
body generates increased numbers of white blood cells during
infection. Thus, leukocytosis may simply be a normal physiological
parameter that reflects infection. Alternatively, leukocytosis may
be an indicator of injury or other disease such as cancer.
Leokocytoses, include but are not limited to, eosinophilia, and
accumulations of macrophages. In specific embodiments, the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention may be useful in
diagnosing, prognosing, preventing, and/or treating leukopenia. In
other specific embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating leukocytosis.
[0827] Leukopenia may be a generalized decreased in all types of
white blood cells, or may be a specific depletion of particular
types of white blood cells. Thus, in specific embodiments, the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may be useful in
diagnosing, prognosing, preventing, and/or treating decreases in
neutrophil numbers, known as neutropenia. Neutropenias that may be
diagnosed, prognosed, prevented, and/or treated by the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention include, but are not
limited to, infantile genetic agranulocytosis, familial
neutropenia, cyclic neutropenia, neutropenias resulting from or
associated with dietary deficiencies (e.g., vitamin B 12 deficiency
or folic acid deficiency), neutropenias resulting from or
associated with drug treatments (e.g., antibiotic regimens such as
penicillin treatment, sulfonamide treatment, anticoagulant
treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer
chemotherapy), and neutropenias resulting from increased neutrophil
destruction that may occur in association with some bacterial or
viral infections, allergic disorders, autoimmune diseases,
conditions in which an individual has an enlarged spleen (e.g.,
Felty syndrome, malaria and sarcoidosis), and some drug treatment
regimens.
[0828] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in diagnosing, prognosing, preventing, and/or
treating lymphocytopenias (decreased numbers of B and/or T
lymphocytes), including, but not limited to, lymphocytopenias
resulting from or associated with stress, drug treatments (e.g.,
drug treatment with corticosteroids, cancer chemotherapies, and/or
radiation therapies), AIDS infection and/or other diseases such as,
for example, cancer, rheumatoid arthritis, systemic lupus
erythematosus, chronic infections, some viral infections and/or
hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich
Syndome, severe combined immunodeficiency, ataxia
telangiectsia).
[0829] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in diagnosing, prognosing, preventing, and/or
treating diseases and disorders associated with macrophage numbers
and/or macrophage function including, but not limited to, Gaucher's
disease, Niemann-Pick disease, Letterer-Siwe disease and
Hand-Schuller-Christian disease.
[0830] In another embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating diseases and disorders associated with
eosinophil numbers and/or eosinophil function including, but not
limited to, idiopathic hypereosinophilic syndrome,
eosinophilia-myalgia syndrome, and Hand-Schuller-Christian
disease.
[0831] In yet another embodiment, the albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating leukemias and lymphomas including, but
not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL),
acute myeloid (myelocytic, myelogenous, myeloblastic, or
myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B
cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell
leukenia), chronic myelocytic (myeloid, myelogenous, or
granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma,
Burkitt's lymphoma, and mycosis fungoides.
[0832] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating diseases and disorders of plasma cells
including, but not limited to, plasma cell dyscrasias, monoclonal
gammaopathies, monoclonal gammopathies of undetermined
significance, multiple myeloma, macroglobulinemia, Waldenstrom's
macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
[0833] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in treating, preventing, and/or
diagnosing myeloproliferative disorders, including but not limited
to, polycythemia vera, relative polycythemia, secondary
polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod
metaplasia, thrombocythemia, (including both primary and seconday
thrombocythemia) and chronic myelocytic leukemia.
[0834] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as a treatment prior to surgery, to
increase blood cell production.
[0835] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to enhance the
migration, phagocytosis, superoxide production, antibody dependent
cellular cytotoxicity of neutrophils, eosionophils and
macrophages.
[0836] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to increase the number
of stem cells in circulation prior to stem cells pheresis. In
another specific embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to increase the number
of stem cells in circulation prior to platelet pheresis.
[0837] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to increase cytokine
production.
[0838] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in preventing, diagnosing, and/or
treating primary hematopoietic disorders.
[0839] Hyperproliferative Disorders
[0840] In certain embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention can be used to treat or detect hyperproliferative
disorders, including neoplasms. Albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may inhibit the proliferation of the disorder
through direct or indirect interactions. Alternatively, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may proliferate other cells which
can inhibit the hyperproliferative disorder.
[0841] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. This immune response
may be increased by either enhancing an existing immune response,
or by initiating a new immune response. Alternatively, decreasing
an immune response may also be a method of treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[0842] Examples of hyperproliferative disorders that can be treated
or detected by fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to neoplasms located in the: colon,
abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
(central and peripheral), lymphatic system, pelvis, skin, soft
tissue, spleen, thorax, and urogenital tract.
[0843] Similarly, other hyperproliferative disorders can also be
treated or detected by fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention.
Examples of such hyperproliferative disorders include, but are not
limited to: Acute Childhood Lymphoblastic Leukemia, Acute
Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid
Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular
Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease,
Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult
Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft
Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies,
Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone
Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of
the Renal Pelvis and Ureter, Central Nervous System (Primary)
Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma,
Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)
Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood
Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia,
Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma,
Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell
Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood Hypothalamic and Visual Pathway Glioma, Childhood
Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood
Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial
Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,
Childhood Visual Pathway and Hypothalamic Glioma, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer,
Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma,
Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal
Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic
Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor,
Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer,
Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ
Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia,
Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease,
Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer,
Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,
Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer,
Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung
Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male
Breast Cancer, Malignant Mesothelioma, Malignant Thymoma,
Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary
Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,
Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous
Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,
Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma
Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant
Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian
Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant
Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary
Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer,
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0844] In another preferred embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to diagnose, prognose, prevent,
and/or treat premalignant conditions and to prevent progression to
a neoplastic or malignant state, including but not limited to those
disorders described above. Such uses are 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, 1976, Basic Pathology, 2d Ed., W. B. Saunders
Co., Philadelphia, pp. 68-79.)
[0845] 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. Hyperplastic
disorders which can be diagnosed, prognosed, prevented, and/or
treated with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to, angiofollicular mediastinal lymph
node hyperplasia, angiolymphoid hyperplasia with eosinophilia,
atypical melanocytic hyperplasia, basal cell hyperplasia, benign
giant lymph node hyperplasia, cementum hyperplasia, congenital
adrenal hyperplasia, congenital sebaceous hyperplasia, cystic
hyperplasia, cystic hyperplasia of the breast, denture hyperplasia,
ductal hyperplasia, endometrial hyperplasia, fibromuscular
hyperplasia, focal epithelial hyperplasia, gingival hyperplasia,
inflammatory fibrous hyperplasia, inflammatory papillary
hyperplasia, intravascular papillary endothelial hyperplasia,
nodular hyperplasia of prostate, nodular regenerative hyperplasia,
pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia,
and verrucous hyperplasia.
[0846] 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. Metaplastic disorders which can be diagnosed,
prognosed, prevented, and/or treated with fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to, agnogenic myeloid
metaplasia, apocrine metaplasia, atypical metaplasia,
autoparenchymatous metaplasia, connective tissue metaplasia,
epithelial metaplasia, intestinal metaplasia, metaplastic anemia,
metaplastic ossification, metaplastic polyps, myeloid metaplasia,
primary myeloid metaplasia, secondary myeloid metaplasia, squamous
metaplasia, squamous metaplasia of amnion, and symptomatic myeloid
metaplasia.
[0847] 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.
Dysplastic disorders which can be diagnosed, prognosed, prevented,
and/or treated with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia,
cleidocranial dysplasia, congenital ectodermal dysplasia,
craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia,
fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic
dysplasia, mammary dysplasia, mandibulofacial dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous
dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculovertebral dysplasia, odontogenic dysplasia,
opthalmomandibulomelic dysplasia, periapical cemental dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial
dysplasia.
[0848] Additional pre-neoplastic disorders which can be diagnosed,
prognosed, prevented, and/or treated with fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to, benign
dysproliferative disorders (e.g., benign tumors, fibrocystic
conditions, tissue hypertrophy, intestinal polyps, colon polyps,
and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease,
Farmer's Skin, solar cheilitis, and solar keratosis.
[0849] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, may be used to diagnose and/or prognose disorders
associated with the tissue(s) in which the polypeptide of the
invention is expressed.
[0850] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention conjugated to a toxin or a radioactive isotope, as
described herein, may be used to treat cancers and neoplasms,
including, but not limited to, those described herein. In a further
preferred embodiment, albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention conjugated to a toxin or a radioactive isotope, as
described herein, may be used to treat acute myelogenous
leukemia.
[0851] Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may affect apoptosis, and therefore, would be useful in treating a
number of diseases associated with increased cell survival or the
inhibition of apoptosis. For example, diseases associated with
increased cell survival or the inhibition of apoptosis that could
be diagnosed, prognosed, prevented, and/or treated by
polynucleotides, polypeptides, and/or agonists or antagonists of
the invention, include cancers (such as follicular lymphomas,
carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders such as, multiple sclerosis,
Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid
arthritis) and viral infections (such as herpes viruses, pox
viruses and adenoviruses), inflammation, graft v. host disease,
acute graft rejection, and chronic graft rejection.
[0852] In preferred embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used to inhibit growth, progression, and/or
metastasis of cancers, in particular those listed above.
[0853] Additional diseases or conditions associated with increased
cell survival that could be diagnosed, prognosed, prevented, and/or
treated by fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, include, but are
not limited to, progression, and/or metastases of malignancies and
related disorders such as leukemia (including acute leukemias
(e.g., acute lymphocytic leukemia, acute myelocytic leukemia
(including myeloblastic, promyelocytic, myelomonocytic, monocytic,
and erythroleukemia)) and chronic leukemias (e.g., chronic
myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease
and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0854] Diseases associated with increased apoptosis that could be
diagnosed, prognosed, prevented, and/or treated by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, include AIDS; neurodegenerative
disorders (such as Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar
degeneration and brain tumor or prior associated disease);
autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's
disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid
arthritis) myelodysplastic syndromes (such as aplastic anemia),
graft v. host disease, ischemic injury (such as that caused by
myocardial infarction, stroke and reperfusion injury), liver injury
(e.g., hepatitis related liver injury, ischemia/reperfusion injury,
cholestosis (bile duct injury) and liver cancer); toxin-induced
liver disease (such as that caused by alcohol), septic shock,
cachexia and anorexia.
[0855] Hyperproliferative diseases and/or disorders that could be
diagnosed, prognosed, prevented, and/or treated by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, include, but are not limited to,
neoplasms located in the liver, abdomen, bone, breast, digestive
system, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye,
head and neck, nervous system (central and peripheral), lymphatic
system, pelvis, skin, soft tissue, spleen, thorax, and urogenital
tract.
[0856] Similarly, other hyperproliferative disorders can also be
diagnosed, prognosed, prevented, and/or treated by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention. Examples of such hyperproliferative
disorders include, but are not limited to: hypergammaglobulinemia,
lymphoproliferative disorders, paraproteinemias, purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia,
Gaucher's Disease, histiocytosis, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0857] Another preferred embodiment utilizes polynucleotides
encoding albumin fusion proteins of the invention to inhibit
aberrant cellular division, by gene therapy using the present
invention, and/or protein fusions or fragments thereof.
[0858] Thus, the present invention provides a method for treating
cell proliferative disorders by inserting into an abnormally
proliferating cell a polynucleotide encoding an albumin fusion
protein of the present invention, wherein said polynucleotide
represses said expression.
[0859] Another embodiment of the present invention provides a
method of treating cell-proliferative disorders in individuals
comprising administration of one or more active gene copies of the
present invention to an abnormally proliferating cell or cells. In
a preferred embodiment, polynucleotides of the present invention is
a DNA construct comprising a recombinant expression vector
effective in expressing a DNA sequence encoding said
polynucleotides. In another preferred embodiment of the present
invention, the DNA construct encoding the fusion protein of the
present invention is inserted into cells to be treated utilizing a
retrovirus, or more preferably an adenoviral vector (See G J.
Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated
by reference). In a most preferred embodiment, the viral vector is
defective and will not transform non-proliferating cells, only
proliferating cells. Moreover, in a preferred embodiment, the
polynucleotides of the present invention inserted into
proliferating cells either alone, or in combination with or fused
to other polynucleotides, can then be modulated via an external
stimulus (i.e. magnetic, specific small molecule, chemical, or drug
administration, etc.), which acts upon the promoter upstream of
said polynucleotides to induce expression of the encoded protein
product. As such the beneficial therapeutic affect of the present
invention may be expressly modulated (i.e. to increase, decrease,
or inhibit expression of the present invention) based upon said
external stimulus.
[0860] Polynucleotides of the present invention may be useful in
repressing expression of oncogenic genes or antigens. By
"repressing expression of the oncogenic genes" is intended the
suppression of the transcription of the gene, the degradation of
the gene transcript (pre-message RNA), the inhibition of splicing,
the destruction of the messenger RNA, the prevention of the
post-translational modifications of the protein, the destruction of
the protein, or the inhibition of the normal function of the
protein.
[0861] For local administration to abnormally proliferating cells,
polynucleotides of the present invention may be administered by any
method known to those of skill in the art including, but not
limited to transfection, electroporation, microinjection of cells,
or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any other method described throughout the
specification. The polynucleotide of the present invention may be
delivered by known gene delivery systems such as, but not limited
to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.
Cell. Biol. 5:3403 (1985) or other efficient DNA delivery systems
(Yates et al., Nature 313:812 (1985)) known to those skilled in the
art. These references are exemplary only and are hereby
incorporated by reference. In order to specifically deliver or
transfect cells which are abnormally proliferating and spare
non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as described in the art and elsewhere herein) delivery
system known to those of skill in the art. Since host DNA
replication is required for retroviral DNA to integrate and the
retrovirus will be unable to self replicate due to the lack of the
retrovirus genes needed for its life cycle. Utilizing such a
retroviral delivery system for polynucleotides of the present
invention will target said gene and constructs to abnormally
proliferating cells and will spare the non-dividing normal
cells.
[0862] The polynucleotides of the present invention may be
delivered directly to cell proliferative disorder/disease sites in
internal organs, body cavities and the like by use of imaging
devices used to guide an injecting needle directly to the disease
site. The polynucleotides of the present invention may also be
administered to disease sites at the time of surgical
intervention.
[0863] By "cell proliferative disease" is meant any human or animal
disease or disorder, affecting any one or any combination of
organs, cavities, or body parts, which is characterized by single
or multiple local abnormal proliferations of cells, groups of
cells, or tissues, whether benign or malignant.
[0864] Any amount of the polynucleotides of the present invention
may be administered as long as it has a biologically inhibiting
effect on the proliferation of the treated cells. Moreover, it is
possible to administer more than one of the polynucleotide of the
present invention simultaneously to the same site. By "biologically
inhibiting" is meant partial or total growth inhibition as well as
decreases in the rate of proliferation or growth of the cells. The
biologically inhibitory dose may be determined by assessing the
effects of the polynucleotides of the present invention on target
malignant or abnormally proliferating cell growth in tissue
culture, tumor growth in animals and cell cultures, or any other
method known to one of ordinary skill in the art.
[0865] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
of the present invention are useful in inhibiting the angiogenesis
of proliferative cells or tissues, either alone, as a protein
fusion, or in combination with other polypeptides directly or
indirectly, as described elsewhere herein. In a most preferred
embodiment, said anti-angiogenesis effect may be achieved
indirectly, for example, through the inhibition of hematopoietic,
tumor-specific cells, such as tumor-associated macrophages (See
Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which
is hereby incorporated by reference).
[0866] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in inhibiting proliferative cells or tissues through
the induction of apoptosis. These fusion proteins and/or
polynucleotides may act either directly, or indirectly to induce
apoptosis of proliferative cells and tissues, for example in the
activation of a death-domain receptor, such as tumor necrosis
factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related
apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See
Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998),
which is hereby incorporated by reference). Moreover, in another
preferred embodiment of the present invention, these fusion
proteins and/or polynucleotides may induce apoptosis through other
mechanisms, such as in the activation of other proteins which will
activate apoptosis, or through stimulating the expression of these
proteins, either alone or in combination with small molecule drugs
or adjuviants, such as apoptonin, galectins, thioredoxins,
anti-inflammatory proteins (See for example, Mutat Res
400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem
Biol Interact. April 24; 111-112:23-34 (1998), J Mol Med.
76(6):402-12 (1998), Int J Tissue React; 20(1):3-15 (1998), which
are all hereby incorporated by reference).
[0867] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
are useful in inhibiting the metastasis of proliferative cells or
tissues. Inhibition may occur as a direct result of administering
these albumin fusion proteins and/or polynucleotides, or
indirectly, such as activating the expression of proteins known to
inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr
Top Microbiol Immunol 1998; 231:125-41, which is hereby
incorporated by reference). Such thereapeutic affects of the
present invention may be achieved either alone, or in combination
with small molecule drugs or adjuvants.
[0868] In another embodiment, the invention provides a method of
delivering compositions containing the albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention to targeted cells expressing the a
polypeptide bound by, that binds to, or associates with an albumin
fusion protein of the invention. Albumin fusion proteins of the
invention may be associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic,
hydrophilic, ionic and/or covalent interactions.
[0869] Albumin fusion proteins of the invention are useful in
enhancing the immunogenicity and/or antigenicity of proliferating
cells or tissues, either directly, such as would occur if the
albumin fusion proteins of the invention `vaccinated` the immune
response to respond to proliferative antigens and immunogens, or
indirectly, such as in activating the expression of proteins known
to enhance the immune response (e.g. chemokines), to said antigens
and immunogens.
[0870] Renal Disorders
[0871] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose disorders
of the renal system. Renal disorders which can be diagnosed,
prognosed, prevented, and/or treated with compositions of the
invention include, but are not limited to, kidney failure,
nephritis, blood vessel disorders of kidney, metabolic and
congenital kidney disorders, urinary disorders of the kidney,
autoimmune disorders, sclerosis and necrosis, electrolyte
imbalance, and kidney cancers.
[0872] Kidney diseases which can be diagnosed, prognosed,
prevented, and/or treated with compositions of the invention
include, but are not limited to, acute kidney failure, chronic
kidney failure, atheroembolic renal failure, end-stage renal
disease, inflammatory diseases of the kidney (e.g., acute
glomerulonephritis, postinfectious glomerulonephritis, rapidly
progressive glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis, familial nephrotic syndrome,
membranoproliferative glomerulonephritis I and II, mesangial
proliferative glomerulonephritis, chronic glomerulonephritis, acute
tubulointerstitial nephritis, chronic tubulointerstitial nephritis,
acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis,
lupus nephritis, chronic nephritis, interstitial nephritis, and
post-streptococcal glomerulonephritis), blood vessel disorders of
the kidneys (e.g., kidney infarction, atheroembolic kidney disease,
cortical necrosis, malignant nephrosclerosis, renal vein
thrombosis, renal underperfusion, renal retinopathy, renal
ischemia-reperfusion, renal artery embolism, and renal artery
stenosis), and kidney disorders resulting form urinary tract
disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal
lithiasis, nephrolithiasis), reflux nephropathy, urinary tract
infections, urinary retention, and acute or chronic unilateral
obstructive uropathy.)
[0873] In addition, compositions of the invention can be used to
diagnose, prognose, prevent, and/or treat metabolic and congenital
disorders of the kidney (e.g., uremia, renal amyloidosis, renal
osteodystrophy, renal tubular acidosis, renal glycosuria,
nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome,
renal fibrocystic osteosis (renal rickets), Hartnup disease,
Bartter's syndrome, Liddle's syndrome, polycystic kidney disease,
medullary cystic disease, medullary sponge kidney, Alport's
syndrome, nail-patella syndrome, congenital nephrotic syndrome,
CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic
diabetes insipidus, analgesic nephropathy, kidney stones, and
membranous nephropathy), and autoimmune disorders of the kidney
(e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome,
IgA nephropathy, and IgM mesangial proliferative
glomerulonephritis).
[0874] Compositions of the invention can also be used to diagnose,
prognose, prevent, and/or treat sclerotic or necrotic disorders of
the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal
segmental glomerulosclerosis (FSGS), necrotizing
glomerulonephritis, and renal papillary necrosis), cancers of the
kidney (e.g., nephroma, hypemephroma, nephroblastoma, renal cell
cancer, transitional cell cancer, renal adenocarcinoma, squamous
cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g.,
nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria,
hyponatremia, hypernatremia, hypokalemia, hyperkalemia,
hypocalcemia, hypercalcemia, hypophosphatemia, and
hyperphosphatemia).
[0875] Compositions of the invention may be administered using any
method known in the art, including, but not limited to, direct
needle injection at the delivery site, intravenous injection,
topical administration, catheter infusion, biolistic injectors,
particle accelerators, gelfoam sponge depots, other commercially
available depot materials, osmotic pumps, oral or suppositorial
solid pharmaceutical formulations, decanting or topical
applications during surgery, aerosol delivery. Such methods are
known in the art. Compositions of the invention may be administered
as part of a Therapeutic, described in more detail below. Methods
of delivering polynucleotides of the invention are described in
more detail herein.
[0876] Cardiovascular Disorders
[0877] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose
cardiovascular disorders, including, but not limited to, peripheral
artery disease, such as limb ischemia.
[0878] Cardiovascular disorders include, but are not limited to,
cardiovascular abnormalities, such as arterio-arterial fistula,
arteriovenous fistula, cerebral arteriovenous malformations,
congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
Congenital heart defects include, but are not limited to, aortic
coarctation, cor triatriatum, coronary vessel anomalies, crisscross
heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of fallot, transposition of great vessels, double outlet
right ventricle, tricuspid atresia, persistent truncus arteriosus,
and heart septal defects, such as aortopulmonary septal defect,
endocardial cushion defects, Lutembacher's Syndrome, trilogy of
Fallot, ventricular heart septal defects.
[0879] Cardiovascular disorders also include, but are not limited
to, heart disease, such as arrhythmias, carcinoid heart disease,
high cardiac output, low cardiac output, cardiac tamponade,
endocarditis (including bacterial), heart aneurysm, cardiac arrest,
congestive heart failure, congestive cardiomyopathy, paroxysmal
dyspnea, cardiac edema, heart hypertrophy, congestive
cardiomyopathy, left ventricular hypertrophy, right ventricular
hypertrophy, post-infarction heart rupture, ventricular septal
rupture, heart valve diseases, myocardial diseases, myocardial
ischemia, pericardial effusion, pericarditis (including
constrictive and tuberculous), pneumopericardium,
postpericardiotomy syndrome, pulmonary heart disease, rheumatic
heart disease, ventricular dysfunction, hyperemia, cardiovascular
pregnancy complications, Scimitar Syndrome, cardiovascular
syphilis, and cardiovascular tuberculosis.
[0880] Arrhythmias include, but are not limited to, sinus
arrhythmia, atrial fibrillation, atrial flutter, bradycardia,
extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine
Syndrome, Mahaim-type pre-excitation syndrome,
Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias,
and ventricular fibrillation. Tachycardias include paroxysmal
tachycardia, supraventricular tachycardia, accelerated
idioventricular rhythm, atrioventricular nodal reentry tachycardia,
ectopic atrial tachycardia, ectopic junctional tachycardia,
sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades
de Pointes, and ventricular tachycardia.
[0881] Heart valve diseases include, but are not limited to, aortic
valve insufficiency, aortic valve stenosis, hear murmurs, aortic
valve prolapse, mitral valve prolapse, tricuspid valve prolapse,
mitral valve insufficiency, mitral valve stenosis, pulmonary
atresia, pulmonary valve insufficiency, pulmonary valve stenosis,
tricuspid atresia, tricuspid valve insufficiency, and tricuspid
valve stenosis.
[0882] Myocardial diseases include, but are not limited to,
alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic
cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular
stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns
Syndrome, myocardial reperfusion injury, and myocarditis.
[0883] Myocardial ischemias include, but are not limited to,
coronary disease, such as angina pectoris, coronary aneurysm,
coronary arteriosclerosis, coronary thrombosis, coronary vasospasm,
myocardial infarction and myocardial stunning.
[0884] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular disorders, diabetic angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-occlusive disease, hypertension, hypotension,
ischemia, peripheral vascular diseases, phlebitis, pulmonary
veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein occlusion, Scimitar syndrome, superior vena cava syndrome,
telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer,
vasculitis, and venous insufficiency.
[0885] Aneurysms include, but are not limited to, dissecting
aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms,
aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart
aneurysms, and iliac aneurysms.
[0886] Arterial occlusive diseases include, but are not limited to,
arteriosclerosis, intermittent claudication, carotid stenosis,
fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya
disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis obliterans.
[0887] Cerebrovascular disorders include, but are not limited to,
carotid artery diseases, cerebral amyloid angiopathy, cerebral
aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral
arteriovenous malformation, cerebral artery diseases, cerebral
embolism and thrombosis, carotid artery thrombosis, sinus
thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural
hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral
infarction, cerebral ischemia (including transient), subclavian
steal syndrome, periventricular leukomalacia, vascular headache,
cluster headache, migraine, and vertebrobasilar insufficiency.
[0888] Embolisms include, but are not limited to, air embolisms,
amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome,
fat embolisms, pulmonary embolisms, and thromoboembolisms.
Thrombosis include, but are not limited to, coronary thrombosis,
hepatic vein thrombosis, retinal vein occlusion, carotid artery
thrombosis, sinus thrombosis, Wallenberg's syndrome, and
thrombophlebitis.
[0889] Ischemic disorders include, but are not limited to, cerebral
ischemia, ischemic colitis, compartment syndromes, anterior
compartment syndrome, myocardial ischemia, reperfusion injuries,
and peripheral limb ischemia. Vasculitis includes, but is not
limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss
Syndrome, mucocutaneous lymph node syndrome, thromboangiitis
obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura,
allergic cutaneous vasculitis, and Wegener's granulomatosis.
[0890] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be administered using any method known in the art, including,
but not limited to, direct needle injection at the delivery site,
intravenous injection, topical administration, catheter infusion,
biolistic injectors, particle accelerators, gelfoam sponge depots,
other commercially available depot materials, osmotic pumps, oral
or suppositorial solid pharmaceutical formulations, decanting or
topical applications during surgery, aerosol delivery. Such methods
are known in the art. Methods of delivering polynucleotides are
described in more detail herein.
[0891] Respiratory Disorders
[0892] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to treat, prevent, diagnose, and/or prognose diseases
and/or disorders of the respiratory system.
[0893] Diseases and disorders of the respiratory system include,
but are not limited to, nasal vestibulitis, nonallergic rhinitis
(e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis,
vasomotor rhinitis), nasal polyps, and sinusitis, juvenile
angiofibromas, cancer of the nose and juvenile papillomas, vocal
cord polyps, nodules (singer's nodules), contact ulcers, vocal cord
paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial),
tonsillitis, tonsillar cellulitis, parapharyngeal abscess,
laryngitis, laryngoceles, and throat cancers (e.g., cancer of the
nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g.,
squamous cell carcinoma, small cell (oat cell) carcinoma, large
cell carcinoma, and adenocarcinoma), allergic disorders
(eosinophilic pneumonia, hypersensitivity pneumonitis (e.g.,
extrinsic allergic alveolitis, allergic interstitial pneumonitis,
organic dust pneumoconiosis, allergic bronchopulmonary
aspergillosis, asthma, Wegener's granulomatosis (granulomatous
vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial
pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal
pneumonia), Staphylococcus aureus (staphylococcal pneumonia),
Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and
Pseudomas spp.), Mycoplasma pneumoniae pneumonia, Hemophilus
influenzae pneumonia, Legionella pneumophila (Legionnaires'
disease), and Chlamydia psittaci (Psittacosis)), and viral
pneumonia (e.g., influenza, chickenpox (varicella).
[0894] Additional diseases and disorders of the respiratory system
include, but are not limited to bronchiolitis, polio
(poliomyelitis), croup, respiratory syncytial viral infection,
mumps, erythema infectiosum (fifth disease), roseola infantum,
progressive rubella panencephalitis, german measles, and subacute
sclerosing panencephalitis), fungal pneumonia (e.g.,
Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal
infections in people with severely suppressed immune systems (e.g.,
cryptococcosis, caused by Cryptococcus neoformans; aspergillosis,
caused by Aspergillus spp.; candidiasis, caused by Candida; and
mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia),
atypical pneumonias (e.g., Mycoplasma and Chlamydia spp.),
opportunistic infection pneumonia, nosocomial pneumonia, chemical
pneumonitis, and aspiration pneumonia, pleural disorders (e.g.,
pleurisy, pleural effusion, and pneumothorax (e.g., simple
spontaneous pneumothorax, complicated spontaneous pneumothorax,
tension pneumothorax)), obstructive airway diseases (e.g., asthma,
chronic obstructive pulmonary disease (COPD), emphysema, chronic or
acute bronchitis), occupational lung diseases (e.g., silicosis,
black lung (coal workers' pneumoconiosis), asbestosis, berylliosis,
occupational asthsma, byssinosis, and benign pneumoconioses),
Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g.,
fibrosing alveolitis, usual interstitial pneumonia), idiopathic
pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid
interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe
disease, Hand-Schuller-Christian disease, eosinophilic granuloma),
idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary
alveolar proteinosis), Acute respiratory distress syndrome (also
called, e.g., adult respiratory distress syndrome), edema,
pulmonary embolism, bronchitis (e.g., viral, bacterial),
bronchiectasis, atelectasis, lung abscess (caused by, e.g.,
Staphylococcus aureus or Legionella pneumophila), and cystic
fibrosis.
[0895] Anti-Angiogenesis Activity
[0896] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. Rastinejad et al., Cell
56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions,
such as wound healing, organ regeneration, embryonic development,
and female reproductive processes, angiogenesis is stringently
regulated and spatially and temporally delimited. Under conditions
of pathological angiogenesis such as that characterizing solid
tumor growth, these regulatory controls fail. Unregulated
angiogenesis becomes pathologic and sustains progression of many
neoplastic and non-neoplastic diseases. A number of serious
diseases are dominated by abnormal neovascularization including
solid tumor growth and metastases, arthritis, some types of eye
disorders, and psoriasis. See, e.g., reviews by Moses et al.,
Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med.,
333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res.
29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein
and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz,
Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science
221:719-725 (1983). In a number of pathological conditions, the
process of angiogenesis contributes to the disease state. For
example, significant data have accumulated which suggest that the
growth of solid tumors is dependent on angiogenesis. Folkman and
Klagsbrun, Science 235:442-447 (1987).
[0897] The present invention provides for treatment of diseases or
disorders associated with neovascularization by administration of
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention. Malignant and metastatic
conditions which can be treated with the polynucleotides and
polypeptides, or agonists or antagonists of the invention include,
but are not limited to, malignancies, solid tumors, and cancers
described herein and otherwise known in the art (for a review of
such disorders, see Fishman et al., Medicine, 2d Ed., J. B.
Lippincott Co., Philadelphia (1985)). Thus, the present invention
provides a method of treating an angiogenesis-related disease
and/or disorder, comprising administering to an individual in need
thereof a therapeutically effective amount of an albumin fusion
protein of the invention and/or polynucleotides encoding an albumin
fusion protein of the invention. For example, fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be utilized in a variety of
additional methods in order to therapeutically treat a cancer or
tumor. Cancers which may be treated with fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to solid tumors,
including prostate, lung, breast, ovarian, stomach, pancreas,
larynx, esophagus, testes, liver, parotid, biliary tract, colon,
rectum, cervix, uterus, endometrium, kidney, bladder, thyroid
cancer; primary tumors and metastases; melanomas; glioblastoma;
Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer;
colorectal cancer; advanced malignancies; and blood born tumors
such as leukemias. For example, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention may be delivered topically, in order to treat cancers
such as skin cancer, head and neck tumors, breast tumors, and
Kaposi's sarcoma.
[0898] Within yet other aspects, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention may be utilized to treat superficial forms of bladder
cancer by, for example, intravesical administration. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be delivered directly into the
tumor, or near the tumor site, via injection or a catheter. Of
course, as the artisan of ordinary skill will appreciate, the
appropriate mode of administration will vary according to the
cancer to be treated. Other modes of delivery are discussed
herein.
[0899] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating other disorders, besides cancers, which
involve angiogenesis. These disorders include, but are not limited
to: benign tumors, for example hemangiomas, acoustic neuromas,
neurofibromas, trachomas, and pyogenic granulomas; artheroscleric
plaques; ocular angiogenic diseases, for example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia
(abnormal blood vessel growth) of the eye; rheumatoid arthritis;
psoriasis; delayed wound healing; endometriosis; vasculogenesis;
granulations; hypertrophic scars (keloids); nonunion fractures;
scleroderma; trachoma; vascular adhesions; myocardial angiogenesis;
coronary collaterals; cerebral collaterals; arteriovenous
malformations; ischemic limb angiogenesis; Osler-Webber Syndrome;
plaque neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's
disease; and atherosclerosis.
[0900] For example, within one aspect of the present invention
methods are provided for treating hypertrophic scars and keloids,
comprising the step of administering albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention to a hypertrophic scar or keloid.
[0901] Within one embodiment of the present invention fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention are directly injected into a
hypertrophic scar or keloid, in order to prevent the progression of
these lesions. This therapy is of particular value in the
prophylactic treatment of conditions which are known to result in
the development of hypertrophic scars and keloids (e.g., burns),
and is preferably initiated after the proliferative phase has had
time to progress (approximately 14 days after the initial injury),
but before hypertrophic scar or keloid development. As noted above,
the present invention also provides methods for treating
neovascular diseases of the eye, including for example, corneal
neovascularization, neovascular glaucoma, proliferative diabetic
retinopathy, retrolental fibroplasia and macular degeneration.
[0902] Moreover, Ocular disorders associated with
neovascularization which can be treated with the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention include, but are not limited to:
neovascular glaucoma, diabetic retinopathy, retinoblastoma,
retrolental fibroplasia, uveitis, retinopathy of prematurity
macular degeneration, corneal graft neovascularization, as well as
other eye inflammatory diseases, ocular tumors and diseases
associated with choroidal or iris neovascularization. See, e.g.,
reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and
Gartner et al., Surv. Ophthal. 22:291-312 (1978).
[0903] Thus, within one aspect of the present invention methods are
provided for treating neovascular diseases of the eye such as
corneal neovascularization (including corneal graft
neovascularization), comprising the step of administering to a
patient a therapeutically effective amount of a compound (e.g.,
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention) to the cornea, such that
the formation of blood vessels is inhibited. Briefly, the cornea is
a tissue which normally lacks blood vessels. In certain
pathological conditions however, capillaries may extend into the
cornea from the pericorneal vascular plexus of the limbus. When the
cornea becomes vascularized, it also becomes clouded, resulting in
a decline in the patient's visual acuity. Visual loss may become
complete if the cornea completely opacitates. A wide variety of
disorders can result in corneal neovascularization, including for
example, corneal infections (e.g., trachoma, herpes simplex
keratitis, leishmaniasis and onchocerciasis), immunological
processes (e.g., graft rejection and Stevens-Johnson's syndrome),
alkali burns, trauma, inflammation (of any cause), toxic and
nutritional deficiency states, and as a complication of wearing
contact lenses.
[0904] Within particularly preferred embodiments of the invention,
may be prepared for topical administration in saline (combined with
any of the preservatives and antimicrobial agents commonly used in
ocular preparations), and administered in eyedrop form. The
solution or suspension may be prepared in its pure form and
administered several times daily. Alternatively, anti-angiogenic
compositions, prepared as described above, may also be administered
directly to the cornea. Within preferred embodiments, the
anti-angiogenic composition is prepared with a muco-adhesive
polymer which binds to cornea. Within further embodiments, the
anti-angiogenic factors or anti-angiogenic compositions may be
utilized as an adjunct to conventional steroid therapy. Topical
therapy may also be useful prophylactically in corneal lesions
which are known to have a high probability of inducing an
angiogenic response (such as chemical burns). In these instances
the treatment, likely in combination with steroids, may be
instituted immediately to help prevent subsequent
complications.
[0905] Within other embodiments, the compounds described above may
be injected directly into the corneal stroma by an ophthalmologist
under microscopic guidance. The preferred site of injection may
vary with the morphology of the individual lesion, but the goal of
the administration would be to place the composition at the
advancing front of the vasculature (i.e., interspersed between the
blood vessels and the normal cornea). In most cases this would
involve perilimbic corneal injection to "protect" the cornea from
the advancing blood vessels. This method may also be utilized
shortly after a corneal insult in order to prophylactically prevent
corneal neovascularization. In this situation the material could be
injected in the perilimbic cornea interspersed between the corneal
lesion and its undesired potential limbic blood supply. Such
methods may also be utilized in a similar fashion to prevent
capillary invasion of transplanted corneas. In a sustained-release
form injections might only be required 2-3 times per year. A
steroid could also be added to the injection solution to reduce
inflammation resulting from the injection itself.
[0906] Within another aspect of the present invention, methods are
provided for treating neovascular glaucoma, comprising the step of
administering to a patient a therapeutically effective amount of an
albumin fusion protein of the invention and/or polynucleotides
encoding an albumin fusion protein of the invention to the eye,
such that the formation of blood vessels is inhibited. In one
embodiment, the compound may be administered topically to the eye
in order to treat early forms of neovascular glaucoma. Within other
embodiments, the compound may be implanted by injection into the
region of the anterior chamber angle. Within other embodiments, the
compound may also be placed in any location such that the compound
is continuously released into the aqueous humor. Within another
aspect of the present invention, methods are provided for treating
proliferative diabetic retinopathy, comprising the step of
administering to a patient a therapeutically effective amount of an
albumin fusion protein of the invention and/or polynucleotides
encoding an albumin fusion protein of the invention to the eyes,
such that the formation of blood vessels is inhibited.
[0907] Within particularly preferred embodiments of the invention,
proliferative diabetic retinopathy may be treated by injection into
the aqueous humor or the vitreous, in order to increase the local
concentration of the polynucleotide, polypeptide, antagonist and/or
agonist in the retina. Preferably, this treatment should be
initiated prior to the acquisition of severe disease requiring
photocoagulation.
[0908] Within another aspect of the present invention, methods are
provided for treating retrolental fibroplasia, comprising the step
of administering to a patient a therapeutically effective amount of
an albumin fusion protein of the invention and/or polynucleotides
encoding an albumin fusion protein of the invention to the eye,
such that the formation of blood vessels is inhibited. The compound
may be administered topically, via intravitreous injection and/or
via intraocular implants.
[0909] Additionally, disorders which can be treated with fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention include, but are not limited to,
hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic
plaques, delayed wound healing, granulations, hemophilic joints,
hypertrophic scars, nonunion fractures, Osler-Weber syndrome,
pyogenic granuloma, scleroderma, trachoma, and vascular
adhesions.
[0910] Moreover, disorders and/or states, which can be treated,
prevented, diagnosed, and/or prognosed with the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention of the invention include, but are
not limited to, solid tumors, blood born tumors such as leukemias,
tumor metastasis, Kaposi's sarcoma, benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular
angiogenic diseases, for example, diabetic retinopathy, retinopathy
of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, and uvietis, delayed wound healing, endometriosis,
vascluogenesis, granulations, hypertrophic scars (keloids),
nonunion fractures, scleroderma, trachoma, vascular adhesions,
myocardial angiogenesis, coronary collaterals, cerebral
collaterals, arteriovenous malformations, ischemic limb
angiogenesis, Osler-Webber Syndrome, plaque neovascularization,
telangiectasia, hemophiliac joints, angiofibroma fibromuscular
dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth control agent by preventing vascularization required for
embryo implantation controlling menstruation, diseases that have
angiogenesis as a pathologic consequence such as cat scratch
disease (Rochele minalia quintosa), ulcers (Helicobacter pylori),
Bartonellosis and bacillary angiomatosis.
[0911] In one aspect of the birth control method, an amount of the
compound sufficient to block embryo implantation is administered
before or after intercourse and fertilization have occurred, thus
providing an effective method of birth control, possibly a "morning
after" method. Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also be used in controlling menstruation or administered as
either a peritoneal lavage fluid or for peritoneal implantation in
the treatment of endometriosis.
[0912] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be incorporated into surgical sutures in order to prevent
stitch granulomas.
[0913] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be utilized in a wide variety of surgical procedures. For
example, within one aspect of the present invention a compositions
(in the form of, for example, a spray or film) may be utilized to
coat or spray an area prior to removal of a tumor, in order to
isolate normal surrounding tissues from malignant tissue, and/or to
prevent the spread of disease to surrounding tissues. Within other
aspects of the present invention, compositions (e.g., in the form
of a spray) may be delivered via endoscopic procedures in order to
coat tumors, or inhibit angiogenesis in a desired locale. Within
yet other aspects of the present invention, surgical meshes which
have been coated with anti-angiogenic compositions of the present
invention may be utilized in any procedure wherein a surgical mesh
might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition
may be utilized during abdominal cancer resection surgery (e.g.,
subsequent to colon resection) in order to provide support to the
structure, and to release an amount of the anti-angiogenic
factor.
[0914] Within further aspects of the present invention, methods are
provided for treating tumor excision sites, comprising
administering albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
to the resection margins of a tumor subsequent to excision, such
that the local recurrence of cancer and the formation of new blood
vessels at the site is inhibited. Within one embodiment of the
invention, the anti-angiogenic compound is administered directly to
the tumor excision site (e.g., applied by swabbing, brushing or
otherwise coating the resection margins of the tumor with the
anti-angiogenic compound). Alternatively, the anti-angiogenic
compounds may be incorporated into known surgical pastes prior to
administration. Within particularly preferred embodiments of the
invention, the anti-angiogenic compounds are applied after hepatic
resections for malignancy, and after neurosurgical operations.
[0915] Within one aspect of the present invention, fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be administered to the resection
margin of a wide variety of tumors, including for example, breast,
colon, brain and hepatic tumors. For example, within one embodiment
of the invention, anti-angiogenic compounds may be administered to
the site of a neurological tumor subsequent to excision, such that
the formation of new blood vessels at the site are inhibited.
[0916] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also be administered along with other anti-angiogenic factors.
Representative examples of other anti-angiogenic factors include:
Anti-Invasive Factor, retinoic acid and derivatives thereof,
paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1,
Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator
Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms
of the lighter "d group" transition metals.
[0917] 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.
[0918] 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.
[0919] 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.
[0920] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include 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)); Thalidomide;
Angostatic steroid; AGM-1470; carboxynaminolmidazole; and
metalloproteinase inhibitors such as BB94.
[0921] Diseases at the Cellular Level
[0922] Diseases associated with increased cell survival or the
inhibition of apoptosis that could be treated, prevented,
diagnosed, and/or prognosed using fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, include cancers (such as follicular lymphomas,
carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders (such as, multiple sclerosis,
Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid
arthritis) and viral infections (such as herpes viruses, pox
viruses and adenoviruses), inflammation, graft v. host disease,
acute graft rejection, and chronic graft rejection.
[0923] In preferred embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used to inhibit growth, progression, and/or metasis
of cancers, in particular those listed above.
[0924] Additional diseases or conditions associated with increased
cell survival that could be treated or detected by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention include, but are not limited to,
progression, and/or metastases of malignancies and related
disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0925] Diseases associated with increased apoptosis that could be
treated, prevented, diagnosed, and/or prognesed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, include, but are not limited to,
AIDS; neurodegenerative disorders (such as Alzheimer's disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis
pigmentosa, Cerebellar degeneration and brain tumor or prior
associated disease); autoimmune disorders (such as, multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) myelodysplastic syndromes (such as
aplastic anemia), graft v. host disease, ischemic injury (such as
that caused by myocardial infarction, stroke and reperfusion
injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and
liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock, cachexia and anorexia.
[0926] Wound Healing and Epithelial Cell Proliferation
[0927] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, for therapeutic purposes, for
example, to stimulate epithelial cell proliferation and basal
keratinocytes for the purpose of wound healing, and to stimulate
hair follicle production and healing of dermal wounds. Albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, may be clinically useful
in stimulating wound healing including surgical wounds, excisional
wounds, deep wounds involving damage of the dermis and epidermis,
eye tissue wounds, dental tissue wounds, oral cavity wounds,
diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,
venous stasis ulcers, burns resulting from heat exposure or
chemicals, and other abnormal wound healing conditions such as
uremia, malnutrition, vitamin deficiencies and complications
associated with systemic treatment with steroids, radiation therapy
and antineoplastic drugs and antimetabolites. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, could be used to promote dermal
reestablishment subsequent to dermal loss
[0928] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used to increase the adherence of skin grafts to a wound
bed and to stimulate re-epithelialization from the wound bed. The
following are types of grafts that fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, could be used to increase adherence to a wound bed:
autografts, artificial skin, allografts, autodermic graft,
autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone
graft, brephoplastic grafts, cutis graft, delayed graft, dermic
graft, epidermic graft, fascia graft, full thickness graft,
heterologous graft, xenograft, homologous graft, hyperplastic
graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch
graft, omenpal graft, patch graft, pedicle graft, penetrating
graft, split skin graft, thick split graft. Albumin fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, can be used to promote skin strength and
to improve the appearance of aged skin.
[0929] It is believed that fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
will also produce changes in hepatocyte proliferation, and
epithelial cell proliferation in the lung, breast, pancreas,
stomach, small intestine, and large intestine. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, could promote proliferation of
epithelial cells such as sebocytes, hair follicles, hepatocytes,
type II pneumocytes, mucin-producing goblet cells, and other
epithelial cells and their progenitors contained within the skin,
lung, liver, and gastrointestinal tract. Albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, may promote proliferation of endothelial
cells, keratinocytes, and basal keratinocytes.
[0930] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could also be used to reduce the side effects of gut toxicity that
result from radiation, chemotherapy treatments or viral infections.
Albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, may have a
cytoprotective effect on the small intestine mucosa. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, may also stimulate healing of
mucositis (mouth ulcers) that result from chemotherapy and viral
infections.
[0931] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could further be used in full regeneration of skin in full and
partial thickness skin defects, including burns, (i.e.,
repopulation of hair follicles, sweat glands, and sebaceous
glands), treatment of other skin defects such as psoriasis. Albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, could be used to treat
epidermolysis bullosa, a defect in adherence of the epidermis to
the underlying dermis which results in frequent, open and painful
blisters by accelerating reepithelialization of these lesions.
Albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, could also be
used to treat gastric and doudenal ulcers and help heal by scar
formation of the mucosal lining and regeneration of glandular
mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel
diseases, such as Crohn's disease and ulcerative colitis, are
diseases which result in destruction of the mucosal surface of the
small or large intestine, respectively. Thus, fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, could be used to promote the resurfacing
of the mucosal surface to aid more rapid healing and to prevent
progression of inflammatory bowel disease. Treatment with fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, is expected to have a significant
effect on the production of mucus throughout the gastrointestinal
tract and could be used to protect the intestinal mucosa from
injurious substances that are ingested or following surgery.
Albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, could be used to
treat diseases associate with the under expression.
[0932] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used to prevent and heal damage to the lungs due to
various pathological states. Albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, which could stimulate proliferation and
differentiation and promote the repair of alveoli and brochiolar
epithelium to prevent or treat acute or chronic lung damage. For
example, emphysema, which results in the progressive loss of
aveoli, and inhalation injuries, i.e., resulting from smoke
inhalation and burns, that cause necrosis of the bronchiolar
epithelium and alveoli could be effectively treated using
polynucleotides or polypeptides, agonists or antagonists of the
present invention. Also fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used to stimulate the proliferation of and differentiation
of type II pneumocytes, which may help treat or prevent disease
such as hyaline membrane diseases, such as infant respiratory
distress syndrome and bronchopulmonary displasia, in premature
infants.
[0933] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could stimulate the proliferation and differentiation of
hepatocytes and, thus, could be used to alleviate or treat liver
diseases and pathologies such as fulminant liver failure caused by
cirrhosis, liver damage caused by viral hepatitis and toxic
substances (i.e., acetaminophen, carbon tetraholoride and other
hepatotoxins known in the art).
[0934] In addition, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used treat or prevent the onset of diabetes mellitus. In
patients with newly diagnosed Types I and II diabetes, where some
islet cell function remains, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, could be used to maintain the islet function so as to
alleviate, delay or prevent permanent manifestation of the disease.
Also, fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, could be used as
an auxiliary in islet cell transplantation to improve or promote
islet cell function.
[0935] Neural Activity and Neurological Diseases
[0936] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used for the diagnosis and/or treatment of diseases,
disorders, damage or injury of the brain and/or nervous system.
Nervous system disorders that can be treated with the compositions
of the invention (e.g., fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention),
include, but are not limited to, nervous system injuries, and
diseases or disorders which result in either a disconnection of
axons, a diminution or degeneration of neurons, or demyelination.
Nervous system lesions which may be treated in a patient (including
human and non-human mammalian patients) according to the methods of
the invention, include but are not limited to, the following
lesions of either the central (including spinal cord, brain) or
peripheral nervous systems: (1) ischemic lesions, in which a lack
of oxygen in a portion of the nervous system results in neuronal
injury or death, including cerebral infarction or ischemia, or
spinal cord infarction or ischemia; (2) traumatic lesions,
including lesions caused by physical injury or associated with
surgery, for example, lesions which sever a portion of the nervous
system, or compression injuries; (3) malignant lesions, in which a
portion of the nervous system is destroyed or injured by malignant
tissue which is either a nervous system associated malignancy or a
malignancy derived from non-nervous system tissue; (4) infectious
lesions, in which a portion of the nervous system is destroyed or
injured as a result of infection, for example, by an abscess or
associated with infection by human immunodeficiency virus, herpes
zoster, or herpes simplex virus or with Lyme disease, tuberculosis,
or syphilis; (5) degenerative lesions, in which a portion of the
nervous system is destroyed or injured as a result of a
degenerative process including but not limited to, degeneration
associated with Parkinson's disease, Alzheimer's disease,
Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6)
lesions associated with nutritional diseases or disorders, in which
a portion of the nervous system is destroyed or injured by a
nutritional disorder or disorder of metabolism including, but not
limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke
disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease
(primary degeneration of the corpus callosum), and alcoholic
cerebellar degeneration; (7) neurological lesions associated with
systemic diseases including, but not limited to, diabetes (diabetic
neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma,
or sarcoidosis; (8) lesions caused by toxic substances including
alcohol, lead, or particular neurotoxins; and (9) demyelinated
lesions in which a portion of the nervous system is destroyed or
injured by a demyelinating disease including, but not limited to,
multiple sclerosis, human immunodeficiency virus-associated
myelopathy, transverse myelopathy or various etiologies,
progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
[0937] In one embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to protect neural cells from the damaging
effects of hypoxia. In a further preferred embodiment, the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used to protect neural
cells from the damaging effects of cerebral hypoxia. According to
this embodiment, the compositions of the invention are used to
treat or prevent neural cell injury associated with cerebral
hypoxia. In one non-exclusive aspect of this embodiment, the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, are used to
treat or prevent neural cell injury associated with cerebral
ischemia. In another non-exclusive aspect of this embodiment, the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention are used to treat
or prevent neural cell injury associated with cerebral
infarction.
[0938] In another preferred embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to treat or prevent neural cell
injury associated with a stroke. In a specific embodiment, albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used to treat or
prevent cerebral neural cell injury associated with a stroke.
[0939] In another preferred embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to treat or prevent neural cell
injury associated with a heart attack. In a specific embodiment,
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention are used to treat
or prevent cerebral neural cell injury associated with a heart
attack.
[0940] The compositions of the invention which are useful for
treating or preventing a nervous system disorder may be selected by
testing for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, compositions of the invention which elicit any of the
following effects may be useful according to the invention: (1)
increased survival time of neurons in culture either in the
presence or absence of hypoxia or hypoxic conditions; (2) increased
sprouting of neurons in culture or in vivo; (3) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or (4) decreased symptoms of neuron
dysfunction in vivo. Such effects may be measured by any method
known in the art. In preferred, non-limiting embodiments, increased
survival of neurons may routinely be measured using a method set
forth herein or otherwise known in the art, such as, for example,
in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in
Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased
sprouting of neurons may be detected by methods known in the art,
such as, for example, the methods set forth in Pestronk et al.,
Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev.
Neurosci., 4:17-42 (1981); increased production of
neuron-associated molecules may be measured by bioassay, enzymatic
assay, antibody binding, Northern blot assay, etc., using
techniques known in the art and depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0941] In specific embodiments, motor neuron disorders that may be
treated according to the invention include, but are not limited to,
disorders such as infarction, infection, exposure to toxin, trauma,
surgical damage, degenerative disease or malignancy that may affect
motor neurons as well as other components of the nervous system, as
well as disorders that selectively affect neurons such as
amyotrophic lateral sclerosis, and including, but not limited to,
progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
[0942] Further, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may play a role in neuronal survival; synapse formation;
conductance; neural differentiation, etc. Thus, compositions of the
invention (including fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention)
may be used to diagnose and/or treat or prevent diseases or
disorders associated with these roles, including, but not limited
to, learning and/or cognition disorders. The compositions of the
invention may also be useful in the treatment or prevention of
neurodegenerative disease states and/or behavioural disorders. Such
neurodegenerative disease states and/or behavioral disorders
include, but are not limited to, Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease, Tourette Syndrome, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, compositions of the invention
may also play a role in the treatment, prevention and/or detection
of developmental disorders associated with the developing embryo,
or sexually-linked disorders.
[0943] Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be useful in protecting neural cells from diseases, damage,
disorders, or injury, associated with cerebrovascular disorders
including, but not limited to, carotid artery diseases (e.g.,
carotid artery thrombosis, carotid stenosis, or Moyamoya Disease),
cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,
cerebral arteriosclerosis, cerebral arteriovenous malformations,
cerebral artery diseases, cerebral embolism and thrombosis (e.g.,
carotid artery thrombosis, sinus thrombosis, or Wallenberg's
Syndrome), cerebral hemorrhage (e.g., epidural or subdural
hematoma, or subarachnoid hemorrhage), cerebral infarction,
cerebral ischemia (e.g., transient cerebral ischemia, Subclavian
Steal Syndrome, or vertebrobasilar insufficiency), vascular
dementia (e.g., multi-infarct), leukomalacia, periventricular, and
vascular headache (e.g., cluster headache or migraines).
[0944] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, for therapeutic purposes, for
example, to stimulate neurological cell proliferation and/or
differentiation. Therefore, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to treat and/or detect neurologic diseases. Moreover,
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, can be used as a marker
or detector of a particular nervous system disease or disorder.
[0945] Examples of neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, brain diseases, such as metabolic brain diseases which
includes phenylketonuria such as maternal phenylketonuria, pyruvate
carboxylase deficiency, pyruvate dehydrogenase complex deficiency,
Wernicke's Encephalopathy, brain edema, brain neoplasms such as
cerebellar neoplasms which include infratentorial neoplasms,
cerebral ventricle neoplasms such as choroid plexus neoplasms,
hypothalamic neoplasms, supratentorial neoplasms, canavan disease,
cerebellar diseases such as cerebellar ataxia which include
spinocerebellar degeneration such as ataxia telangiectasia,
cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph
Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as
infratentorial neoplasms, diffuse cerebral sclerosis such as
encephalitis periaxialis, globoid cell leukodystrophy,
metachromatic leukodystrophy and subacute sclerosing
panencephalitis.
[0946] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include cerebrovascular disorders (such as carotid artery diseases
which include carotid artery thrombosis, carotid stenosis and
Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm,
cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous
malformations, cerebral artery diseases, cerebral embolism and
thrombosis such as carotid artery thrombosis, sinus thrombosis and
Wallenberg's Syndrome, cerebral hemorrhage such as epidural
hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral
infarction, cerebral ischemia such as transient cerebral ischemia,
Subclavian Steal Syndrome and vertebrobasilar insufficiency,
vascular dementia such as multi-infarct dementia, periventricular
leukomalacia, vascular headache such as cluster headache and
migraine.
[0947] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include dementia such as AIDS Dementia Complex, presenile dementia
such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile
dementia such as Alzheimer's Disease and progressive supranuclear
palsy, vascular dementia such as multi-infarct dementia,
encephalitis which include encephalitis periaxialis, viral
encephalitis such as epidemic encephalitis, Japanese Encephalitis,
St. Louis Encephalitis, tick-borne encephalitis and West Nile
Fever, acute disseminated encephalomyelitis, meningoencephalitis
such as uveomeningoencephalitic syndrome, Postencephalitic
Parkinson Disease and subacute sclerosing panencephalitis,
encephalomalacia such as periventricular leukomalacia, epilepsy
such as generalized epilepsy which includes infantile spasms,
absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome,
tonic-clonic epilepsy, partial epilepsy such as complex partial
epilepsy, frontal lobe epilepsy and temporal lobe epilepsy,
post-traumatic epilepsy, status epilepticus such as Epilepsia
Partialis Continua, and Hallervorden-Spatz Syndrome.
[0948] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include hydrocephalus such as Dandy-Walker Syndrome and normal
pressure hydrocephalus, hypothalamic diseases such as hypothalamic
neoplasms, cerebral malaria, narcolepsy which includes cataplexy,
bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's
Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial
tuberculoma and Zellweger Syndrome, central nervous system
infections such as AIDS Dementia Complex, Brain Abscess, subdural
empyema, encephalomyelitis such as Equine Encephalomyelitis,
Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic
Encephalomyelitis, Visna, and cerebral malaria.
[0949] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include meningitis such as arachnoiditis, aseptic meningtitis such
as viral meningtitis which includes lymphocytic choriomeningitis,
Bacterial meningtitis which includes Haemophilus Meningtitis,
Listeria Meningtitis, Meningococcal Meningtitis such as
Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and
meningeal tuberculosis, fungal meningitis such as Cryptococcal
Meningtitis, subdural effusion, meningoencephalitis such as
uvemeningoencephalitic syndrome, myelitis such as transverse
myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which
includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion
diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform
Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and
cerebral toxoplasmosis.
[0950] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include central nervous system neoplasms such as brain neoplasms
that include cerebellar neoplasms such as infratentorial neoplasms,
cerebral ventricle neoplasms such as choroid plexus neoplasms,
hypothalamic neoplasms and supratentorial neoplasms, meningeal
neoplasms, spinal cord neoplasms which include epidural neoplasms,
demyelinating diseases such as Canavan Diseases, diffuse cerebral
sceloris which includes adrenoleukodystrophy, encephalitis
periaxialis, globoid cell leukodystrophy, diffuse cerebral
sclerosis such as metachromatic leukodystrophy, allergic
encephalomyelitis, necrotizing hemorrhagic encephalomyelitis,
progressive multifocal leukoencephalopathy, multiple sclerosis,
central pontine myelinolysis, transverse myelitis, neuromyelitis
optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High
Pressure Nervous Syndrome, Meningism, spinal cord diseases such as
amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular
atrophy such as Werdnig-Hoffmann Disease, spinal cord compression,
spinal cord neoplasms such as epidural neoplasms, syringomyelia,
Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as
Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down
Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff
Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria,
Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup
Urine Disease, mucolipidosis such as fucosidosis, neuronal
ceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria
such as maternal phenylketonuria, Prader-Willi Syndrome, Rett
Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR
Syndrome, nervous system abnormalities such as holoprosencephaly,
neural tube defects such as anencephaly which includes
hydrangencephaly, Arnold-Chairi Deformity, encephalocele,
meningocele, meningomyelocele, spinal dysraphism such as spina
bifida cystica and spina bifida occulta.
[0951] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include hereditary motor and sensory neuropathies which include
Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease,
hereditary spastic paraplegia, Werdnig-Hoffmann Disease, Hereditary
Sensory and Autonomic Neuropathies such as Congenital Analgesia and
Familial Dysautonomia, Neurologic manifestations (such as agnosia
that include Gerstmann's Syndrome, Amnesia such as retrograde
amnesia, apraxia, neurogenic bladder, cataplexy, communicative
disorders such as hearing disorders that includes deafness, partial
hearing loss, loudness recruitment and tinnitus, language disorders
such as aphasia which include agraphia, anomia, broca aphasia, and
Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language
development disorders, speech disorders such as aphasia which
includes anomia, broca aphasia and Wernicke Aphasia, articulation
disorders, communicative disorders such as speech disorders which
include dysarthria, echolalia, mutism and stuttering, voice
disorders such as aphonia and hoarseness, decerebrate state,
delirium, fasciculation, hallucinations, meningism, movement
disorders such as angelman syndrome, ataxia, athetosis, chorea,
dystonia, hypokinesia, muscle hypotonia, myoclonus, tic,
torticollis and tremor, muscle hypertonia such as muscle rigidity
such as stiff-man syndrome, muscle spasticity, paralysis such as
facial paralysis which includes Herpes Zoster Oticus,
Gastroparesis, Hemiplegia, opthalmoplegia such as diplopia, Duane's
Syndrome, Horner's Syndrome, Chronic progressive external
opthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical
Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome,
quadriplegia, respiratory paralysis and vocal cord paralysis,
paresis, phantom limb, taste disorders such as ageusia and
dysgeusia, vision disorders such as amblyopia, blindness, color
vision defects, diplopia, hemianopsia, scotoma and subnormal
vision, sleep disorders such as hypersomnia which includes
Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as
trismus, unconsciousness such as coma, persistent vegetative state
and syncope and vertigo, neuromuscular diseases such as amyotonia
congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic
Syndrome, motor neuron disease, muscular atrophy such as spinal
muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann
Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia
Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy,
Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical
Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous
system diseases such as acrodynia, amyloid neuropathies, autonomic
nervous system diseases such as Adie's Syndrome, Barre-Lieou
Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex
Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve
Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma
which includes Neurofibromatosis 2, Facial Nerve Diseases such as
Facial Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility
disorders which includes amblyopia, nystagmus, oculomotor nerve
paralysis, opthalmoplegia such as Duane's Syndrome, Horner's
Syndrome, Chronic Progressive External Opthalmoplegia which
includes Kearns Syndrome, Strabismus such as Esotropia and
Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as
Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk
Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema,
Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases
such as Neuromyelitis Optica and Swayback, and Diabetic
neuropathies such as diabetic foot.
[0952] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include nerve compression syndromes such as carpal tunnel syndrome,
tarsal tunnel syndrome, thoracic outlet syndrome such as cervical
rib syndrome, ulnar nerve compression syndrome, neuralgia such as
causalgia, cervico-brachial neuralgia, facial neuralgia and
trigeminal neuralgia, neuritis such as experimental allergic
neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and
radiculities such as polyradiculitis, hereditary motor and sensory
neuropathies such as Charcot-Marie Disease, Hereditary Optic
Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and
Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic
Neuropathies which include Congenital Analgesia and Familial
Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and
Tetany).
[0953] Endocrine Disorders
[0954] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose disorders
and/or diseases related to hormone imbalance, and/or disorders or
diseases of the endocrine system.
[0955] Hormones secreted by the glands of the endocrine system
control physical growth, sexual function, metabolism, and other
functions. Disorders may be classified in two ways: disturbances in
the production of hormones, and the inability of tissues to respond
to hormones. The etiology of these hormone imbalance or endocrine
system diseases, disorders or conditions may be genetic, somatic,
such as cancer and some autoimmune diseases, acquired (e.g., by
chemotherapy, injury or toxins), or infectious. Moreover, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention can be used as a marker or
detector of a particular disease or disorder related to the
endocrine system and/or hormone imbalance.
[0956] Endocrine system and/or hormone imbalance and/or diseases
encompass disorders of uterine motility including, but not limited
to: complications with pregnancy and labor (e.g., pre-term labor,
post-term pregnancy, spontaneous abortion, and slow or stopped
labor); and disorders and/or diseases of the menstrual cycle (e.g.,
dysmenorrhea and endometriosis).
[0957] Endocrine system and/or hormone imbalance disorders and/or
diseases include disorders and/or diseases of the pancreas, such
as, for example, diabetes mellitus, diabetes insipidus, congenital
pancreatic agenesis, pheochromocytoma--islet cell tumor syndrome;
disorders and/or diseases of the adrenal glands such as, for
example, Addison's Disease, corticosteroid deficiency, virilizing
disease, hirsutism, Cushing's Syndrome, hyperaldosteronism,
pheochromocytoma; disorders and/or diseases of the pituitary gland,
such as, for example, hyperpituitarism, hypopituitarism, pituitary
dwarfism, pituitary adenoma, panhypopituitarism, acromegaly,
gigantism; disorders and/or diseases of the thyroid, including but
not limited to, hyperthyroidism, hypothyroidism, Plummer's disease,
Graves' disease (toxic diffuse goiter), toxic nodular goiter,
thyroiditis (Hashimoto's thyroiditis, subacute granulomatous
thyroiditis, and silent lymphocytic thyroiditis), Pendred's
syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone
coupling defect, thymic aplasia, Hurthle cell tumours of the
thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid
carcinoma; disorders and/or diseases of the parathyroid, such as,
for example, hyperparathyroidism, hypoparathyroidism; disorders
and/or diseases of the hypothalamus.
[0958] In addition, endocrine system and/or hormone imbalance
disorders and/or diseases may also include disorders and/or
diseases of the testes or ovaries, including cancer. Other
disorders and/or diseases of the testes or ovaries further include,
for example, ovarian cancer, polycystic ovary syndrome,
Klinefelter's syndrome, vanishing testes syndrome (bilateral
anorchia), congenital absence of Leydig's cells, cryptorchidism,
Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the
testis (benign), neoplasias of the testis and neo-testis.
[0959] Moreover, endocrine system and/or hormone imbalance
disorders and/or diseases may also include disorders and/or
diseases such as, for example, polyglandular deficiency syndromes,
pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and
disorders and/or cancers of endocrine tissues.
[0960] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, may be used to diagnose, prognose, prevent,
and/or treat endocrine diseases and/or disorders associated with
the tissue(s) in which the Therapeutic protein corresponding to the
Therapeutic protein portion of the albumin protein of the invention
is expressed,
[0961] Reproductive System Disorders
[0962] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used for the diagnosis, treatment, or prevention of diseases
and/or disorders of the reproductive system. Reproductive system
disorders that can be treated by the compositions of the invention,
include, but are not limited to, reproductive system injuries,
infections, neoplastic disorders, congenital defects, and diseases
or disorders which result in infertility, complications with
pregnancy, labor, or parturition, and postpartum difficulties.
[0963] Reproductive system disorders and/or diseases include
diseases and/or disorders of the testes, including testicular
atrophy, testicular feminization, cryptorchism (unilateral and
bilateral), anorchia, ectopic testis, epididymitis and orchitis
(typically resulting from infections such as, for example,
gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion,
vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell
carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors,
and teratomas), stromal tumors (e.g., Leydig cell tumors),
hydrocele, hematocele, varicocele, spermatocele, inguinal hernia,
and disorders of sperm production (e.g., immotile cilia syndrome,
aspermia, asthenozoospermia, azoospermia, oligospermia, and
teratozoospermia).
[0964] Reproductive system disorders also include disorders of the
prostate gland, such as acute non-bacterial prostatitis, chronic
non-bacterial prostatitis, acute bacterial prostatitis, chronic
bacterial prostatitis, prostatodystonia, prostatosis, granulomatous
prostatitis, malacoplakia, benign prostatic hypertrophy or
hyperplasia, and prostate neoplastic disorders, including
adenocarcinomas, transitional cell carcinomas, ductal carcinomas,
and squamous cell carcinomas.
[0965] Additionally, the compositions of the invention may be
useful in the diagnosis, treatment, and/or prevention of disorders
or diseases of the penis and urethra, including inflammatory
disorders, such as balanoposthitis, balanitis xerotica obliterans,
phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea,
non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV,
AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and
pearly penile papules; urethral abnormalities, such as hypospadias,
epispadias, and phimosis; premalignant lesions, including
Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant
condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile
cancers, including squamous cell carcinomas, carcinoma in situ,
verrucous carcinoma, and disseminated penile carcinoma; urethral
neoplastic disorders, including penile urethral carcinoma,
bulbomembranous urethral carcinoma, and prostatic urethral
carcinoma; and erectile disorders, such as priapism, Peyronie's
disease, erectile dysfunction, and impotence.
[0966] Moreover, diseases and/or disorders of the vas deferens
include vasculititis and CBAVD (congenital bilateral absence of the
vas deferens); additionally, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used in the diagnosis, treatment, and/or
prevention of diseases and/or disorders of the seminal vesicles,
including hydatid disease, congenital chloride diarrhea, and
polycystic kidney disease.
[0967] Other disorders and/or diseases of the male reproductive
system include, for example, Klinefelter's syndrome, Young's
syndrome, premature ejaculation, diabetes mellitus, cystic
fibrosis, Kartagener's syndrome, high fever, multiple sclerosis,
and gynecomastia.
[0968] Further, the polynucleotides, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used in the diagnosis, treatment, and/or
prevention of diseases and/or disorders of the vagina and vulva,
including bacterial vaginosis, candida vaginitis, herpes simplex
virus, chancroid, granuloma inguinale, lymphogranuloma venereum,
scabies, human papillomavirus, vaginal trauma, vulvar trauma,
adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis,
condyloma acuminatum, syphilis, molluscum contagiosum, atrophic
vaginitis, Paget's disease, lichen sclerosus, lichen planus,
vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis,
vulvar vestibulitis, and neoplastic disorders, such as squamous
cell hyperplasia, clear cell carcinoma, basal cell carcinoma,
melanomas, cancer of Bartholin's gland, and vulvar intraepithelial
neoplasia.
[0969] Disorders and/or diseases of the uterus include
dysmenorrhea, retroverted uterus, endometriosis, fibroids,
adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome,
hydatidiform moles, Asherman's syndrome, premature menopause,
precocious puberty, uterine polyps, dysfunctional uterine bleeding
(e.g., due to aberrant hormonal signals), and neoplastic disorders,
such as adenocarcinomas, keiomyosarcomas, and sarcomas.
Additionally, the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful as a marker or detector of, as well as in the
diagnosis, treatment, and/or prevention of congenital uterine
abnormalities, such as bicornuate uterus, septate uterus, simple
unicornuate uterus, unicornuate uterus with a noncavitary
rudimentary horn, unicornuate uterus with a non-communicating
cavitary rudimentary horn, unicornuate uterus with a communicating
cavitary horn, arcuate uterus, uterine didelfus, and T-shaped
uterus.
[0970] Ovarian diseases and/or disorders include anovulation,
polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian
cysts, ovarian hypofunction, ovarian insensitivity to
gonadotropins, ovarian overproduction of androgens, right ovarian
vein syndrome, amenorrhea, hirutism, and ovarian cancer (including,
but not limited to, primary and secondary cancerous growth,
Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian
papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma,
and Ovarian Krukenberg tumors).
[0971] Cervical diseases and/or disorders include cervicitis,
chronic cervicitis, mucopurulent cervicitis, cervical dysplasia,
cervical polyps, Nabothian cysts, cervical erosion, cervical
incompetence, and cervical neoplasms (including, for example,
cervical carcinoma, squamous metaplasia, squamous cell carcinoma,
adenosquamous cell neoplasia, and columnar cell neoplasia).
[0972] Additionally, diseases and/or disorders of the reproductive
system include disorders and/or diseases of pregnancy, including
miscarriage and stillbirth, such as early abortion, late abortion,
spontaneous abortion, induced abortion, therapeutic abortion,
threatened abortion, missed abortion, incomplete abortion, complete
abortion, habitual abortion, missed abortion, and septic abortion;
ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding
during pregnancy, gestational diabetes, intrauterine growth
retardation, polyhydramnios, HELLP syndrome, abruptio placentae,
placenta previa, hyperemesis, preeclampsia, eclampsia, herpes
gestationis, and urticaria of pregnancy. Additionally, the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention may be used in the
diagnosis, treatment, and/or prevention of diseases that can
complicate pregnancy, including heart disease, heart failure,
rheumatic heart disease, congenital heart disease, mitral valve
prolapse, high blood pressure, anemia, kidney disease, infectious
disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious
hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes
mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's
thyroiditis, chronic active hepatitis, cirrhosis of the liver,
primary biliary cirrhosis, asthma, systemic lupus eryematosis,
rheumatoid arthritis, myasthenia gravis, idiopathic
thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder
disorders, and obstruction of the intestine.
[0973] Complications associated with labor and parturition include
premature rupture of the membranes, pre-term labor, post-term
pregnancy, postmaturity, labor that progresses too slowly, fetal
distress (e.g., abnormal heart rate (fetal or maternal), breathing
problems, and abnormal fetal position), shoulder dystocia,
prolapsed umbilical cord, amniotic fluid embolism, and aberrant
uterine bleeding.
[0974] Further, diseases and/or disorders of the postdelivery
period, including endometritis, myometritis, parametritis,
peritonitis, pelvic thrombophlebitis, pulmonary embolism,
endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis,
cystitis, postpartum hemorrhage, and inverted uterus.
[0975] Other disorders and/or diseases of the female reproductive
system that may be diagnosed, treated, and/or prevented by the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention include, for
example, Turner's syndrome, pseudohermaphroditism, premenstrual
syndrome, pelvic inflammatory disease, pelvic congestion (vascular
engorgement), frigidity, anorgasmia, dyspareunia, ruptured
fallopian tube, and Mittelschmerz.
[0976] Infectious Disease
[0977] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used to treat or detect infectious agents. For example, by
increasing the immune response, particularly increasing the
proliferation and differentiation of B and/or T cells, infectious
diseases may be treated. The immune response may be increased by
either enhancing an existing immune response, or by initiating a
new immune response. Alternatively, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may also directly inhibit the infectious agent,
without necessarily eliciting an immune response.
[0978] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention. Examples of
viruses, include, but are not limited to Examples of viruses,
include, but are not limited to the following DNA and RNA viruses
and viral families: Arbovirus, Adenoviridae, Arenaviridae,
Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,
Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,
Hepadnaviridae (Hepatitis), Herpesviridae (such as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus
(e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae (e.g., Influenza A, Influenza B, and
parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,
Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia. Albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, can be used to treat or detect any of these symptoms or
diseases. In specific embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used to treat: meningitis, Dengue, EBV, and/or
hepatitis (e.g., hepatitis B). In an additional specific embodiment
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used to treat patients
nonresponsive to one or more other commercially available hepatitis
vaccines. In a further specific embodiment fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to treat AIDS.
[0979] Similarly, bacterial and fungal agents that can cause
disease or symptoms and that can be treated or detected by albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention include, but not limited
to, the following Gram-Negative and Gram-positive bacteria,
bacterial families, and fungi: Actinomyces (e.g., Norcardia),
Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae
(e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides
fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia
burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia,
Clostridium (e.g., Clostridium botulinum, Clostridium dificile,
Clostridium perfringens, Clostridium tetani), Coccidioides,
Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus,
Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and
Enterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter
aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g.,
Salmonella typhi, Salmonella enteritidis, Salmonella typhi),
Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g.,
Haemophilus influenza type B), Helicobacter, Legionella (e.g.,
Legionella pneumophila), Leptospira, Listeria (e.g., Listeria
monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium
leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio
cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria
meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g.,
Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g.,
Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp.,
Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus,
Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and
Groups A, B, and C Streptococci), and Ureaplasmas. These bacterial,
parasitic, and fungal families can cause diseases or symptoms,
including, but not limited to: antibiotic-resistant infections,
bacteremia, endocarditis, septicemia, eye infections (e.g.,
conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial
diarrhea, opportunistic infections (e.g., AIDS related infections),
paronychia, prosthesis-related infections, dental caries, Reiter's
Disease, respiratory tract infections, such as Whooping Cough or
Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery,
paratyphoid fever, food poisoning, Legionella disease, chronic and
acute inflammation, erythema, yeast infections, typhoid, pneumonia,
gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia,
syphillis, diphtheria, leprosy, brucellosis, peptic ulcers,
anthrax, spontaneous abortions, birth defects, pneumonia, lung
infections, ear infections, deafness, blindness, lethargy, malaise,
vomiting, chronic diarrhea, Crohn's disease, colitis, vaginosis,
sterility, pelvic inflammatory diseases, candidiasis,
paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus,
impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted
diseases, skin diseases (e.g., cellulitis, dermatocycoses),
toxemia, urinary tract infections, wound infections, noscomial
infections. Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
can be used to treat or detect any of these symptoms or diseases.
In specific embodiments, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
are used to treat: tetanus, diptheria, botulism, and/or meningitis
type B.
[0980] Moreover, parasitic agents causing disease or symptoms that
can be treated, prevented, and/or diagnosed by fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention include, but not limited to, the
following families or class: Amebiasis, Babesiosis, Coccidiosis,
Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,
Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis,
Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans
(e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium
malariae and Plasmodium ovale). These parasites can cause a variety
of diseases or symptoms, including, but not limited to: Scabies,
Trombiculiasis, eye infections, intestinal disease (e.g.,
dysentery, giardiasis), liver disease, lung disease, opportunistic
infections (e.g., AIDS related), malaria, pregnancy complications,
and toxoplasmosis. Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
can be used to treat, prevent, and/or diagnose any of these
symptoms or diseases. In specific embodiments, fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to treat, prevent, and/or
diagnose malaria.
[0981] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
could either be by administering an effective amount of an albumin
fusion protein of the invention to the patient, or by removing
cells from the patient, supplying the cells with a polynucleotide
of the present invention, and returning the engineered cells to the
patient (ex vivo therapy). Moreover, the albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention can be used as an antigen in a vaccine to
raise an immune response against infectious disease.
[0982] Regeneration
[0983] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used to differentiate, proliferate, and attract cells,
leading to the regeneration of tissues. (See, Science 276:59-87
(1997)). The regeneration of tissues could be used to repair,
replace, or protect tissue damaged by congenital defects, trauma
(wounds, burns, incisions, or ulcers), age, disease (e.g.
osteoporosis, osteocarthritis, periodontal disease, liver failure),
surgery, including cosmetic plastic surgery, fibrosis, reperfusion
injury, or systemic cytokine damage.
[0984] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous,
hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)
tissue. Preferably, regeneration occurs without or decreased
scarring. Regeneration also may include angiogenesis.
[0985] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may increase regeneration of tissues difficult to heal. For
example, increased tendon/ligament regeneration would quicken
recovery time after damage. Albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention could also be used prophylactically in an effort
to avoid damage. Specific diseases that could be treated include of
tendinitis, carpal tunnel syndrome, and other tendon or ligament
defects. A further example of tissue regeneration of non-healing
wounds includes pressure ulcers, ulcers associated with vascular
insufficiency, surgical, and traumatic wounds.
[0986] Similarly, nerve and brain tissue could also be regenerated
by using fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, to proliferate
and differentiate nerve cells. Diseases that could be treated using
this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated using the albumin fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention.
[0987] Gastrointestinal Disorders
[0988] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose
gastrointestinal disorders, including inflammatory diseases and/or
conditions, infections, cancers (e.g., intestinal neoplasms
(carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of
the small intestine, small bowl lymphoma)), and ulcers, such as
peptic ulcers.
[0989] Gastrointestinal disorders include dysphagia, odynophagia,
inflammation of the esophagus, peptic esophagitis, gastric reflux,
submucosal fibrosis and stricturing, Mallory-Weiss lesions,
leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric
retention disorders, gastroenteritis, gastric atrophy,
gastric/stomach cancers, polyps of the stomach, autoimmune
disorders such as pernicious anemia, pyloric stenosis, gastritis
(bacterial, viral, eosinophilic, stress-induced, chronic erosive,
atrophic, plasma cell, and Menetrier's), and peritoneal diseases
(e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric
lymphadenitis, mesenteric vascular occlusion, panniculitis,
neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).
[0990] Gastrointestinal disorders also include disorders associated
with the small intestine, such as malabsorption syndromes,
distension, irritable bowel syndrome, sugar intolerance, celiac
disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's
disease, intestinal lymphangiectasia, Crohn's disease,
appendicitis, obstructions of the ileum, Meckel's diverticulum,
multiple diverticula, failure of complete rotation of the small and
large intestine, lymphoma, and bacterial and parasitic diseases
(such as Traveler's diarrhea, typhoid and paratyphoid, cholera,
infection by Roundworms (Ascariasis lumbricoides), Hookworms
(Ancylostoma duodenale), Threadworms (Enterobius vermicularis),
Tapeworms (Taenia saginata, Echinococcus granulosus,
Diphyllobothrium spp., and T. solium).
[0991] Liver diseases and/or disorders include intrahepatic
cholestasis (alagille syndrome, biliary liver cirrhosis), fatty
liver (alcoholic fatty liver, reye syndrome), hepatic vein
thrombosis, hepatolentricular degeneration, hepatomegaly,
hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension
(esophageal and gastric varices), liver abscess (amebic liver
abscess), liver cirrhosis (alcoholic, biliary and experimental),
alcoholic liver diseases (fatty liver, hepatitis, cirrhosis),
parasitic (hepatic echinococcosis, fascioliasis, amebic liver
abscess), jaundice (hemolytic, hepatocellular, and cholestatic),
cholestasis, portal hypertension, liver enlargement, ascites,
hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis
(autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced),
toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B,
hepatitis C, hepatitis D, hepatitis E), Wilson's disease,
granulomatous hepatitis, secondary biliary cirrhosis, hepatic
encephalopathy, portal hypertension, varices, hepatic
encephalopathy, primary biliary cirrhosis, primary sclerosing
cholangitis, hepatocellular adenoma, hemangiomas, bile stones,
liver failure (hepatic encephalopathy, acute liver failure), and
liver neoplasms (angiomyolipoma, calcified liver metastases, cystic
liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma,
focal nodular hyperplasia, hepatic adenoma, hepatobiliary
cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma,
liver cancer, liver hemangioendothelioma, mesenchymal hamartoma,
mesenchymal tumors of liver, nodular regenerative hyperplasia,
benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver
disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal
tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma,
Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor,
Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct
hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular
hyperplasia, Nodular regenerative hyperplasia)], malignant liver
tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma,
cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors
of blood vessels, angiosarcoma, Karposi's sarcoma,
hemangioendothelioma, other tumors, embryonal sarcoma,
fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,
teratoma, carcinoid, squamous carcinoma, primary lymphoma]),
peliosis hepatis, erythrohepatic porphyria, hepatic porphyria
(acute intermittent porphyria, porphyria cutanea tarda), Zellweger
syndrome).
[0992] Pancreatic diseases and/or disorders include acute
pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis,
alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas,
cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic
neoplasms, islet-cell tumors, pancreoblastoma), and other
pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic
pseudocyst, pancreatic fistula, insufficiency)).
[0993] Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis), postcholecystectomy syndrome, diverticulosis
of the gallbladder, acute cholecystitis, chronic cholecystitis,
bile duct tumors, and mucocele.
[0994] Diseases and/or disorders of the large intestine include
antibiotic-associated colitis, diverticulitis, ulcerative colitis,
acquired megacolon, abscesses, fungal and bacterial infections,
anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases
(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps
(e.g., villous adenoma), colon carcinoma, colorectal cancer],
colonic diverticulitis, colonic diverticulosis, megacolon
[Hirschsprung disease, toxic megacolon]; sigmoid diseases
[proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease,
diarrhea (infantile diarrhea, dysentery), duodenal diseases
(duodenal neoplasms, duodenal obstruction, duodenal ulcer,
duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal
diseases (ileal neoplasms, ileitis), immunoproliferative small
intestinal disease, inflammatory bowel disease (ulcerative colitis,
Crohn's disease), intestinal atresia, parasitic diseases
(anisakiasis, balantidiasis, blastocystis infections,
cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis),
intestinal fistula (rectal fistula), intestinal neoplasms (cecal
neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms,
intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal
obstruction (afferent loop syndrome, duodenal obstruction, impacted
feces, intestinal pseudo-obstruction [cecal volvulus],
intussusception), intestinal perforation, intestinal polyps
(colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal
diseases (jejunal neoplasms), malabsorption syndromes (blind loop
syndrome, celiac disease, lactose intolerance, short bowl syndrome,
tropical sprue, whipple's disease), mesenteric vascular occlusion,
pneumatosis cystoides intestinalis, protein-losing enteropathies
(intestinal lymphagiectasis), rectal diseases (anus diseases, fecal
incontinence, hemorrhoids, proctitis, rectal fistula, rectal
prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic
esophagitis, hemorrhage, perforation, stomach ulcer,
Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping
syndrome), stomach diseases (e.g., achlorhydria, duodenogastric
reflux (bile reflux), gastric antral vascular ectasia, gastric
fistula, gastric outlet obstruction, gastritis (atrophic or
hypertrophic), gastroparesis, stomach dilatation, stomach
diverticulum, stomach neoplasms (gastric cancer, gastric polyps,
gastric adenocarcinoma, hyperplastic gastric polyp), stomach
rupture, stomach ulcer, stomach volvulus), tuberculosis,
visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,
postoperative nausea and vomiting) and hemorrhagic colitis.
[0995] Further diseases and/or disorders of the gastrointestinal
system include biliary tract diseases, such as, gastroschisis,
fistula (e.g., biliary fistula, esophageal fistula, gastric
fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g.,
biliary tract neoplasms, esophageal neoplasms, such as
adenocarcinoma of the esophagus, esophageal squamous cell
carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such
as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the
pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and
peritoneal neoplasms), esophageal disease (e.g., bullous diseases,
candidiasis, glycogenic acanthosis, ulceration, barrett esophagus
varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum),
fistula (e.g., tracheoesophageal fistula), motility disorders
(e.g., CREST syndrome, deglutition disorders, achalasia, spasm,
gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave
syndrome, Mallory-Weiss syndrome), stenosis, esophagitis,
diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal
diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk
virus infection), hemorrhage (e.g., hematemesis, melena, peptic
ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric
polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g.,
congenital diaphragmatic hernia, femoral hernia, inguinal hernia,
obturator hernia, umbilical hernia, ventral hernia), and intestinal
diseases (e.g., cecal diseases (appendicitis, cecal
neoplasms)).
[0996] Chemotaxis
[0997] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may have chemotaxis activity. A chemotaxic molecule attracts or
mobilizes cells (e.g., monocytes, fibroblasts, neutrophils,
T-cells, mast cells, eosinophils, epithelial and/or endothelial
cells) to a particular site in the body, such as inflammation,
infection, or site of hyperproliferation. The mobilized cells can
then fight off and/or heal the particular trauma or
abnormality.
[0998] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may increase chemotaxic activity of particular cells. These
chemotactic molecules can then be used to treat inflammation,
infection, hyperproliferative disorders, or any immune system
disorder by increasing the number of cells targeted to a particular
location in the body. For example, chemotaxic molecules can be used
to treat wounds and other trauma to tissues by attracting immune
cells to the injured location. Chemotactic molecules of the present
invention can also attract fibroblasts, which can be used to treat
wounds.
[0999] It is also contemplated that fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may inhibit chemotactic activity. These molecules
could also be used to treat disorders. Thus, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention could be used as an inhibitor of chemotaxis.
[1000] Binding Activity
[1001] Albumin fusion proteins of the invention may be used to
screen for molecules that bind to the Therapeutic protein portion
of the fusion protein or for molecules to which the Therapeutic
protein portion of the fusion protein binds. The binding of the
fusion protein and the molecule may activate (agonist), increase,
inhibit (antagonist), or decrease activity of the fusion protein or
the molecule bound. Examples of such molecules include antibodies,
oligonucleotides, proteins (e.g., receptors), or small
molecules.
[1002] Preferably, the molecule is closely related to the natural
ligand of the Therapeutic protein portion of the fusion protein of
the invention, e.g., a fragment of the ligand, or a natural
substrate, a ligand, a structural or functional mimetic. (See,
Coligan et al., Current Protocols in Immunology 1(2): Chapter 5
(1991)). Similarly, the molecule can be closely related to the
natural receptor to which the Therapeutic protein portion of an
albumin fusion protein of the invention binds, or at least, a
fragment of the receptor capable of being bound by the Therapeutic
protein portion of an albumin fusion protein of the invention
(e.g., active site). In either case, the molecule can be rationally
designed using known techniques.
[1003] Preferably, the screening for these molecules involves
producing appropriate cells which express the albumin fusion
proteins of the invention. Preferred cells include cells from
mammals, yeast, Drosophila, or E. coli.
[1004] The assay may simply test binding of a candidate compound to
an albumin fusion protein of the invention, wherein binding is
detected by a label, or in an assay involving competition with a
labeled competitor. Further, the assay may test whether the
candidate compound results in a signal generated by binding to the
fusion protein.
[1005] Alternatively, the assay can be carried out using cell-free
preparations, fusion protein/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing an albumin fusion protein, measuring fusion
protein/molecule activity or binding, and comparing the fusion
protein/molecule activity or binding to a standard.
[1006] Preferably, an ELISA assay can measure fusion protein level
or activity in a sample (e.g., biological sample) using a
monoclonal or polyclonal antibody. The antibody can measure fusion
protein level or activity by either binding, directly or
indirectly, to the albumin fusion protein or by competing with the
albumin fusion protein for a substrate.
[1007] Additionally, the receptor to which a Therapeutic protein
portion of an albumin fusion protein of the invention binds can be
identified by numerous methods known to those of skill in the art,
for example, ligand panning and FACS sorting (Coligan, et al.,
Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example,
in cases wherein the Therapeutic protein portion of the fusion
protein corresponds to FGF, expression cloning may be employed
wherein polyadenylated RNA is prepared from a cell responsive to
the albumin fusion protein, for example, NIH3T3 cells which are
known to contain multiple receptors for the FGF family proteins,
and SC-3 cells, and a cDNA library created from this RNA is divided
into pools and used to transfect COS cells or other cells that are
not responsive to the albumin fusion protein. Transfected cells
which are grown on glass slides are exposed to the albumin fusion
protein of the present invention, after they have been labeled. The
albumin fusion proteins can be labeled by a variety of means
including iodination or inclusion of a recognition site for a
site-specific protein kinase.
[1008] Following fixation and incubation, the slides are subjected
to auto-radiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an iterative
sub-pooling and re-screening process, eventually yielding a single
clones that encodes the putative receptor.
[1009] As an alternative approach for receptor identification, a
labeled albumin fusion protein can be photoaffinity linked with
cell membrane or extract preparations that express the receptor
molecule for the Therapeutoc protein component of an albumin fusion
protein of the invention, the linked material may be resolved by
PAGE analysis and exposed to X-ray film. The labeled complex
containing the receptors of the fusion protein can be excised,
resolved into peptide fragments, and subjected to protein
microsequencing. The amino acid sequence obtained from
microsequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
genes encoding the putative receptors.
[1010] Moreover, the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of the
fusion protein, and/or Therapeutic protein portion or albumin
component of an albumin fusion protein of the present invention,
thereby effectively generating agonists and antagonists of an
albumin fusion protein of the present invention. See generally,
U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and
5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol.
8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82
(1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999);
and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13
(1998); each of these patents and publications are hereby
incorporated by reference). In one embodiment, alteration of
polynucleotides encoding albumin fusion proteins of the invention
and thus, the albumin fusion proteins encoded thereby, may be
achieved by DNA shuffling. DNA shuffling involves the assembly of
two or more DNA segments into a desired molecule by homologous, or
site-specific, recombination. In another embodiment,
polynucleotides encoding albumin fusion proteins of the invention
and thus, the albumin fusion proteins encoded thereby, may be
altered by being subjected to random mutagenesis by error-prone
PCR, random nucleotide insertion or other methods prior to
recombination. In another embodiment, one or more components,
motifs, sections, parts, domains, fragments, etc., of an albumin
fusion protein of the present invention may be recombined with one
or more components, motifs, sections, parts, domains, fragments,
etc. of one or more heterologous molecules. In preferred
embodiments, the heterologous molecules are family members. In
further preferred embodiments, the heterologous molecule is a
growth factor such as, for example, platelet-derived growth factor
(PDGF), insulin-like growth factor (IGF-I), transforming growth
factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast
growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,
BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp),
60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal,
MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and
glial-derived neurotrophic factor (GDNF).
[1011] Other preferred fragments are biologically active fragments
of the Therapeutic protein portion and/or albumin component of the
albumin fusion proteins of the present invention. Biologically
active fragments are those exhibiting activity similar, but not
necessarily identical, to an activity of a Therapeutic protein
portion and/or albumin component of the albumin fusion proteins of
the present invention. The biological activity of the fragments may
include an improved desired activity, or a decreased undesirable
activity.
[1012] Additionally, this invention provides a method of screening
compounds to identify those which modulate the action of an albumin
fusion protein of the present invention. An example of such an
assay comprises combining a mammalian fibroblast cell, an albumin
fusion protein of the present invention, and the compound to be
screened and .sup.3[H] thymidine under cell culture conditions
where the fibroblast cell would normally proliferate. A control
assay may be performed in the absence of the compound to be
screened and compared to the amount of fibroblast proliferation in
the presence of the compound to determine if the compound
stimulates proliferation by determining the uptake of .sup.3[H]
thymidine in each case. The amount of fibroblast cell proliferation
is measured by liquid scintillation chromatography which measures
the incorporation of .sup.3[H] thymidine. Both agonist and
antagonist compounds may be identified by this procedure.
[1013] In another method, a mammalian cell or membrane preparation
expressing a receptor for the Therapeutic protein component of a
fusion protein of the invention is incubated with a labeled fusion
protein of the present invention in the presence of the compound.
The ability of the compound to enhance or block this interaction
could then be measured. Alternatively, the response of a known
second messenger system following interaction of a compound to be
screened and the receptor is measured and the ability of the
compound to bind to the receptor and elicit a second messenger
response is measured to determine if the compound is a potential
fusion protein. Such second messenger systems include but are not
limited to, cAMP guanylate cyclase, ion channels or
phosphoinositide hydrolysis.
[1014] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat disease or to bring about a particular result in a
patient (e.g., blood vessel growth) by activating or inhibiting the
fusion protein/molecule. Moreover, the assays can discover agents
which may inhibit or enhance the production of the albumin fusion
proteins of the invention from suitably manipulated cells or
tissues.
[1015] Therefore, the invention includes a method of identifying
compounds which bind to an albumin fusion protein of the invention
comprising the steps of: (a) incubating a candidate binding
compound with an albumin fusion protein of the present invention;
and (b) determining if binding has occurred. Moreover, the
invention includes a method of identifying agonists/antagonists
comprising the steps of: (a) incubating a candidate compound with
an albumin fusion protein of the present invention, (b) assaying a
biological activity, and (b) determining if a biological activity
of the fusion protein has been altered.
[1016] Targeted Delivery
[1017] In another embodiment, the invention provides a method of
delivering compositions to targeted cells expressing a receptor for
a component of an albumin fusion protein of the invention.
[1018] As discussed herein, fusion proteins of the invention may be
associated with heterologous polypeptides, heterologous nucleic
acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic
and/or covalent interactions. In one embodiment, the invention
provides a method for the specific delivery of compositions of the
invention to cells by administering fusion proteins of the
invention (including antibodies) that are associated with
heterologous polypeptides or nucleic acids. In one example, the
invention provides a method for delivering a Therapeutic protein
into the targeted cell. In another example, the invention provides
a method for delivering a single stranded nucleic acid (e.g.,
antisense or ribozymes) or double stranded nucleic acid (e.g., DNA
that can integrate into the cell's genome or replicate episomally
and that can be transcribed) into the targeted cell.
[1019] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering an albumin fusion protein of the invention
(e.g., polypeptides of the invention or antibodies of the
invention) in association with toxins or cytotoxic prodrugs.
[1020] By "toxin" is meant compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins,
modified toxins, catalytic subunits of toxins, or any molecules or
enzymes not normally present in or on the surface of a cell that
under defined conditions cause the cell's death. Toxins that may be
used according to the methods of the invention include, but are not
limited to, radioisotopes known in the art, compounds such as, for
example, antibodies (or complement fixing containing portions
thereof) that bind an inherent or induced endogenous cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is meant a
non-toxic compound that is converted by an enzyme, normally present
in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may
be used according to the methods of the invention include, but are
not limited to, glutamyl derivatives of benzoic acid mustard
alkylating agent, phosphate derivatives of etoposide or mitomycin
C, cytosine arabinoside, daunorubisin, and phenoxyacetamide
derivatives of doxorubicin.
[1021] Drug Screening
[1022] Further contemplated is the use of the albumin fusion
proteins of the present invention, or the polynucleotides encoding
these fusion proteins, to screen for molecules which modify the
activities of the albumin fusion protein of the present invention
or proteins corresponding to the Therapeutic protein portion of the
albumin fusion protein. Such a method would include contacting the
fusion protein with a selected compound(s) suspected of having
antagonist or agonist activity, and assaying the activity of the
fusion protein following binding.
[1023] This invention is particularly useful for screening
therapeutic compounds by using the albumin fusion proteins of the
present invention, or binding fragments thereof, in any of a
variety of drug screening techniques. The albumin fusion protein
employed in such a test may be affixed to a solid support,
expressed on a cell surface, free in solution, or located
intracellularly. One method of drug screening utilizes eukaryotic
or prokaryotic host cells which are stably transformed with
recombinant nucleic acids expressing the albumin fusion protein.
Drugs are screened against such transformed cells or supernatants
obtained from culturing such cells, in competitive binding assays.
One may measure, for example, the formulation of complexes between
the agent being tested and an albumin fusion protein of the present
invention.
[1024] Thus, the present invention provides methods of screening
for drugs or any other agents which affect activities mediated by
the albumin fusion proteins of the present invention. These methods
comprise contacting such an agent with an albumin fusion protein of
the present invention or a fragment thereof and assaying for the
presence of a complex between the agent and the albumin fusion
protein or a fragment thereof, by methods well known in the art. In
such a competitive binding assay, the agents to screen are
typically labeled. Following incubation, free agent is separated
from that present in bound form, and the amount of free or
uncomplexed label is a measure of the ability of a particular agent
to bind to the albumin fusion protein of the present invention.
[1025] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to an albumin fusion protein of the present invention, and is
described in great detail in European Patent Application 84/03564,
published on Sep. 13, 1984, which is incorporated herein by
reference herein. Briefly stated, large numbers of different small
peptide test compounds are synthesized on a solid substrate, such
as plastic pins or some other surface. The peptide test compounds
are reacted with an albumin fusion protein of the present invention
and washed. Bound peptides are then detected by methods well known
in the art. Purified albumin fusion protein may be coated directly
onto plates for use in the aforementioned drug screening
techniques. In addition, non-neutralizing antibodies may be used to
capture the peptide and immobilize it on the solid support.
[1026] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding an albumin fusion protein of the present invention
specifically compete with a test compound for binding to the
albumin fusion protein or fragments thereof. In this manner, the
antibodies are used to detect the presence of any peptide which
shares one or more antigenic epitopes with an albumin fusion
protein of the invention.
[1027] Binding Peptides and Other Molecules
[1028] The invention also encompasses screening methods for
identifying polypeptides and nonpolypeptides that bind albumin
fusion proteins of the invention, and the binding molecules
identified thereby. These binding molecules are useful, for
example, as agonists and antagonists of the albumin fusion proteins
of the invention. Such agonists and antagonists can be used, in
accordance with the invention, in the therapeutic embodiments
described in detail, below.
[1029] This method comprises the steps of: contacting an albumin
fusion protein of the invention with a plurality of molecules; and
identifying a molecule that binds the albumin fusion protein.
[1030] The step of contacting the albumin fusion protein of the
invention with the plurality of molecules may be effected in a
number of ways. For example, one may contemplate immobilizing the
albumin fusion protein on a solid support and bringing a solution
of the plurality of molecules in contact with the immobilized
polypeptides. Such a procedure would be akin to an affinity
chromatographic process, with the affinity matrix being comprised
of the immobilized albumin fusion protein of the invention. The
molecules having a selective affinity for the albumin fusion
protein can then be purified by affinity selection. The nature of
the solid support, process for attachment of the albumin fusion
protein 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.
[1031] 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" by
an albumin fusion protein of the invention, optionally in the
presence of an inducer should one be required for expression, to
determine if any selective affinity interaction takes place between
the albumin fusion protein and the individual clone. Prior to
contacting the albumin fusion 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 an albumin fusion
protein of the invention. Furthermore, the amino acid sequence of
the polypeptide having a selective affinity for an albumin fusion
protein of the invention 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
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.
[1032] In certain situations, it may be desirable to wash away any
unbound polypeptides from a mixture of an albumin fusion protein of
the invention and the plurality of polypeptides prior to attempting
to determine or to detect the presence of a selective affinity
interaction. Such a wash step may be particularly desirable when
the albumin fusion protein of the invention or the plurality of
polypeptides are bound to a solid support.
[1033] The plurality of molecules provided according to this method
may be provided by way of diversity libraries, such as random or
combinatorial peptide or nonpeptide libraries which can be screened
for molecules that specifically bind an albumin fusion protein of
the invention. 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 No. WO 93/20242; and Brenner
and Lerner, Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992).
[1034] Examples of phage display libraries are described in Scott
et al., Science 249:386-390 (1990); Devlin et al., Science,
249:404-406 (1990); Christian et al., 1992, 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 No. WO
94/18318 dated Aug. 18, 1994.
[1035] In vitro translation-based libraries include but are not
limited to those described in PCT Publication No. WO 91/05058 dated
Apr. 18, 1991; and Mattheakis et al., Proc. Natl. Acad. Sci. USA
91:9022-9026 (1994).
[1036] By way of examples of nonpeptide 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)).
[1037] 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.
[1038] 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.
[1039] 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.
[1040] 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
et al., Adv. Exp. Med. Biol. 251:215-218 (1989); Scott et al.,
Science 249:386-390 (1990); Fowlkes et al., BioTechniques
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 et al., Science 263:671-673 (1993); and
PCT Publication No. WO 94/18318.
[1041] In a specific embodiment, screening to identify a molecule
that binds an albumin fusion protein of the invention can be
carried out by contacting the library members with an albumin
fusion protein of the invention immobilized on a solid phase and
harvesting those library members that bind to the albumin fusion
protein. Examples of such screening methods, termed "panning"
techniques are described by way of example in Parmley et al., Gene
73:305-318 (1988); Fowlkes et al., BioTechniques 13:422-427 (1992);
PCT Publication No. WO 94/18318; and in references cited
herein.
[1042] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields et al., 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
polypeptides of the invention.
[1043] Where the 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.
[1044] 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 or that positions 4, 8, and 9 of a
decapeptide library be fixed to include only arginine. 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.
[1045] As mentioned above, in the case of a 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 binding polypeptide has in the
range of 15-100 amino acids, or 20-50 amino acids.
[1046] The selected binding polypeptide can be obtained by chemical
synthesis or recombinant expression.
[1047] Other Activities
[1048] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention,
may be employed in treatment for stimulating re-vascularization of
ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions.
The albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may also be
employed to stimulate angiogenesis and limb regeneration, as
discussed above.
[1049] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed for treating wounds due to injuries, burns,
post-operative tissue repair, and ulcers since they are mitogenic
to various cells of different origins, such as fibroblast cells and
skeletal muscle cells, and therefore, facilitate the repair or
replacement of damaged or diseased tissue.
[1050] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed stimulate neuronal growth and to treat and
prevent neuronal damage which occurs in certain neuronal disorders
or neuro-degenerative conditions such as Alzheimer's disease,
Parkinson's disease, and AIDS-related complex. An albumin fusion
protein of the invention and/or polynucleotide encoding an albumin
fusion protein of the invention may have the ability to stimulate
chondrocyte growth, therefore, they may be employed to enhance bone
and periodontal regeneration and aid in tissue transplants or bone
grafts.
[1051] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may be also be employed to prevent skin aging due to sunburn by
stimulating keratinocyte growth.
[1052] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed for preventing hair loss. Along the same
lines, an albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may be employed to stimulate growth and differentiation of
hematopoietic cells and bone marrow cells when used in combination
with other cytokines.
[1053] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed to maintain organs before transplantation or
for supporting cell culture of primary tissues. An albumin fusion
protein of the invention and/or polynucleotide encoding an albumin
fusion protein of the invention may also be employed for inducing
tissue of mesodermal origin to differentiate in early embryos.
[1054] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also increase or decrease the differentiation or proliferation
of embryonic stem cells, besides, as discussed above, hematopoietic
lineage.
[1055] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be used to modulate mammalian characteristics, such as
body height, weight, hair color, eye color, skin, percentage of
adipose tissue, pigmentation, size, and shape (e.g., cosmetic
surgery). Similarly, an albumin fusion protein of the invention
and/or polynucleotide encoding an albumin fusion protein of the
invention may be used to modulate mammalian metabolism affecting
catabolism, anabolism, processing, utilization, and storage of
energy.
[1056] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may be used to change a mammal's mental state or physical state by
influencing biorhythms, caricadic rhythms, depression (including
depressive disorders), tendency for violence, tolerance for pain,
reproductive capabilities (preferably by Activin or Inhibin-like
activity), hormonal or endocrine levels, appetite, libido, memory,
stress, or other cognitive qualities.
[1057] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be used as a food additive or preservative, such as to
increase or decrease storage capabilities, fat content, lipid,
protein, carbohydrate, vitamins, minerals, cofactors or other
nutritional components.
[1058] The above-recited applications have uses in a wide variety
of hosts. Such hosts include, but are not limited to, human,
murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat,
non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig,
sheep, dog or cat. In preferred embodiments, the host is a mammal.
In most preferred embodiments, the host is a human.
[1059] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
[1060] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the
alterations detected in the present invention and practice the
claimed methods. The following working examples therefore,
specifically point out preferred embodiments of the present
invention, and are not to be construed as limiting in any way the
remainder of the disclosure.
EXAMPLES
Example 1
Generation of pScNHSA and pScCHSA
[1061] The vectors pScNHSA (ATCC Deposit No. PTA-3279) and pScCHSA
(ATCC Deposit No. PTA-3276) are derivatives of pPPC0005 (ATCC
Deposit No. PTA-3278) and are used as cloning vectors into which
polynucleotides encoding a therapeutic protein or fragment or
variant thereof is inserted adjacent to and in translation frame
with polynucleotides encoding human serum albumin "HSA". pScCHSA
may be used for generating Therapeutic protein-HSA fusions, while
pScNHSA may be used to generate HSA-Therapeutic protein
fusions.
[1062] Generation of pScCHSA: Albumin Fusion with the Albumin
Moiety C-Terminal to the Therapeutic Portion.
[1063] A vector to facilitate cloning DNA encoding a Therapeutic
protein N-terminal to DNA encoding the mature albumin protein was
made by altering the nucleic acid sequence that encodes the
chimeric HSA signal peptide in pPPC0005 to include the Xho I and
Cla I restriction sites.
[1064] First, the Xho I and Cla I sites inherent to pPPC0005
(located 3' of the ADH1 terminator sequence) were eliminated by
digesting pPPC0005 with Xho I and Cla I, filling in the sticky ends
with T4 DNA polymerase, and religating the blunt ends to create
pPPC0006.
[1065] Second, the Xho I and Cla I restriction sites were
engineered into the nucleic acid sequence that encodes the signal
peptide of HSA (a chimera of the HSA leader and a kex2 site from
mating factor alpha, "MAF") in pPPC0006 using two rounds of PCR. In
the first round of PCR, amplification with primers shown as SEQ ID
NO:36 and SEQ ID NO:37 was performed. The primer whose sequence is
shown as SEQ ID NO:36 comprises a nucleic acid sequence that
encodes part of the signal peptide sequence of HSA, a kex2 site
from the mating factor alpha leader sequence, and part of the
amino-terminus of the mature form of HSA. Four point mutations were
introduced in the sequence, creating the Xho I and Cla I sites
found at the junction of the chimeric signal peptide and the mature
form of HSA. These four mutations are underlined in the sequence
shown below. In pPPC0005 the nucleotides at these four positions
from 5' to 3' are T, G, T, and G.
5'-GCCTCGACAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGATTTAAAGATTTGGG-3- '
(SEQ ID NO:36) and
5'-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3'
(SEQ ID NO:37). A second round of PCR was then performed with an
upstream flanking primer, 5'-TACAAACTTAAGAGTCCAATTAGC-3' (SEQ ID
NO:38) and a downstream flanking primer
5'-CACTTCTCTAGAGTGGTTTCATATGTCTT-3' (SEQ ID NO:39). The resulting
PCR product was then purified and digested with Afl II and Xba I
and ligated into the same sites in pPPC0006 creating pScCHSA. The
resulting plasmid has Xho I and Cla I sites engineered into the
signal sequence. The presence of the Xho I site creates a single
amino acid change in the end of the signal sequence from LDKR to
LEKR. The D to E change will not be present in the final albumin
fusion protein expression plasmid when a nucleic acid sequence
comprising a polynucleotide encoding the Therapeutic portion of the
albumin fusion protein with a 5' Sal I site (which is compatible
with the Xho I site) and a 3' Cla I site is ligated into the Xho I
and Cla I sites of pScCHSA. Ligation of Sal I to Xho I restores the
original amino acid sequence of the signal peptide sequence. DNA
encoding the Therapeutic portion of the albumin fusion protein may
be inserted after the Kex2 site (Kex2 cleaves after the dibasic
amino acid sequence KR at the end of the signal peptide) and prior
to the Cla I site.
[1066] Generation of pScNHSA: Albumin Fusion with the Albumin
Moiety N-Terminal to the Therapeutic Portion.
[1067] A vector to facilitate cloning DNA encoding a Therapeutic
protein portion C-terminal to DNA encoding the mature albumin
protein, was made by adding three, eight-base-pair restriction
sites to pScCHSA. The Asc I, Fse I, and Pme I restriction sites
were added in between the Bsu36 I and Hind III sites at the end of
the nucleic acid sequence encoding the mature HSA protein. This was
accomplished through the use of two complementary synthetic primers
containing the Asc I, Fse I, and Pme I restriction sites underlined
(SEQ ID NO:40 and SEQ ID NO:41).
5'-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTTTAAACTAAGCTTAATTCT-3'
(SEQ ID NO:40) and
5-AGAATTAAGCTTAGTTTAAACGGCCGGCCGGCGCGCCTTATTATAAGCCTAAGGCAGCTT-3'
(SEQ ID NO:41). These primers were annealed and digested with Bsu36
I and Hind III and ligated into the same sites in pScCHSA creating
pScNHSA.
Example 2
General Construct Generation for Yeast Transformation
[1068] The vectors pScNHSA and pScCHSA may be used as cloning
vectors into which polynucleotides encoding a therapeutic protein
or fragment or variant thereof is inserted adjacent to
polynucleotides encoding mature human serum albumin "HSA". pScCHSA
is used for generating Therapeutic protein-HSA fusions, while
pScNHSA may be used to generate HSA-Therapeutic protein
fusions.
[1069] Generation of Albumin Fusion Constructs Comprising
HSA-Therapeutic Protein Fusion Products.
[1070] DNA encoding a Therapeutic protein (e.g., sequences shown in
SEQ ID NO:X or known in the art) may be PCR amplified using the
primers which facilitate the generation of a fusion construct
(e.g., by adding restriction sites, encoding seamless fusions,
encoding linker sequences, etc.) For example, one skilled in the
art could design a 5' primer that adds polynucleotides encoding the
last four amino acids of the mature form of HSA (and containing the
Bsu36I site) onto the 5' end of DNA encoding a Therapeutic protein;
and a 3' primer that adds a STOP codon and appropriate cloning
sites onto the 3' end of the Therapeutic protein coding sequence.
For instance, the forward primer used to amplify DNA encoding a
Therapeutic protein might have the sequence,
5'-aagctGCCTTAGGCTTA(N).sub.15-3' (SEQ ID NO:42) where the
underlined sequence is a Bsu36I site, the upper case nucleotides
encode the last four amino acids of the mature HSA protein (ALGL),
and (N).sub.15 is identical to the first 15 nucleotides encoding
the Therapeutic protein of interest. Similarly, the reverse primer
used to amplify DNA encoding a Therapeutic protein might have the
sequence,
##STR00001##
(SEQ ID NO:43) where the italicized sequence is a Pme I site, the
double underlined sequence is an Fse I site, the singly underlined
sequence is an Asc I site, the boxed nucleotides are the reverse
complement of two tandem stop codons, and (N).sub.15 is identical
to the reverse complement of the last 15 nucleotides encoding the
Therapeutic protein of interest. Once the PCR product is amplified
it may be cut with Bsu36I and one of (Asc I, Fse I, or Pme I) and
ligated into pScNHSA.
[1071] The presence of the Xho I site in the HSA chimeric leader
sequence creates a single amino acid change in the end of the
chimeric signal sequence, i.e. the HSA-kex2 signal sequence, from
LDKR (SEQ ID NO:44) to LEKR (SEQ ID NO:45).
[1072] Generation of Albumin Fusion Constructs Comprising Gene-HSA
Fusion Products.
[1073] Similar to the method described above, DNA encoding a
Therapeutic protein may be PCR amplified using the following
primers: A 5' primer that adds polynucleotides containing a SalI
site and encoding the last three amino acids of the HSA leader
sequence, DKR, onto the 5' end of DNA encoding a Therapeutic
protein; and a 3' primer that adds polynucleotides encoding the
first few amino acids of the mature HSA containing a Cla I site
onto the 3' end of DNA encoding a Therapeutic protein. For
instance, the forward primer used to amplify the DNA encoding a
Therapeutic protein might have the sequence,
5'-aggagcgtcGACAAAAGA(N).sub.15-3' (SEQ ID NO:46) where the
underlined sequence is a Sal I site, the upper case nucleotides
encode the last three amino acids of the HSA leader sequence (DKR),
and (N).sub.15 is identical to the first 15 nucleotides encoding
the Therapeutic protein of interest. Similarly, the reverse primer
used to amplify the DNA encoding a Therapeutic protein might have
the sequence, 5'-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATC(N).sub.15-3'
(SEQ ID NO:47) where the italicized sequence is a Cla I site, the
underlined nucleotides are the reverse complement of the DNA
encoding the first 9 amino acids of the mature form of HSA
(DAHKSEVAH, SEQ ID NO:48), and (N).sub.15 is identical to the
reverse complement of the last 15 nucleotides encoding the
Therapeutic protein of interest. Once the PCR product is amplified
it may be cut with Sal I and Cla I and ligated into pScCHSA
digested with Xho I and Cla I. A different signal or leader
sequence may be desired, for example, invertase "INV" (Swiss-Prot
Accession P00724), mating factor alpha "MAF" (Genbank Accession
AAA18405), MPIF (Geneseq AAF82936), Fibulin B (Swiss-Prot Accession
P23142), Clusterin (Swiss-Prot Accession P10909), Insulin-Like
Growth Factor-Binding Protein 4 (Swiss-Prot Accession P22692), and
permutations of the HSA leader sequence can be subcloned into the
appropriate vector by means of standard methods known in the
art.
[1074] Generation of Albumin Fusion Construct Compatible for
Expression in Yeast S. cerevisiae.
[1075] The Not I fragment containing the DNA encoding either an
N-terminal or C-terminal albumin fusion protein generated from
pScNHSA or pScCHSA may then be cloned into the Not I site of pSAC35
which has a LEU2 selectable marker. The resulting vector is then
used in transformation of a yeast S. cerevisiae expression
system.
Example 3
General Expression in Yeast S. cerevisiae
[1076] An expression vector compatible with yeast expression can be
transformed into yeast S. cerevisiae by lithium acetate
transformation, electroporation, or other methods known in the art
and or as described in part in Sambrook, Fritsch, and Maniatis.
1989. "Molecular Cloning: A Laboratory Manual, 2.sup.nd edition",
volumes 1-3, and in Ausubel et al. 2000. Massachusetts General
Hospital and Harvard Medical School "Current Protocols in Molecular
Biology", volumes 1-4. The expression vectors are introduced into
S. cerevisiae strains DXY1, D88, or BXP10 by transformation,
individual transformants can be grown, for example, for 3 days at
30.degree. C. in 10 mL YEPD (1% w/v yeast extract, 2% w/v, peptone,
2% w/v, dextrose), and cells can be collected at stationary phase
after 60 hours of growth. Supernatants are collected by clarifying
cells at 3000 g for 10 minutes.
[1077] pSAC35 (Sleep et al., 1990, Biotechnology 8:42 and see FIG.
3) comprises, in addition to the LEU2 selectable marker, the entire
yeast 2 .mu.m plasmid to provide replication functions, the PRB1
promoter, and the ADH1 termination signal.
Example 4
General Purification of an Albumin Fusion Protein Expressed from an
Albumin Fusion in Yeast S. cerevisiae
[1078] In preferred embodiments, albumin fusion proteins of the
invention comprise the mature form of HSA fused to either the N- or
C-terminus of the mature form of a therapeutic protein or portions
thereof (e.g., the mature form of a therapeutic protein listed in
Table 1, or the mature form of a therapeutic protein shown in Table
2 as SEQ ID NO:Z). In one embodiment of the invention, albumin
fusion proteins of the invention further comprise a signal sequence
which directs the nascent fusion polypeptide in the secretory
pathways of the host used for expression. In a preferred
embodiment, the signal peptide encoded by the signal sequence is
removed, and the mature albumin fusion protein is secreted directly
into the culture medium. Albumin fusion proteins of the invention
preferably comprise heterologous signal sequences (e.g., the
non-native signal sequence of a particular therapeutic protein)
including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin,
Insulin-Like Growth Factor Binding Protein 4, variant HSA leader
sequences including, but not limited to, a chimeric HSA/MAF leader
sequence, or other heterologous signal sequences known in the art.
Especially preferred as those signal sequence listed in Table 2
and/or the signal sequence listed in the "Expression of Fusion
Proteins" and/or "Additional Methods of Recombinant and Synthetic
Production of Albumin Fusion Proteins" section of the
specification, above. In preferred embodiments, the fusion proteins
of the invention further comprise an N-terminal methionine residue.
Polynucleotides encoding these polypeptides, including fragments
and/or variants, are also encompassed by the invention.
[1079] Albumin fusion proteins expressed in yeast as described
above can be purified on a small-scale over a Dyax peptide affinity
column as follows. Supernatants from yeast expressing an albumin
fusion protein is diafiltrated against 3 mM phosphate buffer pH
6.2, 20 mM NaCl and 0.01% Tween 20 to reduce the volume and to
remove the pigments. The solution is then filtered through a 0.22
.mu.m device. The filtrate is loaded onto a Dyax peptide affinity
column. The column is eluted with 100 mM Tris/HCl, pH 8.2 buffer.
The peak fractions containing protein are collected and analyzed on
SDS-PAGE after concentrating 5-fold.
[1080] For large scale purification, the following method can be
utilized. The supernatant in excess of 2 L is diafiltered and
concentrated to 500 mL in 20 mM Tris/HCl pH 8.0. The concentrated
protein solution is loaded onto a pre-equilibrated 50 mL
DEAE-Sepharose Fast Flow column, the column is washed, and the
protein is eluted with a linear gradient of NaCl from 0 to 0.4 M
NaCl in 20 mM Tris/HCl, pH 8.0. Those fractions containing the
protein are pooled, adjusted to pH 6.8 with 0.5 M sodium phosphate
(NaH.sub.2PO.sub.4). A final concentration of 0.9 M
(NH.sub.4).sub.2SO.sub.4 is added to the protein solution and the
whole solution is loaded onto a pre-equilibrated 50 mL Butyl650S
column. The protein is eluted with a linear gradient of ammonium
sulfate (0.9 to 0 M (NH.sub.4).sub.2SO.sub.4). Those fractions with
the albumin fusion are again pooled, diafiltered against 10 mM
Na.sub.2HPO.sub.4/citric acid buffer pH 5.75, and loaded onto a 50
mL pre-equilibrated SP-Sepharose Fast Flow column. The protein is
eluted with a NaCl linear gradient from 0 to 0.5 M. The fractions
containing the protein of interest are combined, the buffer is
changed to 10 mM Na.sub.2HPO citric acid pH 6.25 with an Amicon
concentrator, the conductivity is <2.5 mS/cm. This protein
solution is loaded onto a 15 mL pre-equilibrated Q-Sepharose high
performance column, the column is washed, and the protein is eluted
with a NaCl linear gradient from 0 to 0.15 M NaCl. The purified
protein can then be formulated into a specific buffer composition
by buffer exchange.
Example 5
General Construct Generation for Mammalian Cell Transfection
[1081] Generation of Albumin Fusion Construct Compatible for
Expression in Mammalian Cell-Lines.
[1082] Albumin fusion constructs can be generated in expression
vectors for use in mammalian cell culture systems. DNA encoding a
therapeutic protein can be cloned N-terminus or C-terminus to HSA
in a mammalian expression vector by standard methods known in the
art (e.g., PCR amplification, restriction digestion, and ligation).
Once the expression vector has been constructed, transfection into
a mammalian expression system can proceed. Suitable vectors are
known in the art including, but not limited to, for example, the
pC4 vector, and/or vectors available from Lonza Biologics, Inc.
(Portsmouth, N.H.).
[1083] The DNA encoding human serum albumin has been cloned into
the pC4 vector which is suitable for mammalian culture systems,
creating plasmid pC4:HSA (ATCC Deposit #PTA-3277). This vector has
a DiHydroFolate Reductase, "DHFR", gene that will allow for
selection in the presence of methotrexate.
[1084] The pC4:HSA vector is suitable for expression of albumin
fusion proteins in CHO cells. For expression, in other mammalian
cell culture systems, it may be desirable to subclone a fragment
comprising, or alternatively consisting of, DNA which encodes for
an albumin fusion protein into an alternative expression vector.
For example, a fragment comprising, or alternatively consisting, of
DNA which encodes for a mature albumin fusion protein may be
subcloned into another expression vector including, but not limited
to, any of the mammalian expression vectors described herein.
[1085] In a preferred embodiment, DNA encoding an albumin fusion
construct is subcloned into vectors provided by Lonza Biologics,
Inc. (Portsmouth, N.H.) by procedures known in the art for
expression in NS0 cells.
[1086] Generation of Albumin Fusion Constructs Comprising
HSA-Therapeutic Protein Fusion Products.
[1087] Using pC4:HSA (ATCC Deposit #PTA-3277), albumin fusion
constructs can be generated in which the Therapeutic protein
portion is C terminal to the mature albumin sequence. For example,
one can clone DNA encoding a Therapeutic protein of fragment or
variant thereof between the Bsu 361 and Asc I restriction sites of
the vector. When cloning into the Bsu 361 and Asc I, the same
primer design used to clone into the yeast vector system (SEQ ID
NO:42 and 43) may be employed (see Example 2).
[1088] Generation of Albumin Fusion Constructs Comprising Gene-HSA
Fusion Products.
[1089] Using pC4:HSA (ATCC Deposit #PTA-3277), albumin fusion
constructs can be generated in which a Therapeutic protein portion
is cloned N terminal to the mature albumin sequence. For example,
one can clone DNA encoding a Therapeutic protein that has its own
signal sequence between the Bam HI (or Hind III) and Cla I sites of
pC4:HSA. When cloning into either the Bam HI or Hind III site, it
is preferrable to include a Kozak sequence (CCGCCACCATG, SEQ ID
NO:49) prior to the translational start codon of the DNA encoding
the Therapeutic protein. If a Therapeutic protein does not have a
signal sequence, DNA encoding that Therapeutic protein may be
cloned in between the Xho I and Cla I sites of pC4:HSA. When using
the Xho I site, the following 5' (SEQ ID NO:50) and 3' (SEQ ID
NO:51) exemplary PCR primers may be used:
TABLE-US-00008 (SEQ ID NO:50)
5'-CCGCCGCTCGAGGGGTGTGTTTCGTCGA(N).sub.18-3' (SEQ ID NO:51)
5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATC(N).sub.18-3'
[1090] In the 5' primer (SEQ ID NO:50), the underlined sequence is
a Xho I site; and the Xho I site and the DNA following the Xho I
site code for the last seven amino acids of the leader sequence of
natural human serum albumin. In SEQ ID NO:50, "(N).sub.18" is DNA
identical to the first 18 nucleotides encoding the Therapeutic
protein of interest. In the 3' primer (SEQ ID NO:51), the
underlined sequence is a Cla I site; and the Cla I site and the DNA
following it are the reverse complement of the DNA encoding the
first 10 amino acids of the mature HSA protein (SEQ ID NO:1). In
SEQ ID NO:51 "(N).sub.18" is the reverse complement of DNA encoding
the last 18 nucleotides encoding the Therapeutic protein of
interest. Using these two primers, one may PCR amplify the
Therapeutic protein of interest, purify the PCR product, digest it
with Xho I and Cla I restriction enzymes and clone it into the Xho
I and Cla I sites in the pC4:HSA vector.
[1091] If an alternative leader sequence is desired, the native
albumin leader sequence can be replaced with the chimeric albumin
leader, i.e., the HSA-kex2 signal peptide, or an alternative leader
by standard methods known in the art. (For example, one skilled in
the art could routinely PCR amplify an alternate leader and
subclone the PCR product into an albumin fusion construct in place
of the albumin leader while maintaining the reading frame).
Example 6
General Expression in Mammalian Cell-Lines
[1092] An albumin fusion construct generated in an expression
vector compatible with expression in mammalian cell-lines can be
transfected into appropriate cell-lines by calcium phosphate
precipitation, lipofectamine, electroporation, or other
transfection methods known in the art and/or as described in
Sambrook, Fritsch, and Maniatis. 1989. "Molecular Cloning: A
Laboratory Manual, 2.sup.nd edition" and in Ausubel et al. 2000.
Massachusetts General Hospital and Harvard Medical School "Current
Protocols in Molecular Biology", volumes 1-4. The transfected cells
are then selected for by the presence of a selecting agent
determined by the selectable marker in the expression vector.
[1093] The pC4 expression vector (ATCC Accession No. 209646) is a
derivative of the plasmid pSV2-DHFR (ATCC Accession No. 37146). pC4
contains the strong promoter Long Terminal Repeats "LTR" of the
Rous Sarcoma Virus (Cullen et al., March 1985, Molecular and
Cellular Biology, 438-447) and a fragment of the CytoMegaloVirus
"CMV"-enhancer (Boshart et al., 1985, Cell 41: 521-530). The vector
also contains the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene, and the mouse DHFR gene under
control of the SV40 early promoter. Chinese hamster ovary "CHO"
cells or other cell-lines lacking an active DHFR gene are used for
transfection. Transfection of an albumin fusion construct in pC4
into CHO cells by methods known in the art will allow for the
expression of the albumin fusion protein in CHO cells, followed by
leader sequence cleavage, and secretion into the supernatant. The
albumin fusion protein is then further purified from the
supernatant.
[1094] The pEE12.1 expression vector is provided by Lonza
Biologics, Inc. (Portsmouth, N.H.) and is a derivative of pEE6
(Stephens and Cockett, 1989, Nucl. Acids Res. 17: 7110). This
vector comprises a promoter, enhancer and complete 5'-untranslated
region of the Major Immediate Early gene of the human
CytoMegaloVirus, "hCMV-MIE" (International Publication #
WO89/01036), upstream of a sequence of interest, and a Glutamine
Synthetase gene (Murphy et al., 1991, Biochem J. 227: 277-279;
Bebbington et al., 1992, Bio/Technology 10:169-175; U.S. Pat. No.
5,122,464) for purposes of selection of transfected cells in
selective methionine sulphoximine containing medium. Transfection
of albumin fusion constructs made in pEE12.1 into NS0 cells
(International Publication #WO86/05807) by methods known in the art
will allow for the expression of the albumin fusion protein in NS0
cells, followed by leader sequence cleavage, and secretion into the
supernatant. The albumin fusion protein is then further purified
from the supernatant using techniques described herein or otherwise
known in the art.
[1095] Expression of an albumin fusion protein may be analyzed, for
example, by SDS-PAGE and Western blot, reversed phase HPLC
analysis, or other methods known in the art.
[1096] Stable CHO and NS0 cell-lines transfected with albumin
fusion constructs are generated by methods known in the art (e.g.,
lipofectamine transfection) and selected, for example, with 100 nM
methotrexate for vectors having the DiHydroFolate Reductase `DHFR`
gene as a selectable marker or through growth in the absence of
glutamine. Expression levels can be examined for example, by
immunoblotting, primarily, with an anti-HSA serum as the primary
antibody, or, secondarily, with serum containing antibodies
directed to the Therapeutic protein portion of a given albumin
fusion protein as the primary antibody.
[1097] Expression levels are examined by immunoblot detection with
anti-HSA serum as the primary antibody. The specific productivity
rates are determined via ELISA in which the capture antibody can be
a monoclonal antibody towards the therapeutic protein portion of
the albumin fusion and the detecting antibody can be the monoclonal
anti-HSA-biotinylated antibody (or vice versa), followed by
horseradish peroxidase/streptavidin binding and analysis according
to the manufacturer's protocol.
Example 7
Expression of an Albumin Fusion Protein in Mammalian Cells
[1098] The albumin fusion proteins of the present invention can be
expressed in a mammalian cell. A typical mammalian expression
vector contains a promoter element, which mediates the initiation
of transcription of mRNA, a protein coding sequence, and signals
required for the termination of transcription and polyadenylation
of the transcript. Additional elements include enhancers, Kozak
sequences and intervening sequences flanked by donor and acceptor
sites for RNA splicing. Highly efficient transcription is achieved
with the early and late promoters from SV40, the long terminal
repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early promoter of the cytomegalovirus (CMV). However, cellular
elements can also be used (e.g., the human actin promoter).
[1099] Suitable expression vectors for use in practicing the
present invention include, for example, vectors such as, pSVL and
pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr
(ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport
3.0. Mammalian host cells that could be used include, but are not
limited to, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells
and Chinese hamster ovary (CHO) cells.
[1100] Alternatively, the albumin fusion protein can be expressed
in stable cell lines containing the polynucleotide encoding the
albumin fusion protein integrated into a chromosome. The
co-transfection with a selectable marker such as DHFR, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[1101] The transfected polynucleotide encoding the fusion protein
can also be amplified to express large amounts of the encoded
fusion protein. The DHFR (dihydrofolate reductase) marker is useful
in developing cell lines that carry several hundred or even several
thousand copies of the gene of interest. (See, e.g., Alt et al., J.
Biol. Chem. 253:1357-1370 (1978); Hamlin et al., Biochem. et
Biophys. Acta, 1097:107-143 (1990); Page et al., Biotechnology
9:64-68 (1991)). Another useful selection marker is the enzyme
glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279
(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using
these markers, the mammalian cells are grown in selective medium
and the cells with the highest resistance are selected. These cell
lines contain the amplified gene(s) integrated into a chromosome.
Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
[1102] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No.
37146), the expression vectors pC4 (ATCC Accession No. 209646) and
pC6 (ATCC Accession No. 209647) contain the strong promoter (LTR)
of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular
Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer
(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
also contain the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene, and the mouse DHFR gene under
control of the SV40 early promoter.
[1103] Specifically, the plasmid pC6, for example, is digested with
appropriate restriction enzymes and then dephosphorylated using
calf intestinal phosphates by procedures known in the art. The
vector is then isolated from a 1% agarose gel.
[1104] A polynucleotide encoding an albumin fusion protein of the
present invention is generated using techniques known in the art
and this polynucleotide is amplified using PCR technology known in
the art. If a naturally occurring signal sequence is used to
produce the fusion protein of the present invention, the vector
does not need a second signal peptide. Alternatively, if a
naturally occurring signal sequence is not used, the vector can be
modified to include a heterologous signal sequence. (See, e.g.,
International Publication No. WO 96/34891.)
[1105] The amplified fragment encoding the fusion protein of the
invention is isolated from a 1% agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Calif.). The
fragment then is digested with appropriate restriction enzymes and
again purified on a 1% agarose gel.
[1106] The amplified fragment encoding the albumin fusion protein
of the invention is then digested with the same restriction enzyme
and purified on a 1% agarose gel. The isolated fragment and the
dephosphorylated vector are then ligated with T4 DNA ligase. E.
coli HB101 or XL-1 Blue cells are then transformed and bacteria are
identified that contain the fragment inserted into plasmid pC6
using, for instance, restriction enzyme analysis.
[1107] Chinese hamster ovary cells lacking an active DHFR gene is
used for transfection. Five .mu.g of the expression plasmid pC6 or
pC4 is cotransfected with 0.5 .mu.g of the plasmid pSVneo using
lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a
dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including
G418. The cells are seeded in alpha minus MEM supplemented with 1
mg/ml G418. After 2 days, the cells are trypsinized and seeded in
hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml
G418. After about 10-14 days single clones are trypsinized and then
seeded in 6-well petri dishes or 10 ml flasks using different
concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800
nM). Clones growing at the highest concentrations of methotrexate
are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired fusion protein is analyzed, for instance, by SDS-PAGE and
Western blot or by reversed phase HPLC analysis.
Example 8
General Purification of an Albumin Fusion Protein Expressed from an
Albumin Fusion Construct in Mammalian Cell-Lines
[1108] In preferred embodiments, albumin fusion proteins of the
invention comprise the mature form of HSA fused to either the N- or
C-terminus of the mature form of a therapeutic protein or portions
thereof (e.g., the mature form of a therapeutic protein listed in
Table 1, or the mature form of a therapeutic protein shown in Table
2 as SEQ ID NO:Z). In one embodiment of the invention, albumin
fusion proteins of the invention further comprise a signal sequence
which directs the nascent fusion polypeptide in the secretory
pathways of the host used for expression. In a preferred
embodiment, the signal peptide encoded by the signal sequence is
removed, and the mature albumin fusion protein is secreted directly
into the culture medium. Albumin fusion proteins of the invention
preferably comprise heterologous signal sequences (e.g., the
non-native signal sequence of a particular therapeutic protein)
including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin,
Insulin-Like Growth Factor Binding Protein 4, variant HSA leader
sequences including, but not limited to, a chimeric HSA/MAF leader
sequence, or other heterologous signal sequences known in the art.
Especially preferred as those signal sequence listed in Table 2
and/or the signal sequence listed in the "Expression of Fusion
Proteins" and/or "Additional Methods of Recombinant and Synthetic
Production of Albumin Fusion Proteins" section of the
specification, above. In preferred embodiments, the fusion proteins
of the invention further comprise an N-terminal methionine residue.
Polynucleotides encoding these polypeptides, including fragments
and/or variants, are also encompassed by the invention.
[1109] Albumin fusion proteins from mammalian cell-line
supernatants are purified according to different protocols
depending on the expression system used.
[1110] Purification from CHO and 293T Cell-Lines.
[1111] Purification of an albumin fusion protein from CHO cell
supernatant or from transiently transfected 293T cell supernatant
may involve initial capture with an anionic HQ resin using a sodium
phosphate buffer and a phosphate gradient elution, followed by
affinity chromatography on a Blue Sepharose FF column using a salt
gradient elution. Blue Sepharose FF removes the main BSA/fetuin
contaminants. Further purification over the Poros PI 50 resin with
a phosphate gradient may remove and lower endotoxin contamination
as well as concentrate the albumin fusion protein.
[1112] Purification from NSO Cell-Line.
[1113] Purification of an albumin-fusion protein from NSO cell
supernatant may involve Q-Sepharose anion exchange chromatography,
followed by SP-sepharose purification with a step elution, followed
by Phenyl-650M purification with a step elution, and, ultimately,
diafiltration.
[1114] The purified protein may then be formulated by buffer
exchange.
Example 9
Bacterial Expression of an Albumin Fusion Protein
[1115] A polynucleotide encoding an albumin fusion protein of the
present invention comprising a bacterial signal sequence is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' ends of the DNA sequence, to synthesize insertion fragments.
The primers used to amplify the polynucleotide encoding insert
should preferably contain restriction sites, such as BamHI and
XbaI, at the 5' end of the primers in order to clone the amplified
product into the expression vector. For example, BamHI and XbaI
correspond to the restriction enzyme sites on the bacterial
expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This
plasmid vector encodes antibiotic resistance (Amp.sup.r), a
bacterial origin of replication (ori), an IPTG-regulatable
promoter/operator (P/O), a ribosome binding site (RBS), a
6-histidine tag (6-His), and restriction enzyme cloning sites.
[1116] The pQE-9 vector is digested with BamHI and XbaI and the
amplified fragment is ligated into the pQE-9 vector maintaining the
reading frame initiated at the bacterial RBS. The ligation mixture
is then used to transform the E. coli strain M15/rep4 (Qiagen,
Inc.) which contains multiple copies of the plasmid pREP4, which
expresses the lacI repressor and also confers kanamycin resistance
(Kan.sup.r). Transformants are identified by their ability to grow
on LB plates and ampicillin/kanamycin resistant colonies are
selected. Plasmid DNA is isolated and confirmed by restriction
analysis.
[1117] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100
ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a
large culture at a ratio of 1:100 to 1:250. The cells are grown to
an optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene
expression.
[1118] Cells are grown for an extra 3 to 4 hours. Cells are then
harvested by centrifugation (20 mins at 6000.times.g). The cell
pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl
or preferably in 8 M urea and concentrations greater than 0.14 M
2-mercaptoethanol by stirring for 3-4 hours at 4.degree. C. (see,
e.g., Burton et al., Eur. J. Biochem. 179:379-387 (1989)). The cell
debris is removed by centrifugation, and the supernatant containing
the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin column (available from QIAGEN, Inc.,
supra). Proteins with a 6.times.His tag bind to the Ni-NTA resin
with high affinity and can be purified in a simple one-step
procedure (for details see: The QIAexpressionist (1995) QIAGEN,
Inc., supra).
[1119] Briefly, the supernatant is loaded onto the column in 6 M
guanidine-HCl, pH 8. The column is first washed with 10 volumes of
6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M
guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M
guanidine-HCl, pH 5.
[1120] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column.
Exemplary conditions are as follows: renature using a linear 6M-1M
urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4,
containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins are eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4.degree. C. or frozen at -80.degree. C.
[1121] In addition to the above expression vector, the present
invention further includes an expression vector, called pHE4a (ATCC
Accession Number 209645, deposited on Feb. 25, 1998) which contains
phage operator and promoter elements operatively linked to a
polynucleotide encoding an albumin fusion protein of the present
invention, called pHE4a. (ATCC Accession Number 209645, deposited
on Feb. 25, 1998.) This vector contains: 1) a
neomycinphosphotransferase gene as a selection marker, 2) an E.
coli origin of replication, 3) a T5 phage promoter sequence, 4) two
lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the
lactose operon repressor gene (lacIq). The origin of replication
(oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter
and operator sequences are made synthetically.
[1122] DNA can be inserted into the pHE4a by restricting the vector
with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted
product on a gel, and isolating the larger fragment (the stuffer
fragment should be about 310 base pairs). The DNA insert is
generated according to PCR protocols described herein or otherwise
known in the art, using PCR primers having restriction sites for
NdeI (5' primer) and XbaI, BamHI, XhoI, or Asp718 (3' primer). The
PCR insert is gel purified and restricted with compatible enzymes.
The insert and vector are ligated according to standard
protocols.
[1123] The engineered vector may be substituted in the above
protocol to express protein in a bacterial system.
Example 10
Isolation of a Selected cDNA Clone from the Deposited Sample
[1124] Many of the albumin fusion constructs of the invention have
been deposited with the ATCC as shown in Table 3. The albumin
fusion constructs may comprise any one of the following expression
vectors: the yeast S. cerevisiae expression vector pSAC35, the
mammalian expression vector pC4, or the mammalian expression vector
pEE12.1.
[1125] pSAC35 (Sleep et al., 1990, Biotechnology 8:42), pC4 (ATCC
Accession No. 209646; Cullen et al., Molecular and Cellular
Biology, 438-447 (1985); Boshart et al., Cell 41: 521-530 (1985)),
and pEE12.1 (Lonza Biologics, Inc.; Stephens and Cockett, Nucl.
Acids Res. 17: 7110 (1989); International Publication #WO89/01036;
Murphy et al., Biochem J. 227: 277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992); U.S. Pat. No. 5,122,464;
International Publication #WO86/05807) vectors comprise an
ampicillin resistance gene for growth in bacterial cells. These
vectors and/or an albumin fusion construct comprising them can be
transformed into an E. coli strain such as Stratagene XL-1 Blue
(Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La
Jolla, Calif., 92037) using techniques described in the art such as
Hanahan, spread onto Luria-Broth agar plates containing 100
.mu.g/mL ampicillin, and grown overnight at 37.degree. C.
[1126] The deposited material in the sample assigned the ATCC
Deposit Number cited in Table 3 for any given albumin fusion
construct also may contain one or more additional albumin fusion
constructs, each encoding different albumin fusion proteins. Thus,
deposits sharing the same ATCC Deposit Number contain at least an
albumin fusion construct identified in the corresponding row of
Table 3.
[1127] Two approaches can be used to isolate a particular albumin
fusion construct from the deposited sample of plasmid DNAs cited
for that albumin fusion construct in Table 3.
[1128] Method 1: Screening
[1129] First, an albumin fusion construct may be directly isolated
by screening the sample of deposited plasmid DNAs using a
polynucleotide probe corresponding to SEQ ID NO:X for an individual
construct ID number in Table 1, using methods known in the art. For
example, a specific polynucleotide with 30-40 nucleotides may be
synthesized using an Applied Biosystems DNA synthesizer according
to the sequence reported. The oligonucleotide can be labeled, for
instance, with .sup.32P-.gamma.-ATP using T4 polynucleotide kinase
and purified according to routine methods. (E.g., Maniatis et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
Cold Spring, N.Y. (1982)). The albumin fusion construct from a
given ATCC deposit is transformed into a suitable host, as
indicated above (such as XL-1 Blue (Stratagene)) using techniques
known to those of skill in the art, such as those provided by the
vector supplier or in related publications or patents cited above.
The transformants are plated on 1.5% agar plates (containing the
appropriate selection agent, e.g., ampicillin) to a density of
about 150 transformants (colonies) per plate. These plates are
screened using Nylon membranes according to routine methods for
bacterial colony screening (e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor
Laboratory Press, pages 1.93 to 1.104), or other techniques known
to those of skill in the art.
[1130] Method 2: PCR
[1131] Alternatively, DNA encoding a given albumin fusion protein
may be amplified from a sample of a deposited albumin fusion
construct with SEQ ID NO:X, for example, by using two primers of
17-20 nucleotides that hybridize to the deposited albumin fusion
construct 5' and 3' to the DNA encoding a given albumin fusion
protein. The polymerase chain reaction is carried out under routine
conditions, for instance, in 25 .mu.l of reaction mixture with 0.5
ug of the above cDNA template. A convenient reaction mixture is
1.5-5 mM MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each of dATP,
dCTP, dGTP, dTTP, 25 .mu.mol of each primer and 0.25 Unit of Taq
polymerase. Thirty five cycles of PCR (denaturation at 94.degree.
C. for 1 min; annealing at 55.degree. C. for 1 min; elongation at
72.degree. C. for 1 min) are performed with a Perkin-Elmer Cetus
automated thermal cycler. The amplified product is analyzed by
agarose gel electrophoresis and the DNA band with expected
molecular weight is excised and purified. The PCR product is
verified to be the selected sequence by subcloning and sequencing
the DNA product.
[1132] Several methods are available for the identification of the
5' or 3' non-coding portions of a gene which may not be present in
the deposited clone. These methods include but are not limited to,
filter probing, clone enrichment using specific probes, and
protocols similar or identical to 5' and 3' "RACE" protocols which
are known in the art. For instance, a method similar to 5' RACE is
available for generating the missing 5' end of a desired
full-length transcript. (Fromont-Racine et al., Nucleic Acids Res.,
21(7):1683-1684 (1993)).
[1133] Briefly, a specific RNA oligonucleotide is ligated to the 5'
ends of a population of RNA presumably containing full-length gene
RNA transcripts. A primer set containing a primer specific to the
ligated RNA oligonucleotide and a primer specific to a known
sequence of the gene of interest is used to PCR amplify the 5'
portion of the desired full-length gene. This amplified product may
then be sequenced and used to generate the full length gene.
[1134] This above method starts with total RNA isolated from the
desired source, although poly-A+ RNA can be used. The RNA
preparation can then be treated with phosphatase if necessary to
eliminate 5' phosphate groups on degraded or damaged RNA which may
interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid
pyrophosphatase in order to remove the cap structure present at the
5' ends of messenger RNAs. This reaction leaves a 5' phosphate
group at the 5' end of the cap cleaved RNA which can then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
[1135] This modified RNA preparation is used as a template for
first strand cDNA synthesis using a gene specific oligonucleotide.
The first strand synthesis reaction is used as a template for PCR
amplification of the desired 5' end using a primer specific to the
ligated RNA oligonucleotide and a primer specific to the known
sequence of the gene of interest. The resultant product is then
sequenced and analyzed to confirm that the 5' end sequence belongs
to the desired gene.
Example 11
Multifusion Fusions
[1136] The albumin fusion proteins (e.g., containing a Therapeutic
protein (or fragment or variant thereof) fused to albumin (or a
fragment or variant thereof)) may additionally be fused to other
proteins to generate "multifusion proteins". These multifusion
proteins can be used for a variety of applications. For example,
fusion of the albumin fusion proteins of the invention to His-tag,
HA-tag, protein A, IgG domains, and maltose binding protein
facilitates purification. (See e.g., EP A 394,827; Traunecker et
al., Nature 331:84-86 (1988)). Nuclear localization signals fused
to the polypeptides of the present invention can target the protein
to a specific subcellular localization, while covalent heterodimer
or homodimers can increase or decrease the activity of an albumin
fusion protein. Furthermore, the fusion of additional protein
sequences to the albumin fusion proteins of the invention may
further increase the solubility and/or stability of the fusion
protein. The fusion proteins described above can be made using or
routinely modifying techniques known in the art and/or by modifying
the following protocol, which outlines the fusion of a polypeptide
to an IgG molecule.
[1137] 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. These primers also should have convenient
restriction enzyme sites that will facilitate cloning into an
expression vector, preferably a mammalian or yeast expression
vector.
[1138] For example, if pC4 (ATCC Accession No. 209646) 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 a polynucleotide encoding an albumin
fusion protein of the present invention (generated and isolated
using techniques known in the art), is ligated into this BamHI
site. Note that the polynucleotide encoding the fusion protein of
the invention is cloned without a stop codon, otherwise a Fc
containing fusion protein will not be produced.
[1139] If the naturally occurring signal sequence is used to
produce the albumin fusion protein of the present invention, 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.,
International Publication No. WO 96/34891.)
[1140] Human IgG Fc Region:
TABLE-US-00009 (SEQ ID NO: 52)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT
Example 12
Production of an Antibody from an Albumin Fusion Protein
[1141] Hybridoma Technology
[1142] Antibodies that bind the albumin fusion proteins of the
present invention and portions of the albumin fusion proteins of
the present invention (e.g., the Therapeutic protein portion or
albumin portion of the fusion protein) can be prepared by a variety
of methods. (See, Current Protocols, Chapter 2.) As one example of
such methods, a preparation of an albumin fusion protein of the
invention or a portion of an albumin fusion protein of the
invention is prepared and purified to render it substantially free
of natural contaminants. Such a preparation is then introduced into
an animal in order to produce polyclonal antisera of greater
specific activity.
[1143] Monoclonal antibodies specific for an albumin fusion protein
of the invention, or a portion of an albumin fusion protein of the
invention, are 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., pp. 563-681 (1981)). In general, an animal (preferably a
mouse) is immunized with an albumin fusion protein of the
invention, or a portion of an albumin fusion protein of the
invention. 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 (SP20),
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 an albumin fusion protein of the
invention, or a portion of an albumin fusion protein of the
invention.
[1144] Alternatively, additional antibodies capable of binding to
an albumin fusion protein of the invention, or a portion of an
albumin fusion protein of the invention 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 an albumin
fusion protein of the invention (or portion of an albumin fusion
protein of the invention)-specific antibody can be blocked by the
fusion protein of the invention, or a portion of an albumin fusion
protein of the invention. Such antibodies comprise anti-idiotypic
antibodies to the fusion protein of the invention (or portion of an
albumin fusion protein of the invention)-specific antibody and are
used to immunize an animal to induce formation of further fusion
protein of the invention (or portion of an albumin fusion protein
of the invention)-specific antibodies.
[1145] For in vivo use of antibodies in humans, an antibody is
"humanized". Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric and
humanized antibodies are known in the art and are discussed herein.
(See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., International
Publication No. WO 8702671; Boulianne et al., Nature 312:643
(1984); Neuberger et al., Nature 314:268 (1985)).
[1146] Isolation Of Antibody Fragments Directed Against an albumin
fusion protein of the invention, or a portion of an albumin fusion
protein of the invention From A Library Of scFvs. Naturally
occurring V-genes isolated from human PBLs are constructed into a
library of antibody fragments which contain reactivities against an
albumin fusion protein of the invention, or a portion of an albumin
fusion protein of the 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).
[1147] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in International
Publication No. WO 92/01047. To rescue phage displaying antibody
fragments, approximately 10.sup.9 E. coli harboring the phagemid
are used to inoculate 50 ml of 2.times.TY containing 1% glucose and
100 .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
inoculate 50 ml of 2.times.TY-AMP-GLU, 2.times.108 TU of delta gene
3 helper (M13 delta gene III, see International Publication No. 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 min.
and the pellet resuspended in 2 liters of 2.times.TY containing 100
.mu.g/ml ampicillin and 50 ug/ml kanamycin and grown overnight.
Phage are prepared as described in International Publication No. WO
92/01047.
[1148] 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 spun down (IEC-Centra 8,400 r.p.m. 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 Mm filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[1149] Panning of the Library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
an albumin fusion protein of the invention, or a portion of an
albumin fusion protein of the invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 10.sup.13 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 .mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[1150] Characterization of Binders. 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 an albumin fusion protein of the invention, or a
portion of an albumin fusion protein of the invention, in 50 mM
bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., International
Publication No. WO 92/01047) and then by sequencing. These ELISA
positive clones may also be further characterized by techniques
known in the art, such as, for example, epitope mapping, binding
affinity, receptor signal transduction, ability to block or
competitively inhibit antibody/antigen binding, and competitive
agonistic or antagonistic activity.
Example 13
[.sup.3H]-2-Deoxyglucose Uptake Assay
[1151] Adipose, skeletal muscle, and liver are insulin-sensitive
tissues. Insulin can stimulate glucose uptake/transport into these
tissues. In the case of adipose and skeletal muscle, insulin
initiates the signal transduction that eventually leads to the
translocation of the glucose transporter 4 molecule, GLUT4, from a
specialized intracellular compartment to the cell surface. Once on
the cell surface, GLUT4 allows for glucose uptake/transport.
[1152] [.sup.3H]-2-Deoxyglucose Uptake
[1153] A number of adipose and muscle related cell-lines can be
used to test for glucose uptake/transport activity in the absence
or presence of a combination of any one or more of the therapeutic
drugs listed for the treatment of diabetes mellitus. In particular,
the 3T3-L1 murine fibroblast cells and the L6 murine skeletal
muscle cells can be differentiated into 3T3-L1 adipocytes and into
myotubes, respectively, to serve as appropriate in vitro models for
the [.sup.3H]-2-deoxyglucose uptake assay (Urso et al., J Biol
Chem, 274(43): 30864-73 (1999); Wang et al., J Mol Endocrinol,
19(3): 241-8 (1997); Haspel et al., J Membr Biol, 169 (1): 45-53
(1999); Tsakiridis et al., Endocrinology, 136(10): 4315-22 (1995)).
Briefly, 2.times.10.sup.5 cells/100 .mu.L of adipocytes or
differentiated L6 cells are transferred to 96-well Tissue-Culture,
"TC", treated, i.e., coated with 50 .mu.g/mL of poly-L-lysine,
plates in post-differentiation medium and are incubated overnight
at 37.degree. C. in 5% CO.sub.2. The cells are first washed once
with serum free low glucose DMEM medium and are then starved with
100 .mu.L/well of the same medium and with 100 .mu.L/well of either
buffer or of a combination of any one or more of the therapeutic
drugs listed for the treatment of diabetes mellitus, for example,
increasing concentrations of 1 nM, 10 nM, and 100 nM of the
therapeutics of the subject invention (e.g., specific fusions
disclosed as SEQ ID NO:Y and fragments and variants thereof) for 16
hours at 37.degree. C. in the absence or presence of 1 nM insulin.
The plates are washed three times with 100 .mu.L/well of HEPES
buffered saline. Insulin is added at 1 nM in HEPES buffered saline
for 30 min at 37.degree. C. in the presence of 10 .mu.M labeled
[.sup.3H]-2-deoxyglucose (Amersham, #TRK672) and 10 .mu.M unlabeled
2-deoxyglucose (SIGMA, D-3179). As control, the same conditions are
carried out except in the absence of insulin. A final concentration
of 10 .mu.M cytochalasin B (SIGMA, C6762) is added at 100
.mu.L/well in a separate well to measure the non-specific uptake.
The cells are washed three times with HEPES buffered saline.
Labeled, i.e., 10 .mu.M of [.sup.3H]-2-deoxyglucose, and unlabeled,
i.e., 10 .mu.M of 2-deoxyglucose, are added for 10 minutes at room
temperature. The cells are washed three times with cold Phosphate
Buffered Saline, "PBS". The cells are lysed upon the addition of
150 .mu.L/well of 0.2 N NaOH and subsequent incubation with shaking
for 20 minutes at room temperature. Samples are then transferred to
a scintillation vial to which is added 5 mL of scintillation fluid.
The vials are counted in a Beta-Scintillation counter. Uptake in
duplicate conditions, the difference being the absence or presence
of insulin, is determined with the following equation: [(Insulin
counts per minute "cpm"--Non-Specific cpm)/(No Insulin
cpm-Non-Specific cpm)]. Average responses fall within the limits of
about 5-fold and 3-fold that of controls for adipocytes and
myotubes, respectively.
[1154] Differentiation of Cells
[1155] The cells are allowed to become fully confluent in a T-75
cm.sup.2 flask. The medium is removed and replaced with 25 mL of
pre-differentiation medium for 48 hours. The cells are incubated at
37.degree. C., in 5% CO.sub.2, 85% humidity. After 48 hours, the
pre-differentiation medium is removed and replaced with 25 mL
differentiation medium for 48 hours. The cells are again incubated
at 37.degree. C., in 5% CO.sub.2, 85% humidity. After 48 hours, the
medium is removed and replaced with 30 mL post-differentiation
medium. Post-differentiation medium is maintained for 14-20 days or
until complete differentiation is achieved. The medium is changed
every 2-3 days. Human adipocytes can be purchased from Zen-Bio, INC
(#SA-1096).
Example 14
In Vitro Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-Lines
[1156] It has recently been shown that GLP-1 induces
differentiation of the rat pancreatic ductal epithelial cell-line
ARIP in a time- and dose-dependent manner which is associated with
an increase in Islet Duodenal Homeobox-1 (IDX-1) and insulin mRNA
levels (Hui et al., 2001, Diabetes, 50(4): 785-96). The IDX-1 in
turn increases mRNA levels of the GLP-1 receptor.
[1157] Cells Types Tested
[1158] RIN-M cells: These cells are available from the American
Type Tissue Culture Collection (ATCC Cell Line Number CRL-2057).
The RIN-M cell line was derived from a radiation induced
transplantable rat islet cell tumor. The line was established from
a nude mouse xenograft of the tumor. The cells produce and secrete
islet polypeptide hormones, and produce L-dopa decarboxylase (a
marker for cells having amine precursor uptake and decarboxylation,
or APUD, activity).
[1159] ARIP cells: These are pancreatic exocrine cells of
epithelial morphology available from the American Type Tissue
Culture Collection (ATCC Cell Line Number CRL-1674). See also,
references: Jessop, N. W. and Hay, R. J., "Characteristics of two
rat pancreatic exocrine cell lines derived from transplantable
tumors," In Vitro 16: 212, (1980); Cockell, M. et al.,
"Identification of a cell-specific DNA-binding activity that
interacts with a transcriptional activator of genes expressed in
the acinar pancreas," Mol. Cell. Biol. 9: 2464-2476, (1989); Roux,
E., et al. "The cell-specific transcription factor PTF1 contains
two different subunits that interact with the DNA" Genes Dev. 3:
1613-1624, (1989); and, Hui, H., et al., "Glucagon-like peptide 1
induces differentiation of islet duodenal homeobox-1-positive
pancreatic ductal cells into insulin-secreting cells," Diabetes 50:
785-796 (2001).
[1160] Preparation of Cells
[1161] The RIN-M cell-line is grown in RPMI 1640 medium (Hyclone,
#SH300027.01) with 10% fetal bovine serum (HyClone, #SH30088.03)
and is subcultured every 6 to 8 days at a ratio of 1:3 to 1:6. The
medium is changed every 3 to 4 days.
[1162] The ARIP (ATCC #CRL-1674) cell-line is grown in Ham's F12K
medium (ATCC, #30-2004) with 2 mM L-glutamine adjusted to contain
1.5 g/L sodium bicarbonate and 10% fetal bovine serum. The ARIP
cell-line is subcultured at a ratio of 1:3 to 1:6 twice per week.
The medium is changed every 3 to 4 days.
[1163] Assay Protocol
[1164] The cells are seeded at 4000 cells/well in 96-well plates
and cultured for 48 to 72 hours to 50% confluence. The cells are
switched to serum-free media at 100 .mu.L/well. After incubation
for 48-72 hours, serum and/or the therapeutics of the subject
invention (e.g., albumin fusion proteins of the invention and
fragments and variants thereof) are added to the well. Incubation
persists for an additional 36 hours. [.sup.3H]-Thymidine (5-20
Ci/mmol) (Amersham Pharmacia, #TRK120) is diluted to 1
microCuries/5 microliters. After the 36 hour incubation, 5
microliters is added per well for a further 24 hours. The reaction
is terminated by washing the cells gently with cold
Phosphate-Buffered Saline, "PBS", once. The cells are then fixed
with 100 microliters of 10% ice cold TCA for 15 min at 4.degree. C.
The PBS is removed and 200 microliters of 0.2 N NaOH is added. The
plates are incubated for 1 hour at room temperature with shaking.
The solution is transferred to a scintillation vial and 5 mL of
scintillation fluid compatible with aqueous solutions is added and
mixed vigorously. The vials are counted in a beta scintillation
counter. As negative control, only buffer is used. As a positive
control fetal calf serum is used.
Example 15
Assaying for Glycosuria
[1165] Glycosuria (i.e., excess sugar in the urine), can be readily
assayed to provide an index of the disease state of diabetes
mellitus. Excess urine in a patient sample as compared with a
normal patient sample is symptomatic of IDDM and NIDDM. Efficacy of
treatment of such a patient having IDDM and NIDDM is indicated by a
resulting decrease in the amount of excess glucose in the urine. In
a preferred embodiment for IDDM and NIDDM monitoring, urine samples
from patients are assayed for the presence of glucose using
techniques known in the art. Glycosuria in humans is defined by a
urinary glucose concentration exceeding 100 mg per 100 ml. Excess
sugar levels in those patients exhibiting glycosuria can be
measured even more precisely by obtaining blood samples and
assaying serum glucose.
Example 16
Assays Detecting Stimulation or Inhibition of B cell Proliferation
and Differentiation
[1166] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and
IL-15. Interestingly, these signals are by themselves weak
effectors but can, in combination with various co-stimulatory
proteins, induce activation, proliferation, differentiation,
homing, tolerance and death among B cell populations.
[1167] One of the best studied classes of B-cell co-stimulatory
proteins is the TNF-superfamily. Within this family CD40, CD27, and
CD30 along with their respective ligands CD154, CD70, and CD153
have been found to regulate a variety of immune responses. Assays
which allow for the detection and/or observation of the
proliferation and differentiation of these B-cell populations and
their precursors are valuable tools in determining the effects
various proteins may have on these B-cell populations in terms of
proliferation and differentiation. Listed below are two assays
designed to allow for the detection of the differentiation,
proliferation, or inhibition of B-cell populations and their
precursors.
[1168] In Vitro Assay-Albumin fusion proteins of the invention
(including fusion proteins containing fragments or variants of
Therapeutic proteins and/or albumin or fragments or variants of
albumin) can be assessed for its ability to induce activation,
proliferation, differentiation or inhibition and/or death in B-cell
populations and their precursors. The activity of an albumin fusion
protein of the invention on purified human tonsillar B cells,
measured qualitatively over the dose range from 0.1 to 10,000
ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay
in which purified tonsillar B cells are cultured in the presence of
either formalin-fixed Staphylococcus aureus Cowan I (SAC) or
immobilized anti-human IgM antibody as the priming agent. Second
signals such as IL-2 and IL-15 synergize with SAC and IgM
crosslinking to elicit B cell proliferation as measured by
tritiated-thymidine incorporation. Novel synergizing agents can be
readily identified using this assay. The assay involves isolating
human tonsillar B cells by magnetic bead (MACS) depletion of
CD3-positive cells. The resulting cell population is greater than
95% B cells as assessed by expression of CD45R(B220).
[1169] Various dilutions of each sample are placed into individual
wells of a 96-well plate to which are added 10.sup.5 B-cells
suspended in culture medium (RPMI 1640 containing 10% FBS,
5.times.10.sup.-5M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin,
and 10.sup.-5 dilution of SAC) in a total volume of 150 ul.
Proliferation or inhibition is quantitated by a 20 h pulse (1
uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor
addition. The positive and negative controls are IL2 and medium
respectively.
[1170] In vivo Assay-BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of an albumin fusion protein of the
invention (including fusion proteins containing fragments or
variants of Therapeutic proteins and/or albumin or fragments or
variants of albumin). Mice receive this treatment for 4 consecutive
days, at which time they are sacrificed and various tissues and
serum collected for analyses. Comparison of H&E sections from
normal spleens and spleens treated with the albumin fusion protein
of the invention identify the results of the activity of the fusion
protein on spleen cells, such as the diffusion of peri-arterial
lymphatic sheaths, and/or significant increases in the nucleated
cellularity of the red pulp regions, which may indicate the
activation of the differentiation and proliferation of B-cell
populations. Immunohistochemical studies using a B cell marker,
anti-CD45R(B220), are used to determine whether any physiological
changes to splenic cells, such as splenic disorganization, are due
to increased B-cell representation within loosely defined B-cell
zones that infiltrate established T-cell regions.
[1171] Flow cytometric analyses of the spleens from mice treated
with the albumin fusion protein is used to indicate whether the
albumin fusion protein specifically increases the proportion of
ThB+, CD45R(B220) dull B cells over that which is observed in
control mice.
[1172] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgM and IgA levels are compared between buffer
and fusion protein treated mice.
Example 17
T Cell Proliferation Assay
[1173] A CD3-induced proliferation assay is performed on PBMCs and
is measured by the uptake of .sup.3H-thymidine. The assay is
performed as follows. Ninety-six well plates are coated with 100
.mu.l/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched
control mAb (B33.1) overnight at 4 degrees C. (1 .mu.g/ml in 0.05M
bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC
are isolated by F/H gradient centrifugation from human peripheral
blood and added to quadruplicate wells (5.times.10.sup.4/well) of
mAb coated plates in RPMI containing 10% FCS and P/S in the
presence of varying concentrations of an albumin fusion protein of
the invention (including fusion proteins containing fragments or
variants of Therapeutic proteins and/or albumin or fragments or
variants of albumin) (total volume 200 ul). Relevant protein buffer
and medium alone are controls. After 48 hr. culture at 37 degrees
C., plates are spun for 2 min. at 1000 rpm and 100 .mu.l of
supernatant is removed and stored -20 degrees C. for measurement of
IL-2 (or other cytokines) if effect on proliferation is observed.
Wells are supplemented with 100 ul of medium containing 0.5 uCi of
.sup.3H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells
are harvested and incorporation of .sup.3H-thymidine used as a
measure of proliferation. Anti-CD3 alone is the positive control
for proliferation. IL-2 (100 U/ml) is also used as a control which
enhances proliferation. Control antibody which does not induce
proliferation of T cells is used as the negative control for the
effects of fusion proteins of the invention.
Example 18
Effect of Fusion Proteins of the Invention on the Expression of MHC
Class II, Costimulatory and Adhesion Molecules and Cell
Differentiation of Monocytes and Monocyte-Derived Human Dendritic
Cells
[1174] Dendritic cells are generated by the expansion of
proliferating precursors found in the peripheral blood: adherent
PBMC or elutriated monocytic fractions are cultured for 7-10 days
with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells
have the characteristic phenotype of immature cells (expression of
CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with
activating factors, such as TNF-.alpha., causes a rapid change in
surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of
FC.gamma.RII, upregulation of CD83). These changes correlate with
increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
[1175] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of an
albumin fusion protein of the invention or LPS (positive control),
washed with PBS containing 1% BSA and 0.02 mM sodium azide, and
then incubated with 1:20 dilution of appropriate FITC- or
PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C.
After an additional wash, the labeled cells are analyzed by flow
cytometry on a FACScan (Becton Dickinson).
[1176] Effect on the production of cytokines. Cytokines generated
by dendritic cells, in particular IL-12, are important in the
initiation of T-cell dependent immune responses. IL-12 strongly
influences the development of Th1 helper T-cell immune response,
and induces cytotoxic T and NK cell function. An ELISA is used to
measure the IL-12 release as follows. Dendritic cells (10.sup.6/ml)
are treated with increasing concentrations of an albumin fusion
protein of the invention for 24 hours. LPS (100 ng/ml) is added to
the cell culture as positive control. Supernatants from the cell
cultures are then collected and analyzed for IL-12 content using
commercial ELISA kit (e.g., R & D Systems (Minneapolis,
Minn.)). The standard protocols provided with the kits are
used.
[1177] Effect on the expression of MHC Class II, costimulatory and
adhesion molecules. Three major families of cell surface antigens
can be identified on monocytes: adhesion molecules, molecules
involved in antigen presentation, and Fc receptor. Modulation of
the expression of MHC class II antigens and other costimulatory
molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T
cell activation. Increased expression of Fc receptors may correlate
with improved monocyte cytotoxic activity, cytokine release and
phagocytosis.
[1178] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of an albumin fusion protein of the invention or LPS
(positive control), washed with PBS containing 1% BSA and 0.02 mM
sodium azide, and then incubated with 1:20 dilution of appropriate
FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4
degrees C. After an additional wash, the labeled cells are analyzed
by flow cytometry on a FACScan (Becton Dickinson).
[1179] Monocyte activation and/or increased survival. Assays for
molecules that activate (or alternatively, inactivate) monocytes
and/or increase monocyte survival (or alternatively, decrease
monocyte survival) are known in the art and may routinely be
applied to determine whether a molecule of the invention functions
as an inhibitor or activator of monocytes. Albumin fusion proteins
of the invention can be screened using the three assays described
below. For each of these assays, Peripheral blood mononuclear cells
(PBMC) are purified from single donor leukopacks (American Red
Cross, Baltimore, Md.) by centrifugation through a Histopaque
gradient (Sigma). Monocytes are isolated from PBMC by counterflow
centrifugal elutriation.
[1180] Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or
other stimuli. Their death results from internally regulated
processes (apoptosis). Addition to the culture of activating
factors, such as TNF-alpha dramatically improves cell survival and
prevents DNA fragmentation. Propidium iodide (PI) staining is used
to measure apoptosis as follows. Monocytes are cultured for 48
hours in polypropylene tubes in serum-free medium (positive
control), in the presence of 100 ng/ml TNF-alpha (negative
control), and in the presence of varying concentrations of the
fusion protein to be tested. Cells are suspended at a concentration
of 2.times.10.sup.6/ml in PBS containing PI at a final
concentration of 5 .mu.g/ml, and then incubated at room temperature
for 5 minutes before FACScan analysis. PI uptake has been
demonstrated to correlate with DNA fragmentation in this
experimental paradigm.
[1181] Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other
cellular populations of the immune system through the release of
cytokines after stimulation. An ELISA to measure cytokine release
is performed as follows. Human monocytes are incubated at a density
of 5.times.10.sup.5 cells/ml with increasing concentrations of an
albumin fusion protein of the invention and under the same
conditions, but in the absence of the fusion protein. For IL-12
production, the cells are primed overnight with IFN (100 U/ml) in
the presence of the fusion protein. LPS (10 ng/ml) is then added.
Conditioned media are collected after 24 h and kept frozen until
use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then
performed using a commercially available ELISA kit (e.g., R & D
Systems (Minneapolis, Minn.)) and applying the standard protocols
provided with the kit.
[1182] Oxidative burst. Purified monocytes are plated in 96-w plate
at 2-1.times.10.sup.5 cell/well. Increasing concentrations of an
albumin fusion protein of the invention are added to the wells in a
total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine
and antibiotics). After 3 days incubation, the plates are
centrifuged and the medium is removed from the wells. To the
macrophage monolayers, 0.2 ml per well of phenol red solution (140
mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose,
0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the
stimulant (200 nM PMA). The plates are incubated at 37.degree. C.
for 2 hours and the reaction is stopped by adding 20 .mu.l 1N NaOH
per well. The absorbance is read at 610 nm. To calculate the amount
of H.sub.2O.sub.2 produced by the macrophages, a standard curve of
a H.sub.2O.sub.2 solution of known molarity is performed for each
experiment.
Example 19
The Effect of Albumin Fusion Proteins of the Invention on the
Growth of Vascular Endothelial Cells
[1183] On day 1, human umbilical vein endothelial cells (HUVEC) are
seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199
containing 10% FBS, 8 units/ml heparin. An albumin fusion protein
of the invention, and positive controls, such as VEGF and basic FGF
(bFGF) are added, at varying concentrations. On days 4 and 6, the
medium is replaced. On day 8, cell number is determined with a
Coulter Counter.
[1184] An increase in the number of HUVEC cells indicates that the
fusion protein may proliferate vascular endothelial cells, while a
decrease in the number of HUVEC cells indicates that the fusion
protein inhibits vascular endothelial cells.
Example 20
Rat Corneal Wound Healing Model
[1185] This animal model shows the effect of an albumin fusion
protein of the invention on neovascularization. The experimental
protocol includes:
[1186] Making a 1-1.5 mm long incision from the center of cornea
into the stromal layer.
[1187] Inserting a spatula below the lip of the incision facing the
outer corner of the eye.
[1188] Making a pocket (its base is 1-1.5 mm form the edge of the
eye).
[1189] Positioning a pellet, containing 50 ng-5 ug of an albumin
fusion protein of the invention, within the pocket.
[1190] Treatment with an an albumin fusion protein of the invention
can also be applied topically to the corneal wounds in a dosage
range of 20 mg-500 mg (daily treatment for five days).
Example 21
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[1191] Diabetic db+/db+ Mouse Model.
[1192] To demonstrate that an albumin fusion protein of the
invention accelerates the healing process, the genetically diabetic
mouse model of wound healing is used. The full thickness wound
healing model in the db+/db+ mouse is a well characterized,
clinically relevant and reproducible model of impaired wound
healing. Healing of the diabetic wound is dependent on formation of
granulation tissue and re-epithelialization rather than contraction
(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D.
G. et al., Am. J. Pathol. 136:1235 (1990)).
[1193] The diabetic animals have many of the characteristic
features observed in Type II diabetes mellitus. Homozygous
(db+/db+) mice are obese in comparison to their normal heterozygous
(db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al.
Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show
polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+)
have elevated blood glucose, increased or normal insulin levels,
and suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.
51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55
(1985)). Peripheral neuropathy, myocardial complications, and
microvascular lesions, basement membrane thickening and glomerular
filtration abnormalities have been described in these animals
(Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et
al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.
40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6
(1982)). These homozygous diabetic mice develop hyperglycemia that
is resistant to insulin analogous to human type II diabetes (Mandel
et al., J. Immunol. 120:1375-1377 (1978)).
[1194] The characteristics observed in these animals suggests that
healing in this model may be similar to the healing observed in
human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246
(1990)).
[1195] Genetically diabetic female C57BL/KsJ (db+/db+) mice and
their non-diabetic (db+/+m) heterozygous littermates are used in
this study (Jackson Laboratories). The animals are purchased at 6
weeks of age and are 8 weeks old at the beginning of the study.
Animals are individually housed and received food and water ad
libitum. All manipulations are performed using aseptic techniques.
The experiments are conducted according to the rules and guidelines
of Human Genome Sciences, Inc. Institutional Animal Care and Use
Committee and the Guidelines for the Care and Use of Laboratory
Animals.
[1196] Wounding protocol is performed according to previously
reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med.
172:245-251 (1990)). Briefly, on the day of wounding, animals are
anesthetized with an intraperitoneal injection of Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in
deionized water. The dorsal region of the animal is shaved and the
skin washed with 70% ethanol solution and iodine. The surgical area
is dried with sterile gauze prior to wounding. An 8 mm
full-thickness wound is then created using a Keyes tissue punch.
Immediately following wounding, the surrounding skin is gently
stretched to eliminate wound expansion. The wounds are left open
for the duration of the experiment. Application of the treatment is
given topically for 5 consecutive days commencing on the day of
wounding. Prior to treatment, wounds are gently cleansed with
sterile saline and gauze sponges.
[1197] Wounds are visually examined and photographed at a fixed
distance at the day of surgery and at two day intervals thereafter.
Wound closure is determined by daily measurement on days 1-5 and on
day 8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[1198] An albumin fusion protein of the invention is administered
using at a range different doses, from 4 mg to 500 mg per wound per
day for 8 days in vehicle. Vehicle control groups received 50 mL of
vehicle solution.
[1199] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology and
immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for
further processing.
[1200] Three groups of 10 animals each (5 diabetic and 5
non-diabetic controls) are evaluated: 1) Vehicle placebo control,
2) untreated group, and 3) treated group.
[1201] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total square area of
the wound. Contraction is then estimated by establishing the
differences between the initial wound area (day 0) and that of post
treatment (day 8). The wound area on day 1 is 64 mm.sup.2, the
corresponding size of the dermal punch. Calculations are made using
the following formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
a
[1202] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using a Reichert-Jung microtome. Routine
hematoxylin-eosin (H&E) staining is performed on cross-sections
of bisected wounds. Histologic examination of the wounds are used
to assess whether the healing process and the morphologic
appearance of the repaired skin is altered by treatment with an
albumin fusion protein of the invention. This assessment included
verification of the presence of cell accumulation, inflammatory
cells, capillaries, fibroblasts, re-epithelialization and epidermal
maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235
(1990)). A calibrated lens micrometer is used by a blinded
observer.
[1203] Tissue sections are also stained immunohistochemically with
a polyclonal rabbit anti-human keratin antibody using ABC Elite
detection system. Human skin is used as a positive tissue control
while non-immune IgG is used as a negative control. Keratinocyte
growth is determined by evaluating the extent of
reepithelialization of the wound using a calibrated lens
micrometer.
[1204] Proliferating cell nuclear antigen/cyclin (PCNA) in skin
specimens is demonstrated by using anti-PCNA antibody (1:50) with
an ABC Elite detection system. Human colon cancer served as a
positive tissue control and human brain tissue is used as a
negative tissue control. Each specimen included a section with
omission of the primary antibody and substitution with non-immune
mouse IgG. Ranking of these sections is based on the extent of
proliferation on a scale of 0-8, the lower side of the scale
reflecting slight proliferation to the higher side reflecting
intense proliferation.
[1205] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[1206] Steroid Impaired Rat Model
[1207] The inhibition of wound healing by steroids has been well
documented in various in vitro and in vivo systems (Wahl,
Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid
Action: Basic and Clinical Aspects. 280-302 (1989); Wahl et al., J.
Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med.
147:1684-1694 (1978)). Glucocorticoids retard wound healing by
inhibiting angiogenesis, decreasing vascular permeability (Ebert et
al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation,
and collagen synthesis (Beck et al., Growth Factors. 5: 295-304
(1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and
producing a transient reduction of circulating monocytes (Haynes et
al., J. Clin. Invest. 61: 703-797 (1978); Wahl, "Glucocorticoids
and wound healing", In: Antiinflammatory Steroid Action: Basic and
Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)).
The systemic administration of steroids to impaired wound healing
is a well establish phenomenon in rats (Beck et al., Growth
Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, "Glucocorticoids and wound healing", In:
Antiinflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc.
Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
[1208] To demonstrate that an albumin fusion protein of the
invention can accelerate the healing process, the effects of
multiple topical applications of the fusion protein on full
thickness excisional skin wounds in rats in which healing has been
impaired by the systemic administration of methylprednisolone is
assessed.
[1209] Young adult male Sprague Dawley rats weighing 250-300 g
(Charles River Laboratories) are used in this example. The animals
are purchased at 8 weeks of age and are 9 weeks old at the
beginning of the study. The healing response of rats is impaired by
the systemic administration of methylprednisolone (17 mg/kg/rat
intramuscularly) at the time of wounding. Animals are individually
housed and received food and water ad libitum. All manipulations
are performed using aseptic techniques. This study is conducted
according to the rules and guidelines of Human Genome Sciences,
Inc. Institutional Animal Care and Use Committee and the Guidelines
for the Care and Use of Laboratory Animals.
[1210] The wounding protocol is followed according to that
described above. On the day of wounding, animals are anesthetized
with an intramuscular injection of ketamine (50 mg/kg) and xylazine
(5 mg/kg). The dorsal region of the animal is shaved and the skin
washed with 70% ethanol and iodine solutions. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is created using a Keyes tissue punch. The wounds are left
open for the duration of the experiment. Applications of the
testing materials are given topically once a day for 7 consecutive
days commencing on the day of wounding and subsequent to
methylprednisolone administration. Prior to treatment, wounds are
gently cleansed with sterile saline and gauze sponges.
[1211] Wounds are visually examined and photographed at a fixed
distance at the day of wounding and at the end of treatment. Wound
closure is determined by daily measurement on days 1-5 and on day
8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[1212] The fusion protein of the invention is administered using at
a range different doses, from 4 mg to 500 mg per wound per day for
8 days in vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[1213] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology. Tissue specimens
are placed in 10% neutral buffered formalin in tissue cassettes
between biopsy sponges for further processing.
[1214] Three groups of 10 animals each (5 with methylprednisolone
and 5 without glucocorticoid) are evaluated: 1) Untreated group 2)
Vehicle placebo control 3) treated groups.
[1215] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total area of the
wound. Closure is then estimated by establishing the differences
between the initial wound area (day 0) and that of post treatment
(day 8). The wound area on day 1 is 64 mm.sup.2, the corresponding
size of the dermal punch. Calculations are made using the following
formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
b
[1216] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using an Olympus microtome. Routine hematoxylin-eosin
(H&E) staining is performed on cross-sections of bisected
wounds. Histologic examination of the wounds allows assessment of
whether the healing process and the morphologic appearance of the
repaired skin is improved by treatment with an albumin fusion
protein of the invention. A calibrated lens micrometer is used by a
blinded observer to determine the distance of the wound gap.
[1217] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
Example 22
Lymphedema Animal Model
[1218] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of an albumin fusion protein of the invention
in lymphangiogenesis and re-establishment of the lymphatic
circulatory system in the rat hind limb. Effectiveness is measured
by swelling volume of the affected limb, quantification of the
amount of lymphatic vasculature, total blood plasma protein, and
histopathology. Acute lymphedema is observed for 7-10 days. Perhaps
more importantly, the chronic progress of the edema is followed for
up to 3-4 weeks.
[1219] Prior to beginning surgery, blood sample is drawn for
protein concentration analysis. Male rats weighing approximately
.about.350 g are dosed with Pentobarbital. Subsequently, the right
legs are shaved from knee to hip. The shaved area is swabbed with
gauze soaked in 70% EtOH. Blood is drawn for serum total protein
testing. Circumference and volumetric measurements are made prior
to injecting dye into paws after marking 2 measurement levels (0.5
cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of
both right and left paws are injected with 0.05 ml of 1% Evan's
Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
[1220] Using the knee joint as a landmark, a mid-leg inguinal
incision is made circumferentially allowing the femoral vessels to
be located. Forceps and hemostats are used to dissect and separate
the skin flaps. After locating the femoral vessels, the lymphatic
vessel that runs along side and underneath the vessel(s) is
located. The main lymphatic vessels in this area are then
electrically coagulated or suture ligated.
[1221] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located. The 2 proximal and 2 distal lymphatic
vessels and distal blood supply of the popliteal node are then
ligated by suturing. The popliteal lymph node, and any accompanying
adipose tissue, is then removed by cutting connective tissues.
[1222] Care is taken to control any mild bleeding resulting from
this procedure. After lymphatics are occluded, the skin flaps are
sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin
edges are sealed to the underlying muscle tissue while leaving a
gap of .about.0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[1223] To avoid infection, animals are housed individually with
mesh (no bedding). Recovering animals are checked daily through the
optimal edematous peak, which typically occurred by day 5-7. The
plateau edematous peak are then observed. To evaluate the intensity
of the lymphedema, the circumference and volumes of 2 designated
places on each paw before operation and daily for 7 days are
measured. The effect of plasma proteins on lymphedema is determined
and whether protein analysis is a useful testing perimeter is also
investigated. The weights of both control and edematous limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
[1224] Circumference Measurements: Under brief gas anesthetic to
prevent limb movement, a cloth tape is used to measure limb
circumference. Measurements are done at the ankle bone and dorsal
paw by 2 different people and those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[1225] Volumetric Measurements: On the day of surgery, animals are
anesthetized with Pentobarbital and are tested prior to surgery.
For daily volumetrics animals are under brief halothane anesthetic
(rapid immobilization and quick recovery), and both legs are shaved
and equally marked using waterproof marker on legs. Legs are first
dipped in water, then dipped into instrument to each marked level
then measured by Buxco edema software (Chen/Victor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[1226] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca.sup.2+ comparison.
[1227] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs are amputated using a
quillitine, then both experimental and control legs are cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint is disarticulated and the foot is weighed.
[1228] Histological Preparations: The transverse muscle located
behind the knee (popliteal) area is dissected and arranged in a
metal mold, filled with freezeGel, dipped into cold methylbutane,
placed into labeled sample bags at -80EC until sectioning. Upon
sectioning, the muscle is observed under fluorescent microscopy for
lymphatics.
Example 23
Suppression of TNF Alpha-Induced Adhesion Molecule Expression by an
Albumin Fusion Protein of the Invention
[1229] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[1230] Tumor necrosis factor alpha (TNF-a), a potent
proinflammatory cytokine, is a stimulator of all three CAMs on
endothelial cells and may be involved in a wide variety of
inflammatory responses, often resulting in a pathological
outcome.
[1231] The potential of an albumin fusion protein of the invention
to mediate a suppression of TNF-a induced CAM expression can be
examined. A modified ELISA assay which uses ECs as a solid phase
absorbent is employed to measure the amount of CAM expression on
TNF-a treated ECs when co-stimulated with a member of the FGF
family of proteins.
[1232] To perform the experiment, human umbilical vein endothelial
cell (HUVEC) cultures are obtained from pooled cord harvests and
maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.)
supplemented with 10% FCS and 1% penicillin/streptomycin in a 37
degree C. humidified incubator containing 5% CO.sub.2. HUVECs are
seeded in 96-well plates at concentrations of 1.times.10.sup.4
cells/well in EGM medium at 37 degree C. for 18-24 hrs or until
confluent. The monolayers are subsequently washed 3 times with a
serum-free solution of RPMI-1640 supplemented with 100 U/ml
penicillin and 100 mg/ml streptomycin, and treated with a given
cytokine and/or growth factor(s) for 24 h at 37 degree C. Following
incubation, the cells are then evaluated for CAM expression.
[1233] Human Umbilical Vein Endothelial cells (HUVECs) are grown in
a standard 96 well plate to confluence. Growth medium is removed
from the cells and replaced with 90 ul of 199 Medium (10% FBS).
Samples for testing and positive or negative controls are added to
the plate in triplicate (in 10 ul volumes). Plates are incubated at
37 degree C. for either 5 h (selectin and integrin expression) or
24 h (integrin expression only). Plates are aspirated to remove
medium and 100 .mu.l of 0.1% paraformaldehyde-PBS (with Ca.sup.++
and Mg.sup.++) is added to each well. Plates are held at 4.degree.
C. for 30 min.
[1234] Fixative is then removed from the wells and wells are washed
1.times. with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the
wells to dry. Add 10 .mu.l of diluted primary antibody to the test
and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and
Anti-E-selectin-Biotin are used at a concentration of 10 .mu.g/ml
(1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at
37.degree. C. for 30 min. in a humidified environment. Wells are
washed X3 with PBS(+Ca,Mg)+0.5% BSA.
[1235] Then add 20 .mu.l of diluted ExtrAvidin-Alkaline Phosphotase
(1:5,000 dilution) to each well and incubated at 37.degree. C. for
30 min. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of
p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer
(pH 10.4). 100 .mu.l of pNPP substrate in glycine buffer is added
to each test well. Standard wells in triplicate are prepared from
the working dilution of the ExtrAvidin-Alkaline Phosphotase in
glycine buffer: 1:5,000
(10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-150.5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent must then be added to each of the standard
wells. The plate must be incubated at 37.degree. C. for 4 h. A
volume of 50 .mu.l of 3M NaOH is added to all wells. The results
are quantified on a plate reader at 405 nm. The background
subtraction option is used on blank wells filled with glycine
buffer only. The template is set up to indicate the concentration
of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng;
0.18 ng]. Results are indicated as amount of bound AP-conjugate in
each sample.
Example 24
Construction of GAS Reporter Construct
[1236] One signal transduction pathway involved in the
differentiation and proliferation of cells is called the Jaks-STATs
pathway. Activated proteins in the Jaks-STATs pathway bind to gamma
activation site "GAS" elements or interferon-sensitive responsive
element ("ISRE"), located in the promoter of many genes. The
binding of a protein to these elements alter the expression of the
associated gene.
[1237] GAS and ISRE elements are recognized by a class of
transcription factors called Signal Transducers and Activators of
Transcription, or "STATs." There are six members of the STATs
family. Stat1 and Stat3 are present in many cell types, as is Stat2
(as response to IFN-alpha is widespread). Stat4 is more restricted
and is not in many cell types though it has been found in T helper
class I, cells after treatment with IL-12. Stat5 was originally
called mammary growth factor, but has been found at higher
concentrations in other cells including myeloid cells. It can be
activated in tissue culture cells by many cytokines.
[1238] The STATs are activated to translocate from the cytoplasm to
the nucleus upon tyrosine phosphorylation by a set of kinases known
as the Janus Kinase ("Jaks") family. Jaks represent a distinct
family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2,
and Jak3. These kinases display significant sequence similarity and
are generally catalytically inactive in resting cells.
[1239] The Jaks are activated by a wide range of receptors
summarized in the Table below. (Adapted from review by Schidler and
Darnell, Ann. Rev. Biochem. 64:621-51 (1995)). A cytokine receptor
family, capable of activating Jaks, is divided into two groups: (a)
Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and
thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10.
The Class 1 receptors share a conserved cysteine motif (a set of
four conserved cysteines and one tryptophan) and a WSXWS motif (a
membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID
NO:53)).
[1240] Thus, on binding of a ligand to a receptor, Jaks are
activated, which in turn activate STATs, which then translocate and
bind to GAS elements. This entire process is encompassed in the
Jaks-STATs signal transduction pathway. Therefore, activation of
the Jaks-STATs pathway, reflected by the binding of the GAS or the
ISRE element, can be used to indicate proteins involved in the
proliferation and differentiation of cells. For example, growth
factors and cytokines are known to activate the Jaks-STATs pathway
(See Table 5, below). Thus, by using GAS elements linked to
reporter molecules, activators of the Jaks-STATs pathway can be
identified.
TABLE-US-00010 TABLE 5 JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS
GAS(elements) or ISRE IFN family IFN-a/B + + - - 1, 2, 3 ISRE IFN-g
+ + - 1 GAS (IRF1 > Lys6 > IFP) Il-10 + ? ? - 1, 3 gp130
family IL-6 (Pleiotropic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP)
Il-11 (Pleiotropic) ? + ? ? 1, 3 OnM (Pleiotropic) ? + + ? 1, 3 LIF
(Pleiotropic) ? + + ? 1, 3 CNTF (Pleiotropic) -/+ + + ? 1, 3 G-CSF
(Pleiotropic) ? + ? ? 1, 3 IL-12 (Pleiotropic) + - + + 1, 3 g-C
family IL-2 (lymphocytes) - + - + 1, 3, 5 GAS IL-4 (lymph/myeloid)
- + - + 6 GAS(IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) - +
- + 5 GAS IL-9 (lymphocytes) - + - + 5 GAS IL-13 (lymphocyte) - + ?
? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) - - + - 5
GAS(IRF1 > IFP >> Ly6) IL-5 (myeloid) - - + - 5 GAS GM-CSF
(myeloid) - - + - 5 GAS Growth hormone family GH ? - + - 5 PRL ?
+/- + - 1, 3, 5 EPO ? - + - 5 GAS (B- CAS > IRF1 = IFP >>
Ly6) Receptor Tyrosine Kinases EGF ? + + - 1, 3 GAS (IRF1) PDGF ? +
+ - 1, 3 CSF-1 ? + + - 1, 3 GAS(not IRF1)
[1241] To construct a synthetic GAS containing promoter element,
which is used in the Biological Assays described in Examples 27-29,
a PCR based strategy is employed to generate a GAS-SV40 promoter
sequence. The 5' primer contains four tandem copies of the GAS
binding site found in the IRF1 promoter and previously demonstrated
to bind STATs upon induction with a range of cytokines (Rothman et
al., Immunity 1:457-468 (1994).), although other GAS or ISRE
elements can be used instead. The 5' primer also contains 18 bp of
sequence complementary to the SV40 early promoter sequence and is
flanked with an XhoI site. The sequence of the 5' primer is:
TABLE-US-00011 (SEQ ID NO:54) 5':
GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTC
CCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG: 3'
[1242] The downstream primer is complementary to the SV40 promoter
and is flanked with a Hind III site:
5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:55)
[1243] PCR amplification is performed using the SV40 promoter
template present in the B-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI/Hind III
and subcloned into BLSK2-. (Stratagene.) Sequencing with forward
and reverse primers confirms that the insert contains the following
sequence:
TABLE-US-00012 (SEQ ID NO:56) 5':
CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCG
AAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGT
CCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCC
ATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG
GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGG
AGGCCTAGGCTTTTGCAAAAAGCTT: 3'
[1244] With this GAS promoter element linked to the SV40 promoter,
a GAS:SEAP2 reporter construct is next engineered. Here, the
reporter molecule is a secreted alkaline phosphatase, or "SEAP."
Clearly, however, any reporter molecule can be instead of SEAP, in
this or in any of the other Examples. Well known reporter molecules
that can be used instead of SEAP include chloramphenicol
acetyltransferase (CAT), luciferase, alkaline phosphatase,
B-galactosidase, green fluorescent protein (GFP), or any protein
detectable by an antibody.
[1245] The above sequence confirmed synthetic GAS-SV40 promoter
element is subcloned into the pSEAP-Promoter vector obtained from
Clontech using HindIII and XhoI, effectively replacing the SV40
promoter with the amplified GAS:SV40 promoter element, to create
the GAS-SEAP vector. However, this vector does not contain a
neomycin resistance gene, and therefore, is not preferred for
mammalian expression systems.
[1246] Thus, in order to generate mammalian stable cell lines
expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed
from the GAS-SEAP vector using SalI and NotI, and inserted into a
backbone vector containing the neomycin resistance gene, such as
pGFP-1 (Clontech), using these restriction sites in the multiple
cloning site, to create the GAS-SEAP/Neo vector. Once this vector
is transfected into mammalian cells, this vector can then be used
as a reporter molecule for GAS binding as described in Examples
27-29.
[1247] Other constructs can be made using the above description and
replacing GAS with a different promoter sequence. For example,
construction of reporter molecules containing EGR and NF-KB
promoter sequences are described in Examples 27-31. However, many
other promoters can be substituted using the protocols described in
these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin
promoters can be substituted, alone or in combination (e.g.,
GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS). Similarly,
other cell lines can be used to test reporter construct activity,
such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell),
Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 25
Assay for SEAP Activity
[1248] As a reporter molecule for the assays described in examples
disclosed herein, SEAP activity is assayed using the Tropix
Phospho-light Kit (Cat. BP-400) according to the following general
procedure. The Tropix Phospho-light Kit supplies the Dilution,
Assay, and Reaction Buffers used below.
[1249] Prime a dispenser with the 2.5.times. Dilution Buffer and
dispense 15 ul of 2.5.times. dilution buffer into Optiplates
containing 35 ul of a solution containing an albumin fusion protein
of the invention. Seal the plates with a plastic sealer and
incubate at 65 degree C. for 30 min. Separate the Optiplates to
avoid uneven heating.
[1250] Cool the samples to room temperature for 15 minutes. Empty
the dispenser and prime with the Assay Buffer. Add 50 ml Assay
Buffer and incubate at room temperature 5 min. Empty the dispenser
and prime with the Reaction Buffer (see the Table below). Add 50 ul
Reaction Buffer and incubate at room temperature for 20 minutes.
Since the intensity of the chemiluminescent signal is time
dependent, and it takes about 10 minutes to read 5 plates on a
luminometer, thus one should treat 5 plates at each time and start
the second set 10 minutes later.
[1251] Read the relative light unit in the luminometer. Set H12 as
blank, and print the results. An increase in chemiluminescence
indicates reporter activity.
TABLE-US-00013 TABLE 6 Rxn buffer # of plates diluent (ml) CSPD
(ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16
90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22
120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28
150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34
180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40
210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235
11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13
Example 26
Assay Identifying Neuronal Activity
[1252] When cells undergo differentiation and proliferation, a
group of genes are activated through many different signal
transduction pathways. One of these genes, EGR1 (early growth
response gene 1), is induced in various tissues and cell types upon
activation. The promoter of EGR1 is responsible for such induction.
Using the EGR1 promoter linked to reporter molecules, the ability
of fusion proteins of the invention to activate cells can be
assessed.
[1253] Particularly, the following protocol is used to assess
neuronal activity in PC12 cell lines. PC12 cells (rat
phenochromocytoma cells) are known to proliferate and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF
(epidermal growth factor). The EGR1 gene expression is activated
during this treatment. Thus, by stably transfecting PC12 cells with
a construct containing an EGR promoter linked to SEAP reporter,
activation of PC12 cells by an albumin fusion protein of the
present invention can be assessed.
[1254] The EGR/SEAP reporter construct can be assembled by the
following protocol. The EGR-1 promoter sequence (-633 to +1)
(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified
from human genomic DNA using the following primers:
TABLE-US-00014 First primer: (SEQ ID NO:57) 5'
GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' Second primer: (SEQ ID NO:58)
5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3'
[1255] Using the GAS:SEAP/Neo vector produced in Example 24, EGR1
amplified product can then be inserted into this vector. Linearize
the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII,
removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product
with these same enzymes. Ligate the vector and the EGR1
promoter.
[1256] To prepare 96 well-plates for cell culture, two mls of a
coating solution (1:30 dilution of collagen type I (Upstate Biotech
Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per
one 10 cm plate or 50 ml per well of the 96-well plate, and allowed
to air dry for 2 hr.
[1257] PC12 cells are routinely grown in RPMI-1640 medium (Bio
Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat.
#12449-78P), 5% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 ug/ml
streptomycin on a precoated 10 cm tissue culture dish. One to four
split is done every three to four days. Cells are removed from the
plates by scraping and resuspended with pipetting up and down for
more than 15 times.
[1258] Transfect the EGR/SEAP/Neo construct into PC12 using
techniques known in the art. EGR-SEAP/PC12 stable cells are
obtained by growing the cells in 300 ug/ml G418. The G418-free
medium is used for routine growth but every one to two months, the
cells should be re-grown in 300 ug/ml G418 for couple of
passages.
[1259] To assay for neuronal activity, a 10 cm plate with cells
around 70 to 80% confluent is screened by removing the old medium.
Wash the cells once with PBS (Phosphate buffered saline). Then
starve the cells in low serum medium (RPMI-1640 containing 1% horse
serum and 0.5% FBS with antibiotics) overnight.
[1260] The next morning, remove the medium and wash the cells with
PBS. Scrape off the cells from the plate, suspend the cells well in
2 ml low serum medium. Count the cell number and add more low serum
medium to reach final cell density as 5.times.10.sup.5
cells/ml.
[1261] Add 200 ul of the cell suspension to each well of 96-well
plate (equivalent to 1.times.10.sup.5 cells/well). Add a series of
different concentrations of an albumin fusion protein of the
invention, 37 degree C. for 48 to 72 hr. As a positive control, a
growth factor known to activate PC12 cells through EGR can be used,
such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold
induction of SEAP is typically seen in the positive control wells.
SEAP assay may be routinely performed using techniques known in the
art and/or as described in Example 25.
Example 27
Assay for T-Cell Activity
[1262] The following protocol is used to assess T-cell activity by
identifying factors, and determining whether an albumin fusion
protein of the invention proliferates and/or differentiates
T-cells. T-cell activity is assessed using the GAS/SEAP/Neo
construct produced in Example 24. Thus, factors that increase SEAP
activity indicate the ability to activate the Jaks-STATS signal
transduction pathway. The T-cell used in this assay is Jurkat
T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC
Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No.
CRL-1582) cells can also be used.
[1263] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In
order to generate stable cell lines, approximately 2 million Jurkat
cells are transfected with the GAS-SEAP/neo vector using DMRIE-C
(Life Technologies) (transfection procedure described below). The
transfected cells are seeded to a density of approximately 20,000
cells per well and transfectants resistant to 1 mg/ml genticin
selected. Resistant colonies are expanded and then tested for their
response to increasing concentrations of interferon gamma. The dose
response of a selected clone is demonstrated.
[1264] Specifically, the following protocol will yield sufficient
cells for 75 wells containing 200 ul of cells. Thus, it is either
scaled up, or performed in multiple to generate sufficient cells
for multiple 96 well plates. Jurkat cells are maintained in
RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml
OPTI-MEM containing 50 ul of DMRIE-C and incubate at room
temperature for 15-45 mins.
[1265] During the incubation period, count cell concentration, spin
down the required number of cells (10.sup.7 per transfection), and
resuspend in OPTI-MEM to a final concentration of 10.sup.7
cells/ml. Then add 1 ml of 1.times.10.sup.7 cells in OPTI-MEM to
T25 flask and incubate at 37 degree C. for 6 hrs. After the
incubation, add 10 ml of RPMI+15% serum.
[1266] The Jurkat:GAS-SEAP stable reporter lines are maintained in
RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are
treated with varying concentrations of one or more fusion proteins
of the present invention.
[1267] On the day of treatment with the fusion protein, the cells
should be washed and resuspended in fresh RPMI+10% serum to a
density of 500,000 cells per ml. The exact number of cells required
will depend on the number of fusion proteins and the number of
different concentrations of fusion proteins being screened. For one
96 well plate, approximately 10 million cells (for 10 plates, 100
million cells) are required.
[1268] The well dishes containing Jurkat cells treated with the
fusion protein are placed in an incubator for 48 hrs (note: this
time is variable between 48-72 hrs). 35 ul samples from each well
are then transferred to an opaque 96 well plate using a 12 channel
pipette. The opaque plates should be covered (using sellophene
covers) and stored at -20 degree C. until SEAP assays are performed
according to Example 25. The plates containing the remaining
treated cells are placed at 4 degree C. and serve as a source of
material for repeating the assay on a specific well if desired.
[1269] As a positive control, 100 Unit/ml interferon gamma can be
used which is known to activate Jurkat T cells. Over 30 fold
induction is typically observed in the positive control wells.
[1270] The above protocol may be used in the generation of both
transient, as well as, stable transfected cells, which would be
apparent to those of skill in the art.
Example 28
Assay for T-Cell Activity
[1271] NF-KB (Nuclear Factor KB) is a transcription factor
activated by a wide variety of agents including the inflammatory
cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and
lymphotoxin-beta, by exposure to LPS or thrombin, and by expression
of certain viral gene products. As a transcription factor, NF-KB
regulates the expression of genes involved in immune cell
activation, control of apoptosis (NF-KB appears to shield cells
from apoptosis), B and T-cell development, anti-viral and
antimicrobial responses, and multiple stress responses.
[1272] In non-stimulated conditions, NF-KB is retained in the
cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB
is phosphorylated and degraded, causing NF-KB to shuttle to the
nucleus, thereby activating transcription of target genes. Target
genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and
class 1 MHC.
[1273] Due to its central role and ability to respond to a range of
stimuli, reporter constructs utilizing the NF-KB promoter element
are used to screen the fusion protein. Activators or inhibitors of
NF-KB would be useful in treating, preventing, and/or diagnosing
diseases. For example, inhibitors of NF-KB could be used to treat
those diseases related to the acute or chronic activation of NF-KB,
such as rheumatoid arthritis.
[1274] To construct a vector containing the NF-KB promoter element,
a PCR based strategy is employed. The upstream primer contains four
tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID
NO:59), 18 bp of sequence complementary to the 5' end of the SV40
early promoter sequence, and is flanked with an XhoI site:
TABLE-US-00015 (SEQ ID NO:60) 5':
GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGG
GACTTTCCATCCTGCCATCTCAATTAG: 3'
[1275] The downstream primer is complementary to the 3' end of the
SV40 promoter and is flanked with a Hind III site:
TABLE-US-00016 (SEQ ID NO:55) 5': GCGGCAAGCTTTTTGCAAAGCCTAGGC:
3'
[1276] PCR amplification is performed using the SV40 promoter
template present in the pB-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI and Hind
III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7
and T3 primers confirms the insert contains the following
sequence:
TABLE-US-00017 (SEQ ID NO:61) 5':
CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTT
TCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCC
GCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATG
GCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCT
GAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTG CAAAAAGCTT:
3'
[1277] Next, replace the SV40 minimal promoter element present in
the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40
fragment using XhoI and HindIII. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred
for mammalian expression systems.
[1278] In order to generate stable mammalian cell lines, the
NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP
vector using restriction enzymes SalI and NotI, and inserted into a
vector containing neomycin resistance. Particularly, the
NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech),
replacing the GFP gene, after restricting pGFP-1 with SalI and
NotI.
[1279] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat
T-cells are created and maintained according to the protocol
described in Example 25. Similarly, the method for assaying fusion
proteins with these stable Jurkat T-cells is also described in
Example 25. As a positive control, exogenous TNF alpha (0.1, 1, 10
ng) is added to wells H9, H10, and H11, with a 5-10 fold activation
typically observed.
Example 29
Assay Identifying Myeloid Activity
[1280] The following protocol is used to assess myeloid activity of
an albumin fusion protein of the present invention by determining
whether the fusion protein proliferates and/or differentiates
myeloid cells. Myeloid cell activity is assessed using the
GAS/SEAP/Neo construct produced in Example 24. Thus, factors that
increase SEAP activity indicate the ability to activate the
Jaks-STATS signal transduction pathway. The myeloid cell used in
this assay is U937, a pre-monocyte cell line, although TF-1, HL60,
or KG1 can be used.
[1281] To transiently transfect U937 cells with the GAS/SEAP/Neo
construct produced in Example 24, a DEAE-Dextran method (Kharbanda
et. al., 1994, Cell Growth & Differentiation, 5:259-265) is
used. First, harvest 2.times.10.sup.7 U937 cells and wash with PBS.
The U937 cells are usually grown in RPMI 1640 medium containing 10%
heat-inactivated fetal bovine serum (FBS) supplemented with 100
units/ml penicillin and 100 mg/ml streptomycin.
[1282] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4)
buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid
DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na.sub.2HPO.sub.4.7H.sub.2O, 1
mM MgCl.sub.2, and 675 uM CaCl.sub.2. Incubate at 37 degrees C. for
45 min.
[1283] Wash the cells with RPMI 1640 medium containing 10% FBS and
then resuspend in 10 ml complete medium and incubate at 37 degree
C. for 36 hr.
[1284] The GAS-SEAP/U937 stable cells are obtained by growing the
cells in 400 ug/ml G418. The G418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in
400 ug/ml G418 for couple of passages.
[1285] These cells are tested by harvesting 1.times.10.sup.8 cells
(this is enough for ten 96-well plates assay) and wash with PBS.
Suspend the cells in 200 ml above described growth medium, with a
final density of 5.times.10.sup.5 cells/ml. Plate 200 ul cells per
well in the 96-well plate (or 1.times.10.sup.5 cells/well).
[1286] Add different concentrations of the fusion protein. Incubate
at 37 degee C for 48 to 72 hr. As a positive control, 100 Unit/ml
interferon gamma can be used which is known to activate U937 cells.
Over 30 fold induction is typically observed in the positive
control wells. SEAP assay the supernatant according to methods
known in the art and/or the protocol described in example 25.
Example 30
Assay Identifying Changes in Small Molecule Concentration and
Membrane Permeability
[1287] Binding of a ligand to a receptor is known to alter
intracellular levels of small molecules, such as calcium,
potassium, sodium, and pH, as well as alter membrane potential.
These alterations can be measured in an assay to identify fusion
proteins which bind to receptors of a particular cell. Although the
following protocol describes an assay for calcium, this protocol
can easily be modified to detect changes in potassium, sodium, pH,
membrane potential, or any other small molecule which is detectable
by a fluorescent probe.
[1288] The following assay uses Fluorometric Imaging Plate Reader
("FLIPR") to measure changes in fluorescent molecules (Molecular
Probes) that bind small molecules. Clearly, any fluorescent
molecule detecting a small molecule can be used instead of the
calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.;
catalog no. F-14202), used here.
[1289] For adherent cells, seed the cells at 10,000-20,000
cells/well in a Co-star black 96-well plate with clear bottom. The
plate is incubated in a CO.sub.2 incubator for 20 hours. The
adherent cells are washed two times in Biotek washer with 200 ul of
HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after
the final wash.
[1290] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic
acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4
is added to each well. The plate is incubated at 37 degrees C. in a
CO.sub.2 incubator for 60 min. The plate is washed four times in
the Biotek washer with HBSS leaving 100 ul of buffer.
[1291] For non-adherent cells, the cells are spun down from culture
media. Cells are re-suspended to 2-5.times.10.sup.6 cells/ml with
HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in
10% pluronic acid DMSO is added to each ml of cell suspension. The
tube is then placed in a 37 degrees C. water bath for 30-60 min.
The cells are washed twice with HBSS, resuspended to
1.times.10.sup.6 cells/ml, and dispensed into a microplate, 100
ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate
is then washed once in Denley Cell Wash with 200 ul, followed by an
aspiration step to 100 ul final volume.
[1292] For a non-cell based assay, each well contains a fluorescent
molecule, such as fluo-4. The fusion protein of the invention is
added to the well, and a change in fluorescence is detected.
[1293] To measure the fluorescence of intracellular calcium, the
FLIPR is set for the following parameters: (1) System gain is
300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is
F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular signaling event caused by an albumin fusion protein
of the present invention or a molecule induced by an albumin fusion
protein of the present invention, which has resulted in an increase
in the intracellular Ca.sup.++ concentration.
Example 31
Assay Identifying Tyrosine Kinase Activity
[1294] The Protein Tyrosine Kinases (PTK) represent a diverse group
of transmembrane and cytoplasmic kinases. Within the Receptor
Protein Tyrosine Kinase (RPTK) group are receptors for a range of
mitogenic and metabolic growth factors including the PDGF, FGF,
EGF, NGF, HGF and Insulin receptor subfamilies. In addition there
are a large family of RPTKs for which the corresponding ligand is
unknown. Ligands for RPTKs include mainly secreted small proteins,
but also membrane-bound and extracellular matrix proteins.
[1295] Activation of RPTK by ligands involves ligand-mediated
receptor dimerization, resulting in transphosphorylation of the
receptor subunits and activation of the cytoplasmic tyrosine
kinases. The cytoplasmic tyrosine kinases include receptor
associated tyrosine kinases of the src-family (e.g., src, yes, lck,
lyn, fyn) and non-receptor linked and cytosolic protein tyrosine
kinases, such as the Jak family, members of which mediate signal
transduction triggered by the cytokine superfamily of receptors
(e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[1296] Because of the wide range of known factors capable of
stimulating tyrosine kinase activity, identifying whether an
albumin fusion protein of the present invention or a molecule
induced by a fusion protein of the present invention is capable of
activating tyrosine kinase signal transduction pathways is of
interest. Therefore, the following protocol is designed to identify
such molecules capable of activating the tyrosine kinase signal
transduction pathways.
[1297] Seed target cells (e.g., primary keratinocytes) at a density
of approximately 25,000 cells per well in a 96 well Loprodyne
Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.).
The plates are sterilized with two 30 minute rinses with 100%
ethanol, rinsed with water and dried overnight. Some plates are
coated for 2 hr with 100 ml of cell culture grade type I collagen
(50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can
be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel
purchased from Becton Dickinson (Bedford, Mass.), or calf serum,
rinsed with PBS and stored at 4 degree C. Cell growth on these
plates is assayed by seeding 5,000 cells/well in growth medium and
indirect quantitation of cell number through use of alamarBlue as
described by the manufacturer Alamar Biosciences, Inc. (Sacramento,
Calif.) after 48 hr. Falcon plate covers #3071 from Becton
Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent
Screen Plates. Falcon Microtest III cell culture plates can also be
used in some proliferation experiments.
[1298] To prepare extracts, A431 cells are seeded onto the nylon
membranes of Loprodyne plates (20,000/200 ml/well) and cultured
overnight in complete medium. Cells are quiesced by incubation in
serum-free basal medium for 24 hr. After 5-20 minutes treatment
with EGF (60 ng/ml) or a different concentrations of an albumin
fusion protein of the invention, the medium was removed and 100 ml
of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton
X-100, 0.1% SDS, 2 mM Na.sub.3VO4, 2 mM Na4P2O7 and a cocktail of
protease inhibitors (#1836170) obtained from Boehereinger Mannheim
(Indianapolis, Ind.)) is added to each well and the plate is shaken
on a rotating shaker for 5 minutes at 4.degree. C. The plate is
then placed in a vacuum transfer manifold and the extract filtered
through the 0.45 mm membrane bottoms of each well using house
vacuum. Extracts are collected in a 96-well catch/assay plate in
the bottom of the vacuum manifold and immediately placed on ice. To
obtain extracts clarified by centrifugation, the content of each
well, after detergent solubilization for 5 minutes, is removed and
centrifuged for 15 minutes at 4 degree C. at 16,000.times.g.
[1299] Test the filtered extracts for levels of tyrosine kinase
activity. Although many methods of detecting tyrosine kinase
activity are known, one method is described here.
[1300] Generally, the tyrosine kinase activity of an albumin fusion
protein of the invention is evaluated by determining its ability to
phosphorylate a tyrosine residue on a specific substrate (a
biotinylated peptide). Biotinylated peptides that can be used for
this purpose include PSK1 (corresponding to amino acids 6-20 of the
cell division kinase cdc2-p34) and PSK2 (corresponding to amino
acids 1-17 of gastrin). Both peptides are substrates for a range of
tyrosine kinases and are available from Boehringer Mannheim.
[1301] The tyrosine kinase reaction is set up by adding the
following components in order. First, add 10 ul of 5 uM
Biotinylated Peptide, then 10 ul ATP/Mg.sub.2+ (5 mM ATP/50 mM
MgCl.sub.2), then 10 ul of 5.times. Assay Buffer (40 mM imidazole
hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100
mM MgCl.sub.2, 5 mM MnC.sub.2, 0.5 mg/ml BSA), then 5 ul of Sodium
Vanadate (1 mM), and then 5 ul of water. Mix the components gently
and preincubate the reaction mix at 30 degree C. for 2 min. Initial
the reaction by adding 10 ul of the control enzyme or the filtered
supernatant.
[1302] The tyrosine kinase assay reaction is then terminated by
adding 10 ul of 120 mm EDTA and place the reactions on ice.
[1303] Tyrosine kinase activity is determined by transferring 50 ul
aliquot of reaction mixture to a microtiter plate (MTP) module and
incubating at 37 degree C. for 20 min. This allows the streptavidin
coated 96 well plate to associate with the biotinylated peptide.
Wash the MTP module with 300 ul/well of PBS four times. Next add 75
ul of anti-phosphotyrosine antibody conjugated to horse radish
peroxidase(anti-P-Tyr-POD(0.5u/ml)) to each well and incubate at 37
degree C. for one hour. Wash the well as above.
[1304] Next add 100 ul of peroxidase substrate solution (Boehringer
Mannheim) and incubate at room temperature for at least 5 mins (up
to 30 min). Measure the absorbance of the sample at 405 nm by using
ELISA reader. The level of bound peroxidase activity is quantitated
using an ELISA reader and reflects the level of tyrosine kinase
activity.
Example 32
Assay Identifying Phosphorylation Activity
[1305] As a potential alternative and/or complement to the assay of
protein tyrosine kinase activity described in Example 31, an assay
which detects activation (phosphorylation) of major intracellular
signal transduction intermediates can also be used. For example, as
described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However,
phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map
kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase
(MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,
phosphotyrosine, or phosphothreonine molecule, can be detected by
substituting these molecules for Erk-1 or Erk-2 in the following
assay.
[1306] Specifically, assay plates are made by coating the wells of
a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr
at room temp, (RT). The plates are then rinsed with PBS and blocked
with 3% BSA/PBS for 1 hr at RT. The protein G plates are then
treated with 2 commercial monoclonal antibodies (100 ng/well)
against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology).
(To detect other molecules, this step can easily be modified by
substituting a monoclonal antibody detecting any of the above
described molecules.) After 3-5 rinses with PBS, the plates are
stored at 4 degree C. until use.
[1307] A431 cells are seeded at 20,000/well in a 96-well Loprodyne
filterplate and cultured overnight in growth medium. The cells are
then starved for 48 hr in basal medium (DMEM) and then treated with
EGF (6 ng/well) or varying concentrations of the fusion protein of
the invention for 5-20 minutes. The cells are then solubilized and
extracts filtered directly into the assay plate.
[1308] After incubation with the extract for 1 hr at RT, the wells
are again rinsed. As a positive control, a commercial preparation
of MAP kinase (10 ng/well) is used in place of A431 extract. Plates
are then treated with a commercial polyclonal (rabbit) antibody (1
ug/ml) which specifically recognizes the phosphorylated epitope of
the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is
biotinylated by standard procedures. The bound polyclonal antibody
is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent
in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased fluorescent signal over background indicates a
phosphorylation by the fusion protein of the present invention or a
molecule induced by an albumin fusion protein of the present
invention.
Example 33
Phosphorylation Assay
[1309] In order to assay for phosphorylation activity of an albumin
fusion protein of the invention, a phosphorylation assay as
described in U.S. Pat. No. 5,958,405 (which is herein incorporated
by reference) is utilized. Briefly, phosphorylation activity may be
measured by phosphorylation of a protein substrate using
gamma-labeled .sup.32P-ATP and quantitation of the incorporated
radioactivity using a gamma radioisotope counter. The fusion
protein of the invention is incubated with the protein substrate,
.sup.32P-ATP, and a kinase buffer. The .sup.32P incorporated into
the substrate is then separated from free .sup.32P-ATP by
electrophoresis, and the incorporated .sup.32P is counted and
compared to a negative control. Radioactivity counts above the
negative control are indicative of phosphorylation activity of the
fusion protein.
Example 34
Detection of Phosphorylation Activity (Activation) of an Albumin
Fusion Protein of the Invention in the Presence of Polypeptide
Ligands
[1310] Methods known in the art or described herein may be used to
determine the phosphorylation activity of an albumin fusion protein
of the invention. A preferred method of determining phosphorylation
activity is by the use of the tyrosine phosphorylation assay as
described in U.S. Pat. No. 5,817,471 (incorporated herein by
reference).
Example 35
Assay for the Stimulation of Bone Marrow CD34+ Cell
Proliferation
[1311] This assay is based on the ability of human CD34+ to
proliferate in the presence of hematopoietic growth factors and
evaluates the ability of fusion proteins of the invention to
stimulate proliferation of CD34+ cells.
[1312] It has been previously shown that most mature precursors
will respond to only a single signal. More immature precursors
require at least two signals to respond. Therefore, to test the
effect of fusion proteins of the invention on hematopoietic
activity of a wide range of progenitor cells, the assay contains a
given fusion protein of the invention in the presence or absence of
hematopoietic growth factors. Isolated cells are cultured for 5
days in the presence of Stem Cell Factor (SCF) in combination with
tested sample. SCF alone has a very limited effect on the
proliferation of bone marrow (BM) cells, acting in such conditions
only as a "survival" factor. However, combined with any factor
exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will
cause a synergistic effect. Therefore, if the tested fusion protein
has a stimulatory effect on hematopoietic progenitors, such
activity can be easily detected. Since normal BM cells have a low
level of cycling cells, it is likely that any inhibitory effect of
a given fusion protein might not be detected. Accordingly, assays
for an inhibitory effect on progenitors is preferably tested in
cells that are first subjected to in vitro stimulation with
SCF+IL+3, and then contacted with the compound that is being
evaluated for inhibition of such induced proliferation.
[1313] Briefly, CD34+ cells are isolated using methods known in the
art. The cells are thawed and resuspended in medium (QBSF 60
serum-free medium with 1% L-glutamine (500 ml) Quality Biological,
Inc., Gaithersburg, Md. Cat#160-204-101). After several gentle
centrifugation steps at 200.times.g, cells are allowed to rest for
one hour. The cell count is adjusted to 2.5.times.10.sup.5
cells/ml. During this time, 100 .mu.l of sterile water is added to
the peripheral wells of a 96-well plate. The cytokines that can be
tested with an albumin fusion protein of the invention in this
assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat#255-SC) at
50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D
Systems, Minneapolis, Minn., Cat#203-ML) at 30 ng/ml. After one
hour, 10 .mu.l of prepared cytokines, varying concentrations of an
albumin fusion protein of the invention, and 20 .mu.l of diluted
cells are added to the media which is already present in the wells
to allow for a final total volume of 100 .mu.l. The plates are then
placed in a 37.degree. C./5% CO.sub.2 incubator for five days.
[1314] Eighteen hours before the assay is harvested, 0.5
.mu.Ci/well of [3H] Thymidine is added in a 10 .mu.l volume to each
well to determine the proliferation rate. The experiment is
terminated by harvesting the cells from each 96-well plate to a
filtermat using the Tomtec Harvester 96. After harvesting, the
filtermats are dried, trimmed and placed into OmniFilter assemblies
consisting of one OmniFilter plate and one OmniFilter Tray. 60
.mu.l Microscint is added to each well and the plate sealed with
TopSeal-A press-on sealing film A bar code 15 sticker is affixed to
the first plate for counting. The sealed plates are then loaded and
the level of radioactivity determined via the Packard Top Count and
the printed data collected for analysis. The level of radioactivity
reflects the amount of cell proliferation.
[1315] The studies described in this example test the activity of a
given fusion protein to stimulate bone marrow CD34+ cell
proliferation. One skilled in the art could easily modify the
exemplified studies to test the activity of fusion porteins and
polynucleotides of the invention (e.g., gene therapy) as well as
agonists and antagonists thereof. The ability of an albumin fusion
protein of the invention to stimulate the proliferation of bone
marrow CD34+ cells indicates that the albumin fusion protein and/or
polynucleotides corresponding to the fusion protein are useful for
the diagnosis and treatment of disorders affecting the immune
system and hematopoiesis. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections above, and
elsewhere herein.
Example 36
Assay for Extracellular Matrix Enhanced Cell Response (EMECR)
[1316] The objective of the Extracellular Matrix Enhanced Cell
Response (EMECR) assay is to evaluate the ability of fusion
proteins of the invention to act on hematopoietic stem cells in the
context of the extracellular matrix (ECM) induced signal.
[1317] Cells respond to the regulatory factors in the context of
signal(s) received from the surrounding microenvironment. For
example, fibroblasts, and endothelial and epithelial stem cells
fail to replicate in the absence of signals from the ECM.
Hematopoietic stem cells can undergo self-renewal in the bone
marrow, but not in in vitro suspension culture. The ability of stem
cells to undergo self-renewal in vitro is dependent upon their
interaction with the stromal cells and the ECM protein fibronectin
(fn). Adhesion of cells to fn is mediated by the
.alpha..sub.5..beta..sub.1 and .alpha..sub.4..beta..sub.1 integrin
receptors, which are expressed by human and mouse hematopoietic
stem cells. The factor(s) which integrate with the ECM environment
and are responsible for stimulating stem cell self-renewal have a
not yet been identified. Discovery of such factors should be of
great interest in gene therapy and bone marrow transplant
applications
[1318] Briefly, polystyrene, non tissue culture treated, 96-well
plates are coated with fn fragment at a coating concentration of
0.2 .mu.g/cm.sup.2. Mouse bone marrow cells are plated (1,000
cells/well) in 0.2 ml of serum-free medium. Cells cultured in the
presence of IL-3 (5 ng/ml)+SCF (50 ng/ml) would serve as the
positive control, conditions under which little self-renewal but
pronounced differentiation of the stem cells is to be expected.
Albumin fusion proteins of the invention are tested with
appropriate negative controls in the presence and absence of SCF
(5.0 ng/ml), where volume of the administered composition
containing the albumin fusion protein of the invention represents
10% of the total assay volume. The plated cells are then allowed to
grow by incubating in a low oxygen environment (5% CO.sub.2, 7%
O.sub.2, and 88% N.sub.2) tissue culture incubator for 7 days. The
number of proliferating cells within the wells is then quantitated
by measuring thymidine incorporation into cellular DNA.
Verification of the positive hits in the assay will require
phenotypic characterization of the cells, which can be accomplished
by scaling up of the culture system and using appropriate antibody
reagents against cell surface antigens and FACScan.
[1319] If a particular fusion protein of the present invention is
found to be a stimulator of hematopoietic progenitors, the fusion
protein and polynucleotides corresponding to the fusion protein may
be useful for example, in the diagnosis and treatment of disorders
affecting the immune system and hematopoiesis. Representative uses
are described in the "Immune Activity" and "Infectious Disease"
sections above, and elsewhere herein. The fusion protein may also
be useful in the expansion of stem cells and committed progenitors
of various blood lineages, and in the differentiation and/or
proliferation of various cell types.
[1320] Additionally, the albumin fusion proteins of the invention
and polynucleotides encoding albumin fusion proteins of the
invention, may also be employed to inhibit the proliferation and
differentiation of hematopoietic cells and therefore may be
employed to protect bone marrow stem cells from chemotherapeutic
agents during chemotherapy. This antiproliferative effect may allow
administration of higher doses of chemotherapeutic agents and,
therefore, more effective chemotherapeutic treatment.
[1321] Moreover, fusion proteins of the invention and
polynucleotides encoding albumin fusion proteins of the invention
may also be useful for the treatment and diagnosis of hematopoietic
related disorders such as, anemia, pancytopenia, leukopenia,
thrombocytopenia or leukemia, since stromal cells are important in
the production of cells of hematopoietic lineages. The uses include
bone marrow cell ex-vivo culture, bone marrow transplantation, bone
marrow reconstitution, radiotherapy or chemotherapy of
neoplasia.
Example 37
Human Dermal Fibroblast and Aortic Smooth Muscle Cell
Proliferation
[1322] An albumin fusion protein of the invention is added to
cultures of normal human dermal fibroblasts (NHDF) and human aortic
smooth muscle cells (AoSMC) and two co-assays are performed with
each sample. The first assay examines the effect of the fusion
protein on the proliferation of normal human dermal fibroblasts
(NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of
fibroblasts or smooth muscle cells is a part of several
pathological processes, including fibrosis, and restenosis. The
second assay examines IL6 production by both NHDF and SMC. IL6
production is an indication of functional activation. Activated
cells will have increased production of a number of cytokines and
other factors, which can result in a proinflammatory or
immunomodulatory outcome. Assays are run with and without co-TNFa
stimulation, in order to check for costimulatory or inhibitory
activity.
[1323] Briefly, on day 1, 96-well black plates are set up with 1000
cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 .mu.l culture
media. NHDF culture media contains: Clonetics FB basal media, 1
mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while
AoSMC culture media contains Clonetics SM basal media, 0.5 .mu.g/ml
hEGF, 5 mg/ml insulin, 1 .mu.g/ml hFGF, 50 mg/ml gentamycin, 50
.mu.g/ml Amphotericin B, 5% FBS. After incubation at 37.degree. C.
for at least 4-5 hours culture media is aspirated and replaced with
growth arrest media. Growth arrest media for NHDF contains
fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth
arrest media for AoSMC contains SM basal media, 50 mg/ml
gentamycin, 50 .mu.g/ml Amphotericin B, 0.4% FBS. Incubate at
37.degree. C. until day 2.
[1324] On day 2, serial dilutions and templates of an albumin
fusion protein of the invention are designed such that they always
include media controls and known-protein controls. For both
stimulation and inhibition experiments, proteins are diluted in
growth arrest media. For inhibition experiments, TNFa is added to a
final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Add 1/3
vol media containing controls or an albumin fusion protein of the
invention and incubate at 37 degrees C./5% CO.sub.2 until day
5.
[1325] Transfer 60 .mu.l from each well to another labeled 96-well
plate, cover with a plate-sealer, and store at 4 degrees C. until
Day 6 (for IL6 ELISA). To the remaining 100 .mu.l in the cell
culture plate, aseptically add Alamar Blue in an amount equal to
10% of the culture volume (10 .mu.l). Return plates to incubator
for 3 to 4 hours. Then measure fluorescence with excitation at 530
nm and emission at 590 nm using the CytoFluor. This yields the
growth stimulation/inhibition data.
[1326] On day 5, the IL6 ELISA is performed by coating a 96 well
plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody
diluted in PBS, pH 7.4, incubate ON at room temperature.
[1327] On day 6, empty the plates into the sink and blot on paper
towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the
plates with 200 .mu.l/well of Pierce Super Block blocking buffer in
PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05%
Tween-20). Blot plates on paper towels. Then add 50 .mu.l/well of
diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50
mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0
ng/ml). Add duplicate samples to top row of plate. Cover the plates
and incubate for 2 hours at RT on shaker.
[1328] Plates are washed with wash buffer and blotted on paper
towels. Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and
add 100 .mu.l/well. Cover the plate and incubate 1 h at RT. Plates
are again washed with wash buffer and blotted on paper towels.
[1329] Add 100 .mu.l/well of Enhancement Solution. Shake for 5
minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings
from triplicate samples in each assay were tabulated and
averaged.
[1330] A positive result in this assay suggests AoSMC cell
proliferation and that the albumin fusion protein may be involved
in dermal fibroblast proliferation and/or smooth muscle cell
proliferation. A positive result also suggests many potential uses
of the fusion protein and polynucleotides encoding the albumin
fusion protein. For example, inflammation and immune responses,
wound healing, and angiogenesis, as detailed throughout this
specification. Particularly, fusion proteins may be used in wound
healing and dermal regeneration, as well as the promotion of
vasculogenesis, both of the blood vessels and lymphatics. The
growth of vessels can be used in the treatment of, for example,
cardiovascular diseases. Additionally, fusion proteins showing
antagonistic activity in this assay may be useful in treating
diseases, disorders, and/or conditions which involve angiogenesis
by acting as an anti-vascular agent (e.g., anti-angiogenesis).
These diseases, disorders, and/or conditions are known in the art
and/or are described herein, such as, for example, malignancies,
solid tumors, benign tumors, for example hemangiomas, acoustic
neuromas, neurofibromas, trachomas, and pyogenic granulomas;
artheroscleric plaques; ocular angiogenic diseases, for example,
diabetic retinopathy, retinopathy of prematurity, macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and
Pterygia (abnormal blood vessel growth) of the eye; rheumatoid
arthritis; psoriasis; delayed wound healing; endometriosis;
vasculogenesis; granulations; hypertrophic scars (keloids);
nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial angiogenesis; coronary collaterals; cerebral
collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular
dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
Moreover, albumin fusion proteins that act as antagonists in this
assay may be useful in treating anti-hyperproliferative diseases
and/or anti-inflammatory known in the art and/or described
herein.
Example 38
Cellular Adhesion Molecule (CAM) Expression on Endothelial
Cells
[1331] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[1332] Briefly, endothelial cells (e.g., Human Umbilical Vein
Endothelial cells (HUVECs)) are grown in a standard 96 well plate
to confluence, growth medium is removed from the cells and replaced
with 100 .mu.l of 199 Medium (10% fetal bovine serum (FBS)).
Samples for testing (containing an albumin fusion protein of the
invention) and positive or negative controls are added to the plate
in triplicate (in 10 .mu.l volumes). Plates are then incubated at
37.degree. C. for either 5 h (selectin and integrin expression) or
24 h (integrin expression only). Plates are aspirated to remove
medium and 100 .mu.l of 0.1% paraformaldehyde-PBS (with Ca++ and
Mg++) is added to each well. Plates are held at 4.degree. C. for 30
min. Fixative is removed from the wells and wells are washed
1.times. with PBS(+Ca,Mg)+0.5% BSA and drained. 10 .mu.l of diluted
primary antibody is added to the test and control wells.
Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin
are used at a concentration of 10 .mu.g/ml (1:10 dilution of 0.1
mg/ml stock antibody). Cells are incubated at 37.degree. C. for 30
min a humidified environment. Wells are washed three times with
PBS(+Ca,Mg)+0.5% BSA. 20 .mu.l of diluted ExtrAvidin-Alkaline
Phosphatase (1:5,000 dilution, referred to herein as the working
dilution) are added to each well and incubated at 37.degree. C. for
30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA.
Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of
glycine buffer (pH 10.4). 100 .mu.l of pNPP substrate in glycine
buffer is added to each test well. Standard wells in triplicate are
prepared from the working dilution of the ExtrAvidin-Alkaline
Phosphotase in glycine buffer: 1:5,000
(10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-1.50.5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent is then added to each of the standard wells.
The plate is incubated at 37.degree. C. for 4 h. A volume of 50
.mu.l of 3M NaOH is added to all wells. The plate is read on a
plate reader at 405 nm using the background subtraction option on
blank wells filled with glycine buffer only. Additionally, the
template is set up to indicate the concentration of AP-conjugate in
each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results
are indicated as amount of bound AP-conjugate in each sample.
Example 39
Alamar Blue Endothelial Cells Proliferation Assay
[1333] This assay may be used to quantitatively determine protein
mediated inhibition of bFGF-induced proliferation of Bovine
Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells
(BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs).
This assay incorporates a fluorometric growth indicator based on
detection of metabolic activity. A standard Alamar Blue
Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF
added as a source of endothelial cell stimulation. This assay may
be used with a variety of endothelial cells with slight changes in
growth medium and cell concentration. Dilutions of protein batches
to be tested are diluted as appropriate. Serum-free medium (GIBCO
SFM) without bFGF is used as a non-stimulated control and
Angiostatin or TSP-1 are included as a known inhibitory
controls.
[1334] Briefly, LEC, BAECs or UTMECs are seeded in growth media at
a density of 5000 to 2000 cells/well in a 96 well plate and placed
at 37 degrees C. overnight. After the overnight incubation of the
cells, the growth media is removed and replaced with GIBCO EC-SFM.
The cells are treated with the appropriate dilutions of an albumin
fusion protein of the invention or control protein sample(s)
(prepared in SFM) in triplicate wells with additional bFGF to a
concentration of 10 ng/ml. Once the cells have been treated with
the samples, the plate(s) is/are placed back in the 37.degree. C.
incubator for three days. After three days 10 ml of stock alamar
blue (Biosource Cat# DAL1100) is added to each well and the
plate(s) is/are placed back in the 37.degree. C. incubator for four
hours. The plate(s) are then read at 530 nm excitation and 590 nm
emission using the CytoFluor fluorescence reader. Direct output is
recorded in relative fluorescence units.
[1335] Alamar blue is an oxidation-reduction indicator that both
fluoresces and changes color in response to chemical reduction of
growth medium resulting from cell growth. As cells grow in culture,
innate metabolic activity results in a chemical reduction of the
immediate surrounding environment. Reduction related to growth
causes the indicator to change from oxidized (non-fluorescent blue)
form to reduced (fluorescent red) form (i.e., stimulated
proliferation will produce a stronger signal and inhibited
proliferation will produce a weaker signal and the total signal is
proportional to the total number of cells as well as their
metabolic activity). The background level of activity is observed
with the starvation medium alone. This is compared to the output
observed from the positive control samples (bFGF in growth medium)
and protein dilutions.
Example 40
Detection of Inhibition of a Mixed Lymphocyte Reaction
[1336] This assay can be used to detect and evaluate inhibition of
a Mixed Lymphocyte Reaction (MLR) by fusion proteins of the
invention. Inhibition of a MLR may be due to a direct effect on
cell proliferation and viability, modulation of costimulatory
molecules on interacting cells, modulation of adhesiveness between
lymphocytes and accessory cells, or modulation of cytokine
production by accessory cells. Multiple cells may be targeted by
the albumin fusion proteins that inhibit MLR since the peripheral
blood mononuclear fraction used in this assay includes T, B and
natural killer lymphocytes, as well as monocytes and dendritic
cells.
[1337] Albumin fusion proteins of the invention found to inhibit
the MLR may find application in diseases associated with lymphocyte
and monocyte activation or proliferation. These include, but are
not limited to, diseases such as asthma, arthritis, diabetes,
inflammatory skin conditions, psoriasis, eczema, systemic lupus
erythematosus, multiple sclerosis, glomerulonephritis, inflammatory
bowel disease, crohn's disease, ulcerative colitis,
arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft
disease, hepatitis, leukemia and lymphoma.
[1338] Briefly, PBMCs from human donors are purified by density
gradient centrifugation using Lymphocyte Separation Medium
(LSM.RTM., density 1.0770 g/ml, Organon Teknika Corporation, West
Chester, Pa.). PBMCs from two donors are adjusted to
2.times.10.sup.6 cells/ml in RPMI-1640 (Life Technologies, Grand
Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs
from a third donor is adjusted to 2.times.10.sup.5 cells/ml. Fifty
microliters of PBMCs from each donor is added to wells of a 96-well
round bottom microtiter plate. Dilutions of the fusion protein test
material (50 .mu.l) is added in triplicate to microtiter wells.
Test samples (of the protein of interest) are added for final
dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn.,
catalog number 202-IL) is added to a final concentration of 1
.mu.g/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog
number MAB379) is added to a final concentration of 10 .mu.g/ml.
Cells are cultured for 7-8 days at 37.degree. C. in 5% CO.sub.2,
and 1 .mu.C of [.sup.3H] thymidine is added to wells for the last
16 hrs of culture. Cells are harvested and thymidine incorporation
determined using a Packard TopCount. Data is expressed as the mean
and standard deviation of triplicate determinations.
[1339] Samples of the fusion protein of interest are screened in
separate experiments and compared to the negative control
treatment, anti-CD4 mAb, which inhibits proliferation of
lymphocytes and the positive control treatment, IL-2 (either as
recombinant material or supernatant), which enhances proliferation
of lymphocytes.
Example 41
Assays for Protease Activity
[1340] The following assay may be used to assess protease activity
of an albumin fusion protein of the invention.
[1341] Gelatin and casein zymography are performed essentially as
described (Heusen et al., Anal. Biochem., 102:196-202 (1980);
Wilson et al., Journal of Urology, 149:653-658 (1993)). Samples are
run on 10% polyacryamide/0.1% SDS gels containing 1% gelain
orcasein, soaked in 2.5% triton at room temperature for 1 hour, and
in 0.1M glycine, pH 8.3 at 37.degree. C. 5 to 16 hours. After
staining in amido black areas of proteolysis appear as clear areas
agains the blue-black background. Trypsin (Sigma T8642) is used as
a positive control.
[1342] Protease activity is also determined by monitoring the
cleavage of n-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma
B-4500. Reactions are set up in (25 mMNaPO.sub.4, 1 mM EDTA, and 1
mM BAEE), pH 7.5. Samples are added and the change in absorbance at
260 nm is monitored on the Beckman DU-6 spectrophotometer in the
time-drive mode. Trypsin is used as a positive control.
[1343] Additional assays based upon the release of acid-soluble
peptides from casein or hemoglobin measured as absorbance at 280 nm
or colorimetrically using the Folin method are performed as
described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5
(1984). Other assays involve the solubilization of chromogenic
substrates (Ward, Applied Science, 251-317 (1983)).
Example 42
Identifying Serine Protease Substrate Specificity
[1344] Methods known in the art or described herein may be used to
determine the substrate specificity of the albumin fusion proteins
of the present invention having serine protease activity. A
preferred method of determining substrate specificity is by the use
of positional scanning synthetic combinatorial libraries as
described in GB 2 324 529 (incorporated herein in its
entirety).
Example 43
Ligand Binding Assays
[1345] The following assay may be used to assess ligand binding
activity of an albumin fusion protein of the invention.
[1346] Ligand binding assays provide a direct method for
ascertaining receptor pharmacology and are adaptable to a high
throughput format. The purified ligand for an albumin fusion
protein of the invention is radiolabeled to high specific activity
(50-2000 Ci/mmol) for binding studies. A determination is then made
that the process of radiolabeling does not diminish the activity of
the ligand towards the fusion protein. Assay conditions for
buffers, ions, pH and other modulators such as nucleotides are
optimized to establish a workable signal to noise ratio for both
membrane and whole cell polypeptide sources. For these assays,
specific polypeptide binding is defined as total associated
radioactivity minus the radioactivity measured in the presence of
an excess of unlabeled competing ligand. Where possible, more than
one competing ligand is used to define residual nonspecific
binding.
Example 44
Functional Assay in Xenopus Oocytes
[1347] Capped RNA transcripts from linearized plasmid templates
encoding an albumin fusion protein of the invention is synthesized
in vitro with RNA polymerases in accordance with standard
procedures. In vitro transcripts are suspended in water at a final
concentration of 0.2 mg/ml. Ovarian lobes are removed from adult
female toads, Stage V defolliculated oocytes are obtained, and RNA
transcripts (10 ng/oocytc) are injected in a 50 nl bolus using a
microinjection apparatus. Two electrode voltage clamps are used to
measure the currents from individual Xenopus oocytes in response
fusion protein and polypeptide agonist exposure. Recordings are
made in Ca2+ free Barth's medium at room temperature. The Xenopus
system can be used to screen known ligands and tissue/cell extracts
for activating ligands.
Example 45
Microphysiometric Assays
[1348] Activation of a wide variety of secondary messenger systems
results in extrusion of small amounts of acid from a cell. The acid
formed is largely as a result of the increased metabolic activity
required to fuel the intracellular signaling process. The pH
changes in the media surrounding the cell are very small but are
detectable by the CYTOSENSOR microphysiometer (Molecular Devices
Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of
detecting the ability of an albumin fusion protein of the invention
to activate secondary messengers that are coupled to an energy
utilizing intracellular signaling pathway.
Example 46
Extract/Cell Supernatant Screening
[1349] A large number of mammalian receptors exist for which there
remains, as yet, no cognate activating ligand (agonist). Thus,
active ligands for these receptors may not be included within the
ligands banks as identified to date. Accordingly, the albumin
fusion proteins of the invention can also be functionally screened
(using calcium, cAMP, microphysiometer, oocyte electrophysiology,
etc., functional screens) against tissue extracts to identify
natural ligands for the Therapeutic protein portion and/or albumin
protein portion of an albumin fusion protein of the invention.
Extracts that produce positive functional responses can be
sequentially subfractionated until an activating ligand is isolated
and identified.
Example 47
ATP-Binding Assay
[1350] The following assay may be used to assess ATP-binding
activity of fusion proteins of the invention.
[1351] ATP-binding activity of an albumin fusion protein of the
invention may be detected using the ATP-binding assay described in
U.S. Pat. No. 5,858,719, which is herein incorporated by reference
in its entirety. Briefly, ATP-binding to an albumin fusion protein
of the invention is measured via photoaffinity labeling with
8-azido-ATP in a competition assay. Reaction mixtures containing 1
mg/ml of ABC transport protein are incubated with varying
concentrations of ATP, or the non-hydrolyzable ATP analog
adenyl-5'-imidodiphosphate for 10 minutes at 4.degree. C. A mixture
of 8-azido-ATP (Sigma Chem. Corp., St. Louis, Mo.) plus 8-azido-ATP
(.sup.32P-ATP) (5 mCi/.mu.mol, ICN, Irvine Calif.) is added to a
final concentration of 100 .mu.M and 0.5 ml aliquots are placed in
the wells of a porcelain spot plate on ice. The plate is irradiated
using a short wave 254 nm UV lamp at a distance of 2.5 cm from the
plate for two one-minute intervals with a one-minute cooling
interval in between. The reaction is stopped by addition of
dithiothreitol to a final concentration of 2 mM. The incubations
are subjected to SDS-PAGE electrophoresis, dried, and
autoradiographed. Protein bands corresponding to the albumin fusion
proteins of the invention are excised, and the radioactivity
quantified. A decrease in radioactivity with increasing ATP or
adenly-5'-imidodiphosphate provides a measure of ATP affinity to
the fusion protein.
Example 48
Identification of Signal Transduction Proteins that Interact with
an Albumin Fusion Protein of the Present Invention
[1352] Albumin fusion proteins of the invention may serve as
research tools for the identification, characterization and
purification of signal transduction pathway proteins or receptor
proteins. Briefly, a labeled fusion protein of the invention is
useful as a reagent for the purification of molecules with which it
interacts. In one embodiment of affinity purification, an albumin
fusion protein of the invention is covalently coupled to a
chromatography column. Cell-free extract derived from putative
target cells, such as carcinoma tissues, is passed over the column,
and molecules with appropriate affinity bind to the albumin fusion
protein. The protein complex is recovered from the column,
dissociated, and the recovered molecule subjected to N-terminal
protein sequencing. This amino acid sequence is then used to
identify the captured molecule or to design degenerate
oligonucleotide probes for cloning the relevant gene from an
appropriate cDNA library.
Example 49
IL-6 Bioassay
[1353] A variety of assays are known in the art for testing the
proliferative effects of an albumin fusion protein of the
invention. For example, one such assay is the IL-6 Bioassay as
described by Marz et al. (Proc. Natl. Acad. Sci., U.S.A.,
95:3251-56 (1998), which is herein incorporated by reference).
After 68 hrs. at 37.degree. C., the number of viable cells is
measured by adding the tetrazolium salt thiazolyl blue (MTT) and
incubating for a further 4 hrs. at 37.degree. C. B9 cells are lysed
by SDS and optical density is measured at 570 nm. Controls
containing IL-6 (positive) and no cytokine (negative) are Briefly,
IL-6 dependent B9 murine cells are washed three times in IL-6 free
medium and plated at a concentration of 5,000 cells per well in 50
.mu.l, and 50 .mu.l of fusion protein of the invention is added.
utilized. Enhanced proliferation in the test sample(s) (containing
an albumin fusion protein of the invention) relative to the
negative control is indicative of proliferative effects mediated by
the fusion protein.
Example 50
Support of Chicken Embryo Neuron Survival
[1354] To test whether sympathetic neuronal cell viability is
supported by an albumin fusion protein of the invention, the
chicken embryo neuronal survival assay of Senaldi et al may be
utilized (Proc. Natl. Acad. Sci., U.S.A., 96:11458-63 (1998), which
is herein incorporated by reference). Briefly, motor and
sympathetic neurons are isolated from chicken embryos, resuspended
in L15 medium (with 10% FCS, glucose, sodium selenite,
progesterone, conalbumin, putrescine, and insulin; Life
Technologies, Rockville, Md.) and Dulbecco's modified Eagles medium
[with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH
7.2); Life Technologies, Rockville, Md.], respectively, and
incubated at 37.degree. C. in 5% CO.sub.2 in the presence of
different concentrations of the purified fusion protein of the
invention, as well as a negative control lacking any cytokine.
After 3 days, neuron survival is determined by evaluation of
cellular morphology, and through the use of the colorimetric assay
of Mosmann (Mosmann, T., J. Immunol. Methods, 65:55-63 (1983)).
Enhanced neuronal cell viability as compared to the controls
lacking cytokine is indicative of the ability of the albumin fusion
protein to enhance the survival of neuronal cells.
Example 51
Assay for Phosphatase Activity
[1355] The following assay may be used to assess serine/threonine
phosphatase (PTPase) activity of an albumin fusion protein of the
invention.
[1356] In order to assay for serine/threonine phosphatase (PTPase)
activity, assays can be utilized which are widely known to those
skilled in the art. For example, the serine/threonine phosphatase
(PSPase) activity of an albumin fusion protein of the invention may
be measured using a PSPase assay kit from New England Biolabs, Inc.
Myelin basic protein (MyBP), a substrate for PSPase, is
phosphorylated on serine and threonine residues with cAMP-dependent
Protein Kinase in the presence of [.sup.32P]ATP. Protein
serine/threonine phosphatase activity is then determined by
measuring the release of inorganic phosphate from .sup.32P-labeled
MyBP.
Example 52
Interaction of Serine/Threonine Phosphatases with Other
Proteins
[1357] Fusion proteins of the invention having serine/threonine
phosphatase activity (e.g., as determined in Example 51) are
useful, for example, as research tools for the identification,
characterization and purification of additional interacting
proteins or receptor proteins, or other signal transduction pathway
proteins. Briefly, a labeled fusion protein of the invention is
useful as a reagent for the purification of molecules with which it
interacts. In one embodiment of affinity purification, an albumin
fusion protein of the invention is covalently coupled to a
chromatography column. Cell-free extract derived from putative
target cells, such as neural or liver cells, is passed over the
column, and molecules with appropriate affinity bind to the fusion
protein. The fusion protein-complex is recovered from the column,
dissociated, and the recovered molecule subjected to N-terminal
protein sequencing. This amino acid sequence is then used to
identify the captured molecule or to design degenerate
oligonucleotide probes for cloning the relevant gene from an
appropriate cDNA library.
Example 53
Assaying for Heparanase Activity
[1358] There a numerous assays known in the art that may be
employed to assay for heparanase activity of an albumin fusion
protein of the invention. In one example, heparanase activity of an
albumin fusion protein of the invention, is assayed as described by
Vlodavsky et al., (Vlodavsky et al., Nat. Med., 5:793-802 (1999)).
Briefly, cell lysates, conditioned media, intact cells
(1.times.10.sup.6 cells per 35-mm dish), cell culture supernatant,
or purified fusion protein are incubated for 18 hrs at 37.degree.
C., pH 6.2-6.6, with .sup.35S-labeled ECM or soluble ECM derived
peak I proteoglycans. The incubation medium is centrifuged and the
supernatant is analyzed by gel filtration on a Sepharose CL-6B
column (0.9.times.30 cm). Fractions are eluted with PBS and their
radioactivity is measured. Degradation fragments of heparan sulfate
side chains are eluted from Sepharose 6B at 0.5<K.sub.av<0.8
(peak II). Each experiment is done at least three times.
Degradation fragments corresponding to "peak II," as described by
Vlodavsky et al., is indicative of the activity of an albumin
fusion protein of the invention in cleaving heparan sulfate.
Example 54
Immobilization of Biomolecules
[1359] This example provides a method for the stabilization of an
albumin fusion protein of the invention in non-host cell lipid
bilayer constucts (see, e.g., Bieri et al., Nature Biotech
17:1105-1108 (1999), hereby incorporated by reference in its
entirety herein) which can be adapted for the study of fusion
proteins of the invention in the various functional assays
described above. Briefly, carbohydrate-specific chemistry for
biotinylation is used to confine a biotin tag to an albumin fusion
protein of the invention, thus allowing uniform orientation upon
immobilization. A 50 uM solution of an albumin fusion protein of
the invention in washed membranes is incubated with 20 mM NaIO4 and
1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr
at room temperature (reaction volume, 150 ul). Then the sample is
dialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce
Chemical Co., Rockford Ill.) at 4 C first for 5 h, exchanging the
buffer after each hour, and finally for 12 h against 500 ml buffer
R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodium phosphate, pH7). Just
before addition into a cuvette, the sample is diluted 1:5 in buffer
ROG50 (Buffer R supplemented with 50 mM octylglucoside).
Example 55
Assays for Metalloproteinase Activity
[1360] Metalloproteinases are peptide hydrolases which use metal
ions, such as Zn.sup.2+, as the catalytic mechanism.
Metalloproteinase activity of an albumin fusion protein of the
present invention can be assayed according to methods known in the
art. The following exemplary methods are provided:
[1361] Proteolysis of Alpha-2-Macroglobulin
[1362] To confirm protease activity, a purified fusion protein of
the invention is mixed with the substrate alpha-2-macroglobulin
(0.2 unit/ml; Boehringer Mannheim, Germany) in 1.times. assay
buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl.sub.2, 25 .mu.M
ZnCl.sub.2 and 0.05% Brij-35) and incubated at 37.degree. C. for
1-5 days. Trypsin is used as positive control. Negative controls
contain only alpha-2-macroglobulin in assay buffer. The samples are
collected and boiled in SDS-PAGE sample buffer containing 5%
2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide
gel. After electrophoresis the proteins are visualized by silver
staining. Proteolysis is evident by the appearance of lower
molecular weight bands as compared to the negative control.
[1363] Inhibition of Alpha-2-Macroglobulin Proteolysis by
Inhibitors of Metalloproteinases
[1364] Known metalloproteinase inhibitors (metal chelators (EDTA,
EGTA, AND HgCl.sub.2), peptide metalloproteinase inhibitors (TIMP-1
and TIMP-2), and commercial small molecule MMP inhibitors) may also
be used to characterize the proteolytic activity of an albumin
fusion protein of the invention. Three synthetic MMP inhibitors
that may be used are: MMP inhibitor I, [IC.sub.50=1.0 .mu.M against
MMP-1 and MMP-8; IC.sub.50=30 .mu.M against MMP-9; IC.sub.50=150
.mu.M against MMP-3]; MMP-3 (stromelysin-1) inhibitor I
[IC.sub.50=5 .mu.M against MMP-3], and MMP-3 inhibitor II
[K.sub.i=130 nM against MMP-3]; inhibitors available through
Calbiochem, catalog #444250, 444218, and 444225, respectively).
Briefly, different concentrations of the small molecule MMP
inhibitors are mixed with a purified fusion protein of the
invention (50 .mu.g/ml) in 22.9 .mu.l of 1.times.HEPES buffer (50
mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl.sub.2, 25 .mu.M ZnCl.sub.2
and 0.05% Brij-35) and incubated at room temperature (24.degree.
C.) for 2-hr, then 7.1 .mu.l of substrate alpha-2-macroglobulin
(0.2 unit/ml) is added and incubated at 37.degree. C. for 20-hr.
The reactions are stopped by adding 4.times. sample buffer and
boiled immediately for 5 minutes. After SDS-PAGE, the protein bands
are visualized by silver stain.
[1365] Synthetic Fluorogenic Peptide Substrates Cleavage Assay
[1366] The substrate specificity for fusion proteins of the
invention with demonstrated metalloproteinase activity may be
determined using techniques known in the art, such as using
synthetic fluorogenic peptide substrates (purchased from BACHEM
Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105,
M-2110, and M-2255. The first four are MMP substrates and the last
one is a substrate of tumor necrosis factor-.alpha. (TNF-.alpha.)
converting enzyme (TACE). These substrastes are preferably prepared
in 1:1 dimethyl sulfoxide (DMSO) and water. The stock solutions are
50-500 .mu.M. Fluorescent assays are performed by using a Perkin
Elmer LS 50B luminescence spectrometer equipped with a constant
temperature water bath. The excitation .lamda. is 328 nm and the
emission .lamda. is 393 nm. Briefly, the assay is carried out by
incubating 176 .mu.l 1.times.HEPES buffer (0.2 M NaCl, 10 mM
CaCl.sub.2, 0.05% Brij-35 and 50 mM HEPES, pH 7.5) with 4 .mu.l of
substrate solution (50 .mu.M) at 25.degree. C. for 15 minutes, and
then adding 20 .mu.l of a purified fusion protein of the invention
into the assay cuvett. The final concentration of substrate is 1
.mu.M. Initial hydrolysis rates are monitored for 30-min.
Example 56
Occurrence of Diabetes in NOD Mice
[1367] Female NOD (non-obese diabetic) mice are characterized by
displaying IDDM with a course which is similar to that found in
humans, although the disease is more pronounced in female than male
NOD mice. Hereinafter, unless otherwise stated, the term "NOD
mouse" refers to a female NOD mouse. NOD mice have a progressive
destruction of beta cells which is caused by a chronic autoimmune
disease. Thus, NOD mice begin life with euglycemia, or normal blood
glucose levels. By about 15 to 16 weeks of age, however, NOD mice
start becoming hyperglycemic, indicating the destruction of the
majority of their pancreatic beta cells and the corresponding
inability of the pancreas to produce sufficient insulin. Thus, both
the cause and the progression of the disease are similar to human
IDDM patients.
[1368] In vivo assays of efficacy of the immunization regimens can
be assessed in female NOD/LtJ mice (commercially available from The
Jackson Laboratory, Bar Harbor, Me.). In the literature, it's
reported that 80% of female mice develop diabetes by 24 weeks of
age and onset of insulitis begins between 6-8 weeks age. NOD mice
are inbred and highly responsive to a variety of immunoregulatory
strategies. Adult NOD mice (6-8 weeks of age) have an average mass
of 20-25 g.
[1369] These mice can be either untreated (control), treated with
the therapeutics of the subject invention (e.g., albumin fusion
proteins of the invention and fragments and variants thereof),
alone or in combination with other therapeutic compounds stated
above. The effect of these various treatments on the progression of
diabetes can be measured as follows:
[1370] At 14 weeks of age, the female NOD mice can be phenotyped
according to glucose tolerance. Glucose tolerance can be measured
with the intraperitoneal glucose tolerance test (IPGTT). Briefly,
blood is drawn from the paraorbital plexus at 0 minutes and 60
minutes after the intraperitoneal injection of glucose (1 g/kg body
weight). Normal tolerance is defined as plasma glucose at 0 minutes
of less than 144 mg %, or at 60 minutes of less than 160 mg %.
Blood glucose levels are determined with a Glucometer Elite
apparatus.
[1371] Based upon this phenotypic analysis, animals can be
allocated to the different experimental groups. In particular,
animals with more elevated blood glucose levels can be assigned to
the impaired glucose tolerance group. The mice can be fed ad
libitum and can be supplied with acidified water (pH 2.3).
[1372] The glucose tolerant and intolerant mice can be further
subdivided into control, albumin fusion proteins of the subject
invention, and albumin fusion proteins/therapeutic compounds
combination groups. Mice in the control group can receive an
interperitoneal injection of vehicle daily, six times per week.
Mice in the albumin fusion group can receive an interperitoneal
injection of the therapeutics of the subject invention (e.g.,
albumin fusion proteins of the invention and fragments and variants
thereof) in vehicle daily, six times per week. Mice in the albumin
fusion proteins/therapeutic compounds combination group can receive
both albumin fusion proteins and combinations of therapeutic
compounds as described above.
[1373] The level of urine glucose in the NOD mice can be determined
on a bi-weekly basis using Labstix (Bayer Diagnostics, Hampshire,
England). Weight and fluid intake can also be determined on a
bi-weekly basis. The onset of diabetes is defined after the
appearance of glucosuria on two consecutive determinations. After
10 weeks of treatment, an additional IPGTT can be performed and
animals can be sacrificed the following day.
[1374] Over the 10 week course of treatment, control animals in
both the glucose tolerant and glucose intolerant groups develop
diabetes at a rate of 60% and 86%, respectively (see U.S. Pat. No.
5,866,546, Gross et al.). Thus, high rates of diabetes occur even
in NOD mice which are initially glucose tolerant if no intervention
is made.
[1375] Results can be confirmed by the measurement of blood glucose
levels in NOD mice, before and after treatment. Blood glucose
levels are measured as described above in both glucose tolerant and
intolerant mice in all groups described.
[1376] In an alternative embodiment, the therapeutics of the
subject invention (e.g., specific fusions disclosed as SEQ ID NO:Y
and fragments and variants thereof) can be quantified using
spectrometric analysis and appropriate protein quantities can be
resuspended prior to injection in 50 .mu.l phosphate buffered
saline (PBS) per dose. Two injections, one week apart, can be
administered subcutaneously under the dorsal skin of each mouse.
Monitoring can be performed on two separate occasions prior to
immunization and can be performed weekly throughout the treatment
and continued thereafter. Urine can be tested for glucose every
week (Keto-Diastix..RTM.; Miles Inc., Kankakee, Ill.) and
glycosuric mice can be checked for serum glucose (ExacTech..RTM.,
MediSense, Inc., Waltham, Mass.). Diabetes is diagnosed when
fasting glycemia is greater than 2.5 g/L.
Example 57
Histological Examination of NOD Mice
[1377] Histological examination of tissue samples from NOD mice can
demonstrate the ability of the compositions of the present
invention, and/or a combination of the compositions of the present
invention with other therapeutic agents for diabetes, to increase
the relative concentration of beta cells in the pancreas. The
experimental method is as follows:
[1378] The mice from Example 56 can be sacrificed at the end of the
treatment period and tissue samples can be taken from the pancreas.
The samples can be fixed in 10% formalin in 0.9% saline and
embedded in wax. Two sets of 5 serial 5 .mu.m sections can be cut
for immunolabelling at a cutting interval of 150 .mu.m. Sections
can be immunolabelled for insulin (guinea pig anti-insulin antisera
dilution 1:1000, ICN Thames U.K.) and glucagon (rabbit
anti-pancreatic glucagon antisera dilution 1:2000) and detected
with peroxidase conjugated anti-guinea pig (Dako, High Wycombe,
U.K.) or peroxidase conjugated anti-rabbit antisera (dilution 1:50,
Dako).
[1379] The composition of the present invention may or may not have
as strong an effect on the visible mass of beta cells as it does on
the clinical manifestations of diabetes in glucose tolerant and
glucose intolerant animals.
Example 58
In Vivo Mouse Model of NIDDM
[1380] Male C57BL/6J mice from Jackson Laboratory (Bar Harbor, Me.)
can be obtained at 3 weeks of age and fed on conventional chow or
diets enriched in either fat (35.5% wt/wt; Bioserv. Frenchtown,
N.J.) or fructose (60% wt/wt; Harlan Teklad, Madison, Wis.). The
regular chow is composed of 4.5% wt/wt fat, 23% wt/wt protein,
31.9% wt/wt starch, 3.7% wt/wt fructose, and 5.3% wt/wt fiber. The
high-fat (lard) diet is composed of 35.5% wt/wt fat, 20% wt/wt
protein, 36.4% wt/wt starch, 0.0% wt/wt fructose, and 0.1% wt/wt
fiber. The high-fructose diet is composed of 5% wt/wt fat, 20%
wt/wt protein, 0.0% wt/wt starch, 60% wt/wt fructose, and 9.4%
wt/wt fiber. The mice may be housed no more than five per cage at
22.degree.+/-3.degree. C. temperature- and 50%+/-20%
humidity-controlled room with a 12-hour light (6 am to 6 pm)/dark
cycle (Luo et al., 1998, Metabolism 47(6): 663-8, "Nongenetic mouse
models of non-insulin-dependent diabetes mellitus"; Larsen et al.,
Diabetes 50(11): 2530-9 (2001), "Systemic administration of the
long-acting GLP-1 derivative NN2211 induces lasting and reversible
weight loss in both normal and obese rats"). After exposure to the
respective diets for 3 weeks, mice can be injected
intraperitoneally with either streptozotocin, "STZ" (Sigma, St.
Louis, Mo.), at 100 mg/kg body weight or vehicle (0.05 mol/L citric
acid, pH 4.5) and kept on the same diet for the next 4 weeks. Under
nonfasting conditions, blood is obtained 1, 2, and 4 weeks post-STZ
by nipping the distal part of the tail. Samples are used to measure
nonfasting plasma glucose and insulin concentrations. Body weight
and food intake are recorded weekly.
[1381] To directly determine the effect of the high-fat diet on the
ability of insulin to stimulate glucose disposal, the experiments
can be initiated on three groups of mice, fat-fed, chow-fed
injected with vehicle, and fat-fed injected with STZ at the end of
the 7-week period described above. Mice can be fasted for 4 hours
before the experiments. In the first series of experiments, mice
can be anesthetized with methoxyflurane (Pitman-Moor, Mundelein,
Ill.) inhalation. Regular insulin (Sigma) can be injected
intravenously ([IV] 0.1 U/kg body weight) through a tail vein, and
blood can be collected 3, 6, 9, 12, and 15 minutes after the
injection from a different tail vein. Plasma glucose concentrations
can be determined on these samples, and the half-life (t1/2) of
glucose disappearance from plasma can be calculated using WinNonlin
(Scientific Consulting, Apex, N.C.), a
pharmacokinetics/pharmacodynamics software program.
[1382] In the second series of experiments, mice can be
anesthetized with intraperitoneal sodium pentobarbital (Sigma). The
abdominal cavity is opened, and the main abdominal vein is exposed
and catheterized with a 24-gauge IV catheter (Johnson-Johnson
Medical, Arlington, Tex.). The catheter is secured to muscle tissue
adjacent to the abdominal vein, cut on the bottom of the syringe
connection, and hooked to a prefilled PE50 plastic tube, which in
turn is connected to a syringe with infusion solution. The
abdominal cavity is then sutured closed. With this approach, there
would be no blockage of backflow of the blood from the lower part
of the body. Mice can be infused continuously with glucose (24.1
mg/kg/min) and insulin (10 mU/kg/min) at an infusion volume of 10
.mu.L/min. Retro-orbital blood samples (70 .mu.L each) can be taken
90, 105, 120, and 135 minutes after the start of infusion for
measurement of plasma glucose and insulin concentrations. The mean
of these four samples is used to estimate steady-state plasma
glucose (SSPG) and insulin (SSPI) concentrations for each
animal.
[1383] Finally, experiments to evaluate the ability of the albumin
fusion proteins, the therapeutic compositions of the instant
application, either alone or in combination with any one or more of
the therapeutic drugs listed for the treatment of diabetes
mellitus, to decrease plasma glucose can be performed in the
following two groups of "NIDDM" mice models that are STZ-injected:
(1) fat-fed C57BL/6J, and (2) fructose-fed C57BL/6J. Plasma glucose
concentrations of the mice for these studies may range from 255 to
555 mg/dL. Mice are randomly assigned to treatment with either
vehicle, albumin fusion therapeutics of the present invention
either alone or in combination with any one or more of the
therapeutic drugs listed for the treatment of diabetes mellitus. A
total of three doses can be administered. Tail vein blood samples
can be taken for measurement of the plasma glucose concentration
before the first dose and 3 hours after the final dose.
[1384] Plasma glucose concentrations can be determined using the
Glucose Diagnostic Kit from Sigma (Sigma No. 315), an enzyme
calorimetric assay. Plasma insulin levels can be determined using
the Rat Insulin RIA Kit from Linco Research (#RI-13K; St. Charles,
Mo.).
Example 59
In Vitro H4IIe-SEAP Reporter Assays Establishing Involvement in
Insulin Action
[1385] The Various H4IIe Reporters
[1386] H4IIe/rMEP-SEAP: The malic enzyme promoter isolated from rat
(rMEP) contains a PPAR-gamma element which is in the insulin
pathway. This reporter construct is stably transfected into the
liver H4IIe cell-line.
[1387] H4IIe/SREBP-SEAP: The sterol regulatory element binding
protein (SREBP-1c) is a transcription factor which acts on the
promoters of a number of insulin-responsive genes, for example,
fatty acid synthetase (FAS), and which regulates expression of key
genes in fatty acid metabolism in fibroblasts, adipocytes, and
hepatocytes. SREBP-1c, also known as the adipocyte determination
and differentiation factor 1 (ADD-1), is considered as the primary
mediator of insulin effects on gene expression in adipose cells.
It's activity is modulated by the levels of insulin, sterols, and
glucose. This reporter construct is stably transfected into the
liver H4IIe cell-line.
[1388] H4IIe/FAS-SEAP: The fatty acid synthetase reporter
constructs contain a minimal SREBP-responsive FAS promoter. This
reporter construct is stably transfected into the liver H4IIe
cell-line.
[1389] H4IIe/PEPCK-SEAP: The phosphoenolpyruvate carboxykinase
(PEPCK) promoter is the primary site of hormonal regulation of
PEPCK gene transcription modulating PEPCK activity. PEPCK catalyzes
a committed and rate-limiting step in hepatic gluconeogenesis and
must therefore be carefully controlled to maintain blood glucose
levels within normal limits. This reporter construct is stably
transfected into the liver H4IIe cell-line.
[1390] These reporter constructs can also be stably transfected
into 3T3-L1 fibroblasts and L6 myoblasts. These stable cell-lines
are then differentiated into 3T3-L1 adipocytes and L6 myotubes as
previously described in Example 13. The differentiated cell-lines
can then be used in the SEAP assay described below.
[1391] Growth and Assay Medium
[1392] The growth medium comprises 10% Fetal Bovine Serum (FBS),
10% Calf Serum, 1% NEAA, 1.times. penicillin/streptomycin, and 0.75
mg/mL G418 (for H4IIe/rFAS-SEAP and H4IIe/SREBP-SEAP) or 0.50 mg/mL
G418 (for H4IIe/rMEP-SEAP). For H4IIe/PEPCK-SEAP, the growth medium
consists of 10% FBS, 1% penicillin/streptomycin, 15 mM HEPES
buffered saline, and 0.50 mg/mL G418.
[1393] The assay medium consists of low glucose DMEM medium (Life
Technologies), 1% NEAA, 1.times. penicillin/streptomycin for the
H4IIe/rFAS-SEAP, H4IIe/SREBP-SEAP, H4IIe/rMEP-SEAP reporters. The
assay medium for H4IIe/PEPCK-SEAP reporter consists of 0.1% FBS, 1%
penicillin/streptomycin, and 15 mM HEPES buffered saline.
[1394] Method
[1395] The 96-well plates are seeded at 75,000 cells/well in 100
.mu.L/well of growth medium until cells in log growth phase become
adherent. Cells are starved for 48 hours by replacing growth medium
with assay medium, 200 .mu.L/well. (For H4IIe/PEPCK-SEAP cells,
assay medium containing 0.5 .mu.M dexamethasone is added at 100
.mu.L/well and incubated for approximately 20 hours). The assay
medium is replaced thereafter with 100 .mu.L/well of fresh assay
medium, and a 50 .mu.L aliquot of cell supernatant obtained from
transfected cell-lines expressing the therapeutics of the subject
invention (e.g., albumin fusion proteins of the invention and
fragments and variants thereof) is added to the well. Supernatants
from empty vector transfected cell-lines are used as negative
control. Addition of 10 nM and/or 100 nM insulin to the wells is
used as positive control. After 48 hours of incubation, the
conditioned media are harvested and SEAP activity measured
(Phospha-Light System protocol, Tropix #BP2500). Briefly, samples
are diluted 1:4 in dilution buffer and incubated at 65.degree. C.
for 30 minutes to inactivate the endogenous non-placental form of
SEAP. An aliquot of 50 .mu.L of the diluted samples is mixed with
50 .mu.L of SEAP Assay Buffer which contains a mixture of
inhibitors active against the non-placental SEAP isoenzymes and is
incubated for another 5 minutes. An aliquot of 50 .mu.L of CSPD
chemiluminescent substrate which is diluted 1:20 in Emerald
luminescence enhancer is added to the mixture and incubated for
15-20 minutes. Plates are read in a Dynex plate luminometer.
Example 60
Transgenic Animals
[1396] The albumin fusion proteins of the invention can also be
expressed in transgenic animals. Animals of any species, including,
but not limited to, mice, rats, rabbits, hamsters, guinea pigs,
pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express fusion
proteins of the invention in humans, as part of a gene therapy
protocol.
[1397] Any technique known in the art may be used to introduce the
polynucleotides encoding the albumin fusion proteins of the
invention into animals to produce the founder lines of transgenic
animals. Such techniques include, but are not limited to,
pronuclear microinjection (Paterson et al., Appl. Microbiol.
Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY)
11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834
(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989));
retrovirus mediated gene transfer into germ lines (Van der Putten
et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),
blastocysts or embryos; gene targeting in embryonic stem cells
(Thompson et al., Cell 56:313-321 (1989)); electroporation of cells
or embryos (Lo, 1983, Mol. Cell. Biol. 3:1803-1814 (1983));
introduction of the polynucleotides of the invention using a gene
gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing
nucleic acid constructs into embryonic pleuripotent stem cells and
transferring the stem cells back into the blastocyst; and
sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723
(1989); etc. For a review of such techniques, see Gordon,
"Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989), which
is incorporated by reference herein in its entirety.
[1398] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides encoding albumin
fusion proteins of the invention, for example, nuclear transfer
into enucleated oocytes of nuclei from cultured embryonic, fetal,
or adult cells induced to quiescence (Campell et al., Nature
380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
[1399] The present invention provides for transgenic animals that
carry the polynucleotides encoding the albumin fusion proteins of
the invention in all their cells, as well as animals which carry
these polynucleotides in some, but not all their cells, i.e.,
mosaic animals or chimeric. The transgene may be integrated as a
single transgene or as multiple copies such as in concatamers,
e.g., head-to-head tandems or head-to-tail tandems. The transgene
may also be selectively introduced into and activated in a
particular cell type by following, for example, the teaching of
Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236
(1992)). The regulatory sequences required for such a cell-type
specific activation will depend upon the particular cell type of
interest, and will be apparent to those of skill in the art. When
it is desired that the polynucleotide encoding the fusion protein
of the invention be integrated into the chromosomal site of the
endogenous gene corresponding to the Therapeutic protein portion or
albumin portion of the fusion protein of the invention, gene
targeting is preferred. Briefly, when such a technique is to be
utilized, vectors containing some nucleotide sequences homologous
to the endogenous gene are designed for the purpose of integrating,
via homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene may also be selectively introduced
into a particular cell type, thus inactivating the endogenous gene
in only that cell type, by following, for example, the teaching of
Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory
sequences required for such a cell-type specific inactivation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[1400] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the polynucleotide encoding the fusion protein of
the invention has taken place. The level of mRNA expression of the
polynucleotide encoding the fusion protein of the invention in the
tissues of the transgenic animals may also be assessed using
techniques which include, but are not limited to, Northern blot
analysis of tissue samples obtained from the animal, in situ
hybridization analysis, and reverse transcriptase-PCR (rt-PCR).
Samples of fusion protein-expressing tissue may also be evaluated
immunocytochemically or immunohistochemically using antibodies
specific for the fusion protein.
[1401] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene (i.e.,
polynucleotide encoding an albumin fusion protein of the invention)
on a distinct background that is appropriate for an experimental
model of interest.
Transgenic animals of the invention have uses which include, but
are not limited to, animal model systems useful in elaborating the
biological function of fusion proteins of the invention and the
Therapeutic protein and/or albumin component of the fusion protein
of the invention, studying conditions and/or disorders associated
with aberrant expression, and in screening for compounds effective
in ameliorating such conditions and/or disorders.
Example 61
Method of Treatment Using Gene Therapy-Ex Vivo
[1402] One method of gene therapy transplants fibroblasts, which
are capable of expressing an albumin fusion protein of the present
invention, onto a patient. Generally, fibroblasts are obtained from
a subject by skin biopsy. The resulting tissue is placed in
tissue-culture medium and separated into small pieces. Small chunks
of the tissue are placed on a wet surface of a tissue culture
flask, approximately ten pieces are placed in each flask. The flask
is turned upside down, closed tight and left at room temperature
over night. After 24 hours at room temperature, the flask is
inverted and the chunks of tissue remain fixed to the bottom of the
flask and fresh media (e.g., Ham's F12 media, with 10% FBS,
penicillin and streptomycin) is added. The flasks are then
incubated at 37 degree C. for approximately one week.
[1403] At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and
scaled into larger flasks.
[1404] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[1405] Polynucleotides encoding an albumin fusion protein of the
invention can be generated using techniques known in the art
amplified using PCR primers which correspond to the 5' and 3' end
sequences and optionally having appropriate restriction sites and
initiation/stop codons, if necessary. Preferably, the 5' primer
contains an EcoRI site and the 3' primer includes a HindIII site.
Equal quantities of the Moloney murine sarcoma virus linear
backbone and the amplified EcoRI and HindIII fragment are added
together, in the presence of T4 DNA ligase. The resulting mixture
is maintained under conditions appropriate for ligation of the two
fragments. The ligation mixture is then used to transform bacteria
HB101, which are then plated onto agar containing kanamycin for the
purpose of confirming that the vector has the gene of interest
properly inserted.
[1406] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells transduced with the vector. The
packaging cells now produce infectious viral particles containing
the gene (the packaging cells are now referred to as producer
cells).
[1407] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether the albumin fusion protein is
produced.
[1408] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads.
Example 62
Method of Treatment Using Gene Therapy--In Vivo
[1409] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences
encoding an albumin fusion protein of the invention into an animal.
Polynucleotides encoding albumin fusion proteins of the present
invention may be operatively linked to (i.e., associated with) a
promoter or any other genetic elements necessary for the expression
of the polypeptide by the target tissue. Such gene therapy and
delivery techniques and methods are known in the art, see, for
example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622,
5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479
(1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff,
Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene
Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation
94(12):3281-3290 (1996) (incorporated herein by reference).
[1410] The polynucleotide constructs may be delivered by any method
that delivers injectable materials to the cells of an animal, such
as, injection into the interstitial space of tissues (heart,
muscle, skin, lung, liver, intestine and the like). The
polynucleotide constructs can be delivered in a pharmaceutically
acceptable liquid or aqueous carrier.
[1411] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, lipofectin or
precipitating agents and the like. However, polynucleotides
encoding albumin fusion proteins of the present invention may also
be delivered in liposome formulations (such as those taught in
Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and
Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be
prepared by methods well known to those skilled in the art.
[1412] The polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Any strong promoter known to those skilled in the art
can be used for driving the expression of DNA. Unlike other gene
therapy techniques, one major advantage of introducing naked
nucleic acid sequences into target cells is the transitory nature
of the polynucleotide synthesis in the cells. Studies have shown
that non-replicating DNA sequences can be introduced into cells to
provide production of the desired polypeptide for periods of up to
six months.
[1413] The polynucleotide construct can be delivered to the
interstitial space of tissues within an animal, including muscle,
skin, brain, lung, liver, spleen, bone marrow, thymus, heart,
lymph, blood, bone, cartilage, pancreas, kidney, gall bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system,
eye, gland, and connective tissue. Interstitial space of the
tissues comprises the intercellular fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[1414] For the naked polynucleotide injection, an effective dosage
amount of DNA or RNA will be in the range of from about 0.05 g/kg
body weight to about 50 mg/kg body weight. Preferably the dosage
will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration. The
preferred route of administration is by the parenteral route of
injection into the interstitial space of tissues. However, other
parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
polynucleotide constructs can be delivered to arteries during
angioplasty by the catheter used in the procedure.
[1415] The dose response effects of injected polynucleotide in
muscle in vivo is determined as follows. Suitable template DNA for
production of mRNA coding for polypeptide of the present invention
is prepared in accordance with a standard recombinant DNA
methodology. The template DNA, which may be either circular or
linear, is either used as naked DNA or complexed with liposomes.
The quadriceps muscles of mice are then injected with various
amounts of the template DNA.
[1416] Five to six week old female and male Balb/C mice are
anesthetized by intraperitoneal injection with 0.3 ml of 2.5%
Avertin. A 1.5 cm incision is made on the anterior thigh, and the
quadriceps muscle is directly visualized. The template DNA is
injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge
needle over one minute, approximately 0.5 cm from the distal
insertion site of the muscle into the knee and about 0.2 cm deep. A
suture is placed over the injection site for future localization,
and the skin is closed with stainless steel clips.
[1417] After an appropriate incubation time (e.g., 7 days) muscle
extracts are prepared by excising the entire quadriceps. Every
fifth 15 um cross-section of the individual quadriceps muscles is
histochemically stained for protein expression. A time course for
fusion protein expression may be done in a similar fashion except
that quadriceps from different mice are harvested at different
times. Persistence of DNA in muscle following injection may be
determined by Southern blot analysis after preparing total cellular
DNA and HIRT supernatants from injected and control mice. The
results of the above experimentation in mice can be used to
extrapolate proper dosages and other treatment parameters in humans
and other animals using naked DNA.
Example 63
Biological Effects of Fusion Proteins of the Invention
[1418] Astrocyte and Neuronal Assays.
[1419] Albumin fusion proteins of the invention can be tested for
activity in promoting the survival, neurite outgrowth, or
phenotypic differentiation of cortical neuronal cells and for
inducing the proliferation of glial fibrillary acidic protein
immunopositive cells, astrocytes. The selection of cortical cells
for the bioassay is based on the prevalent expression of FGF-1 and
FGF-2 in cortical structures and on the previously reported
enhancement of cortical neuronal survival resulting from FGF-2
treatment. A thymidine incorporation assay, for example, can be
used to elucidate an albumin fusion protein of the invention's
activity on these cells.
[1420] Moreover, previous reports describing the biological effects
of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro
have demonstrated increases in both neuron survival and neurite
outgrowth (Walicke et al., "Fibroblast growth factor promotes
survival of dissociated hippocampal neurons and enhances neurite
extension." Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay
herein incorporated by reference in its entirety). However, reports
from experiments done on PC-12 cells suggest that these two
responses are not necessarily synonymous and may depend on not only
which FGF is being tested but also on which receptor(s) are
expressed on the target cells. Using the primary cortical neuronal
culture paradigm, the ability of an albumin fusion protein of the
invention to induce neurite outgrowth can be compared to the
response achieved with FGF-2 using, for example, a thymidine
incorporation assay.
[1421] Fibroblast and Endothelial Cell Assays.
[1422] Human lung fibroblasts are obtained from Clonetics (San
Diego, Calif.) and maintained in growth media from Clonetics.
Dermal microvascular endothelial cells are obtained from Cell
Applications (San Diego, Calif.). For proliferation assays, the
human lung fibroblasts and dermal microvascular endothelial cells
can be cultured at 5,000 cells/well in a 96-well plate for one day
in growth medium. The cells are then incubated for one day in 0.1%
BSA basal medium. After replacing the medium with fresh 0.1% BSA
medium, the cells are incubated with the test fusion protein of the
invention proteins for 3 days. Alamar Blue (Alamar Biosciences,
Sacramento, Calif.) is added to each well to a final concentration
of 10%. The cells are incubated for 4 hr. Cell viability is
measured by reading in a CytoFluor fluorescence reader. For the
PGE.sub.2 assays, the human lung fibroblasts are cultured at 5,000
cells/well in a 96-well plate for one day. After a medium change to
0.1% BSA basal medium, the cells are incubated with FGF-2 or fusion
protein of the invention with or without IL-1.alpha. for 24 hours.
The supernatants are collected and assayed for PGE.sub.2 by EIA kit
(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung
fibroblasts are cultured at 5,000 cells/well in a 96-well plate for
one day. After a medium change to 0.1% BSA basal medium, the cells
are incubated with FGF-2 or with or without an albumin fusion
protein of the invention and/or IL-1.alpha. for 24 hours. The
supernatants are collected and assayed for IL-6 by ELISA kit
(Endogen, Cambridge, Mass.).
[1423] Human lung fibroblasts are cultured with FGF-2 or an albumin
fusion protein of the invention for 3 days in basal medium before
the addition of Alamar Blue to assess effects on growth of the
fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which
can be used to compare stimulation with the fusion protein of the
invention.
[1424] Cell Proliferation Based on [3H]Thymidine Incorporation
[1425] The following [3H]Thymidine incorporation assay can be used
to measure the effect of a Therapeutic proteins, e.g., growth
factor proteins, on the proliferation of cells such as fibroblast
cells, epithelial cells or immature muscle cells.
[1426] Sub-confluent cultures are arrested in G1 phase by an 18 h
incubation in serum-free medium. Therapeutic proteins are then
added for 24 h and during the last 4 h, the cultures are labeled
with [3H]thymidine, at a final concentration of 0.33 .mu.M (25
Ci/mmol, Amersham, Arlington Heights, Ill.). The incorporated
[3H]thymidine is precipitated with ice-cold 10% trichloroacetic
acid for 24 h. Subsequently, the cells are rinsed sequentially with
ice-cold 10% trichloroacetic acid and then with ice-cold water.
Following lysis in 0.5 M NaOH, the lysates and PBS rinses (500 ml)
are pooled, and the amount of radioactivity is measured.
[1427] Parkinson Models.
[1428] The loss of motor function in Parkinson's disease is
attributed to a deficiency of striatal dopamine resulting from the
degeneration of the nigrostriatal dopaminergic projection neurons.
An animal model for Parkinson's that has been extensively
characterized involves the systemic administration of 1-methyl-4
phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is
taken-up by astrocytes and catabolized by monoamine oxidase B to
1-methyl-4-phenyl pyridine (MPP.sup.+) and released. Subsequently,
MPP.sup.+ is actively accumulated in dopaminergic neurons by the
high-affinity reuptake transporter for dopamine. MPP.sup.+ is then
concentrated in mitochondria by the electrochemical gradient and
selectively inhibits nicotidamide adenine disphosphate: ubiquinone
oxidoreductionase (complex I), thereby interfering with electron
transport and eventually generating oxygen radicals.
[1429] It has been demonstrated in tissue culture paradigms that
FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic
neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's
group has demonstrated that administering FGF-2 in gel foam
implants in the striatum results in the near complete protection of
nigral dopaminergic neurons from the toxicity associated with MPTP
exposure (Otto and Unsicker, J. Neuroscience, 1990).
[1430] Based on the data with FGF-2, an albumin fusion protein of
the invention can be evaluated to determine whether it has an
action similar to that of FGF-2 in enhancing dopaminergic neuronal
survival in vitro and it can also be tested in vivo for protection
of dopaminergic neurons in the striatum from the damage associated
with MPTP treatment. The potential effect of an albumin fusion
protein of the invention is first examined in vitro in a
dopaminergic neuronal cell culture paradigm. The cultures are
prepared by dissecting the midbrain floor plate from gestation day
14 Wistar rat embryos. The tissue is dissociated with trypsin and
seeded at a density of 200,000 cells/cm.sup.2 on
polyorthinine-laminin coated glass coverslips. The cells are
maintained in Dulbecco's Modified Eagle's medium and F12 medium
containing hormonal supplements (N1). The cultures are fixed with
paraformaldehyde after 8 days in vitro and are processed for
tyrosine hydroxylase, a specific marker for dopaminergic neurons,
immunohistochemical staining. Dissociated cell cultures are
prepared from embryonic rats. The culture medium is changed every
third day and the factors are also added at that time.
[1431] Since the dopaminergic neurons are isolated from animals at
gestation day 14, a developmental time which is past the stage when
the dopaminergic precursor cells are proliferating, an increase in
the number of tyrosine hydroxylase immunopositive neurons would
represent an increase in the number of dopaminergic neurons
surviving in vitro. Therefore, if a therapeutic protein of the
invention acts to prolong the survival of dopaminergic neurons, it
would suggest that the fusion protein may be involved in
Parkinson's Disease.
Example 64
Pancreatic Beta-Cell Transplantation Combination Therapy
[1432] Transplantation is a common form of treatment of autoimmune
disease, especially when the target self tissue has been severely
damaged. For example, and not by way of limitation, pancreas
transplantation and islet cell transplantation are common treatment
options for IDDM (See, e.g., Stewart et al., Journal of Clinical
Endocrinology & Metabolism 86 (3): 984-988 (2001); Brunicardi,
Transplant. Proc. 28: 2138-40 (1996); Kendall & Robertson,
Diabetes Metab. 22: 157-163 (1996); Hamano et al., Kobe J. Med.
Sci. 42: 93-104 (1996); Larsen & Stratta, Diabetes Metab. 22:
139-146 (1996); and Kinkhabwala, et al., Am. J. Surg. 171: 516-520
(1996)). As with any transplantation method, transplantation
therapies for autoimmune disease patients include treatments to
minimize the risk of host rejection of the transplanted tissue.
However, autoimmune disease involves the additional, independent
risk that the pre-existing host autoimmune response which damaged
the original self tissue will exert the same damaging effect on the
transplanted tissue. Accordingly, the present invention encompasses
methods and compositions for the treatment of autoimmune pancreatic
disease using the albumin fusion proteins of the subject invention
in combination with immunomodulators/immunosuppressants in
individuals undergoing transplantation therapy of the autoimmune
disease.
[1433] In accordance with the invention, the albumin fusion-based
compositions and formulations described above, are administered to
prevent and treat damage to the transplanted organ, tissue, or
cells resulting from the host individual's autoimmune response
initially directed against the original self tissue. Administration
may be carried out both prior and subsequent to transplantation in
2 to 4 doses each one week apart.
[1434] The following immunomodulators/immunosuppressants including,
but not limited to, AI-401, CDP-571 (anti-TNF monoclonal antibody),
CG-1088, Diamyd (diabetes vaccine), ICM3 (anti-ICAM-3 monoclonal
antibody), linomide (Roquinimex), NBI-6024 (altered peptide
ligand), TM-27, VX-740 (HMR-3480), caspase 8 protease inhibitors,
thalidomide, hOKT3gamma1 (Ala-ala) (anti-CD3 monoclonal antibody),
Oral Interferon-Alpha, oral lactobacillus, and LymphoStat-B.TM. can
be used together with the albumin fusion therapeutics of the
subject invention in islet cell or pancreas transplantation.
Example 65
Identification and Cloning of VH and VL Domains
[1435] One method to identify and clone VH and VL domains from cell
lines expressing a particular antibody is to perform PCR with VH
and VL specific primers on cDNA made from the antibody expressing
cell lines. Briefly, RNA is isolated from the cell lines and used
as a template for RT-PCR designed to amplify the VH and VL domains
of the antibodies expressed by the EBV cell lines. Cells may be
lysed in the TRIzol.RTM. reagent (Life Technologies, Rockville. MD)
and extracted with one fifth volume of chloroform. After addition
of chloroform, the solution is allowed to incubate at room
temperature for 10 minutes, and the centrifuged at 14,000 rpm for
15 minutes at 4.degree. C. in a tabletop centrifuge. The
supernatant is collected and RNA is precipitated using an equal
volume of isopropanol. Precipitated RNA is pelleted by centrifuging
at 14,000 rpm for 15 minutes at 4.degree. C. in a tabletop
centrifuge. Following centrifugation, the supernatant is discarded
and washed with 75% ethanol. Following washing, the RNA is
centrifuged again at 800 rpm for 5 minutes at 4.degree. C. The
supernatant is discarded and the pellet allowed to air dry. RNA is
the dissolved in DEPC water and heated to 60.degree. C. for 10
minutes. Quantities of RNA can determined using optical density
measurements.
[1436] cDNA may be synthesized, according to methods well-known in
the art, from 1.5-2.5 micrograms of RNA using reverse transciptase
and random hexamer primers. cDNA is then used as a template for PCR
amplification of VH and VL domains. Primers used to amplify VH and
VL genes are shown in Table 7. Typically a PCR reaction makes use
of a single 5' primer and a single 3' primer. Sometimes, when the
amount of available RNA template is limiting, or for greater
efficiency, groups of 5' and/or 3' primers may be used. For
example, sometimes all five VH-5' primers and all JH3' primers are
used in a single PCR reaction. The PCR reaction is carried out in a
50 microliter volume containing 1.times.PCR buffer, 2 mM of each
dNTP, 0.7 units of High Fidelity Taq polymerse, 5' primer mix, 3'
primer mix and 7.5 microliters of cDNA. The 5' and 3' primer mix of
both VH and VL can be made by pooling together 22 pmole and 28
pmole, respectively, of each of the individual primers. PCR
conditions are: 96.degree. C. for 5 minutes; followed by 25 cycles
of 94.degree. C. for 1 minute, 50.degree. C. for 1 minute, and
72.degree. C. for 1 minute; followed by an extension cycle of
72.degree. C. for 10 minutes. After the reaction is completed,
sample tubes are stored 4.degree. C.
TABLE-US-00018 TABLE 7 Primer Sequences Used to Amplify VH and VL
domains. Primer name SEQ ID NO Primer Sequence (5'-3') VH Primers
Hu VH1-5' 62 CAGGTGCAGCTGGTGCAGTCTGG Hu VH2-5' 63
CAGGTCAACTTAAGGGAGTCTGG Hu VH3-5' 64 GAGGTGCAGCTGGTGGAGTCTGG Hu
VH4-5' 65 CAGGTGCAGCTGCAGGAGTCGGG Hu VH5-5' 66
GAGGTGCAGCTGTTGCAGTCTGC Hu VH6-5' 67 CAGGTACAGCTGCAGCAGTCAGG Hu
JH1,2-5' 68 TGAGGAGACGGTGACCAGGGTGCC Hu JH3-5' 69
TGAAGAGACGGTGACCATTGTCCC Hu JH4,5-5' 70 TGAGGAGACGGTGACCAGGGTTCC Hu
JH6-5' 71 TGAGGAGACGGTGACCGTGGTCCC VL Primers Hu Vkappa1-5' 72
GACATCCAGATGACCCAGTCTCC Hu Vkappa2a-5' 73 GATGTTGTGATGACTCAGTCTCC
Hu Vkappa2b-5' 74 GATATTGTGATGACTCAGTCTCC Hu Vkappa3-5' 75
GAAATTGTGTTGACGCAGTCTCC Hu Vkappa4-5' 76 GACATCGTGATGACCCAGTCTCC Hu
Vkappa5-5' 77 GAAACGACACTCACGCAGTCTCC Hu Vkappa6-5' 78
GAAATTGTGCTGACTCAGTCTCC Hu Vlambda1-5' 79 CAGTCTGTGTTGACGCAGCCGCC
Hu Vlambda2-5' 80 CAGTCTGCCCTGACTCAGCCTGC Hu Vlambda3-5' 81
TCCTATGTGCTGACTCAGCCACC Hu Vlambda3b-5' 82 TCTTCTGAGCTGACTCAGGACCC
Hu Vlambda4-5' 83 CACGTTATACTGACTCAACCGCC Hu Vlambda5-5' 84
CAGGCTGTGCTCACTCAGCCGTC Hu Vlambda6-5' 85 AATTTTATGCTGACTCAGCCCCA
Hu Jkappa1-3' 86 ACGTTTGATTTCCACCTTGGTCCC Hu Jkappa2-3' 87
ACGTTTGATCTCCAGCTTGGTCCC Hu Jkappa3-3' 88 ACGTTTGATATCCACTTTGGTCCC
Hu Jkappa4-3' 89 ACGTTTGATCTCCACCTTGGTCCC Hu Jkappa5-3' 90
ACGTTTAATCTCCAGTCGTGTCCC Hu Jlambda1-3' 91 CAGTCTGTGTTGACGCAGCCGCC
Hu Jlambda2-3' 92 CAGTCTGCCCTGACTCAGCCTGC Hu Jlambda3-3' 93
TCCTATGTGCTGACTCAGCCACC Hu Jlambda3b-3' 94 TCTTCTGAGCTGACTCAGGACCC
Hu Jlambda4-3' 95 CACGTTATACTGACTCAACCGCC Hu Jlambda5-3' 96
CAGGCTGTGCTCACTCAGCCGTC Hu Jlambda6-3' 97
AATTTTATGCTGACTCAGCCCCA
PCR samples are then electrophoresed on a 1.3% agarose gel. DNA
bands of the expected sizes (.about.506 base pairs for VH domains,
and 344 base pairs for VL domains) can be cut out of the gel and
purified using methods well known in the art. Purified PCR products
can be ligated into a PCR cloning vector (TA vector from Invitrogen
Inc., Carlsbad, Calif.). Individual cloned PCR products can be
isolated after transfection of E. coli and blue/white color
selection. Cloned PCR products may then be sequenced using methods
commonly known in the art.
[1437] The PCR bands containing the VH domain and the VL domains
can also be used to create full-length Ig expression vectors. VH
and VL domains can be cloned into vectors containing the nucleotide
sequences of a heavy (e.g., human IgG1 or human IgG4) or light
chain (human kappa or human lambda) constant regions such that a
complete heavy or light chain molecule could be expressed from
these vectors when transfected into an appropriate host cell.
Further, when cloned heavy and light chains are both expressed in
one cell line (from either one or two vectors), they can assemble
into a complete functional antibody molecule that is secreted into
the cell culture medium. Methods using polynucleotides encoding VH
and VL antibody domain to generate expression vectors that encode
complete antibody molecules are well known within the art.
Example 66
Preparation of Ha-Cytokine or Ha-Growth Factor Fusion Proteins
(Such as NGF, BDNFa, BDNFb and BDNFc)
[1438] The cDNA for the cytokine or growth factor of interest, such
as NGF, can be isolated by a variety of means including from cDNA
libraries, by RT-PCR and by PCR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods. The
nucleotide sequences for all of these proteins are known and
available. The cDNA can be tailored at the 5' and 3' ends to
generate restriction sites, such that oligonucleotide linkers can
be used, for cloning of the cDNA into a vector containing the cDNA
for HA. This can be at the N or C-terminus with or without the use
of a spacer sequence. NGF (or other cytokine) cDNA is cloned into a
vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from
which the complete expression cassette is then excised and inserted
into the plasmid pSAC35 to allow the expression of the albumin
fusion protein in yeast. The albumin fusion protein secreted from
the yeast can then be collected and purified from the media and
tested for its biological activity. For expression in mammalian
cell lines, a similar procedure is adopted except that the
expression cassette used employs a mammalian promoter, leader
sequence and terminator (See Example 1). This expression cassette
is then excised and inserted into a plasmid suitable for the
transfection of mammalian cell lines.
Example 67
Preparation of HA-IFN Fusion Proteins (Such as IFN.alpha.)
[1439] The cDNA for the interferon of interest such as IFN.alpha.
can be isolated by a variety of means including but not
exclusively, from cDNA libraries, by RT-PCR and by PCR using a
series of overlapping synthetic oligonucleotide primers, all using
standard methods. The nucleotide sequences for interferons, such as
IFN.alpha. are known and available, for instance, in U.S. Pat. Nos.
5,326,859 and 4,588,585, in EP 32 134, as well as in public
databases such as GenBank. The cDNA can be tailored at the 5' and
3' ends to generate restriction sites, such that oligonucleotide
linkers can be used to clone the cDNA into a vector containing the
cDNA for HA. This can be at the N or C-terminus of the HA sequence,
with or without the use of a spacer sequence. The IFN.alpha. (or
other interferon) cDNA is cloned into a vector such as pPPC0005
(FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which the complete
expression cassette is then excised and inserted into the plasmid
pSAC35 to allow the expression of the albumin fusion protein in
yeast. The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
biological activity. For expression in mammalian cell lines a
similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
[1440] Maximum Protein Recovery from Vials
[1441] The albumin fusion proteins of the invention have a high
degree of stability even when they are packaged at low
concentrations. In addition, in spite of the low protein
concentration, good fusion-protein recovery is observed even when
the aqueous solution includes no other protein added to minimize
binding to the vial walls. The recovery of vial-stored HA-IFN
solutions was compared with a stock solution. 6 or 30 .mu.g/ml
HA-IFN solutions were placed in vials and stored at 4.degree. C.
After 48 or 72 hrs a volume originally equivalent to 10 ng of
sample was removed and measured in an IFN sandwich ELISA. The
estimated values were compared to that of a high concentration
stock solution. As shown, there is essentially no loss of the
sample in these vials, indicating that addition of exogenous
material such as albumin is not necessary to prevent sample loss to
the wall of the vials
[1442] In Vivo Stability and Bioavailability of HA-.alpha.-IFN
Fusions
[1443] To determine the in vivo stability and bioavailability of a
HA-.alpha.-IFN fusion molecule, the purified fusion molecule (from
yeast) was administered to monkeys. Pharmaceutical compositions
formulated from HA-.alpha.-IFN fusions may account for the extended
serum half-life and bioavailability. Accordingly, pharmaceutical
compositions may be formulated to contain lower dosages of
alpha-interferon activity compared to the native alpha-interferon
molecule.
[1444] Pharmaceutical compositions containing HA-.alpha.-IFN
fusions may be used to treat or prevent disease in patients with
any disease or disease state that can be modulated by the
administration of .alpha.-IFN. Such diseases include, but are not
limited to, hairy cell leukemia, Kaposi's sarcoma, genital and anal
warts, chronic hepatitis B, chronic non-A, non-B hepatitis, in
particular hepatitis C, hepatitis D, chronic myelogenous leukemia,
renal cell carcinoma, bladder carcinoma, ovarian and cervical
carcinoma, skin cancers, recurrent respirator papillomatosis,
non-Hodgkin's and cutaneous T-cell lymphomas, melanoma, multiple
myeloma, AIDS, multiple sclerosis, gliobastoma, etc. (see
Interferon Alpha, In: AHFS Drug Information, 1997.
[1445] Accordingly, the invention includes pharmaceutical
compositions containing a HA-.alpha.-IFN fusion protein,
polypeptide or peptide formulated with the proper dosage for human
administration. The invention also includes methods of treating
patients in need of such treatment comprising at least the step of
administering a pharmaceutical composition containing at least one
HA-.alpha.-IFN fusion protein, polypeptide or peptide.
[1446] Bifunctional HA-.quadrature.-IFN Fusions
[1447] A HA-.alpha.-IFN expression vector may be modified to
include an insertion for the expression of bifunctional
HA-.alpha.-IFN fusion proteins. For instance, the cDNA for a second
protein of interest may be inserted in frame downstream of the
"rHA-IFN" sequence after the double stop codon has been removed or
shifted downstream of the coding sequence.
[1448] In one version of a bifunctional HA-.alpha.-IFN fusion
protein, an antibody or fragment against B-lymphocyte stimulator
protein (GenBank Acc 4455139) or polypeptide may be fused to one
end of the HA component of the fusion molecule. This bifunctional
protein is useful for modulating any immune response generated by
the .alpha.-IFN component of the fusion.
Example 68
Preparation of HA-Hormone Fusion Protein
[1449] The cDNA for the hormone of interest can be isolated by a
variety of means including but not exclusively, from cDNA
libraries, by RT-PCR and by PCR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods. The
nucleotide sequences for all of these proteins are known and
available, for instance, in public databases such as GenBank. The
cDNA can be tailored at the 5' and 3' ends to generate restriction
sites, such that oligonucleotide linkers can be used, for cloning
of the cDNA into a vector containing the cDNA for HA. This can be
at the N or C-terminus with or without the use of a spacer
sequence. The hormone cDNA is cloned into a vector such as pPPC0005
(FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which the complete
expression cassette is then excised and inserted into the plasmid
pSAC35 to allow the expression of the albumin fusion protein in
yeast. The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
biological activity. For expression in mammalian cell lines a
similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
Example 69
Preparation of HA-Soluble Receptor or HA-Binding Protein Fusion
Protein
[1450] The cDNA for the soluble receptor or binding protein of
interest can be isolated by a variety of means including but not
exclusively, from cDNA libraries, by RT-PCR and by PCR using a
series of overlapping synthetic oligonucleotide primers, all using
standard methods. The nucleotide sequences for all of these
proteins are known and available, for instance, in GenBank. The
cDNA can be tailored at the 5' and 3' ends to generate restriction
sites, such that oligonucleotide linkers can be used, for cloning
of the cDNA into a vector containing the cDNA for HA. This can be
at the N or C-terminus with or without the use of a spacer
sequence. The receptor cDNA is cloned into a vector such as
pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which the
complete expression cassette is then excised and inserted into the
plasmid pSAC35 to allow the expression of the albumin fusion
protein in yeast. The albumin fusion protein secreted from the
yeast can then be collected and purified from the media and tested
for its biological activity. For expression in mammalian cell lines
a similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
Example 70
Preparation of HA-Growth Factors
[1451] The cDNA for the growth factor of interest can be isolated
by a variety of means including but not exclusively, from cDNA
libraries, by RT-PCR and by PCR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods (see
GenBank Acc. No. NP.sub.--000609). The cDNA can be tailored at the
5' and 3' ends to generate restriction sites, such that
oligonucleotide linkers can be used, for cloning of the cDNA into a
vector containing the cDNA for HA. This can be at the N or
C-terminus with or without the use of a spacer sequence. The growth
factor cDNA is cloned into a vector such as pPPC0005 (FIG. 2),
pScCHSA, pScNHSA, or pC4:HSA from which the complete expression
cassette is then excised and inserted into the plasmid pSAC35 to
allow the expression of the albumin fusion protein in yeast. The
albumin fusion protein secreted from the yeast can then be
collected and purified from the media and tested for its biological
activity. For expression in mammalian cell lines a similar
procedure is adopted except that the expression cassette used
employs a mammalian promoter, leader sequence and terminator (See
Example 1). This expression cassette is then excised and inserted
into a plasmid suitable for the transfection of mammalian cell
lines.
Example 71
Preparation of HA-Single Chain Antibody Fusion Proteins
[1452] Single chain antibodies are produced by several methods
including but not limited to: selection from phage libraries,
cloning of the variable region of a specific antibody by cloning
the cDNA of the antibody and using the flanking constant regions as
the primer to clone the variable region, or by synthesizing an
oligonucleotide corresponding to the variable region of any
specific antibody. The cDNA can be tailored at the 5' and 3' ends
to generate restriction sites, such that oligonucleotide linkers
can be used, for cloning of the cDNA into a vector containing the
cDNA for HA. This can be at the N or C-terminus with or without the
use of a spacer sequence. The cell cDNA is cloned into a vector
such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which
the complete expression cassette is then excised and inserted into
the plasmid pSAC35 to allow the expression of the albumin fusion
protein in yeast.
[1453] In fusion molecules of the invention, the V.sub.H and
V.sub.L can be linked by one of the following means or a
combination thereof: a peptide linker between the C-terminus of the
V.sub.H and the N-terminus of the V.sub.L; a Kex2p protease
cleavage site between the V.sub.H and V.sub.L such that the two are
cleaved apart upon secretion and then self associate; and cystine
residues positioned such that the V.sub.H and V.sub.L can form a
disulphide bond between them to link them together. An alternative
option would be to place the V.sub.H at the N-terminus of HA or an
HA domain fragment and the V.sub.L at the C-terminus of the HA or
HA domain fragment.
[1454] The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
activity. For expression in mammalian cell lines a similar
procedure is adopted except that the expression cassette used
employs a mammalian promoter, leader sequence and terminator (See
Example 1). This expression cassette is then excised and inserted
into a plasmid suitable for the transfection of mammalian cell
lines. The antibody produced in this manner can be purified from
media and tested for its binding to its antigen using standard
immunochemical methods.
Example 72
Preparation of HA-Cell Adhesion Molecule Fusion Proteins
[1455] The cDNA for the cell adhesion molecule of interest can be
isolated by a variety of means including but not exclusively, from
cDNA libraries, by RT-PCR and by PCR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods. The
nucleotide sequences for the known cell adhesion molecules are
known and available, for instance, in GenBank. The cDNA can be
tailored at the 5' and 3' ends to generate restriction sites, such
that oligonucleotide linkers can be used, for cloning of the cDNA
into a vector containing the cDNA for HA. This can be at the N or
C-terminus with or without the use of a spacer sequence. The cell
adhesion molecule cDNA is cloned into a vector such as pPPC0005
(FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which the complete
expression cassette is then excised and inserted into the plasmid
pSAC35 to allow the expression of the albumin fusion protein in
yeast. The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
biological activity. For expression in mammalian cell lines a
similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
Example 73
Preparation of Inhibitory Factors and Peptides as HA Fusion
Proteins (Such as HA-Antiviral, HA-Antibiotic, HA-Enzyme Inhibitor
and HA-Anti-Allergic Proteins)
[1456] The cDNA for the peptide of interest such as an antibiotic
peptide can be isolated by a variety of means including but not
exclusively, from cDNA libraries, by RT-PCR and by PCR using a
series of overlapping synthetic oligonucleotide primers, all using
standard methods. The cDNA can be tailored at the 5' and 3' ends to
generate restriction sites, such that oligonucleotide linkers can
be used, for cloning of the cDNA into a vector containing the cDNA
for HA. This can be at the N or C-terminus with or without the use
of a spacer sequence. The peptide cDNA is cloned into a vector such
as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which the
complete expression cassette is then excised and inserted into the
plasmid pSAC35 to allow the expression of the albumin fusion
protein in yeast. The albumin fusion protein secreted from the
yeast can then be collected and purified from the media and tested
for its biological activity. For expression in mammalian cell lines
a similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
Example 74
Preparation of Targeted HA Fusion Proteins
[1457] The cDNA for the protein of interest can be isolated from
cDNA library or can be made synthetically using several overlapping
oligonucleotides using standard molecular biology methods. The
appropriate nucleotides can be engineered in the cDNA to form
convenient restriction sites and also allow the attachment of the
protein cDNA to albumin cDNA. Also a targeting protein or peptide
cDNA such as single chain antibody or peptides, such as nuclear
localization signals, that can direct proteins inside the cells can
be fused to the other end of albumin. The protein of interest and
the targeting peptide is cloned into a vector such as pPPC0005
(FIG. 2), pScCHSA, pScNHSA, or pC4:HSA which allows the fusion with
albumin cDNA. In this manner both N- and C-terminal end of albumin
are fused to other proteins. The fused cDNA is then excised from
pPPC0005 and is inserted into a plasmid such as pSAC35 to allow the
expression of the albumin fusion protein in yeast. All the above
procedures can be performed using standard methods in molecular
biology. The albumin fusion protein secreted from yeast can be
collected and purified from the media and tested for its biological
activity and its targeting activity using appropriate biochemical
and biological tests.
Example 75
Preparation of HA-Enzymes Fusions
[1458] The cDNA for the enzyme of interest can be isolated by a
variety of means including but not exclusively, from cDNA
libraries, by RT-PCR and by PCR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods. The
cDNA can be tailored at the 5' and 3' ends to generate restriction
sites, such that oligonucleotide linkers can be used, for cloning
of the cDNA into a vector containing the cDNA for HA. This can be
at the N or C-terminus with or without the use of a spacer
sequence. The enzyme cDNA is cloned into a vector such as pPPC0005
(FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which the complete
expression cassette is then excised and inserted into the plasmid
pSAC35 to allow the expression of the albumin fusion protein in
yeast. The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
biological activity. For expression in mammalian cell lines a
similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
Example 76
Construct ID 2249, IFNa2-HSA, Generation
[1459] Construct ID 2249, pSAC35:IFNa2.HSA, comprises DNA encoding
an IFNa2 albumin fusion protein which has the HSA chimeric leader
sequence, followed by the mature form of IFNa2 protein, i.e.,
C1-E165, fused to the amino-terminus of the mature form of HSA in
the yeast S. cerevisiae expression vector pSAC35.
[1460] Cloning of IFNa2 cDNA
[1461] The polynucleotide encoding IFNa2 was PCR amplified using
primers IFNa2-1 and IFNa2-2, described below. The PCR amplimer was
cut with Sal I/Cla I, and ligated into Xho I/Cla I cut pScCHSA.
Construct ID #2249 encodes an albumin fusion protein containing the
chimeric leader sequence of HSA, the mature form of IFNa2, followed
by the mature HSA protein.
[1462] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the mature form of IFNa2, IFNa2-1 and
IFNa2-2, were synthesized:
TABLE-US-00019 IFNa2-1: (SEQ ID NO:348)
5'-CGCGCGCGTCGACAAAAGATGTGATCTGCCTCAAACCCACA-3' IFNa2-2: (SEQ ID
NO:349) 5'-GCGCGCATCGATGAGCAACCTCACTCTTGTGTGCATCTTCCTTACTT
CTTAAACTTTCT-3'
[1463] The IFNa2-1 primer incorporates a Sal I cloning site (shown
underlined), nucleotides encoding the last three amino acid
residues of the chimeric HSA leader sequence, as well as 22
nucleotides (shown in bold) encoding the first 7 amino acid
residues of the mature form of IFNa2. In IFNa2-2, the Cla I site
(shown underlined) and the DNA following it are the reverse
complement of DNA encoding the first 10 amino acids of the mature
HSA protein and the last 22 nucleotides (shown in bold) are the
reverse complement of DNA encoding the last 7 amino acid residues
of IFNa2 (see Example 2). A PCR amplimer of IFNa2-HSA was generated
using these primers, purified, digested with Sal I and Cla I
restriction enzymes, and cloned into the Xho I and Cla I sites of
the pScCHSA vector. After the sequence was confirmed, the
expression cassette encoding this IFNa2 albumin fusion protein was
subcloned into Not I digested pSAC35.
[1464] Further, analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing can confirm the presence of
the expected IFNa2 sequence (see below).
[1465] Other IFNa2 albumin fusion proteins using different leader
sequences have been constructed by methods known in the art (see
Example 2). Examples of the various leader sequences include, but
are not limited to, invertase "INV" (constructs 2343 and 2410) and
mating alpha factor "MAF" (construct 2366). These IFNa2 albumin
fusion proteins can be subcloned into mammalian expression vectors
such as pC4 (constructs 2382) and pEE12.1 as described previously
(see Example 5). IFNa2 albumin fusion proteins with the therapeutic
portion fused C-terminus to HSA can also be constructed (construct
2381).
[1466] IFNa2 albumin fusion proteins of the invention preferably
comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to
either the N- or C-terminus of the mature form of IFNa2, i.e.,
Cys-1 to Glu-165. In one embodiment of the invention, IFNa2 albumin
fusion proteins of the invention further comprise a signal sequence
which directs the nascent fusion polypeptide in the secretory
pathways of the host used for expression. In a further preferred
embodiment, the signal peptide encoded by the signal sequence is
removed, and the mature IFNa2 albumin fusion protein is secreted
directly into the culture medium. IFNa2 albumin fusion proteins of
the invention may comprise heterologous signal sequences including,
but not limited to, MAF, INV, Ig, Fibulin B, Clusterin,
Insulin-Like Growth Factor Binding Protein 4, variant HSA leader
sequences including, but not limited to, a chimeric HSA/MAF leader
sequence, or other heterologous signal sequences known in the art.
In a preferred embodiment, IFNa2 albumin fusion proteins of the
invention comprise the native IFNa2. In further preferred
embodiments, the IFNa2 albumin fusion proteins of the invention
further comprise an N-terminal methionine residue. Polynucleotides
encoding these polypeptides, including fragments and/or variants,
are also encompassed by the invention.
[1467] Expression and Purification of Construct ID 2249.
[1468] Expression in Yeast S. cerevisiae.
[1469] Transformation of construct 2249 into yeast S. cerevisiae
strain BXP10 was carried out by methods known in the art (see
Example 3). Cells can be collected at stationary phase after 72
hours of growth. Supernatants are collected by clarifying cells at
3000 g for 10 min. Expression levels are examined by immunoblot
detection with anti-HSA serum (Kent Laboratories) or as the primary
antibody. The IFNa2 albumin fusion protein of approximate molecular
weight of 88.5 kDa can be obtained.
[1470] Purification from Yeast S. cerevisiae Cell Supernatant.
[1471] The cell supernatant containing IFNa2 albumin fusion protein
expressed from construct ID #2249 in yeast S. cerevisiae cells can
be purified either small scale over a Dyax peptide affinity column
(see Example 4) or large scale by following 5 steps: diafiltration,
anion exchange chromatography using DEAE-Sepharose Fast Flow
column, hydrophobic interaction chromatography (HIC) using Butyl
650S column, cation exchange chromatography using an
SP-SepharoseFast Flow column or a Blue-Sepharose chromatography,
and high performance chromatography using Q-sepharose high
performance column chromatography (see Example 4). The IFNa2
albumin fusion protein may elute from the DEAE-Sepharose Fast Flow
column with 100-250 mM NaCl, from the SP-Sepharose Fast Flow column
with 150-250 mM NaCl, and from the Q-Sepharose High Performance
column at 5-7.5 mS/cm. N-terminal sequencing should yield the
sequence CDLPQ (SEQ ID NO:98) which corresponds to the mature form
of IFNa2.
[1472] The Activity of IFNa2 can be Assayed Using an In Vitro
ISRE-SEAP Assay
[1473] Method
[1474] The IFNa2 albumin fusion protein encoded by construct ID
#2249 can be tested for activity in the ISRE-SEAP assay as
previously described in Example 76. Briefly, conditioned yeast
supernatants were tested at a 1:1000 dilution for their ability to
direct ISRE signal transduction on the ISRE-SEAP/293F reporter
cell-line. The ISRE-SEAP/293F reporter cells were plated at
3.times.10.sup.4 cell/well in 96-well, poly-D-lysine coated,
plates, one day prior to treatment. The reporter cells were then
incubated for 18 or 24 hours prior to removing 40 .mu.L for use in
the SEAP Reporter Gene Chemiluminescent Assay (Roche catalog
#1779842). Recombinant human Interferon beta, "rhIFNb" (Biogen),
was used as a positive control.
[1475] Result
[1476] The purified preparation of IFNa2-HSA demonstrated a
relatively linear increase in the ISRE-SEAP assay over
concentrations ranging from 10.sup.-1 to 10.sup.1 ng/mL (see FIG.
4) or 10.sup.-10 to 10.sup.-8 ng/mL (see FIG. 5).
[1477] In Vivo Induction of OAS by Interferon Alpha Fusion Encoded
by Construct ID 2249.
[1478] Method
[1479] The OAS enzyme, 2'-5'-OligoAdenylate Synthetase, is
activated at the transcriptional level by interferon in response to
antiviral infection. The effect of interferon constructs can be
measured by obtaining blood samples from treated monkeys and
analyzing these samples for transcriptional activation of two OAS
mRNA, p41 and p69. A volume of 0.5 mL of whole blood was obtained
from 4 animals per group at 7 different time points, day 0, day 1,
day 2, day 4, day 8, day 10, and day 14 per animal. The various
groups include vehicle control, intravenous injection of 30
.mu.g/kg HSA-IFN on day 1, subcutaneous injection of 30 .mu.g/kg of
HSA-IFN on day 1, subcutaneous injection of 300 .mu.g/kg of HSA-IFN
on day 1, and subcutaneous injection of 40 .mu.g/kg of Interferon
alpha (Schering-Plough) as a positive control on days 1, 3, and 5.
The levels of the p41 and the p69 mRNA transcripts were determined
by real-time quantitative PCR (Taqman) using probes specific for
p41-OAS and p69-OAS. OAS mRNA levels were quantitated relative to
18S ribosomal RNA endogenous control. The albumin fusion encoded by
construct 2249 can be subjected to similar experimentation.
[1480] Results
[1481] A significant increase in mRNA transcript levels for both
p41 and p69 OAS was observed in HSA-interferon treated monkeys in
contrast to IFNa treated monkeys (see FIG. 6 for p41 data). The
effect lasted nearly 10 days.
Example 77
Indications for IFNa2 Albumin Fusion Proteins
[1482] IFN alpha albumin fusion protein (including, but not limited
to, those encoded by constructs 2249, 2343, 2410, 2366, 2382, and
2381) can be used to treat, prevent, ameliorate, and/or detect
multiple sclerosis. Other indications include, but are not limited
to viral infections including Severe Acute Respiratory Syndrome
(SARS) and other coronavirus infections; filoviruses, including but
not limited to Ebola viruses and Marburg virus; Arenaviruses,
including but not limited to Pichende virus, Lassa virus, Junin
virus, Machupo virus, Guanarito virus; and lymphocytic
choriomeningitis virus (LCMV); Bunyaviruses, including but not
limited to Punta toro virus, Crimean-Congo hemorrhagic fever virus,
sandfly fever viruses, Rift Valley fever virus, La Crosse virus,
and hantaviruses; Flaviviruses, including but not limited to Yellow
Fever, Banzi virus, West Nile virus, Dengue viruses, Japanese
Encephalitis virus, Tick-borne encephalitis, Omsk Hemorrhagic
Fever, and Kyasanur Forest Disease virus; Togaviruses, including
but not limited to Venezuelan, eastern, and western equine
encephalitis viruses, Ross River virus, and Rubella virus; Orthopox
viruses, including but not limited to Vaccinia, Cowpox, Smallpox,
and Monkeypox; Herpesviruses; FluA/B; Respiratory Sincytial virus
(RSV); paraflu; measles; rhinoviruses; adenoviruses; Semliki Forest
virus; Viral Hemorrhagic fevers; Rhabdoviruses; Paramyxoviruses,
including but not limited to Nipah virus and Hendra virus; and
other viral agents identified by the U.S. Centers for Disease
Control and Prevention as high-priority disease agents (i.e.,
Category A, B, and C agents; see, e.g., Moran, Emerg. Med. Clin.
North. Am. 2002; 20(2):311-30 and Darling et al., Emerg. Med. Clin.
North Am. 2002; 20(2):273-309).
[1483] Preferably, the IFN.alpha.-albumin fusion protein or IFN
hybrid fusion protein is administered in combination with a CCR5
antagonist, further in association with at least one of ribavirin,
IL-2, IL-12, pentafuside alone or in combination with an anti-HIV
drug therapy, e.g., HAART, for preparation of a medicament for the
treatment of HIV-1 infections, HCV, or HIV-1 and HCV co-infections
in treatment-naive as well as treatment-experienced adult and
pediatric patients.
Example 78
Construct ID #3691, BNP-HSA, Generation
[1484] Construct ID #3691, pC4:SPCON.BNP1-32/HSA, comprises DNA
encoding a BNP albumin fusion protein which has a consensus leader
sequence, secrecon, followed by the processed, active BNP peptide
(amino acids 1-32) fused to the amino-terminus of the mature form
of HSA in the mammalian expression vector pC4.
[1485] Cloning of BNP cDNA for Construct 3691
[1486] The polynucleotide encoding BNP was PCR amplified using
primers BNP-1 and BNP-2, described below, cut with Bam HI/Cla I,
and ligated into Bam HI/Cla I cut pC4:HSA resulting in construct ID
#3691. Construct ID #3691 encodes an albumin fusion protein
containing a consensus leader sequence (SEQ ID No:111) and the
processed, active form of BNP, followed by the mature HSA protein
(see SEQ ID NO:204 for construct 3691 in Table 2).
[1487] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the active, processed form of BNP, BNP-1
and BNP-2, were synthesized:
TABLE-US-00020 BNP-1: (SEQ ID NO:364)
5'-GAGCGCGGATCCAAGCTTCCGCCATCATGTGGTGGCGCCTGTGGTGG
CTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCAGCCCCAA GCTGGTGCAAGG-3'
BNP-2: (SEQ ID NO:365)
5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCATGCCGCCTC
AGCACTTTGC-3'.
[1488] BNP-1 incorporates a Bam HI cloning site (underlined),
polynucleotides encoding a consensus leader sequence (SEQ ID
NO:111) (italicized), and polynucleotides encoding the first seven
amino acid sequence of BNP (bolded). In BNP-2, the underlined
sequence is a Cla I site, and the polynucleotides that follow it
contains the reverse complement of DNA encoding the last 6 amino
acids of BNP (bolded) and the first 10 amino acids of the mature
HSA protein. Using these two primers the BNP protein was PCR
amplified. Annealing and extension temperatures and times must be
empirically determined for each specific primer pair and
template.
[1489] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bam HI and Cla I. After further purification of the Bam HI-Cla
I fragment by gel electrophoresis, the product was cloned into Bam
HI/Cla I digested pC4:HSA to produce construct ID #3691. The
expression construct was sequence verified.
[1490] Expression and Purification of Construct ID 3691.
[1491] Expression in 293F Cells.
[1492] Construct ID #3691, pC4:SPCON.BNP1-32/HSA, was transfected
into 293F cells by methods known in the art (see Example 6).
[1493] Purification from 293F Cell Supernatant.
[1494] Two liters of supernatant were collected 3 days
post-transfection. The recombinant protein was captured by 5 ml
Blue Sepharose CL-6B column (Amersham Biosciences, Piscataway,
N.J., USA) and eluted by 2 M NaCl. The material was bound to HiPrep
16/10 Phenyl FF (high sub) column and eluted by 20 mM MES, pH 6.7.
BNP-HSA was further purified by hydroxyapatite column
chromatography in sodium phosphate buffer gradient (0-20 mS/cm in
200 ml) at pH 6.8. The final product was exchanged into PBS pH 7.2
by a HiPrep 26/10 desalting column (Amersham Biosciences).
[1495] The Activity of BNP-HSA can be Assayed Using an In Vitro
NPR-A/cGMP Assay.
[1496] Natriuretic peptide receptor-A (NPR-A) is the signaling
receptor for BNP, and as such, is responsible for most of BNP's
biological effects. BNP bioactivity is mediated by NPR-A guanylyl
cyclase domain that converts GTP to cGMP upon activation. A
convenient assay for BNP activity is to measure the BNP stimulation
of a 293F cell line that stably over-expresses NPR-A. The cGMP
production in the cells after exposure to BNP can be measured by
cGMP ELISA.
[1497] Method of Screening NPR-A 293F Stable Clones.
[1498] The open reading frame of human NPR-A was constructed into
pcDNA3.1 expression vector (Invitrogen). 293F cells were stably
transfected with the plasmid DNA by Lipofectamine method and
selected by 0.8 .mu.g/ml G418. 293F/NPR-A stable clones were
screened for best response to recombinant BNP.
[1499] Measurement of cGMP Activation.
[1500] cGMP activation by BNP was carried out in 293F/NPR-A cells
and measured by CatchPoint cyclic-GMP fluorescent assay kit
(Molecular Devices, Sunnyvale, Calif., USA). Briefly, 50,000
cells/well of 293F/NPR-A cells cultured in a 96-well plate were
washed into 80 .mu.l prestimulation buffer (Krebs-Ringer
Bicarbonate Buffer with 10 mM glucose, pH 7.4, 15 nM sodium
bicarbonate, and 0.75 mM 3-isobutyl-1-methylxanthine). BNP-HSA or
recombinant BNP in 40 .mu.l prestimulation buffer was added to the
cells at 37.degree. C. for 10 min. The cells were lysed with 40
.mu.l Lysis Buffer for 10 min with shaking. The amounts of cGMP in
the lysates were quantitated as per the manufacturer's
instruction.
[1501] Result
[1502] The dose-response relationship of BNP-HSA and recombinant
BNP were determined (see FIG. 7). The maximal activities of
Construct ID #3691 and recombinant BNP were similar (1.63.+-.0.016
vs. 1.80.+-.0.016 pm, respectively), with EC50 values of
28.4.+-.1.2, and 0.46.+-.1.1 nM, respectively.
[1503] BNP-HSA Decreases Blood Pressure In Vivo.
[1504] Method
[1505] BNP reduces blood pressure by direct vasodilation as well as
by suppression of renin/angiotensin/endothelin/aldosterone systems.
The ability of BNP-HSA to decrease arterial blood pressure was
tested in three-month old male spontaneously hypertensive rats
purchased from Taconic (Germantown, N.Y., USA). Spontaneously
hypertensive rats are genetically hypertensive with onset of high
blood pressure after three months of age. BNP-HSA or recombinant
BNP was reconstituted in 0.3 cc PBS per rat. The drugs were
delivered via tail vein injection. Systolic and diastolic blood
pressures were recorded by cuff-tail method using XBP-1000 System
(Kent Scientific, Torrington, Conn., USA). For each blood pressure
data point, 4-5 consecutive readings were taken and averaged. Mean
arterial pressure (MAP) was calculated as 1/3 systolic pressure
+2/3 diastolic pressure. For dose-response determination, blood
pressures were measured 20 h after pC4:SPCON.BNP1-32/HSA
administration at doses of 0.5, 2, 6, and 18 nmol/kg.
[1506] Result
[1507] The typical systolic pressure of spontaneously hypertensive
rats was 180-200 mmHg before dosing. A single bolus of 6 nmol/kg
BNP-HSA delivered via tail vein intravenous injection lowered both
systolic and diastolic pressure, which accounted for more than 30
mmHg mean arterial pressure (MAP) reduction. The lowered blood
pressure was steady and continued for a day and then gradually
returned to the baseline over several days (see FIG. 8). In
contrast, due to its instantaneous clearance, a single 6 nmol/kg
bolus of recombinant BNP, produced only a very transient MAP
decrease of about .about.15 mmHg.
[1508] In addition, the dose-response 20 hours post injection of a
bolus of BNP-HSA was determined in four spontaneously hypertensive
rats. 0.5 nmol/kg BNP-HSA had an average of 7 mmHg MAP reduction,
while 6 nmol/kg BNP-HSA had an average of 30 mmHg MAP reduction,
and a high dose of 18 nmol/kg BNP-HSA only lowered the blood
pressure slightly more than 6 nmol/kg.
[1509] In Vivo Induction of Plasma cGMP by BNP-HSA.
[1510] Method
[1511] The intracellular cGMP activation by BNP results in its
release from the cell to circulation. The plasma cGMP level
correlates with BNP-induced cardiovascular and renal physiology.
Plasma cGMP has been used as a biomarker for in vivo BNP action. To
test the induction of plasma cGMP by BNP-HSA in vivo, eleven- to
12-week-old male C57/BL6 mice received a single bolus of
recombinant BNP or BNP-HSA at a 6 nmol/kg dose via tail vein.
Plasma was prepared from the tail bleeds at 5, 10, 20, 40, and 80
min time points for the recombinant BNP dosing group and at
additional 640, 1440, 2880, and 5760 min for the BNP-HSA group.
Plasma samples from mice treated with PBS as the vehicle control
were collected as the zero time points. cGMP levels were determined
by CatchPoint cyclic-GMP fluorescent assay kit according to the
manufacture's instruction.
[1512] Result
[1513] Following a single intravenous bolus of 6 nmol/kg
recombinant BNP or BNP-HSA, peak plasma cGMP levels over the
baseline were increased 3.9- or 5.6-fold, respectively (see FIG.
9). In addition, the one-phase exponential decay half-life of cGMP
following recombinant BNP treatment was 16 min (10 to 42 min, 95%
CI), while the one-phase exponential decay half-life of cGMP
following BNP-HSA administration was 1538 min (1017 to 3153 min,
95% CI).
[1514] In Vivo Pharmacokinetic Analysis of BNP Albumin Fusion
Encoded by Construct ID 3691.
[1515] Method
[1516] Eleven- to 12-week-old male C57/BL6 mice (obtained from Ace
Animals, Boyertown, Pa., USA) weighed 25.1.+-.0.12 g at the time of
the study. All animals were dosed at a volume of 10 ml/kg body
weight. Predose animals were injected with PBS. Recombinant BNP was
injected intravenously in the tail or subcutaneously in the
mid-scapular region.
[1517] Pharmacokinetic analysis was performed on the following
groups:
TABLE-US-00021 TABLE 8 Dose N/ Group Drug (mg/kg) Route time Time
(hours) 1 BNP-HSA 2.19 Subcutaneous 3 0.5, 2, 6, 16, 24, 32, 48,
72, 96 2 BNP-HSA 2.19 Intravenous 3 0.083, 2, 6, 16, 24, 32, 48,
72, 96 3 Vehicle 0 Subcutaneous 3 predose 4 Vehicle 0 Intravenous 3
predose
[1518] Blood was sampled from the inferior vena cava, placed into
an EDTA-coated microtainer, and stored on ice. The samples were
centrifuged in a microcentrifuge at 14,000 rpm (16,000.times.g) for
10 minutes at room temperature. The plasma was transferred into
cluster tubes and stored at -80.degree. C.
[1519] BNP-HSA concentrations in plasma samples were determined
using BNP EIA Kit (Phoenix Pharmaceutical, Belmont, Calif., USA).
The standard curves were generated at the same time on the same
plate with testing samples. The detection limit was 0.11 ng/mL for
recombinant BNP. The assay detects recombinant BNP and does not
cross react to mouse BNP.
[1520] Analysis was conducted by noncompartmental methods
(WinNonlin; version 4.1; Pharsight Corp., Mountain View, Calif.,
USA). The mean plasma concentration at each time was used in the
analysis. A linear up/log down trapezoidal method was used to
calculate the AUC0-.sub.t. Extrapolation to infinity
AUC.sub.0-.infin. was done by dividing the last observed
concentration by the terminal elimination rate constant. Data were
uniformly weighted for these analyses.
[1521] Result
[1522] The mean baseline concentration of BNP-HSA in plasma as
detected in the pre-dose samples was approximately 0.081-0.095
.mu.g/ml. Following a single intravenous or subcutaneous injection,
BNP-HSA had terminal elimination half-lives of 11.2 (intravenous
delivery) or 19.3 h (subcutaneous delivery), while the half-life of
recombinant BNP in mice was 3.1 min. Non compartmental analysis of
BNP-HSA revealed that BNP-HSA had the following
characteristics:
TABLE-US-00022 TABLE 9 Unit Intravenous Subcutaneous t.sub.max h NA
16 C.sub.max .mu.g/ml NA 11.2 t.sub.1/2,term h 11.2 19.3
AUC.sub.0-.infin. (h .mu.g/ml)/(mg/kg) 658.9 227.9 V.sub.ss ml/kg
37 NA V.sub.z or V.sub.z/F ml/kg 53.5 268 CL or CL/F ml/h/kg 3.3
9.6 MRT h 11.2 19.8 Bioavailability % 34.6 C.sub.max, peak plasma
concentration of the drug; t.sub.max, time of maximum plasma
concentration; AUC.sub.0-.infin., area under the plasma drug
concentration-time curve from time 0 to infinite time;
t.sub.1/2,term, terminal elimination phase half-life; CL, clearance
after intravenous dosing; CL/F, apparent clearance after
subcutaneous dosing; V.sub.ss, volume of distribution at
steady-state after intravenous dosing; V.sub.z: volume of
distribution during the terminal phase after intravenous dosing;
V.sub.z/F, volume of distribution during the terminal phase after
subcutaneous dosing; NA, not applicable.
[1523] Five points at the terminal phase of the intravenous profile
and four points at the terminal phase of the subcutaneous profile
were selected for the terminal half-life calculation. The resulting
AUC during this terminal phase was approximately 10% of the total
AUC for the intravenous and subcutaneous profiles, respectively.
This is compared to only 2% and 4% of the total AUC for the
intravenous and subcutaneous profile, respectively, when the last
three points were selected for the terminal half-life
calculation.
Example 79
Construct ID #3618, BNP(2.times.)-HSA, Generation
[1524] Construct ID #3618, pC4:SPCON.BNP1-32(2.times.)/HSA,
comprises DNA encoding a BNP albumin fusion protein which has a
consensus leader sequence, secretion, followed by two processed,
active BNP peptides (amino acids 1-32) in tandem fused to the
amino-terminus of the mature form of HSA in the mammalian
expression vector pC4.
[1525] Cloning of BNP cDNA for Construct 3618
[1526] The polynucleotide encoding the duplicate BNP moiety was
first PCR amplified from the processed active form of BNP (amino
acids 1-32) using four primers BNP-1, BNP-2, BNP-3, and BNP-4,
described below, to create two fragments A and B. Following
amplification, two purified fragments (A and B) were mixed in an
equal molar amount and used as a PCR template and amplified with
primers BNP-5 and BNP-6, as described below. The BNP(2.times.)
insert was then digested with BamHI and ClaI and ligated into
pC4HSA vector pre-digested with BamHI and ClaI resulting in
construct 3618. Construct ID #3618 encodes an albumin fusion
protein containing a consensus leader sequence, secrecon (SEQ ID
NO:111), and two, tandem copies of the processed, active form of
BNP, followed by the mature HSA protein (see SEQ ID NO:226 for
construct 3618 in Table 2).
[1527] Four oligonucleotides suitable for PCR amplification of the
polynucleotides encoding two fragments of BNP protein were first
synthesized:
TABLE-US-00023 BNP-1 (SEQ ID NO:460)
5'-AGCCCCAAGATGGTGCAAGGGTCTGGCTGCTTTGGGAGGAAGATGGA
CCGGATCAGCTCCTCCAGTGGCTGGGCTGCAAAGTGCTGAGGCGGCA T-3' BNP-2 (SEQ ID
NO:461) 5'-CCTTGCACCATCTTGGGGCTATGCCGCCTCAGCACTTTGC-3' BNP-3 (SEQ
ID NO:462) 5'-GCAAAGTGCTGAGGCGGCATAGCCCCAAGATGGTGCAAGG-3' BNP-4
(SEQ ID NO:463) 5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCATGCCGCCTC
AGCACTTTGC-3'
[1528] Using primer sets BNP-1/BNP-2 and BNP-3/BNP-4, two BNP
proteins fragments (A and B, respectively) were PCR amplified.
Annealing and extension temperatures and times must be empirically
determined for each specific primer pair and template. Fragments A
and B were purified (for example, using Wizard PCR Preps DNA
Purification System (Promega Corp)), mixed at equal molar amounts,
and used as a template for PCR amplification using two additional
oligonucleotides suitable for PCR amplification, BNP-5 and
BNP-6:
TABLE-US-00024 BNP-5: (SEQ ID NO:382)
5'-GAGCGCGGATCCAAGCTTCCGCCATCATGTGGTGGCGCCTGTGGTGG
CTGCTGCTGCTCTGCTGCTGCTGTGGCCCATGGTGTGGGCCAGCCCCAAG CTGGTGCAAGG-3'
BNP-6: (SEQ ID NO:383)
5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCATGCCGCCTC
AGCACTTTGC-3'
[1529] BNP-5 incorporates a Bam HI cloning site (underlined),
polynucleotides encoding a consensus leader sequence (SEQ ID
No:111) (italicized), and polynucleotides encoding the first seven
amino acid sequence of BNP (bolded). In BNP-6, the underlined
sequence is a Cla I site, and the polynucleotides that follow it
contains the reverse complement of DNA encoding the last 6 amino
acids of BNP (bolded) and the first 10 amino acids of the mature
HSA protein. Using these two primers, a consensus leader sequence
and two tandem copies of active BNP peptides were PCR amplified.
Annealing and extension temperatures and times must be empirically
determined for each specific primer pair and template.
[1530] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bam HI and Cla I. After further purification of the Bam HI-Cla
I fragment by gel electrophoresis, the product was cloned into Bam
HI/Cla I digested pC4:HSA to produce construct ID #3618. The
expression construct was sequence verified.
[1531] Expression and Purification of Construct ID #3618.
[1532] Expression in 293F Cells.
[1533] Construct ID #3618, pC4:SPCON.BNP1-32(2.times.)/HSA, was
transfected into 293F cells by methods known in the art (see
Example 6).
[1534] Purification from 293F Cell Supernatant.
[1535] pC4:SPCON.BNP1-32(2.times.)/HSA encoded by Construct ID
#3618 was purified as previously described above in Example 78
under subsection heading "Purification from 203F cell
supernatant."
[1536] The Activity of BNP(2.times.)-HSA can be Assayed Using an In
Vitro NPR-A/cGMP Assay.
[1537] The activity of BNP(2.times.)-HSA encoded by Construct ID
#3618 can be assayed in vitro using an NPR-A/cGMP assay as
previously described in Example 78 under subsection heading, "The
activity of BNP-HSA can be assayed using an in vitro NPR-A/cGMP
Assay," and "Method of Screening NPR-A 203F Stable Clones."
[1538] Result
The dose-response relationship of BNP(2.times.)-HSA and recombinant
BNP were determined (see FIG. 7). The maximal activities of
BNP(2.times.)-HSA encoded by Construct ID #3618, and recombinant
BNP were similar (1.68.+-.0.021, vs. 1.80.+-.0.016 pm,
respectively), with EC50 values of 9.8.+-.1.1, and 0.46.+-.1.1 nM
respectively.
Example 80
In Vitro NPR-A/cGMP Assay for BNP
[1539] Background and Methods:
[1540] Natriuretic peptide receptor-A (NPR-A) is the signaling
receptor for BNP, and as such, is responsible for most of BNP's
biological effects. BNP bioactivity is mediated by NPR-A guanylyl
cyclase domain that converts GTP to cGMP upon activation. A
convenient assay for BNP activity is to measure the BNP stimulation
of a 293F cell line that stably over-expresses NPR-A. The cGMP
production in the cells after exposure to BNP can be measured by
cGMP ELISA.
[1541] Method of Screening NPR-A 293F Stable Clones.
[1542] The open reading frame of human NPR-A was constructed into
pcDNA3.1 expression vector (Invitrogen). 293F cells were stably
transfected with the plasmid DNA by Lipofectamine method and
selected by 0.8 .mu.g/ml G418. 293F/NPR-A stable clones were
screened for best response to recombinant BNP.
[1543] Measurement of cGMP Activation.
[1544] cGMP activation by BNP was carried out in 293F/NPR-A cells
and measured by CatchPoint cyclic-GMP fluorescent assay kit
(Molecular Devices, Sunnyvale, Calif., USA). Briefly, 50,000
cells/well of 293F/NPR-A cells cultured in a 96-well plate were
washed into 80 .mu.l prestimulation buffer (Krebs-Ringer
Bicarbonate Buffer with 10 mM glucose, pH 7.4, 15 nM sodium
bicarbonate, and 0.75 mM 3-isobutyl-1-methylxanthine). BNP-HSA or
recombinant BNP in 40 .mu.l prestimulation buffer was added to the
cells at 37.degree. C. for 10 min. The cells were lysed with 40
.mu.l Lysis Buffer for 10 min with shaking. The amounts of cGMP in
the lysates were quantitated as per the manufacturer's instruction
and EC50 values were determined. In this assay, higher cGMP levels
result in lower signals (relative fluorescent units or RFUs).
[1545] Generation of Construct ID #3796
[1546] Construct ID #3796, pSAC35:HSA.BNP(1-32), comprises DNA
encoding a BNP albumin fusion protein which has a HSAsp/KEX2 leader
sequence, followed by the processed, mature form of HSA, fused to
the N-terminus of the procecessed, mature form of BNP peptide
(amino acids 1-32) in the yeast expression vector pSAC35 (see SEQ
ID NO:214 for construct 3796 in Table 2).
[1547] Cloning of BNP cDNA for Construct 3796
[1548] The polynucleotide encoding BNP was PCR amplified using
primers BNP-102689 and BNP-102692, described below, cut with
Bsu36I/AscI, and ligated into Bsu36I/AscI, cut pSAC-NEC resulting
in construct ID #3796. The template for PCR amplification was
polynucleotides encoding the entire mature BNP(1-32 sequence).
[1549] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the active, processed form of BNP,
BNP-102689 and BNP-102692 were synthesized:
TABLE-US-00025 BNP-102689: (SEQ ID NO:378)
5'-AAGCTGCCTTAGGCTTAAGCCCCAAGATGGTGCAAGGGTC-3' BNP-102692: (SEQ ID
NO:379) 5'-GCACCGGGCGCGCCTTAATGCCGCCTCAGCACTTTGCAGC-3'
[1550] BNP-102689 incorporates a Bsu36I cloning site (underlined),
polynucleotides encoding the last five amino acids of HSA
(italicized), and polynucleotides encoding the first eight amino
acid sequence of BNP (bolded). In BNP-102692, the underlined
sequence is a Asc I site, and the polynucleotides that follow it
contains the reverse complement of DNA encoding the last 8 amino
acids of BNP (bolded) and a termination codon (italicized). Using
these two primers the HSA/BNP fusion region was PCR amplified.
Annealing and extension temperatures and times must be empirically
determined for each specific primer pair and template.
[1551] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bsu36I and Asc I. After further purification of the Bsu36I/Asc
I fragment by gel electrophoresis, the product was cloned into
Bsu36I/Asc I digested pSAC35-NEC to produce construct ID #3796. The
expression construct was sequence verified.
[1552] Expression and Purification of Construct ID 3796.
[1553] Expression in S. cerevisiae.
[1554] Construct ID #3796, pSAC35:HSA.BNP(1-32), was transfected
into BXP10 cells by methods known in the art (see Example 4).
[1555] Purification from BXP10 Cell Supernatant.
[1556] After approximately 84 hours, the culture was harvested and
clarified of cells through centrifugation and 0.2 .mu.m filtration.
The recombinant protein was captured by 5 ml Blue Sepharose fast
flow column (GE Healthcare) and eluted by a combination of sodium
chloride and sodium octanoate. Some preparation were completed by
binding the protein to a ceramic hydroxyl appetite column (BioRad)
and eluting with an increased concentration of phosphate. Other
preparations were bound to a DEAE Sepharose fast flow column (GE
Heathcare) and eluted by a linear sodium chloride gradient. The
elution pool was then bound to a Q sepharose high performance
column (GE Healthcare) and eluted with a sodium chloride gradient.
The final product was concentrated and exchanged in formulation
buffer by tangential flow filtration.
[1557] Generation and Cloning of Construct ID #3959
[1558] Construct ID #3959, pSAC35:HSA.BNP(1-29), comprises DNA
encoding a BNP albumin fusion protein which has a HSAsp/KEX2 leader
sequence, followed by the processed, mature form of HSA, fused to
the N-terminus of the procecessed, mature form of BNP peptide
lacking the last three amino acids (S1-L29) in the yeast expression
vector pSAC35 (see SEQ ID NO:501 for construct 3959 in Table
2).
[1559] The polynucleotide encoding BNP was PCR amplified using
primers BNP-103801 and BNP-104315, described below, cut with
Bsu36I/AscI, and ligated into Bsu36I/AscI, cut pSAC-NEC resulting
in construct ID #3959. The template for PCR amplification was
primer construct ID 3796 (see below).
[1560] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the active, processed form of BNP,
BNP-103801 and BNP-104315 were synthesized:
TABLE-US-00026 BNP-103801: (SEQ ID NO:578)
5'-CAGGAGCCCCTTAGGCTTAAGCCCCAAGATGGTGCAAGGGTCT-3' BNP-104315: (SEQ
ID NO:579) 5'-CCTCACTCGGCGCGCCTTACAGCACTTTGCAGCCCAGGCCACTGG
A-3'
[1561] BNP-103801 incorporates a Bsu36I cloning site (underlined),
polynucleotides encoding the last five amino acids of HSA
(italicized), and polynucleotides encoding the first eight amino
acid sequence of BNP (bolded). In BNP-104315, the underlined
sequence is a Asc I site, and the polynucleotides that follow it
contains the reverse complement of DNA encoding the S21 through L29
amino acids of BNP (bolded) and a termination codon (italicized).
Using these two primers the HSA/BNP fusion region was PCR
amplified. Annealing and extension temperatures and times must be
empirically determined for each specific primer pair and
template.
[1562] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bsu36I and Asc I. After further purification of the Bsu36I/Asc
I fragment by gel electrophoresis, the product was cloned into
Bsu36I/Asc I digested pSAC35-NEC to produce construct ID #3959. The
expression construct was sequence verified.
[1563] Expression and Purification of Construct ID 3959.
[1564] Expression in S. cerevisiae.
[1565] Construct ID #3959, pSAC35:HSA.BNP(S1-L29), was transfected
into BXP10 cells by methods known in the art (see Example 4).
[1566] Purification from BXP10 Cell Supernatant.
[1567] After approximately 84 hours, the culture was harvested and
clarified of cells through centrifugation and 0.2 .mu.m filtration.
The recombinant protein was captured by 5 ml Blue Sepharose fast
flow column (GE Healthcare) and eluted by a combination of sodium
chloride and sodium octanoate. Some preparation were completed by
binding the protein to a ceramic hydroxyl appetite column (BioRad)
and eluting with an increased concentration of phosphate. Other
preparations were bound to a DEAE Sepharose fast flow column (GE
Heathcare) and eluted by a linear sodium chloride gradient. The
elution pool was then bound to a Q sepharose high performance
column (GE Healthcare) and eluted with a sodium chloride gradient.
The final product was concentrated and exchanged in formulation
buffer by tangential flow filtration.
[1568] Result
[1569] The dose-response relationship of HSA-BNP(1-29) and
HSA-BNP(1-32) and recombinant BNP were determined (see FIG. 10).
The EC50 value of HSA-BNP(1-29) and HSA-BNP(1-32) were several fold
greater than the EC50 value of recombinant BNP(467.9, 45.06, and
0.2227, respectively). In addition, the HSA-BNP(1-29) fusion
protein had a 10 fold greater EC50 value than HSA-BNP(1-32) fusion
protein.
Example 81
In Vitro Assay for Degradation of ANP and BNP
[1570] Background and Methods:
[1571] Neprilysin, also known as MME, CALLA, CD10, Common acute
lymphocytic leukemia antigen, Enkephalinase, EPN, NEP, Neutral
endopeptidase, or Neutral endopeptidase 24.11, is a 743 amino acid
(MW 90,000-110,000 kD) cell-surface metallopeptidase expressed by
numerous tissues, including, but not limited to, prostate, kidney,
intestine, endometrium, adrenal glands, and lung. Neprilysin
inactivates a variety of physiologically active peptides,
including, but not limited to, ANP, BNP, CNP, substance P,
bradykinin, oxytocin, Leu- and Met-enkephalins, neurotensin,
bombesin, endothelin-1, and bombesin-like peptides, by cleaving the
amino side of hydrophobic residues.
[1572] The relative susceptibility to neprilysin hydrolysis has
been determined to be approximately 4-5 minutes for CNP, 8 minutes
for ANP, and 2 hours for BNP. (Kenny, A. J. et al., Biochem J.
291(1): 83-8 (1993)).
[1573] The Effect of Neprilysin on ANP and BNP Peptides
[1574] ANP and BNP peptides were assayed for activity in the
CatchPoint cGMP assay (Molecular Devises) with or without exposure
to the protease neprilysin. Particuarly, 5 .mu.M of ANP or BNP was
incubated for 24 hours at 37.degree. C. in MES buffer (0.1M
2-(N-morpholino)ethanesulfonic acid (Sigma)) with or without 10 nM
neprilysin (R&D Systems). 293F cells, which have been stably
transfected with NPR-A as described in Example 80, were then
stimulated with various concentrations of protease-treated ANP or
BNP. Cell lysates were then analyzed for cGMP activation using the
CatchPoint cGMP assay (Molecular Devices) as described in Example
80.
[1575] Results
[1576] Dose-response curves were calculated for BNP and ANP with or
without incubation with neprilysin. (See, FIG. 11A). BNP with or
without incubation with neprilysin exhibited similar BNP activity
with EC50 values of 0.2966 and 0.2702, respectively. However,
incubation of ANP with neprilysin resulted in a significant
reduction in ANP activity compared to untreated ANP with EC50
values of 0.2965 and 60.47, respectively. Select samples were
further analyzed by reverse-phase HPLC, using techniques known in
the art. Percent comparisons are to samples incubated for the same
period of time in the absence of neprilysin. (See, FIG. 11D)
Although ANP proteolysis occurs within 20 minutes of treatment with
neprilysin, significant BNP proteolysis is not observed even after
24 hours of incubation with neprilysin.
[1577] The Effect of Neprilysin on ANP-HSA Fusion Proteins
[1578] ANP and ANP-HSA (CID 3484) were incubated in MES buffer
(0.1M 2-(N-morpholino)ethanesulfonic acid (Sigma)) with or without
10 nM neprilysin (R&D Systems) for 20 minutes, 1 hour, or 24
hours at 37.degree. C. 293F cells, which have been stably
transfected with NPR-A as described in Example 80, were then
stimulated with various concentrations of protease-treated ANP or
ANP-HSA. Cell lysates were then analyzed for cGMP activation using
the CatchPoint cGMP assay (Molecular Devices) as described in
Example 80.
[1579] Results
[1580] Dose-response curves were calculated for ANP and ANP-HSA
with or without incubation with neprilysin. (See, FIGS. 11B and
11C, respectively). ANP peptide demonstrated a significant
reduction in activity within 1 hour of treatment with neprilysin.
However, ANP-HSA (CID 3484) demonstrated no significant reduction
in activity even after 24 hours of treatment with neprilysin.
Select samples were further analyzed by reverse-phase HPLC, using
techniques known in the art. Percent comparisons are to samples
incubated for the same period of time in the absence of neprilysin.
(See, FIG. 11D). Although ANP proteolysis occurs within 20 minutes
of treatment with neprilysin, no proteolysis of ANP-HSA (CID 3484)
was observed even after 24 hours of incubation with neprilysin.
Example 82
Anti-Viral Activity of HSA-IFN.alpha.2b, in Combination with
Ribavirin in Genotype 1, Interferon-Naive Chronic Hepatitis C(HCV)
Patients
[1581] Background
[1582] Conventional treatment for Genotype 1, interferon-naive
(IFN-naive) HCV patients utilizes interferon .alpha. in combination
with ribavirin (RBV) for 48 weeks. However, this treatment has
significant practical limitations. Due to the well-known side
effects of current interferon therapy, patients' quality of life is
substantially decreased after each administration of interferon.
Current protocols require dosing at least weekly, resulting in a
prolonged period of decreased quality of life. Large numbers of
patients discontinue treatment as a result, with some studies
reporting discontinuation rates over 50%. Moreover, current
interferon therapy also has a considerable rate of significant
hematological reductions, which can require a reduction in RBV
dose, or more significantly, in a temporary termination of the
interferon treatment regimen until the hematological values
normalize. Thus, there is a clear need for improved therapeutic
protocols for the treatment of genotype 1 HCV in IFN-naive
patients.
[1583] Rationale:
[1584] HSA-IFN.alpha.2b was generated by genetically fusing mature
albumin at its C-terminus to the N-terminus of mature interferon
.alpha.-2b. The safety and efficacy of treatment with
HSA-IFN.alpha.2b in combination with RBV was evaluated in an active
controlled clinical study in genotype 1, IFN-naive HCV human
patients, and compared to conventional treatment with
PEG-IFN.alpha.-2a (PEG-IFN) in combination with RBV as an active
control.
[1585] Methods:
[1586] Human HCV patients who were genotype 1, IFN-naive were
treated with either HSA-IFN.alpha.2b or active control, PEG-IFN, in
combination with ribavirin (RBV). More particularly, 458 human
subjects were randomized into 4 subcutaneous (sc) treatment groups:
(a) PEG-IFN dosed at 180 .mu.g once weekly (Q1w); (b)
HSA-IFN.alpha.2B dosed at 900 .mu.g once every 2 weeks (Q2w); (c)
HSA-IFN.alpha.2b dosed at 1200 .mu.g Q2w; or (d) HSA-IFN.alpha.2b
dosed at 1200 .mu.g once every 4 weeks (Q4w)).
[1587] Each subject in each treatment group also received 1000-1200
mg/day RBV based on body weight. The basis for stratification
included body mass index (BMI) (<25 kg/m.sup.2 or .gtoreq.25
kg/m.sup.2) and HCV RNA titer (<800,000 IU/ml or >800,000
IU/ml).
[1588] Treatment duration of the study is 48 weeks with a 24 week
follow-up. The primary efficacy endpoint of the study is sustained
virologic response (SVR).
[1589] HCV RNA titer was measured using a real-time PCR assay,
Quantasure.TM. (Labcorp) with a sensitivity range (level of
quantification (LOQ)) of 43 IU to 69 million IU/mL and a level of
detection (LOD) of 10 IU/mL. Alanine Transferase (ALT) and
hematologic effects, including absolute neutrophil count (ANC),
hemoglobin, and platelet count were measured using standard
techniques known in the art.
[1590] Intent to treat (ITT) patients are defined as all randomized
and treated subjects of each treatment group, regardless of whether
or not the patient has any missing data points or has dropped out
of the study. Modified intent to treat (MITT) patients are defined
as those patients that could conceivably have a Week 24 visit based
on their date of enrollment in the study.
[1591] Results and Discussion:
[1592] Subject demographics, antiviral response and hematologic
reductions are summarized in Table 10 (preliminary interim
analysis) and Table 11 (final interim analysis). Overall, all four
treatment protocols were well tolerated and there were no
significant difference between the treatment groups with respect to
grade 3-4 lab values or discontinuations due to adverse events.
TABLE-US-00027 TABLE 10 Preliminary Interim Analysis of
Demographic, Anti-viral Response, and Hematological Effect HSA-
HSA- PEG-IFN IFN.alpha.2b HSA- IFN.alpha.2b 180 .mu.g 1200 .mu.g
IFN.alpha.2b 1200 .mu.g Q1w Q2w 900 .mu.g Q2w Q4w (n = 114) (n =
110) (n = 118) (n = 116) Demographics Male 58% 58% 55% 67% Mean BMI
(kg/m.sup.2) 25.1 25.7 25.4 26.1 BMI .gtoreq. 25 kg/m.sup.2 53% 49%
49% 51% Median HCV RNA 6.0 6.0 6.1 6.1 (log IU/ml) RNA .gtoreq.
800,000 62% 55% 56% 62% IU/ml Efficacy at Week 12 HCV RNA Negative
70/112 77/104 74/112 57/109 (<LOQ) (62.5%).sup. (74.0%).sup.
(66.1%) (52.3%)* BMI <25 kg/m.sup.2 vs. 71.1% vs. 74.1% vs.
70.7% vs. 55.6% vs. .gtoreq.25 kg/m.sup.2 54.2% 74.0% 61.1% 49.1%
EVR12 (.gtoreq.2 log 96/112 91/104 90/112 80/109 reduction) (85.7%)
(87.5%) (80.4%) (73.4%) 2.sup.nd phase slope >0.6 49% 58% 52%
42% log/wk Normalization of ALT 12/73 17/80 18.81 27/83 (16.4%)
(21.2%) (22.2%) (32.5%).sup.# Laboratory at Week 12 ANC <
750/mm.sup.3 17.5% 20.0% 22.0% 4.3%** ANC < 500/mm.sup.3 2.6%
3.6% 3.4% 0.9% Hb < 12 g/dL 64% 70.0% 69.5% 49.1%** Platelet
< 50,000/mm.sup.3 3.5% 2.7% 1.7% 0.9% *p-value < 0.05
HSA-IFN.alpha.2b 1200 .mu.g Q2w vs. Q4w; .sup. p-value 0.068
HSA-IFN.alpha.2b 1200 .mu.g Q2w vs. PEG-IFN; .sup.#p-value <
0.05 HSA-IFNA2B 1200 .mu.g Q4w vs. PEG-IFN; **p-value < 0.05
HSA-IFN.alpha.2b 1200 .mu.g Q4w vs. all other groups
TABLE-US-00028 TABLE 11 Final Interim Analysis Demographic,
Anti-viral Response, and Hematological Effect HSA- HSA- PEG-IFN
IFN.alpha.2b HSA- IFN.alpha.2b 180 .mu.g 1200 .mu.g IFN.alpha.2b
1200 .mu.g Q1w Q2w 900 .mu.g Q2w Q4w (n = 114) (n = 110) (n = 118)
(n = 116) Demographics Male 58% 58% 56% 67% Mean BMI (kg/m.sup.2)
25.1 25.7 25.6 26.0 BMI .gtoreq. 25 kg/m.sup.2 50% 48% 51% 50%
Median HCV RNA 6.1 6.0 6.1 6.1 (log IU/ml) RNA .gtoreq. 800,000 62%
55% 56% 62% IU/ml Efficacy at Week 12 ITT HCV RNA Negative 75/114
82/110 82/118 62/116 (<LOQ) (65.8%).sup. (74.5%).sup. (69.5%)
(53.4%)* BMI <25 kg/m.sup.2 vs. 71.9% vs. 77.2% vs. 74.1% vs.
56.9% vs. .gtoreq.25 kg/m.sup.2 59.6% 71.7% 65.0% 50.0% EVR12
(.gtoreq.2 log 101/114 99/110 99/118 88/116 reduction) (88.6%)
(90.0%) (83.9%) (75.9%) 2.sup.nd phase slope >0.6 49% 58% 52%
42% log/wk Normalization of ALT 11/73 17/79 17/80 26/83 (15.1%)
(21.5%) (21.3%) (31.3%).sup.# Efficacy at Week 20 ITT HCV RNA <
LOD 77/114 82/110 83/118 70/116 (undetectable) (67.5%) (74.5%)
(70.3%) (60.3%) Efficacy at Week 24 MITT HCV RNA < LOD 57/90
64/91 58/87 58/83 (undetectable) (63.3%) (70.3%) (66.7%) (69.9%)
Laboratory at Week 12 ANC < 750/mm.sup.3 20.2% 21.8% 22.0%
6.0%** ANC < 500/mm.sup.3 3.5% 3.6% 3.4% 1.7% Hb < 12 g/dL
65% 74% 70% 52%** Platelet < 50,000/mm.sup.3 3.5% 2.7% 1.7% 0.9%
*p-value < 0.05 HSA-IFN.alpha.2b 1200 .mu.g Q2w vs. Q4w; .sup.
p-value 0.15 HSA-IFN.alpha.2b 1200 .mu.g Q2w vs. PEG-IFN;
.sup.#p-value < 0.05 HSA-IFNA2B 1200 .mu.g Q4w vs. PEG-IFN;
**p-value < 0.05 HSA-IFN.alpha.2b 1200 .mu.g Q4w vs. all other
groups
[1593] Antiviral Response Predictors of SVR
[1594] An antiviral response predictor of SVR was defined as a
negative HCV RNA titer (i.e., having an HCV RNA titer <LOQ) at
week 12 of treatment with a second phase slope >0.6 log/wk
(2.sup.nd slope). The phases of the antiviral response curve slope
are indicative of two activities. The first phase shows the direct
antiviral activity of the response. The second phase predicts the
destruction of the HCV infected cells by the treated compound. Thus
a value of the second phase slope at >0.6 log/wk is a good
predictor of SVR (positive predictive value (PPV)>90%).
[1595] Antiviral response predictors of SVR at week 12 ITT were
greatest in the HSA-IFN.alpha.2b 1200 .mu.g Q2w treatment group
where 82/110 subjects or 74.5% ((final interim analysis) (77/104 or
74.0% (preliminary analysis))) had HCV RNA negative levels (i.e.,
levels below the LOQ (<43 IU/mL)) and 58% exhibited a second
phase slope >0.6 log/wk, as compared to 75/114 subjects or 65.8%
((final interim analysis) (70/112 or 62.5% (preliminary interim
analysis))) and 49% in the PEG-IFN control treatment. These data
indicate that the HSA-IFN.alpha.2b 1200 .mu.g Q2w treatment
protocol has antiviral activity that is at least comparable to
conventional treatment with PEG-IFN at week 12. Because RNA
negativity (i.e., the number of patients having HCV RNA titer
levels below the LOQ) at week 12 in the HSA-IFN.alpha.2b 1200 .mu.g
Q2w treatment protocol is approximately 9% (final interim analysis)
and 12% greater (preliminary interim analysis) and the second phase
slope is approximately 9% greater (for both the final and
preliminary interim analysis) compared to the PEG-IFN control
treatment, it is likely that the HSA-IFN.alpha.2b 1200 .mu.g Q2w
treatment protocol may result in superior efficacy over the
conventional PEG-IFN treatment. The number of patients having HCV
RNA negativity in the HSA-IFN.alpha.2b 900 .mu.g Q2w and
HSA-IFN.alpha.2b 1200 .mu.g Q4w treatment groups were similar to
conventional treatment with PEG-IFN.
[1596] Antiviral response predictors of SVR at weeks 20 and 24 are
indicated as subjects having HCV RNA titers below the level of
detection (LOD) (i.e., <10 IU/mL). Antiviral response predictors
of SVR at week 20 was greatest in the HSA-IFN.alpha.2b 1200 .mu.g
Q2w treatment group, where 82/110 subjects or 74.5% (final interim
analysis) had undetectable HCV RNA levels (i.e., <10 IU/mL) as
compared to 77/114 or 67.5% (final interim analysis) in the PEG-IFN
control treatment. Similarly, antiviral response predictors of SVR
at week 24 was greatest in the IFN.alpha.2b 1200 .mu.g Q2w
treatment group where 64/91 or 70.3% (final interim analysis) had
undetectable HCV RNA levels whereas the PEG-IFN control treatment
had 57/90 or 63.3% with undetectable HCV RNA levels. Both the week
20 and 24 data indicate that the HSA-IFN.alpha.2b 1200 .mu.g Q2w
treatment protocol has antiviral activity and safety that is at
least comparable to conventional treatment with PEG-IFN with an
improved dosing schedule.
[1597] Normalization of ALT Levels
[1598] A common measurement of liver function in a patient is the
level of alanine transferase (ALT). One of the hallmarks of HCV
infected patients is high serum ALT levels that are indicative of
liver damage. Thus, the normalization of ALT levels corresponds
with improvement in liver function and has a favorable prognosis
for responding to treatment. Although all treatment protocols
exhibited some ability to normalize ALT levels, the most dramatic
effect was observed in the HSA-IFN.alpha.2b 1200 .mu.g Q4w
treatment protocol, where over twice as many patients (final and
preliminary interim analysis) achieved normalized ALT levels as
compared to conventional treatment with PEG-IFN. Thus,
HSA-IFN.alpha.2b dosed at 1200 .mu.g Q4w is surprisingly more
effective than conventional PEG-IFN treatment in normalizing liver
function in genotype 1, IFN-naive HCV patients.
[1599] Hematological Effects
[1600] Ensuring compliance and exposure to full doses of IFN and
RBV in combination treatment protocols is critical for maximizing
SVR rates.
[1601] Hematological reductions are common during combination
treatment with IFN and RBV. Reductions in hemoglobin (Hb) due to
RBV-induced hemolysis require dose reduction in RBV. In particular,
Hb <12 g/dL requires reducing RBV from 1000-1200 mg/day to 800
mg/day. RBV dose is critical to prevent HCV relapse. The
HSA-IFN.alpha.2b 1200 .mu.g Q4W treatment protocol surprisingly had
significantly fewer reductions in Hb <12 g/dL (52% vs. 65% for
PEG-IFN (final interim analysis); 49.1 vs. 64% for PEG-IFN
(preliminary interim analysis)). This may translate to a lower
relapse rate with the HSA-IFN.alpha.2b 1200 .mu.g Q4W treatment
protocol, allowing for improved SVR.
[1602] Reductions in ANC <750/mm.sup.3 requires dose reducing
the IFN component of the combination treatment. Surprisingly, the
HSA-IFN.alpha.2b 1200 .mu.g Q4W treatment protocol had
significantly fewer ANC <750/mm.sup.3 compared to PEG-IFN (6%
vs. 20.2%, respectively (final interim analysis); 4.3% vs. 17.5%
(preliminary interim analysis)). This again may translate to higher
SVE rates given the fewer dose reductions required with the
HSA-IFN.alpha.2b 1200 .mu.g Q4W treatment protocol.
[1603] Similar hematological reductions occurred across the
HSA-IFN.alpha.2b Q2w and PEG-IFN treatment groups at week 12.
Surprisingly, however, the hematological reductions observed in the
HSA-IFN.alpha.2b 1200 .mu.g Q4w treatment group at week 12 were
approximately 75% lower than those observed in the PEG-IFN
treatment group. These results indicate that HSA-IFN.alpha.2b Q4w
may offer a superior safety profile and improved relapse rate as
compared to conventional PEG-IFN treatment.
[1604] Conclusion
[1605] At week 12, the maximal antiviral activity in genotype 1,
IFN-naive HCV was observed in the HSA-IFN.alpha.2b 1200 .mu.g Q2w
treatment group. Similar effects on hematological reductions were
also observed in the HSA-IFN.alpha.2b 1200 .mu.g Q2w and PEG-IFN
treatment groups. Moreover, at weeks 20 and 24, the maximal
antiviral activity was also observed in the HSA-IFN.alpha.2b 1200
.mu.g Q2w treatment group. Comparable antiviral activity was
continued to be observed at weeks 20 and 24 in the HSA-IFN.alpha.2b
900 .mu.g Q2w treatment group. Accordingly, HSA-IFN.alpha.2b 900
.mu.g Q2w may offer at least a comparable efficacy and safety
profile to the current standard of care, PEG-IFN, with an improved
dosing schedule, translating into a greater convenience for the
patient. Additionally, 1200 .mu.g Q2w may offer at least a
comparable safety profile to the current standard of care, PEG-IFN,
with a possible superior efficacy and an improved dosing schedule,
translating into a greater convenience for the patient.
[1606] Treatment with the HSA-IFN.alpha.2b 1200 .mu.g Q4w protocol
surprisingly showed comparable efficacy at week 12 compared to the
conventional PEG-IFN treatment even though subjects receiving the
PEG-IFN conventional treatment received three additional doses due
to the dosing schedule. Comparable efficacy of the HSA-IFN.alpha.2b
1200 .mu.g Q4w compared to the conventional PEG-IFN treatment
continued through weeks 20 and 24. Improved ability to stabilize
liver function and dramatically reduce the reduction in
hematological factors was also observed with HSA-IFN.alpha.2b 1200
.mu.g Q4w treatment group at week 12, indicating an improvement in
liver injury and a reduction in incidence of dose-reduction or
temporary termination, and possibly relapse following treatment, in
these patients. Thus, these results suggest that the treatment with
HSA-IFN.alpha.2b 1200 .mu.g Q4w may offer efficacy comparable to
combination treatment with PEG-IFN with the advantage of an
improved dosing schedule, a greater ability to normalize liver
function, and lower hematological reductions, resulting in greater
compliance and convenience for the patient, and more favorable
outcome post-therapy. In summary, given recent advances in the
field of HCV therapeutics, an HSA-IFN.alpha.2b Q4W treatment
protocol would have the ideal attributes (e.g., comparable
efficacy, superior tolerability, superior convenience resulting in
greater compliance) to become the interferon-backbone-of-choice for
an interferon-antiviral combination therapy.
[1607] Taken together, these results suggest that combination
treatment of genotype 1, IFN-naive HCV patients with
HSA-IFN.alpha.2b and RBV is at least as effective as treatment with
the conventional PEG-IFN and RBV combination treatment with the
advantage of an improved dosing schedule. Particularly, these
results suggest that treatment with HSA-IFN.alpha.2b in combination
with RBV can have either an superior efficacy with a similar safety
profile, a similar efficacy with an superior safety profile, or
both a superior efficacy and safety profile compared to
conventional PEG-IFN combination treatment with RBV, but with an
improved and highly advantageous dosing schedule.
Example 83
Response of Chronic Hepatitis C(HCV) Non-Responder Patients to
HSA-IFN.alpha.2b, in Combination with Ribavirin
[1608] Background
[1609] Over four million people in the United States have been
infected with the hepatitis C virus (HCV), making the virus the
most common cause of liver disease in the United States. Interferon
alpha (IFN.alpha.) with or without concurrent treatment of the
antiviral molecule, ribavirin (RBV), has historically been
recognized as the most effective treatment for patients. More
recently, pegylated forms of interferon alpha have been approved
for treatment of HCV in combination with RBV. These peglyated
interferons have been shown to be more effective in treating HCV
than the standard interferon or interferon in combination with
ribavirin therapies and have become the conventional treatment for
HCV.
[1610] However, this treatment has significant practical
limitations. In addition to the well-known side effects that
substantially decrease a patient's quality of life during therapy
and the considerable rate of significant hematological reductions
associated with the conventional therapy, the conventional therapy
is ineffective for a large proportion of the patients who undergo
the current treatment for HCV. Clinical studies of treatment of HCV
patients who have not previously received an IFN.alpha.-RBV therapy
have shown that approximately 45% of patients who commence the
conventional treatment fail to clear HCV and remain chronically
infected (e.g., non-responders). In the community, the proportion
of patients responding to therapy is considerably less.
[1611] Clinical investigators have responded to the need of the
non-responder population of HCV patients who had failed to respond
to a previous treatment with IFN.alpha. or in combination with RBV
by retreating these patients with the conventional therapy of
pegylated IFN.alpha. and RBV. See, Shiffman et al., "Peginterferon
alfa-2a and ribavirin in patients with chronic hepatitis C who have
failed prior treatment," Gastroenterology 126(4):1015-23 (2004).
Although 35% of patients enrolled in this trial had no evidence of
HCV RNA after 20 weeks of retreatment with the conventional
therapy, many of these patients relapsed after treatment was
discontinued. Thus, only 18% of the patients actually achieved a
sustained viral response (SVR) and were cured of HCV. Similarly,
when non-responder patients who had failed a previous treatment
with conventional pegylated IFN.alpha. therapy were retreated with
another pegylated IFN.alpha., only .about.5-10% of these patients
were able to achieve SVR based on anecdotal evidence. Thus, the
population of patients who have not only failed one interferon
therapy but have failed all current interferon therapies continues
to significantly grow. Accordingly, there is a clear need for
improved therapeutic protocols not only for the treatment of HCV in
general but also for alternative therapies for treatment of those
patients who have been previously treated with an interferon
therapy (e.g., IFN.alpha. treatment-experienced) and who are
non-responders, particularly those non-responder patients who are
the most difficult to treat (e.g., patients who have failed
previous treatment or retreatment with the current conventional
care).
[1612] Rationale
[1613] HSA-IFN.alpha.2b was generated by genetically fusing mature
albumin at its C-terminus to the N-terminus of mature interferon
.alpha.-2b. The safety, tolerability, and efficacy of treatment
with HSA-IFN.alpha.2b in combination with RBV was evaluated in
randomized, open label clinical study in IFN.alpha.
treatment-experienced, non-responder HCV human patients. For the
purposes of this study, non-responders were defined as either those
HCV patients who had stopped previous therapy at week 12 due a
failure to achieve a 2-log reduction in HCV RNA levels (e.g., early
viral response, week 12 or EVR12) or those patients who failed to
achieve SVR after completion of the treatment protocol. Patients
who relapsed after discontinuation of therapy were excluded from
the study. In addition, at least 50% of the patients in the study
previously failed a pegylated IFN.alpha. treatment protocol.
[1614] Methods
[1615] Human IFN.alpha. treatment-experienced, non-responder HCV
patients who previously failed at least one IFN.alpha. treatment
protocol were randomized into 3 subcutaneous (sc) HSA-IFN.alpha.2b
treatment groups: (a) 900 .mu.g once every 2 weeks (Q2w); (b) 1200
.mu.g Q2w and (c) 1200 .mu.g once every 4 weeks (Q4w). Each subject
in each treatment group also received 1000-1200 mg/day RBV. After
evaluation of safety data from these initial three cohorts,
HSA-IFN.alpha.2b was dose escalated with the sequential addition of
two additional cohorts who received HSA-IFN.alpha.2b at either 1500
.mu.g Q2w or 1800 .mu.g Q2w in combination with 1000-1200 mg/day
RBV.
[1616] Treatment duration of the study is 48 weeks with a 24 week
follow-up. The primary efficacy endpoint of the study is sustained
virologic response (SVR).
[1617] HCV RNA titer was measured using a real-time PCR assay,
Quantasure.TM. (Labcorp) with a sensitivity range of 10 IU to 100
million IU/mL). Alanine Transferase (ALT) and hematologic effects,
including absolute neutrophil count (ANC), hemoglobin, and platelet
count were measured using standard techniques known in the art.
[1618] Results and Discussion
[1619] Demographics
[1620] Numerous demographic characteristics have been identified
that serve as independent indicators of those patients who have a
prevalence toward non-responding to treatment. These key
pre-treatment predictors of non-responsiveness include (1) genotype
1, (2) high baseline median HCV RNA level, (3) prior non response
to PEG+RBV therapy, (4) African-American, (5) an advanced Fibrosis
level of F3-F4 (using the METAVIR.RTM. classification), and (6)
high BMI (e.g., .gtoreq.25 mg/kg). Perhaps, the best overall
indicator for non-responsiveness is previous failure of PEG-RBV
treatment and in the context of the number of previous IFN based
regimens failed.
[1621] Subject demographics are summarized in Table 12. Overall,
all the subject demographics were similar in all the treatment
groups. The majority of subjects had been exposed to more than one
IFN.alpha. containing regimen and had failed previous therapy with
PEG+RBV. In addition, while the baseline disease characteristics
were comparable across the 5 treatment groups, the 1800 .mu.g Q2w
treatment group had a significantly higher pre-treatment HCV RNA
and the highest proportion of prior PEG+RBV failures. Thus, the
subjects in the 1800 .mu.g Q2w treatment group represented the most
treatment-refractory patient population.
TABLE-US-00029 TABLE 12 Demographics and Baseline Characteristics
900 .mu.g 1200 .mu.g 1200 .mu.g 1500 .mu.g 1800 .mu.g Q2w Q2w Q4w
Q2w Q2w N = 23 N = 24 N = 24 N = 22 N = 22 P value Genotype 1 20
(87.0%) 24 (100%) 22 (91.7%) 21 (95.5%) 21 (95.5%) 0.4531 Med HCV
7.1 6.6 6.2 7.0 7.6 <0.0001 RNA (log.sub.10IU/mL) PEG + RBV 14
(60.9%) 16 (66.7%) 15 (62.5%) 16 (72.2%) 20 (90.9%) 0.1370 African
2 (8.7%) 3 (12.5%) 1 (4.2%) 7 (31.8%) 2 (9.1%) 0.0940 American
F3-F4 7 (30.4%) 7 (29.2%) 4 (16.7%) 9 (40.9%) 7 (31.8%) 0.5012 BMI
.gtoreq. 25 kg/m2 20 (87.0%) 21 (87.5%) 18 (75.0%) 18 (81.8%) 17
(77.3%) 0.7637
[1622] Efficacy and Biological Activity
[1623] Reductions in HCV RNA from pre-treatment levels over the
treatment duration are shown in Table 13 for genotype 1, PEG+RBV
nonresponders, the most refractory HCV patient population. At weeks
2-12, the magnitude of HCV RNA reduction was comparable across the
900-1500 .mu.g treatment groups. However, the maximal viral load
reductions were observed in the 1800 .mu.g treatment group. This
was surprising considering the higher levels of pre-treatment HCV
RNA and the highest proportion of PEG+RBV failures in this
treatment group. The magnitude of antiviral response over the first
12 weeks of therapy reflects the second phase slope of viral
kinetics and is a positive predictor of SVR.
[1624] As shown in FIG. 12, the slopes of HCV RNA reduction are
comparable at week 12 for the 900-1500 .mu.g treatment groups in
genotype 1, PEG+RBV nonresponders. Surprisingly, the magnitude of
HCV RNA reduction is greatest for the 1800 .mu.g treatment group.
The HCV RNA reductions for the 1500 and 1800 .mu.g treatment groups
are comparable at week 24 in this sub-group of patients.
[1625] At week 24, the proportion of subjects who are HCV RNA
negative was comparable across the 900-1500 .mu.g treatment groups.
Subjects were allowed to discontinue at week 24 for lack of
efficacy at the discretion of the investigator and given the
cumulative data from interferon-based regimens demonstrating the
high negative predictive value of lack of EVR12 and week 24 RNA
negativity for SVR. The overall end-of-treatment response (ETR, HCV
negative at week 48) was 30% (22/73) for the 900-1200 .mu.g
treatment groups. Thus, a high proportion of subjects who became
HCV RNA negative at week 12 (e.g., EVR12) or at week 24 achieved
ETR. In addition, the majority of subjects (13/22) continued to be
HCV RNA negative at the week 12 follow-up after 48 weeks of
treatment. This indicates that the potential SVR following
treatment with HSA-IFN.alpha.2b/RBV is 18%.
[1626] In summary, these data indicate that treatment of IFN.alpha.
treatment-experienced, non-responder HCV patients, with a high
percentage of PEG+RBV failures with 900-1200 .mu.g of
HSA-IFN.alpha.2b in combination with RBV results in robust and
comparable antiviral activity. A low viral breakthrough (e.g., HCV
RNA undetectable but subsequently positive at two or more time
points) and relapse rate was also observed in this treatment
refractory non-responder population. In addition, a significantly
greater reduction was also observed in the 1800 .mu.g treatment
group over the first 12 weeks of therapy, indicating that patients
treated at this dose with HSA-IFN.alpha.2b in combination with
ribavirin may have a significant increase in SVR rates.
TABLE-US-00030 TABLE 13 Antiviral Response for GT1 and PEG+RBV
Non-responders 900 .mu.g 1200 .mu.g 1200 .mu.g 1500 .mu.g 1800
.mu.g Q2w Q2w Q4w Q2w Q2w N = 12 N = 16 N = 13 N = 15 N = 19 Week
12 EVR12.sup.a 5 (41.7%) 4 (25%) 3 (23.1%) 5 (33.3) 12 (63.2%) 95%
C.I..sup.b (15.1%, 72.3%) (7.3%, 52.4%) (7.2%, 52.4%) (11.8%,
61.6%) (38.4%, 83.7%) Week 24 Undetectable 2 (16.7%) 3 (18.8%) 2
(15.4%) 4 (26.7%) 6 (31.6%) HCV RNA 95% C.I..sup.b (2.1%, 48.4%)
(4.0%, 45.6%) (1.9%, 45.4%) (7.8%, 55.1%) (12.6%, 56.6%) Week 48
ETR.sup.a 3 (25%) 3 (18.8%) 2 (15.4%) 95% C.I..sup.b (5.5%, 57.2%)
(4.0%, 45.6%) (1.9%, 45.4%) .sup.aDefined as undetectable HCV RNA
or a .gtoreq.2-log reduction in HCV RNA .sup.bExact 95% confidence
interval.
[1627] Hematological Effects
[1628] Ensuring compliance and exposure to full doses of IFN and
RBV in combination treatment protocols is critical for maximizing
SVR rates.
[1629] Hematological reductions are common during combination
treatment with IFN and RBV. RBV dose is critical to prevent HCV
relapse. However, reductions in hemoglobin (Hb) and platelet (PLT)
count due to RBV-induced hemolysis require dose reduction in RBV.
Reductions in absolute neutrophil count (ANC)<750/mm.sup.3
requires dose reducing the IFN component of the combination
treatment.
[1630] Although some ANC and PLT reductions were observed, these
reductions were comparable across all Q2w treatment groups and
reached a plateau around weeks 4 to 8. Likewise, the Hb reductions
from baseline were comparable across all the Q2w treatment groups
(including the 1800 .mu.g treatment group) until week 12 and
beyond. Reductions in hematologic values were less in the Q4w
treatment group. Overall, there were 12/115 subjects who were dose
reduced for the management of adverse events. Most dose reductions
of HSA-IFN.alpha.2b resulted from reductions in ANC as outlined in
the treatment protocol. There was no dose response observed between
the Q2w treatment groups. Thus, there was no increased need for
dose reduction observed in the higher dose treatment groups.
[1631] In summary, although there were some reductions in
hematologic values observed with treatment of HSA-IFN.alpha.2b in
combination with RBV, these reductions were comparable across the
treatment groups, indicating that there was no significant
difference in safety between treating IFN.alpha.
treatment-experienced, non-responder HCV patients with 900-1800
.mu.g of HSA-IFN.alpha.2b in combination with RBV.
[1632] Conclusion
[1633] Taken together, these results suggest that treatment with
HSA-IFN.alpha.2b and RBV may be effective in achieving SVR, and
thus eradicating HCV, in a significant portion of IFN.alpha.
treatment-experienced, non-responder HCV patients, including those
patients who previously failed a PEG+RBV treatment protocol.
Particularly, these results suggest that treatment with
HSA-IFN.alpha.2b 900-1200 .mu.g in combination with RBV may result
in 18% of patients achieving SVR, even after previously failing
PEG+RBV. Moreover, the 1800 .mu.g treatment group showed the
greatest week 24 HCV RNA negativity rates in the most refractory
patient population, indicating that this treatment may result in an
even greater SVR rate for these patients. In addition, the safety
profile was similar across all of the treatment groups of
HSA-IFN.alpha.2b. Moreover, these results indicate that
HSA-IFN.alpha.2b may be efficaciously administered every two to
four weeks, providing an improved dosing schedule. Thus, these
results indicate that the treatment of HSA-IFN.alpha.2b in
combination with RBV provides an efficacious and safe treatment
alternative with a high advantageous and improved dosing schedule,
for those patients who fail an interferon-based therapy,
particularly those that have failed the conventional pegylated
interferon-RBV therapy that is currently lacking in the art.
Example 84
Anti-Viral Activity of HSA-IFN.alpha.2b, in Combination with
Ribavirin in Genotype 2 or 3, Interferon-Naive Chronic Hepatitis
C(HCV) Patients
[1634] Background
[1635] With more than 170 million people infected with the
Hepatitis C virus (HCV) worldwide, this virus has emerged as a
significant public health concern and has rapidly become the most
common cause of liver disease throughout the world. Acute HCV
infection is usually asymptomatic, making early diagnosis
problematic. In fact, HCV infection tends to be a chronic
condition, where approximately 70% of acute infections becoming
persistent. Thus, although the incidence of new infections has been
on the decline, the prevalence of HCV infection is predicted to
remain constant in the near future.
[1636] Interferon alpha (IFN.alpha.) with or without concurrent
treatment of an antiviral molecule, ribavirin (RBV), has
historically been recognized as the most effective treatment for
patients with chronic hepatitis C(CHC). More recently, pegylated
forms of interferon alpha have been approved for treatment of HCV
in combination with RBV. These peglyated interferons have been
shown to be more effective in treating HCV than the standard
interferon or interferon in combination with ribavirin therapies
and have become the conventional treatment for HCV.
[1637] Overall sustained anti-viral responses (SVR) to the
currently recommended therapy vary greatly in CHC patients,
depending on viral and host characteristics, particularly viral
genotype. For example, SVR rates range from approximately 42-46% in
patients with the more common genotype 1. On the other hand,
patients with the less common genotypes 2 or 3 experience SVR rates
at 76-80%. In addition, genotype 2 or 3 patients, who are
considerably less difficult to treat than genotype 1 patients can
be treated for a shorter duration of therapy with a lower dose of
ribavirin.
[1638] Although the currently recommended therapy for genotype 2 or
3 of pegylated interferon in combination with RBV for 24 weeks
followed by a 24 week follow-up period results in a significant
proportion of patients achieving SVR, this treatment protocol still
retains significant practical limitations common to IFN-based
therapies. In particular, the currently recommended therapy remains
plagued by adverse effects that substantially decrease the
patient's quality of life after each administration. Thus, there is
a continued need for new treatment regimens that are efficacious
and better tolerated by patients infected with genotype 2 or 3
HCV.
[1639] Rationale
[1640] HSA-IFN.alpha.2b was generated by genetically fusing mature
albumin at its C-terminus to the N-terminus of mature interferon
.alpha.-2b. The safety and efficacy of treatment with
HSA-IFN.alpha.2b in combination with RBV was evaluated in a
randomized, multi-center, open-label clinical study in genotype 2
or 3, IFN-naive CHC human patients.
[1641] Methods
[1642] 43 human HCV patients who were either genotype 2 or 3,
IFN-naive were randomized into two subcutaneous (sc)
HSA-IFN.alpha.2b treatment groups: (a) 1500 .mu.g dosed every two
weeks (Q2w) or (2) 1500 .mu.g dosed every four weeks (Q4w). Each
subject in each treatment group also received in 800 mg/day of RBV.
The primary basis for stratification included genotype (2 or 3) and
HCV RNA (<800,000 IU/mL or .gtoreq.800,000 IU/mL).
[1643] Treatment duration of the study is 24 weeks with a 24 week
follow-up. The primary efficacy end-point is sustained virologic
response (SVR).
[1644] HCV RNA titer was measured using real-time PCR,
Quantasure.TM. (Labcorp) with a sensitivity range (limit of
quantitation (LOQ)) of 43 IU to 69 million IU/mL. Insulin
resistance was assessed using Homeostasis Assessment Model
(HOMA).
[1645] Intent to treat (ITT) patients are defined as all randomized
and treated subjects of each treatment group, regardless of whether
or not the patient has any missing data points or has dropped out
of the study. Modified intent to treat (MITT) patients are defined
as those patients that could conceivably have a Week 12 visit based
on their date of enrollment in the study.
[1646] Results and Discussion
[1647] Subject demographics and antiviral response at weeks 4 and
12 are summarized in Table 14. Overall, HSA-IFN.alpha.2b was
well-tolerated in both treatment groups.
TABLE-US-00031 TABLE 14 Demographics and Anti-Viral Response.
HSA-IFN.alpha.2b HSA-IFN.alpha.2b 1500 .mu.g Q2w 1500 .mu.g Q4w (N
= 21) (N = 22) Demographics % male 71.4% 68.2% % HCV RNA .gtoreq.
800,000 71.4% 59.1% Mean HOMA pre-treatment 2.2 2.5 Week 4
(<LOQ) ITT 16/21 (76.2%) 15/22 (68.2%) Genotype 2 8/10 6/10
Genotype 3 8/11 9/12 Week 12 (<LOQ) MITT 14/17 (82.4%) 16/18
(88.9%) Genotype 2 7/9 9/9 Genotype 3 7/8 7/9 ITT = intent to
treat; MITT = modified intent to treat (subjects eligible for week
12 visit)
[1648] The magnitude of HCV RNA reduction and the proportion of
genotype 2 or 3 patients with HCV RNA<LOQ were comparable across
both the HSA-IFN.alpha.2b Q2w and HSA-IFN.alpha.2b Q4w treatment
groups. At week 4, the proportion of genotype 2 or 3 patients with
HCV RNA<LOQ was 76.2% in the 1500 .mu.g Q2w and 68.2% in the
1500 .mu.g Q4w treatment group. At week 12, a high proportion of
genotype 2 or 3 patients in both treatment groups had HCV
RNA<LOQ (82.4% in the 1500Q2w and 88.9% in the 1500Q4w).
[1649] Thus, these results indicate that the treatment of genotype
2 or 3 CHC patients with 1500 .mu.g HSA-IFN.alpha.2b at either Q2w
or Q4w weeks results in a robust antiviral response rate. Moreover,
treatment with the HSA-IFN.alpha.2b 1500 .mu.g Q4w protocol showed
comparable efficacy to treatment with the HSA-IFN.alpha.2b 1500
.mu.g Q2w protocol in genotype 2 or 3 patients. Thus, these results
suggest that the treatment with HSA-IFN.alpha.2b 1500 .mu.g at
either Q2w or Q4w is at least as effective as the currently
recommended therapy for patients infected with HCV genotype 2 or 3
with the advantage of a vastly improved dosing schedule,
potentially resulting in superior tolerability and convenience for
the patient.
Example 85
Quality of Life (QOL) of Genotype 1, Interferon-Naive Chronic
Hepatitis C(HCV) Patients Treated with HSA-IFN.alpha.2b, in
Combination with Ribavirin Patients
[1650] Background
[1651] As previously noted, the conventional treatment for genotype
1, interferon-naive (IFN-naive) HCV patients with pegylated
interferon .alpha. in combination with ribavirin (RBV) for 48 weeks
has significant practical limitations. Due to the well-known side
effects of currently recommended interferon therapy, patients'
quality of life is substantially decreased after each
administration of interferon. Current protocols require dosing at
least weekly, resulting in a prolonged period of decreased quality
of life and increase in disability days due to treatment. Large
numbers of patients discontinue treatment as a result, with some
studies reporting discontinuation rates over 50%. Thus, there is an
clear need for improved therapeutic protocols for the treatment of
genotype 1 HCV in IFN-naive patients with improved impact on
quality of life compared to the current standard of care.
[1652] Rationale
[1653] The safety and efficacy of treatment with HSA-IFN.alpha.2b
in combination with RBV was evaluated in an active controlled
clinical study in genotype 1, IFN-naive HCV human patients, and
compared to conventional treatment with PEG-IFN.alpha.-2a (PEG-IFN)
in combination with RBV as an active control as described in
Example 82. During the first twelve weeks of therapy, the effects
on QOL and disability days (e.g., days missed from work) of
treatment with treatment with HSA-IFN.alpha.2b in combination with
RBV were compared with PEG-IFN in combination with RBV.
[1654] Methods
[1655] 458 human, genotype 1 HCV patients were randomized and
treated as described in Example 82. QOL as determined by the
SF-36v2.RTM. measurement model (QualityMetric, Lincoln, R.I.) and
disability days were assessed pre-treatment, and at week 4 and week
12 of treatment. In particular, eight SF-36v2 domains were
assessed: physical functioning (PF), role-physical (RP), bodily
pain (BP), general health (GH), vitality (VT), social functioning
(SF), role-emotional (RE), and mental health (MH). The first four
domains (PF, RP, BP, and GH) correspond to the Physical Health
Component and the remaining four domains (VT, SF, RE, and MH)
correspond to the Mental Health Component of the QOL model.
[1656] Transformed (raw) scores of the eight SF-36v2 domains, as
well as norm-based physical component summary (PCS) score and
mental component summary (MCS) score were evaluated through week 12
of treatment.
[1657] Results and Discussion
TABLE-US-00032 TABLE 15 Quality of Life Measurements PEG-IFN
HSA-IFN.alpha.2b HSA-IFN.alpha.2b HSA-IFN.alpha.2b 180 .mu.g Qw 900
.mu.g Q2w 1200 .mu.g Q2w 1200 .mu.g Q4w (n = 114) (n = 118) (n =
110) (n = 116) Mean change (worsening) from baseline to Week 12 in
SF-36 SF-36 Domain (MCID) QOL parameters PCS (2.6) -8.0 -5.6* -6.5
-6.0 Physical Functioning (5) -18 -14 .sup. -16 -13 Bodily Pain (4)
-21 -11*.sup.# .sup. -14.sup.# .sup. -15.sup.# MCS (2.7) -6.3
-3.6*.sup.# -4.9 -5.0 Vitality (4.2) -19 -13*.sup.# -17 -16 Mental
Health (4) -11 -4*.sup.# .sup. -7.sup.# .sup. -4*.sup.# Social
Functioning (6) -20 -10*.sup.# -15 -19 Number of days missed in the
prior month among subjects working for pay at the time of
assessment Missed work at Week 4 Mean days missed 4 .sup. 1* 3 3
.gtoreq.3 days 21/63 5/66 (8%)* 17/61 (28%) 15/62 (24%) (33%)
.gtoreq.7 days 14/63 3/66 (5%)* 8/61 (13%) 7/62 (11%) (22%) Missed
work at Week 12 Mean days missed 4 .sup. 1* 3 3 .gtoreq.3 days
21/69 7/70 (10%)* 13/59 (22%) 17/72 (24%) (30%) .gtoreq.7 days
12/69 3/70 (4%)* 7/59 (12%) 8/72 (11%) (17%) *p-value<0.05 vs.
Peg-IFN; .sup.#exceeds (MCID) minimal clinically important
difference
[1658] At week 12, QOL for patients receiving 900 .mu.g
HSA-IFN.alpha.2b Q2w was improved relative to PEG-IFN for every
measure, achieving statistical significance in MCS and PCS, as well
as 5 of the 8 individual domains. In the 1200 .mu.g Q2w and 1200
.mu.g Q4w HSA-IFN.alpha.2b treatment groups, worsening of QOL was
reduced relative to PEG-IFN in virtually every measure, with
clinically significant differences observed in both bodily pain and
mental health.
[1659] Overall, genotype 1 HCV patients in any of the
HSA-IFN.alpha.2b treatment groups missed fewer days of work (MDW)
due to their HCV infection and subsequent therapy than genotype 1
HCV patients in the PEG-IFN treatment group. In particular,
patients receiving 900 .mu.g HSA-IFN.alpha.2b Q2w had 75% fewer MDW
and patients receiving 1200 .mu.g HSA-IFN.alpha.2b Q2w or 1200
.mu.g HSA-IFN.alpha.2b Q4w had 25% fewer MDW than patients
receiving PEG-IFN.
[1660] Taken together with the antiviral activity of
HSA-IFN.alpha.2b shown in Example 82, these results suggest that
combination treatment of genotype 1, IFN-naive HCV patients with
HSA-IFN.alpha.2b and RBV is at least as effective as treatment with
the conventional PEG-IFN and RBV combination treatment with the
advantage of an improved dosing schedule and a improved QOL.
Particularly, these results suggest that treatment with
HSA-IFN.alpha.2b in combination with RBV can provide an improved
and highly advantageous treatment protocol over the conventional
PEG-IFN combination treatment with RBV by providing patients with a
reduction in worsening of QOL indicia and fewer missed days of work
over that obtainable with the PEG-IFN combination treatment with
RBV.
Example 86
In Vivo Induction of cGMP by HSA-BNP (Construct ID #3959) in a
Normal Pig Model
[1661] Rationale
[1662] The ability of Brain (B type) natriuretic peptide (BNP) to
mediate cellular effects such as modification of renal function and
vascular tone is well-known in the art. The activity of BNP is
dependent upon its binding to and subsequent activation of
natriuretic receptor A (NPR-A) which is a guanylyl cyclase. The
activation of NPR-A by BNP leads to an elevation of intracellular
cGMP levels that can be measured by assays known in the art, such
as, for example, ELISA. The ability of HSA-BNP (CID 3959) to induce
the production of cGMP in vivo was tested in a normal pig
model.
[1663] Methods
[1664] HSA-BNP (CID 3959) was generated by genetically fusing
mature albumin at its C-terminus to the N-terminus of a
C-terminally truncated form of BNP (amino acids 1 to 29).
[1665] Normotensive, healthy pigs (n=4-6/group) were administered a
single bolus of 5 mg/kg HSA-BNP (CID 3959) or formulation vehicle
alone at time 0. Plasma and urine were collected at 1, 8, 16, 24,
48, and 72 hours post injection. cGMP levels in the collected
plasma and urine were measured by commercially available ELISA
(Molecular Dynamics).
[1666] Results
[1667] Single IV bolus of 5 mg/kg HSA-BNP (CID 3959) resulted in a
significant elevation of both plasma (FIG. 13A) and urine (FIG.
13B) cGMP levels at 1 hour after IV administration. cGMP levels
declined gradually to baseline levels at 24 hours in the plasma and
by 48-72 hours in the urine.
Example 87
Natriuresis Activity of BNP-HSA Fusion Construct in a In Vivo
Pacing Model of Heart Failure
[1668] Background
[1669] Administration of exogenous BNP has been utilized to
facilitate natriuresis in congestive heart failure (CHF). However,
recent studies have suggested that BNP administration can adversely
affect renal function. The effect on renal or left ventricular (LV)
function of HSA-BNP (CID 3959) was assessed in an in vivo severe
CHF pig model. Surprisingly, the results of this study demonstrate
that unlike BNP administration, acute infusion of HSA-BNP (CID
3959) can induce natriuresis without adversely affecting left
ventricular or renal function.
[1670] Methods
[1671] HSA-BNP (CID 3959) was generated by genetically fusing
mature albumin at its C-terminus to the N-terminus of a
C-terminally truncated form of BNP (amino acids 1 to 29).
[1672] CHF was induced in 18 pigs by chronic pacing at 240 bpm for
3 weeks. 8 pigs served as reference controls. The following
baseline characteristics were significantly reduced with CHF
compared to reference controls. Baseline measurements were taken
using techniques known in the art.
TABLE-US-00033 TABLE 16 Baseline measurements in CHF-induced pigs
vs. reference control pigs CHF-Induced Pigs Control Pigs Left
Ventricular Fractional Shortening 21 .+-. 2 32 .+-. 2 by
Echocardiography (LVFS) (%) Renal Vascular Blood Flow using 1.73
.+-. 0.09 2.23 .+-. 0.26 Microspheres (RenFlow) (mL/min/g) Sodium
Clearance (Na.sub.CL) (mL/min) 0.43 .+-. 0.11 4.35 .+-. 2.45
Fractional Excretion of Sodium (FE.sub.Na) 0.25 .+-. 0.07 0.80 .+-.
0.32 (%)
[1673] Following baseline measurements, animals with evidence of
heart failure (e.g., LV dilation occurred with a subsequent decline
in fractional shortening) were randomized for this study. The
animals were anesthetized (n=10/group), administered either
vehicle, 2 mg/kg HSA-BNP (CID 3959) or 6 mg/kg HSA-BNP (CID 3959)
IV and monitored for 4 hours. End diastolic diameter was measured
by echocardiography.
[1674] Results and Conclusions
[1675] Neither dose of HSA-BNP (CID 3959) had a significant effect
on heart rate, mean arterial blood pressure, left ventricular end
diastolic pressure, mean pulmonary arterial pressure, left
ventricular peak pressure, peak positive dp/dt or cardiac output
(data not shown). No change in coronary blood flow was seen in
animals infused with either dose of HSA-BNP (CID 3959) (data not
shown). In addition, although infusing the animals with 6 mg/kg of
HSA-BNP (CID 3959) resulted in increased creatinine clearance and
fractional sodium excretion 30 minutes post-infusion, neither
reached statistical significance over baseline (data not shown).
Similarly, infusion of 6 mg/kg of HSA-BNP (CID 3959) resulted in a
non-significant reduction in plasma renin activity and endothelin
plasma levels compared to vehicle (data not shown).
[1676] Significant increases in sodium clearance (492.+-.281% at 30
minutes post-infusion and 950.+-.483% at 60 minutes post-infusion)
over vehicle were seen in animals infused with 6 mg/kg HSA-BNP (CID
3959). Additionally, end-diastolic diameter changes caused by the
pacing were significantly reduced after either dose of HSA-BNP (CID
3959) compared to vehicle (FIG. 14A). Moreover, changes in left
ventricular fractional shortening were caused by the pacing were
reduced after either dose of HSA-BNP (CID 3959) compared to
vehicle, with the reduction caused by the 2 mg/kg dose of HSA-BNP
(CID 3959) being significant over vehicle (FIG. 14B).
[1677] Thus, taken together, these results demonstrate that acute
infusion of HSA-BNP (CID 3959) can induce natriuresis without
adversely affecting left ventricular or renal function in an in
vivo CHF model.
Example 88
Effect of HSA-BNP (Construct ID #3959) on Cardiorenal Function in
Anesthetized Normal Dogs
[1678] Rationale
[1679] The ability of Brain (B type) natriuretic peptide (BNP) to
mediate modification of renal function and vascular tone is
well-known in the art. A particularly useful model for studying the
effects of BNP on cardiorenal function is the dog. Accordingly, an
extensive assessment of cardiorenal effects, including hemodynamic,
renal, and hormonal effects, of HSA-BNP (CID 3959) was performed in
an anesthetized normal dog model.
[1680] Methods
[1681] HSA-BNP (CID 3959) was generated by genetically fusing
mature albumin at its C-terminus to the N-terminus of a
C-terminally truncated form of BNP (amino acids 1 to 29).
[1682] Hemodynamic, renal, and hormonal parameters of cardiorenal
function were evaluated in this study. In particular, the
hemodynamic parameters included measurement of cardiac output, mean
arterial pressure, pulmonary capillary wedge pressure, and mean
pulmonary artery pressure. Renal parameters included measurement of
urine flow, sodium excretion, glomerular filtration rate (GFR), and
renal blood flow. Hormonal parameters included measurement of
plasma cGMP, renin, angiotensin II, aldosterone, and urinary
cGMP.
[1683] Normal healthy mongrels (n=8/group) were fed a fixed sodium
diet for 5 days prior to the start of the study. On the night
before the acute experiment, the animals were fasted and given 300
mg of lithium carbonate for assessment of renal tubular function.
On the day of the acute experiment, the dogs were anesthetized via
IV with sodium pentobarbital (15 mg/kg), intubated, and
mechanically ventilated with supplemental oxygen.
[1684] A flow-directed balloon-tipped thermodilution catheter was
advanced into the pulmonary artery via the external jugular vein
for cardiac hemodynamic measurement. The femoral artery was
cannulated for blood pressure monitoring, blood sampling, and for
insulin and normal saline infusion. The ureter of the left kidney
was cannulated for urine collection. A calibrated electromagnetic
flow probe was placed around the renal artery to measure renal
blood flow (RBF).
[1685] On the day of the acute experiments, the dogs were
administered a single IV bolus of HSA-BNP (CID 3959) at either 0.5
mg/kg or 5 mg/kg (n=8/group). Effects on cardiorenal function were
monitored for 4.5 hours.
[1686] Cardiovascular parameters measured included mean arterial
pressure (MAP), renal artery pressure (RAP), pulmonary artery
pressure (PAP), cardiac output (CO), and pulmonary capillary wedge
pressure (PCWP). CO was determined by thermodilution. MAP was
assessed via direct measurement from the femoral arterial catheter.
GFR was measured by insulin clearance.
[1687] Cardiovascular hemodynamics were measured at the start of
each clearance. Arterial blood was collected in heparin and EDTA
tubes and immediately placed on ice midway through each clearance.
After centrifugation at 2,500 rpm at 4.degree. C., plasma was
decanted and stored at -20.degree. C. until analysis. Urine was
collected on ice during the entire period of each clearance for
assessment of urine volume, electrolytes and insulin. Urine
collected for cGMP analysis was heated to more than 90.degree. C.
before storage.
[1688] Results and Discussion
[1689] Hemodynamic Effects
[1690] FIGS. 15A-H show the effect of HSA-BNP (CID 3959)
administered at 0.5 mg/kg (FIGS. 15A, C, E, and G) or 5 mg/kg
(FIGS. 15B, D, F, and H) on hemodynamic performance over 4.5 hours
post-infusion compared with baseline readings. Hemodynamic
parameters were measure at baseline prior to the IV bolus of
HSA-BNP (CID 3959) and at 30, 60, 90, 150, 210, and 270 minutes
post-infusion.
[1691] The hemodynamic effects of HSA-BNP (CID 3959) were sustained
over the 4.5 hr observation period. Both 0.5 and 5 mg/kg IV bolus
of HSA-BNP (CID 3959) resulted in a statistically significant and
sustained reduction in pulmonary capillary wedge pressure (PCWP)
(FIGS. 15E and F). The magnitude of reduction in PCWP with these
two doses was not significantly different.
[1692] Effects of HSA-BNP (CID 3959) were dose-related for
pulmonary arterial pressure (PAP) (FIGS. 15G and H) and mean
arterial pressure (MAP) (FIGS. 15C and D). Significant reductions
in PAP were observed when the animals were dosed at 5 mg/kg (FIG.
15H). Likewise, a significant effect on MAP where observed at 270
minutes post-infusion in the 5 mg/kg treatment group (FIG.
15D).
[1693] Renal Effects
[1694] FIGS. 16A-H show the effect of HSA-BNP (CID 3959)
administered at 0.5 mg/kg (FIGS. 16A, C, E, and G) or 5 mg/kg
(FIGS. 16B, D, F, and H) on renal output and blood flood over the
4.5 hours post-infusion compared with baseline readings. Renal
performance parameters were measured at baseline prior to IV bolus
of HSA-BNP (CID 3959) and at 30, 60, 90, 150, 210, and 270 minutes
post-infusion.
[1695] Administration of HSA-BNP (CID 3959) resulted in significant
effects on renal function. Significantly elevated renal blood flow
(FIGS. 16E and F), diuresis (FIGS. 16A and B), natriuresis (Figures
C and d) were observed at both 0.5 and 5 mg/kg HSA-BNP (CID 3959).
A dose-related trend in increased GFR was also apparent (FIGS. 16G
and H).
[1696] The time to maximal effect of HSA-BNP (CID 3959) on the
renal parameters tended to be slightly delayed compared with the
hemodynamic effects. In addition, the magnitude of the increase in
natriuresis and diuresis was significantly higher in the 5 mg/kg
treatment group than in the 0.5 mg/kg treatment group.
[1697] Hormonal Effects
[1698] FIGS. 17A-F show the effect of HSA-BNP (CID 3959)
administered at 0.5 mg/kg (FIGS. 17A, C, and E) or 5 mg/kg (FIGS.
17B, D, and F) on the RAAS hormones over 4.5 hours post-infusion
compared with baseline readings. Plasma aldosterone, renin, and
angiotensin II levels were measured at baseline prior to IV bolus
of HSA-BNP (CID 3959) and at 30, 60, 90, 150, 210, and 270
minutes.
[1699] Administration with both 0.5 and 5 mg/kg IV bolus of HSA-BNP
(CID 3959) resulted in a reduction of renin, angiotensin and
aldosterone levels during the 4.5 hours post-infusion observation
period. The effect on aldosterone levels was significant and
sustained throughout the 270 minute study. Effects on renin and
angiotensin II were significant between 30 and 90 minutes after
administration of HSA-BNP (CID 3959) at both doses, but rebounded
toward the end of the observation period.
[1700] Conclusion
[1701] This study demonstrates that HSA-BNP (CID 3959) behaves in a
similar pharmacological manner as unfused BNP. Administration of
HSA-BNP (CID 3959) in a single IV bolus at 0.5 and 5 mg/kg resulted
in dose-dependent, significant, and sustained changes in multiple
cardiorenal parameters that are consistent with its action as a
long-acting form of BNP. In particular, administration of HSA-BNP
(CID 3959) resulted in increased plasma and urine cGMP levels,
reduced PCWP and PAP, increased natriuresis, diuresis, renal blood
flow and glomerular filtration rate, decreased plasma, aldosterone,
renin, and angiotensin II, and a slight reduction in MAP at the 5
mg/kg dose. Differential effect on component of the
renin-angiotensin-adosterone system is somewhat unexpected result
and may be a favorable attribute of HSA-BNP (CID 3959).
[1702] Taken as a whole, these results suggest that HSA-BNP (CID
3959) may administered at a dose that improves cardiorenal function
without substantial unwanted reductions in systemic blood
pressure.
Example 89
Effect of HSA-BNP (Construct ID #3959) on Blood Pressure in
Telemeterized Beagles
[1703] Rationale
[1704] The ability of Brain (B type) natriuretic peptide (BNP) to
mediate vascular tone is well-known in the art. As noted
previously, a particularly useful model for studying the effects of
BNP on cardiovascular function, including blood pressure, is the
dog. Accordingly, the effect of intravenous (IV) administration of
HSA-BNP (CID 3959) on cardiovascular function, including systolic
and mean arterial blood pressure and heart rate was evaluated in
conscious normal beagle dogs. Additionally, the effectiveness and
duration of response of subcutaneous (SC) administration of HSA-BNP
(CID 3959) was also evaluated.
[1705] Methods
[1706] Healthy beagles (n=4/group) were surgically implanted with a
Data Sciences International radiotelemetry transmitter, which had
systemic arterial blood pressure, heart rate, and ECG data
collection capabilities. Following implantation, the dogs received
either a single IV bolus of 0.1, 0.5, or 5 mg/kg of HSA-BNP (CID
3959) or vehicle.
[1707] Continuous recording of ECG parameters and systemic blood
pressure were monitored for 48 hours following infusion. The dogs
were followed for an additional 9 days (total time=11 days) with
intermittent monitoring.
[1708] The effectiveness and duration of response of subcutaneous
administration of HSA-BNP (CID 3959) on systemic blood pressure was
evaluated by comparing the effect of administration of an IV bolus
of unfused BNP(0.02 mg/kg) to administration of an SC
administration of HSA-BNP (CID 3959) (10 mg/kg).
[1709] Results and Discussion
[1710] Effect of HSA-BNP (CID 3959) on Systolic and Mean Arterial
Blood Pressure
[1711] FIGS. 18A-C show the effect of a single IV bolus of HSA-BNP
(CID 3959) on the systolic and mean arterial blood pressure in
awake dogs. Administration of 5 mg/kg HSA-BNP (CID 3959) resulted
in gradual reductions in systolic blood pressure with a peak effect
at approximately 16 hours and a return to baseline by 48 hours. A
sustained reduction in systolic blood pressure of approximately 15
mmHg was apparent starting at 8 hours following drug administration
and continuing through 20 hours following administration.
[1712] Administration of HSA-BNP (CID 3959) did not have any
obvious effect on diastolic blood pressure or heart rate over the
same observation period (data not shown).
[1713] Lower doses (e.g., 0.5 and 0.1 mg/kg) of HSA-BNP (CID 3959)
were without obvious effect on either blood pressure or heart rate.
In addition, there were no treatment-related changes on ECG
parameters observed at any dose of HSA-BNP (CID 3959).
[1714] Comparison of IV Administered Unfused BNP Peptide to SC
Administered HSA-BNP (CID 3959)
[1715] FIGS. 19A and B show the effect of an IV bolus of unfused
BNP on systemic blood pressure in normal healthy beagles compared
to a SC injection of HSA-BNP (CID 3959). Both HSA-BNP (CID 3959)
and unfused BNP reduced systolic blood pressure in healthy beagles.
The effect of unfused BNP was maximal at approximately 30 minutes
and returned to baseline within hours. In contrast, the effect of
HSA-BNP (CID 3959) was apparent approximately 10 hours after SC
administration, reached a maximum at approximately 40 hours and
returned to baseline between 48 and 72 hours post-injection. The
slow onset effect of HSA-BNP (CID 3959) on blood pressure is
consistent with its slow absorption (T.sub.max.about.36 hours in
the dog). The long duration of effect of HSA-BNP (CID 3959) is
consistent with its long half life (72 hours in the dog).
[1716] Taken as a whole, these results suggest that HSA-BNP (CID
3959) may administered to heart failure patients at a dose low
enough to improve cardiorenal function without having an effect on
systemic blood pressure.
Example 90
Use of BChE Albumin Fusions for Cocaine Breakdown and
Detoxification
[1717] The purpose of this experiment was to determine the affect
of an albumin BChE fusion on cocaine breakdown and
detoxification.
[1718] A. Construction and Purification of BChE-Albumin Fusion:
Albu-CocH
[1719] A mutated, c-terminal-truncated form of human BChE (deleting
the last 45 amino acids of the C-terminus, the tetramerization
domain) was fused to the N-terminus of the mature form of HSA
(gi:28592). FIG. 20 (termed "Albu-CoCH" in this example). A signal
peptide consensus sequence (amino acids 1-23 in FIG. 20) was also
fused to the N-terminus of the BChE portion of the fusion. The
first amino acid in the mature BChE protein in the fusion
corresponds to E29 of the unprocessed, full-length BChE protein.
The mutations in the BChE portion of the fusion are as follows (the
amino acid positions are relative to the unprocessed, full-length
BChE): A227S, S315G, A356W, Y360G (bold, underlined amino acids in
FIG. 20). The HSA portion of the fusion begins at amino acid 553 of
FIG. 20.
[1720] Monomeric protein was expressed in Chinese hamster ovary
cells and purified to near homogeneity using blue affinity and ion
exchange chromatography. Protein purity was assessed as greater
than 95% based on N-terminal sequencing, high performance
size-exclusion chromatography, and active-site titration.
[1721] B. Materials and Methods
[1722] 1. Animals
[1723] Animals were handled according to the Principles of
Laboratory Animal Care (National Research Council, 2003) in
facilities accredited by the American Association for the
Accreditation of Laboratory Animal Care, under IACUC protocols
A9306 (Mayo Clinic) and 0410A64760 (University of Minnesota).
Wistar rats were obtained from Harlan Sprague-Dawley (Madison
Wis.). Females (20 total, weighing approximately 225 at study
onset) were used in behavioral experiments. Males (94, weighing
250-300 g) were used for the other experiments.
[1724] On arrival at the behavior laboratory, rats were pair-housed
in plastic cages and allowed to acclimate at least 3 days.
Initially, all rats had free access to food (Purina Laboratory
Chow, Purina Mills, Minneapolis, Minn.) and water. During the
experiments they received 16 g of food at 3:00 pm daily, which held
them to 85% of the weight of free-fed age-matched controls. Room
lights were on from 6:00 am to 6:00 pm. Temperature (24.degree. C.)
and humidity were kept within a narrow range. The rats for
locomotor study remained pair-housed except for the testing
procedures (activity at 9:00-9:30 am; food reinforcement at 1:00
pm-3:00 pm). For the reinstatement task rats were permanently
transferred to operant chambers (see Behavioral apparatus).
[1725] 2. Drug, Reagents, and Enzymes
[1726] Drugs were prepared in 0.9% NaCl (saline). Cocaine HCl was
from Mallinckrodt (St. Louis Mo.). Other drugs including atropine
sulfate, ketamine, amphetamine sulfate, diisopropylfluorophosphate
(DFP), and sodium pentobarbital were from Sigma Aldrich (St.
Louis). Each batch of enzyme was titrated by incubation for 24 hr
with varying amounts of the irreversible inhibitor, DFP, followed
by determination of residual activity (Sun et al., 2002a).
[1727] 3. Radiometric Assay of Plasma and Tissue Samples
[1728] Blood (100-300 .mu.l) was collected from tail vein or
femoral artery into heparin-treated tubes and centrifuged (10 min
at 8,000 g) to obtain plasma. Brains and hearts were homogenized in
10 volumes of 10 mM sodium phosphate, pH 7.4 with 0.5% Tween-20,
and centrifuged as above. Cocaine hydrolase activity in 50 .mu.l
supernatant aliquots was assayed by incubating 30 min with .sup.3H
cocaine (18 .mu.M, except for substrate kinetics) and measuring
liberated .sup.3H-benzoic acid (Brimijoin et al., (2002) Analytical
Biochemisty 309:200-205.).
[1729] To assess drug metabolism, pentobarbital-anesthetized rats
(45 mg/kg, i.p.) received .sup.3H-cocaine (3.5 mg/kg, 30 .mu.Ci,
i.v.). At times from 30 sec to 2 hr, blood (.apprxeq.150 .mu.l) was
collected from the femoral artery into tubes with DFP (10 .mu.l,
10.sup.-2 M) added to inhibit BChE and carboxylesterase. Plasma
samples were frozen on liquid nitrogen for assay later that day.
Brains and hearts, frozen after aortic perfusion with 100 ml of
isotonic NaCl plus DFP, 10.sup.-5 M, were later homogenized in
phosphate-Tween buffer with the same inhibitor. Aliquots for
determination of cocaine (acidified with 1 ml of 0.2 N HCl) or
benzoic acid (alkalinized with 300 .mu.l of 1M Na2CO3) were
extracted directly into toluene for scintillation counting.
[1730] 4. Blood Pressure Recording
[1731] For continuous monitoring of blood pressure, the rats were
anesthetized with urethane (1.45 g/kg, i.p.). A sterile PE-50
cannula was then placed in one femoral artery and connected to a
calibrated pressure transducer (Gould TA240). Body temperature was
maintained by a heat lamp. Animals stabilized for 30 min before
drug administration.
[1732] 5. Toxicity of Cocaine Overdose
[1733] Rats were dosed with i.p. cocaine from 30 to 1000 mg/kg.
Challenges with 100 mg/kg had been found regularly lethal in
unprotected animals. Death was not an endpoint for the present
studies, which instead used seizures as an index of serious
toxicity. A battery of cage-side observations noted posture, gait,
locomotor activity, paw licking, head bobbing, piloerection, and
labored breathing. Onsets of convulsions were recorded. Rats were
euthanized (sodium pentobarbital, 250 mg/kg i.p.) if they convulsed
once for 60 sec or twice within 2 min.
[1734] 6. Behavioral Apparatus
[1735] The operant chambers were custom octagonal units with
alternating panels of stainless steel or Plexiglas and a steel grid
floor. They contained 2 response levers with stimulus lights above
them, a ceiling light, a food pellet dispenser, and a water bottle
mounted outside. Tygon tubing connected a tether and swivel at the
top of the cage to a syringe pump mounted outside a wooden
enclosure that surrounded the test chamber. Data collection and
experimental programming were controlled by MED-PC software (Med
Associates, St. Albans, Vt.).
[1736] As previously described (Perry et al., (2005)
Psychopharmacology (Berl) 178:193-201), the locomotor track was
circular stainless steel, 71 cm diameter. It was equipped with 4
infrared sensors around the inner perimeter, at 0.degree.,
90.degree., 180.degree. and 270.degree., and connected to a
VersaMax programmable logic controller (IC200UDR001, GE Fanuc
Automation, Charlottesville, Va.).
[1737] 7. Surgical Preparation for Cocaine Self-Administration
[1738] Under anesthesia with ketamine (60 mg/kg) and xylazine (10
mg/kg), with atropine (0.15 ml) and doxapram (5 mg/kg) to
facilitate respiration, 15 rats were implanted with a 15-cm
silastic catheter (0.51 mm i.d., 0.94 mm o.d.; Helix Medical Inc.,
Carpinteria, Calif.). The catheter, with 2 beads of prosthetic
silicone elastomer, 3 and 3.5 cm from one end (MDX4-4210; Factor
II, Inc. Lakeside, Ariz.), was introduced into the right jugular
vein and anchored with sterile silk sutures. The free end was led
to an exit incision medial and 1 cm caudal to the scapulae. Heparin
(10 IU/kg i.v.) and gentomycin (12.0 mg/kg, i.v.) were administered
for 3 days to prevent clotting.
[1739] 8. Behavioral Training and Reinstatement
[1740] The catheterized rats learned to lever press under a fixed
ratio 1 (FR 1) schedule, which delivered 1 infusion of cocaine
contingent upon 1 lever-press. Behavioral sessions began daily at 1
pm and ended at 3 pm. When responding had stabilized for 14 days,
cocaine was replaced with physiologic saline. Self-administration
behavior was then allowed to extinguish for 21-days, during which
responses continued to produce infusion-related stimuli and saline
infusions. Next followed a 3-day cue-extinction period in which the
infusion pump and house light were disconnected in order to
completely extinguish responding and ensure that reinstatement
specifically reflected drug priming.
[1741] Subsequent reinstatement sessions involved no
self-administration of cocaine or saline, or infusion-related
stimuli. To begin each session, the experimenter administered an
i.p. priming injection of saline (S), cocaine (C), or amphetamine
(A) daily at 1 pm for 12 days according to the following sequence:
S C S C S A S A S C S A. On the fourth (C) and sixth (A) day, the
rats were pretreated at 9 am with 2 mg/kg Albu-CocH, administered
through the infusion apparatus and flushed with 0.3 ml sterile
saline.
[1742] 9. Locomotor Activity and Food Reinforced Behavior
[1743] Locomotor activity was assessed daily for 30 min beginning
at 11:00 am by detecting infrared beam breaks, as described by
Perry et al. (2005). Two or more breaks of one beam occurring
before another beam was broken were counted as a single response.
After behavior stabilized (no steadily increasing or decreasing
trends for 3 days), rats were injected at 9 am with saline (tail
vein) for 4 days, then Albu-CocH for 1 day and saline again on the
final day.
[1744] Rats were placed in the operant conditioning chambers for a
3-hr food session at 1:00 pm daily. Food pellets (45 mg) were
contingent upon responses on either lever under a FR 1 adjusting
delay discounting schedule (Perry et al. 2005). A response on the
"immediate lever" produced 1 pellet immediately, while a "delay
lever" response resulted in 3 pellets after a delay that altered
during the session based on the animal's behavior. The delay
started at 6 sec, and it increased by 1 sec after a "delay lever"
response lever and decreased by 1 sec after an "immediate lever"
response. The session ended on completion of 60 trials or after 3
hr, whichever came first. Following the session, rats were given
additional food to reach a total of 16 g per day. Each day the
delay started at the value it ended with the day before.
[1745] Each session was divided into fifteen 4-trial blocks. The
first and second trials of each block were forced exposure to each
lever (immediate and delayed condition in counterbalanced order),
while the third and fourth trials were free choice, immediate or
delayed, and a response on either lever yielded 1 or 3 pellets,
respectively. A mean adjusted delay (MAD) was calculated by
averaging the delays that were in effect on all of the free choice
trials (maximum=30) completed on the "delay" lever. MAD values
served as a quantitative measure of impulsivity for food and
provided another dimension of food-rewarded behavior in addition to
amount of food earned.
[1746] 10. Statistical Analysis and Pharmacokinetic
Calculations
[1747] Blood pressure changes were analyzed statistically with
StatView 4.5 (Abacus Concepts, Berkeley, Calif.). Treatment effects
were subjected to 2-way analysis of variance with time and
treatment as factors; p<0.05 was considered statistically
significant. Self-administration and other behavioral data were
analyzed by one and two way analysis of variance followed by
post-hoc testing. Enzyme plasma concentration-time profiles were
analyzed with t Sigma Plot 4.1 (Jandel Scientific, Temecula
Calif.), fitting the data to a bi-exponential decay curve that
estimated terminal half-life. Enzyme kinetic data (V=velocity,
S=substrate concentration) were analyzed by direct fit to the
Michaelis-Menten equation: V=Vmax*(S/(S+Km)).
[1748] C. Results
[1749] Radiometric assay (see Methods) revealed a cocaine kcat
(2700.+-.190 min-1) and Km (2.1.+-.0.1 .mu.M) similar to the
unfused mutant (Pan et al., 2005). By comparison, the bacterial
enzyme is reported to have a kcat of 470 min-1, and a Km of 0.64
.mu.M (Turner et al., 2002). Catalytic efficiency, measured as the
ratio of kcat/Km, is therefore 75% higher in the mutated BChE.
[1750] When pure Albu-CocH was injected through the tail vein into
male Wistar rats (250-300 g), it had no discernable effect at doses
up to 3 mg/kg, while 10 mg/kg caused mild lethargy for 1 hour.
Enzyme assays of repeated blood samples from 5 rats showed that the
injected activity was stable, with a plasma half-life of 8.+-.0.5
hr (FIG. 23). Furthermore, Albu-CocH blocked pressor responses to a
moderate dose of cocaine without lowering blood pressure on its own
or opposing the pressor effects of norepinephrine (FIG. 24). These
findings led to the hypothesis that Albu-CocH would be able to
alleviate toxicity and overdose in humans. Testing that possibility
in animals required the exposure of awake, unrestrained rats to
doses of cocaine with a potential to evoke serious toxicity
(defined as seizures).
[1751] Experience had shown that an i.p. challenge with cocaine
(100 mg/kg) regularly induced convulsions that ended in death
within 2 minutes unless euthanasia was administered. To minimize
distress here, we followed an IACUC-approved protocol calling for
euthanasia of any rat exhibiting continuous convulsion for 60 sec
or convulsions of over 15 sec within a 2 min period. After
receiving the cocaine challenge, each of ten unprotected rats
developed head bobbing and hyperlocomotion followed by convulsions
(onset time, 170.+-.30 sec), which met the euthanasia criterion.
Pretreatment with i.v. Albu-CocH, however, provided dramatic,
dose-dependent protection (FIG. 25). A small dose (1 mg/kg) delayed
but did not prevent arousal or seizures in 4 of 4 rats (onset,
380.+-.70 sec); a mid-dose (3 mg/kg) prevented seizures but not
signs of arousal (6 of 6 rats); a large dose (10 mg/kg) eliminated
arousal and seizures (6 of 6 rats), and it raised cocaine's ED50
for this toxicity by nearly a factor of 10.
[1752] Not wishing to be bound by any theory, it seems that
accelerated cocaine hydrolysis is the simplest explanation for this
protection because the same cocaine challenge caused convulsions in
all sham-treated animals (3 given 10 mg/kg human serum albumin and
2 given Albu-CocH inactivated by di-isopropylfluorophosphate,
10.sup.-4 M, 1 hr). The protection was specific to cocaine, as
active enzyme at 10 mg/kg failed to delay or prevent convulsions in
3 rats challenged with amphetamine at the threshold dose (150
mg/kg) for producing uniform seizures in our unprotected rats.
Protection against cocaine was lasting. No seizures occurred in any
rat challenged up to 12 hr after receiving 10 mg/kg Albu-CocH (4
rats between 1 and 6 hr, and 4 at 12 hr). Of 5 rats challenged
after 24 hr, 2 escaped seizures, 1 experienced seizures for 15 sec
before recovering, and 2 experienced seizures that met the
criterion for euthanasia. Thus, in this experiment, 24 hours marked
the outer limit of protection.
[1753] Human overdose requires rescue. To evaluate rescue potential
in rats 100 mg/kg of cocaine was injected into rats. When the onset
of convulsions began, the rats were then rapidly administered
Albu-CocH (3 or 10 mg/kg). Each of three rats given 10 mg/kg ceased
convulsing within 1 min, resumed an upright posture within 2 min,
and showed no further signs of cocaine-induced arousal. Thereafter,
apart from lethargy and mild paw swelling for 1 to 2 hour ("hr"),
they resembled untreated controls. Three of three rats given 3
mg/kg also ceased convulsing within 1 min and quickly regained
upright posture. These animals exhibited head bobbing and
hyperlocomotion for approximately 1 hr. On the next day both
treated groups were indistinguishable from rats that never received
cocaine. At 10 mg/kg, Albu-CocH was also partially effective
against a larger cocaine overdose, saving 4 of 6 rats challenged
with 300 mg/kg of i.p. cocaine. In contrast, wild-type BChE, 3
mg/kg, was ineffective at rescue in 3 of 3 rats even after standard
cocaine challenge (100 mg/kg), all of which met our criterion for
early euthanasia.
[1754] These results established dose-dependent and
hydrolase-specific rescue from cocaine intoxication. The rescue was
rapid for an agent largely excluded from the brain (tissue activity
<1% of plasma activity) and thought to act by eliminating free
cocaine and promoting drug dissociation from tissues. Additionally,
Albu-CocH removed cocaine from plasma almost instantly. In 4
control rats treated with .sup.3H-cocaine (3.5 mg/kg, i.v.), plasma
drug half-life was 50.+-.5 min, but in 4 rats given 3 mg/kg
Albu-CocH 10 min beforehand, 98% of the free drug was converted to
benzoic acid within 30 sec (FIG. 26), and the drug burden in heart
and brain was greatly reduced (FIG. 27).
[1755] An enzyme powerful enough to rescue rats from cocaine
toxicity might also be useful in reducing drug-reward and managing
cocaine addiction. To evaluate that possibility, Albu-CocH was
tested in rats that had been trained to self-administer cocaine.
One of the most refractory and troublesome aspects of cocaine
addiction is relapse after abstinence. One goal was to determine
whether fast metabolism of cocaine en route to brain reward centers
could prevent relapse triggered by an i.v. priming injection of
cocaine. The effect of Albu-CocH pretreatment on the cocaine-primed
reinstatement of drug-seeking behavior in rats that had previously
self-administered cocaine and subsequently extinguished their
responding when saline replaced cocaine, was examined.
[1756] Rats were trained to emit one lever press for each cocaine
infusion (0.4 mg/kg, i.v.) during daily sessions under a
fixed-ratio 1 (FR 1) schedule. After behavior stabilized for 14
days, saline was substituted for cocaine for 21 days, and behavior
was allowed to extinguish. In a subsequent reinstatement phase,
priming injections of cocaine were given (alternating daily with
saline priming injections). On selected days, rats received
Albu-CocH (2 mg/kg) 2 hr before the reinstatement session. Cocaine
priming injections (10 mg/kg, i.p.) generated 30-40 responses on
the 2 days with no pretreatment (FIG. 28). After Albu-CocH,
however, cocaine priming caused negligible responding. Saline
priming on intervening days resulted in minimal responses (2-5) on
the lever previously associated with cocaine. To control for
possible nonspecific behavioral suppression, we also tested priming
injections of d-amphetamine, which is not a hydrolase substrate.
Amphetamine primes (2 mg/kg, i.p.) elicited 60 reinstatement
responses, which, consistent with an effect that depended on
selective metabolism, were not significantly reduced by
Albu-CocH.
[1757] To further confirm that Albu-CocH did not cause generalized
behavioral suppression that might impair reinstatement after
cocaine-priming, the effect of Albu-CocH on locomotor activity and
responding for food was investigated. For these studies, 5 rats
were treated alternately with i.v. saline, Albu-CocH, and saline.
Two hr after each day's injection, locomotion was monitored in a
circular open field with 4 infrared beams equally spaced around the
perimeter (Piazza et al., (1989) Behav Brain Res 31:267-271). The
beam break data showed no treatment effect (Table 17).
[1758] Four hours after the saline or enzyme injections, the same
rats were studied in an operant conditioning experiment with food
delivery contingent upon FR 1 lever-press responding in a paradigm
designed to assess impulsivity for reward (Perry et al., 2005).
This task involved 2 levers. One lever immediately produced a
single 45 mg food pellet and the other produced 3 pellets after a
delay. The delay began at 6 sec, and it increased by 1 sec after
each response on the delay lever, and decreased by 1 sec after each
response on the immediate lever. Results after Albu-CocH showed no
significant differences (vs. saline) in trials completed, number of
pellets earned, food intake, or mean adjusted delay for the 60
choice trials (Table 17).
TABLE-US-00034 TABLE 17 Albu-CocH does not alter locomotor activity
or food-reinforced responding. Five rats received saline or
Albu-CocH at 9 am or 3 consecutive days. Mean values (.+-.SEM)
shown for locomotor activity (11 am) and food-rewarded behaviour (1
pm). The enzyme treatment had no statistically significant effect.
Food-reinforced Behavior Locomotor Trials No. of Food Mean adjusted
Injections Beam Breaks Completed Pellets Intake (g) delay* (sec)
Saline 23.8 .+-. 2.4 57.2 .+-. 2.8 110 .+-. 4.9 4.93 .+-. 2.2 14.0
.+-. 5.0 Albu-CocH 21.2 .+-. 4.2 57.8 .+-. 1.4 115 .+-. 6.2 5.15
.+-. 2.7 7.44 .+-. 0.45 Saline 21.0 .+-. 2.6 54.2 .+-. 5.8 108 .+-.
10.3 4.88 .+-. 4.6 6.84 .+-. 1.5 *self-determined measure of
impulsivity for food
[1759] BChE-catalyzed cocaine hydrolysis generates two breakdown
products, benzoic acid and ecgonine methyl ester. As compared with
other metabolites, including norcocaine and benzylecgonine, these
products have greatly reduced biologic activity. Thus, the reaction
is detoxifying.
[1760] These results illustrate that BChE-albumin fusions can
prevent cocaine access to critical biological targets, including
heart and brain, and, while not wishing to be bound by any theory,
suggest that rescue injections create steep diffusion gradients
favoring the loss of cocaine from sites throughout the body and
especially from brain, with its high regional blood flow. Hence
Albu-CocH is well suited for the emergency treatment of cocaine
overdose.
[1761] In addition to emergency uses, the repeated or continuous
delivery of Albu-CocH would help addicts avoid the full relapse
that commonly follows a brief lapse. Long plasma half-life is
desirable for such a purpose. Allometric scaling from the rat data,
in light of experience with other albumin fusion proteins, is
compatible with a several-day half-life of Albu-CocH in humans,
allowing sustained acceleration of cocaine hydrolysis with a view
to preventing relapse.
[1762] Relapse or "reinstatement" is perhaps the greatest challenge
in treating drug abuse. Drug-seeking behavior in established animal
models of reinstatement is regularly triggered by exposure to
drug-associated environmental cues. Factors that predict or enhance
reinstatement include female sex, estrogen status, higher drug
dose, addiction-prone phenotypes such as impulsivity or
sweet-preference, and food restriction. Especially powerful are
"priming exposures" of drugs with related pharmacological
mechanisms. The above experiments show that Albu-CocH was fully
effective in blocking reinstatement provoked by cocaine-priming
injections.
[1763] Under conditions like those that produced reinstatement of
cocaine-primed, cocaine-seeking behavior, Albu-CocH did not affect
locomotor behavior, food-rewarded behavior, impulsivity for food,
or amphetamine-primed reinstatement. These results from an animal
model of relapse support the hypothesis that, by preventing cocaine
access to the brain, accelerated metabolism can blunt not only
toxicity, but also the reward-seeking effects of this drug. The
virtual elimination of cocaine-primed reinstatement suggests that
sustained delivery of an efficient hydrolase would reduce the
probability of relapse in recovering addicts even though it did not
suppress craving. As to administration, it is likely that weekly
administration of BChE-albumin fusions could provide continuous,
rapid elimination of cocaine as a therapy of cocaine addiction.
Furthermore, additional modifications of the protein may provide
even better pharmacokinetics.
[1764] Additionally or alternatively, viral gene transduction of a
BChE albumin fusion may be used as a therapeutic strategy. It has
been shown that standard E1-deleted adenoviral vectors sustain
effective levels of a cocaine hydrolase for days in the rat
bloodstream and brain, while ongoing work with a helper-dependent
adenoviral vector is showing expression windows of months or
more.
[1765] The entire disclosure of each document cited (including
patents, patent applications, patent publications, journal
articles, abstracts, laboratory manuals, books, or other
disclosures) as well as information available through Identifiers
specific to databases such as GenBank, GeneSeq, or the CAS
Registry, referred to in this application are herein incorporated
by reference in their entirety.
[1766] Furthermore, the specification and sequence listing of each
of the following International applications and U.S. applications
are herein incorporated by reference in their entirety:
International Application No. PCT/US02/40891, filed Dec. 23, 2002;
International Application No. PCT/US2004/001369, filed Jan. 20,
2004; International Application No. PCT/US2005/004041, filed Feb.
9, 2005; U.S. application Ser. No. 10/775,204, filed Feb. 11, 2004;
U.S. application Ser. No. 11/175,690, filed Jul. 7, 2005; U.S.
application Ser. No. 11/429,373, filed May 8, 2006; U.S.
application Ser. No. 11/429,276, filed May 8, 2006; U.S.
application Ser. No. 11/429,374, filed May 8, 2006; and U.S.
Provisional Application Nos. 60/707,521, filed Aug. 12, 2005;
60/712,386, filed Aug. 31, 2005; 60/732,724, filed Nov. 3, 2005;
60/776,914, filed Feb. 28, 2006; 60/781,361, filed Mar. 13, 2006;
and 60/810,182, filed Jun. 2, 2006, and 60/813,682, filed Jun. 15,
2006.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080194481A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080194481A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References