U.S. patent application number 10/156424 was filed with the patent office on 2003-02-27 for b7 related protein-2 molecules and uses thereof.
This patent application is currently assigned to Amgen, Inc.. Invention is credited to Mak, Tak Wah, Suh, Woong-Kyung, Yoshinaga, Steven Kiyoshi.
Application Number | 20030039999 10/156424 |
Document ID | / |
Family ID | 23129865 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030039999 |
Kind Code |
A1 |
Yoshinaga, Steven Kiyoshi ;
et al. |
February 27, 2003 |
B7 related protein-2 molecules and uses thereof
Abstract
The present invention provides B7 Related Protein-2 (B7RP-2)
polypeptides and nucleic acid molecules encoding the same. The
invention also provides selective binding agents, vectors, host
cells, and methods for producing B7RP-2 polypeptides. The invention
further provides pharmaceutical compositions and methods for the
diagnosis, treatment, amelioration, and/or prevention of diseases,
disorders, and conditions associated with B7RP-2 polypeptides.
Inventors: |
Yoshinaga, Steven Kiyoshi;
(Thousand Oaks, CA) ; Suh, Woong-Kyung; (Toronto,
CA) ; Mak, Tak Wah; (Toronto, CA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Amgen, Inc.
|
Family ID: |
23129865 |
Appl. No.: |
10/156424 |
Filed: |
May 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60293629 |
May 25, 2001 |
|
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|
Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 1/04 20180101; A61P 7/04 20180101; A61P 19/10 20180101; A61P
3/10 20180101; A61P 31/04 20180101; A61P 37/06 20180101; A61P 19/08
20180101; A61P 7/00 20180101; A61P 19/00 20180101; A61P 19/02
20180101; A61P 17/06 20180101; A61P 3/14 20180101; A61P 31/00
20180101; A61P 37/02 20180101; A61P 5/14 20180101; C07K 14/70532
20130101; A61P 25/00 20180101; A61P 29/00 20180101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/320.1; 435/325; 530/350; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/435 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising (a) the nucleotide
sequence as set forth in any of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ
ID NO: 5; (b) a nucleotide sequence encoding the polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; (c) a
nucleotide sequence that hybridizes under at least moderately
stringent conditions to the complement of the nucleotide sequence
of either (a) or (b), wherein the encoded polypeptide has an
activity of the polypeptide as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6; or (d) a nucleotide sequence
complementary to the nucleotide sequence of any of (a)-(c).
2. An isolated nucleic acid molecule comprising: (a) a nucleotide
sequence encoding a polypeptide that is at least about 70 percent
identical to the polypeptide as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6, wherein the encoded polypeptide has
an activity of the polypeptide set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6; (b) a nucleotide sequence encoding
an allelic variant or splice variant of the nucleotide sequence as
set forth in any of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or
the nucleotide sequence of (a); (c) a region of the nucleotide
sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or
the nucleotide sequence of (a) or (b), encoding a polypeptide
fragment of at least about 25 amino acid residues, wherein the
polypeptide fragment has an activity of the encoded polypeptide as
set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or
is antigenic; (d) a region of the nucleotide sequence of any of SEQ
ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or the nucleotide sequence
of any of (a)-(c) comprising a fragment of at least about 16
nucleotides; (e) a nucleotide sequence that hybridizes under at
least moderately stringent conditions to the complement of the
nucleotide sequence of any of (a)-(d), wherein the encoded
polypeptide has an activity of the polypeptide as set forth in any
of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (f) a nucleotide
sequence complementary to the nucleotide sequence of any of
(a)-(e).
3. An isolated nucleic acid molecule comprising: (a) a nucleotide
sequence encoding a polypeptide as set forth in any of SEQ ID NO:
2, SEQ ID NO: 4, or SEQ ID NO: 6 with at least one conservative
amino acid substitution, wherein the encoded polypeptide has an
activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ
ID NO: 4, or SEQ ID NO: 6; (b) a nucleotide sequence encoding a
polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or
SEQ ID NO: 6 with at least one amino acid insertion, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; (c) a
nucleotide sequence encoding a polypeptide as set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at least one amino
acid deletion, wherein the encoded polypeptide has an activity of
the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or
SEQ ID NO: 6; (d) a nucleotide sequence encoding a polypeptide as
set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6
that has a C- and/or N-terminal truncation, wherein the encoded
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; (e) a nucleotide
sequence encoding a polypeptide as set forth in any of SEQ ID NO:
2, SEQ ID NO: 4, or SEQ ID NO: 6 with at least one modification
that is an amino acid substitution, amino acid insertion, amino
acid deletion, C-terminal truncation, or N-terminal truncation,
wherein the encoded polypeptide has an activity of the polypeptide
set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;
(f) a nucleotide sequence of any of (a)-(e) comprising a fragment
of at least about 16 nucleotides; (g) a nucleotide sequence that
hybridizes under at least moderately stringent conditions to the
complement of the nucleotide sequence of any of (a)-(f), wherein
the encoded polypeptide has an activity of the polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (h)
a nucleotide sequence complementary to the nucleotide sequence of
any of (a)-(g).
4. A vector comprising the nucleic acid molecule of any of claims
1, 2, or 3.
5. A host cell comprising the vector of claim 4.
6. The host cell of claim 5 that is a eukaryotic cell.
7. The host cell of claim 5 that is a prokaryotic cell.
8. A process of producing a B7RP-2 polypeptide comprising culturing
the host cell of claim 5 under suitable conditions to express the
polypeptide, and optionally isolating the polypeptide from the
culture.
9. A polypeptide produced by the process of claim 8.
10. The process of claim 8, wherein the nucleic acid molecule
comprises promoter DNA other than the promoter DNA for the native
B7RP-2 polypeptide operatively linked to the DNA encoding the
B7RP-2 polypeptide.
11. The isolated nucleic acid molecule according to claim 2,
wherein the percent identity is determined using a computer program
that is GAP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, or the
Smith-Waterman algorithm.
12. A process for determining whether a compound inhibits B7RP-2
polypeptide activity or B7RP-2 polypeptide production comprising
exposing a cell according to any of claims 5, 6, or 7 to the
compound and measuring B7RP-2 polypeptide activity or B7RP-2
polypeptide production in the cell.
13. An isolated polypeptide comprising the amino acid sequence as
set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
6.
14. An isolated polypeptide comprising: (a) an amino acid sequence
for an ortholog of any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
6; (b) an amino acid sequence that is at least about 70 percent
identical to the amino acid sequence of any of SEQ ID NO: 2, SEQ
II) NO: 4, or SEQ ID NO: 6, wherein the polypeptide has an activity
of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4,
or SEQ ID NO: 6; (c) a fragment of the amino acid sequence set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6
comprising at least about 25 amino acid residues, wherein the
fragment has an activity of the polypeptide set forth in any of SEQ
ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or is antigenic; or (d) an
amino acid sequence for an allelic variant or splice variant of the
amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO:
4, or SEQ ID NO: 6, or the amino acid sequence of either (a) or
(b).
15. An isolated polypeptide comprising: (a) the amino acid sequence
as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6
with at least one conservative amino acid substitution, wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; (b) the amino acid
sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6 with at least one amino acid insertion, wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; (c) the amino acid
sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6 with at least one amino acid deletion, wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; (d) the amino acid
sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6 that has a C- and/or N-terminal truncation, wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (e) the amino acid
sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6 with at least one modification that is an amino acid
substitution, amino acid insertion, amino acid deletion, C-terminal
truncation, or N-terminal truncation, wherein the polypeptide has
an activity of the polypeptide set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6.
16. An isolated polypeptide encoded by the nucleic acid molecule of
any of claims 1, 2, or 3, wherein the polypeptide has an activity
of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4,
or SEQ ID NO: 6.
17. The isolated polypeptide according to claim 14, wherein the
percent identity is determined using a computer program that is
GAP, BLASTP, FASTA, BLASTA, BLASTX, BestFit, or the Smith-Waterman
algorithm.
18. A selective binding agent or fragment thereof that specifically
binds the polypeptide of any of claims 13, 14, or 15.
19. The selective binding agent or fragment thereof of claim 18
that specifically binds the polypeptide comprising the amino acid
sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6, or a fragment thereof.
20. The selective binding agent of claim 18 that is an antibody or
fragment thereof.
21. The selective binding agent of claim 18 that is a humanized
antibody.
22. The selective binding agent of claim 18 that is a human
antibody or fragment thereof.
23. The selective binding agent of claim 18 that is a polyclonal
antibody or fragment thereof.
24. The selective binding agent claim 18 that is a monoclonal
antibody or fragment thereof.
25. The selective binding agent of claim 18 that is a chimeric
antibody or fragment thereof.
26. The selective binding agent of claim 18 that is a CDR-grafted
antibody or fragment thereof.
27. The selective binding agent of claim 18 that is an
antiidiotypic antibody or fragment thereof.
28. The selective binding agent of claim 18 that is a variable
region fragment.
29. The variable region fragment of claim 28 that is a Fab or a
Fab' fragment.
30. A selective binding agent or fragment thereof comprising at
least one complementarity determining region with specificity for a
polypeptide having the amino acid sequence of any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6.
31. The selective binding agent of claim 18 that is bound to a
detectable label.
32. The selective binding agent of claim 18 that antagonizes B7RP-2
polypeptide biological activity.
33. A method for treating, preventing, or ameliorating a medical
disease, condition, or disorder comprising administering to a
patient an effective amount of a selective binding agent according
to claim 18.
34. The method of claim 33, wherein the medical disease, condition,
or disorder is osteoporosis, Paget's disease, osteomyelitis,
hypercalcemia, osteopenia, or osteonecrosis.
35. The method of claim 33, wherein the medical disease, condition,
or disorder is an autoimmune disease.
36. The method of claim 35, wherein the autoimmune disease is
systemic lupus erythematosis, rheumatoid arthritis, multiple
sclerosis, osteoarthritis, immune thrombocytopenic purpura (ITP),
or psoriasis.
37. The method of claim 33, wherein the medical disease, condition,
or disorder is a chronic inflammatory disease.
38. The method of claim 37, wherein the chronic inflammatory
disease is an inflammatory bowel disease, Grave's disease,
Hashimoto's thyroiditis, or diabetes mellitus.
39. The method of claim 33, wherein the medical disease, condition,
or disorder is cancer.
40. The method of claim 33, wherein the medical disease, condition,
or disorder is an infectious disease.
41. A selective binding agent produced by immunizing an animal with
a polypeptide comprising an amino acid sequence of any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
42. A hybridoma that produces a selective binding agent capable of
binding a polypeptide according to any of claims 1, 2, or 3.
43. A method of detecting or quantitating the amount of B7RP-2
polypeptide using the selective binding agent or fragment of claim
18.
44. A kit for detecting or quantitating the amount of GPCR
polypeptide in a biological sample, comprising the selective
binding agent of claim 18.
45. A composition comprising the polypeptide of any of claims 13,
14, or 15, and a pharmaceutically acceptable formulation agent.
46. The composition of claim 45, wherein the pharmaceutically
acceptable formulation agent is a carrier, adjuvant, solubilizer,
stabilizer, or anti-oxidant.
47. A polypeptide comprising a derivative of the polypeptide of any
of claims 13, 14, or 15.
48. The polypeptide of claim 47 that is covalently modified with a
water-soluble polymer.
49. The polypeptide of claim 48, wherein the water-soluble polymer
is polyethylene glycol, monomethoxy-polyethylene glycol, dextran,
cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol,
propylene glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols, or polyvinyl alcohol.
50. A composition comprising a nucleic acid molecule of any of
claims 1, 2, or 3 and a pharmaceutically acceptable formulation
agent.
51. The composition of claim 50, wherein the nucleic acid molecule
is contained in a viral vector.
52. A viral vector comprising a nucleic acid molecule of any of
claims 1, 2, or 3.
53. A fusion polypeptide comprising the polypeptide of any of
claims 13, 14, or 15 fused to a heterologous amino acid
sequence.
54. The fusion polypeptide of claim 53, wherein the heterologous
amino acid sequence is an IgG constant domain or fragment
thereof.
55. A method for treating, preventing, or ameliorating a medical
disease, condition, or disorder comprising administering to a
patient an effective amount of the polypeptide of any of claims 13,
14, or 15, or the polypeptide encoded by the nucleic acid of any of
claims 1, 2, or 3.
56. The method of claim 55, wherein the medical disease, condition,
or disorder is osteoporosis, Paget's disease, osteomyelitis,
hypercalcemia, osteopenia, or osteonecrosis.
57. The method of claim 55, wherein the medical disease, condition,
or disorder is an autoimmune disease.
58. The method of claim 57, wherein the autoimmune disease is
systemic lupus erythematosis, rheumatoid arthritis, multiple
sclerosis, osteoarthritis, immune thrombocytopenic purpura (ITP),
or psoriasis.
59. The method of claim 55, wherein the medical disease, condition,
or disorder is a chronic inflammatory disease.
60. The method of claim 59, wherein the chronic inflammatory
disease is inflammatory bowel disease, Grave's disease, Hashimoto's
thyroiditis, or diabetes mellitus.
61. The method of claim 55, wherein the medical disease, condition,
or disorder is cancer.
62. The method of claim 55, wherein the medical disease, condition,
or disorder is an infectious disease.
63. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the
polypeptide of any of claims 13, 14, or 15, or the polypeptide
encoded by the nucleic acid molecule of any of claims 1, 2, or 3 in
a sample; and (b) diagnosing a pathological condition or a
susceptibility to a pathological condition based on the presence or
amount of expression of the polypeptide.
64. A device, comprising: (a) a membrane suitable for implantation;
and (b) cells encapsulated within the membrane, wherein the cells
secrete a protein of any of claims 13, 14, or 15; and wherein the
membrane is permeable to the protein and impermeable to materials
detrimental to the cells.
65. A method of identifying a compound that binds to a B7RP-2
polypeptide comprising: (a) contacting the polypeptide of any of
claims 13, 14, or 15 with a compound; and (b) determining the
extent of binding of the B7RP-2 polypeptide to the compound.
66. The method of claim 65, further comprising determining the
activity of the polypeptide when bound to the compound.
67. A method of modulating levels of a polypeptide in an animal
comprising administering to the animal the nucleic acid molecule of
any of claims 1, 2, or 3.
68. A transgenic non-human mammal comprising the nucleic acid
molecule of any of claims 1, 2, or 3.
69. A process for determining whether a compound inhibits B7RP-2
polypeptide activity or B7RP-2 polypeptide production comprising
exposing a transgenic mammal according to claim 68 to the compound,
and measuring B7RP-2 polypeptide activity or B7RP-2 polypeptide
production in the transgenic mammal.
70. A nucleic acid molecule of any of claims 1, 2, or 3 attached to
a solid support.
71. An array of nucleic acid molecules comprising at least one
nucleic acid molecule of any of claims 1, 2, or 3.
72. An isolated polypeptide comprising the amino acid sequence as
set forth in SEQ ID NO: 2 with at least one conservative amino acid
substitution that is a valine at position 20; valine at position
29; valine at position 101; tyrosine at position 120; leucine at
position 184; valine at position 260; valine or isoleucine at
position 261; aspartic acid at position 291; or glutamic acid at
position 306; wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2.
73. An isolated polypeptide comprising the amino acid sequence as
set forth in SEQ ID NO: 32.
Description
[0001] This application claims the benefit of priority from U.S.
Provisional Patent Application No. 60/293,629, filed on May 25,
2001, the disclosure of which is explicitly incorporated by
reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to B7 Related Protein-2
(B7RP-2) polypeptides and nucleic acid molecules encoding the same.
The invention also relates to selective binding agents, vectors,
host cells, and methods for producing B7RP-2 polypeptides. The
invention further relates to pharmaceutical compositions and
methods for the diagnosis, treatment, amelioration, and/or
prevention of diseases, disorders, and conditions associated with
B7RP-2 polypeptides.
BACKGROUND OF THE INVENTION
[0003] Technical advances in the identification, cloning,
expression, and manipulation of nucleic acid molecules and the
deciphering of the human genome have greatly accelerated the
discovery of novel therapeutics. Rapid nucleic acid sequencing
techniques can now generate sequence information at unprecedented
rates and, coupled with computational analyses, allow the assembly
of overlapping sequences into partial and entire genomes and the
identification of polypeptide-encoding regions. A comparison of a
predicted amino acid sequence against a database compilation of
known amino acid sequences allows one to determine the extent of
homology to previously identified sequences and/or structural
landmarks. The cloning and expression of a polypeptide-encoding
region of a nucleic acid molecule provides a polypeptide product
for structural and functional analyses. The manipulation of nucleic
acid molecules and encoded polypeptides may confer advantageous
properties on a product for use as a therapeutic.
[0004] In spite of the significant technical advances in genome
research over the past decade, the potential for the development of
novel therapeutics based on the human genome is still largely
unrealized. Many genes encoding potentially beneficial polypeptide
therapeutics or those encoding polypeptides, which may act as
"targets" for therapeutic molecules, have still not been
identified. Accordingly, it is an object of the invention to
identify novel polypeptides, and nucleic acid molecules encoding
the same, which have diagnostic or therapeutic benefit.
SUMMARY OF THE INVENTION
[0005] The present invention relates to novel B7RP-2 nucleic acid
molecules and encoded polypeptides.
[0006] The invention provides for an isolated nucleic acid molecule
comprising:
[0007] (a) the nucleotide sequence as set forth in any of SEQ ID
NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5;
[0008] (b) a nucleotide sequence encoding the polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;
[0009] (c) a nucleotide sequence that hybridizes under at least
moderately stringent conditions to the complement of the nucleotide
sequence of either (a) or (b), wherein the encoded polypeptide has
an activity of the polypeptide as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6; or
[0010] (d) a nucleotide sequence complementary to the nucleotide
sequence of any of (a)-(c).
[0011] The invention also provides for an isolated nucleic acid
molecule comprising:
[0012] (a) a nucleotide sequence encoding a polypeptide that is at
least about 70 percent identical to the polypeptide as set forth in
any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;
[0013] (b) a nucleotide sequence encoding an allelic variant or
splice variant of the nucleotide sequence as set forth in any of
SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or the nucleotide
sequence of (a);
[0014] (c) a region of the nucleotide sequence of any of SEQ ID NO:
1, SEQ ID NO: 3, or SEQ ID NO: 5, or the nucleotide sequence of (a)
or (b), encoding a polypeptide fragment of at least about 25 amino
acid residues, wherein the polypeptide fragment has an activity of
the encoded polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID
NO: 4, or SEQ ID NO: 6, or is antigenic;
[0015] (d) a region of the nucleotide sequence of any of SEQ ID NO:
1, SEQ ID NO: 3, or SEQ ID NO: 5 or the nucleotide sequence of any
of (a)-(c) comprising a fragment of at least about 16
nucleotides;
[0016] (e) a nucleotide sequence that hybridizes under at least
moderately stringent conditions to the complement of the nucleotide
sequence of any of (a)-(d), wherein the encoded polypeptide has an
activity of the polypeptide as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6; or
[0017] (f) a nucleotide sequence complementary to the nucleotide
sequence of any of (a)-(e).
[0018] The invention further provides for an isolated nucleic acid
molecule comprising:
[0019] (a) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at
least one conservative amino acid substitution, wherein the encoded
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;
[0020] (b) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at
least one amino acid insertion, wherein the encoded polypeptide has
an activity of the polypeptide set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6;
[0021] (c) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at
least one amino acid deletion, wherein the encoded polypeptide has
an activity of the polypeptide set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6;
[0022] (d) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 that
has a C- and/or N-terminal truncation, wherein the encoded
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;
[0023] (e) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at
least one modification that is an amino acid substitution, amino
acid insertion, amino acid deletion, C-terminal truncation, or
N-terminal truncation, wherein the encoded polypeptide has an
activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ
ID NO: 4, or SEQ ID NO: 6;
[0024] (f) a nucleotide sequence of any of (a)-(e) comprising a
fragment of at least about 16 nucleotides;
[0025] (g) a nucleotide sequence that hybridizes under at least
moderately stringent conditions to the complement of the nucleotide
sequence of any of (a)-(f), wherein the encoded polypeptide has an
activity of the polypeptide as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6; or
[0026] (h) a nucleotide sequence complementary to the nucleotide
sequence of any of (a)-(g).
[0027] The present invention provides for an isolated polypeptide
comprising the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
[0028] The invention also provides for an isolated polypeptide
comprising:
[0029] (a) an amino acid sequence for an ortholog of any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;
[0030] (b) an amino acid sequence which is at least about 70
percent identical to the amino acid sequence of any of SEQ ID NO:
2, SEQ ID NO: 4, or SEQ ID NO: 6, wherein the polypeptide has an
activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ
ID NO: 4, or SEQ ID NO: 6;
[0031] (c) a fragment of the amino acid sequence set forth in any
of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 comprising at least
about 25 amino acid residues, wherein the fragment has an activity
of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4,
or SEQ ID NO: 6, or is antigenic; or
[0032] (d) an amino acid sequence for an allelic variant or splice
variant of the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or the amino acid sequence of
either (a) or (b).
[0033] The invention further provides for an isolated polypeptide
comprising:
[0034] (a) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at least one conservative
amino acid substitution, wherein the polypeptide has an activity of
the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or
SEQ ID NO: 6;
[0035] (b) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at least one amino acid
insertion, wherein the polypeptide has an activity of the
polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6;
[0036] (c) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at least one amino acid
deletion, wherein the polypeptide has an activity of the
polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6;
[0037] (d) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 that has a C- and/or
N-terminal truncation, wherein the polypeptide has an activity of
the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or
SEQ ID NO: 6; or
[0038] (e) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 with at least one modification
that is an amino acid substitution, amino acid insertion, amino
acid deletion, C-terminal truncation, or N-terminal truncation,
wherein the polypeptide has an activity of the polypeptide set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
[0039] The invention still further provides for an isolated
polypeptide comprising the amino acid sequence as set forth in SEQ
ID NO: 2 with at least one conservative amino acid substitution
that is a valine at position 20; valine at position 29; valine at
position 101; tyrosine at position 120; leucine at position 184;
valine at position 260; valine or isoleucine at position 261;
aspartic acid at position 291; or glutamic acid at position 306;
wherein the polypeptide has an activity of the polypeptide set
forth in SEQ ID NO: 2.
[0040] Also provided are fusion polypeptides comprising B7RP-2
amino acid sequences.
[0041] The present invention also provides for an expression vector
comprising the isolated nucleic acid molecules as set forth herein,
recombinant host cells comprising the recombinant nucleic acid
molecules as set forth herein, and a method of producing a B7RP-2
polypeptide comprising culturing the host cells and optionally
isolating the polypeptide so produced.
[0042] A transgenic non-human animal comprising a nucleic acid
molecule encoding a B7RP-2 polypeptide is also encompassed by the
invention. The B7RP-2 nucleic acid molecules are introduced into
the animal in a manner that allows expression and increased levels
of a B7RP-2 polypeptide, which may include increased circulating
levels. Alternatively, the B7RP-2 nucleic acid molecules are
introduced into the animal in a manner that prevents expression of
endogenous B7RP-2 polypeptide (i.e., generates a transgenic animal
possessing a B7RP-2 polypeptide gene knockout). The transgenic
non-human animal is preferably a mammal, and more preferably a
rodent, such as a rat or a mouse.
[0043] Also provided are derivatives of the B7RP-2 polypeptides of
the present invention.
[0044] Additionally provided are selective binding agents such as
antibodies and peptides capable of specifically binding the B7RP-2
polypeptides of the invention. Such antibodies and peptides may be
agonistic or antagonistic.
[0045] Pharmaceutical compositions comprising the nucleotides,
polypeptides, or selective binding agents of the invention and one
or more pharmaceutically acceptable formulation agents are also
encompassed by the invention. The pharmaceutical compositions are
used to provide therapeutically effective amounts of the
nucleotides or polypeptides of the present invention. The invention
is also directed to methods of using the polypeptides, nucleic acid
molecules, and selective binding agents.
[0046] The B7RP-2 polypeptides and nucleic acid molecules of the
present invention may be used to treat, prevent, ameliorate, and/or
detect diseases and disorders, including those recited herein.
[0047] The present invention also provides a method of assaying
test molecules to identify a test molecule that binds to a B7RP-2
polypeptide. The method comprises contacting a B7RP-2 polypeptide
with a test molecule to determine the extent of binding of the test
molecule to the polypeptide. The method further comprises
determining whether such test molecules are agonists or antagonists
of a B7RP-2 polypeptide. The present invention further provides a
method of testing the impact of molecules on the expression of
B7RP-2 polypeptide or on the activity of B7RP-2 polypeptide.
[0048] Methods of regulating expression and modulating (i.e.,
increasing or decreasing) levels of a B7RP-2 polypeptide are also
encompassed by the invention. One method comprises administering to
an animal a nucleic acid molecule encoding a B7RP-2 polypeptide. In
another method, a nucleic acid molecule comprising elements that
regulate or modulate the expression of a B7RP-2 polypeptide may be
administered. Examples of these methods include gene therapy, cell
therapy, and anti-sense therapy as further described herein.
[0049] In another aspect of the present invention, the B7RP-2
polypeptides may be used for identifying receptors thereof ("B7RP-2
polypeptide receptors"). Various forms of "expression cloning" have
been extensively used to clone receptors for protein ligands. See,
e.g., Simonsen and Lodish, 1994, Trends Pharmacol. Sci. 15:437-41
and Tartaglia et al., 1995, Cell 83:1263-71. The isolation of a
B7RP-2 polypeptide receptor is useful for identifying or developing
novel agonists and antagonists of the B7RP-2 polypeptide signaling
pathway. Such agonists and antagonists include soluble B7RP-2
polypeptide receptors, anti-B7RP-2 polypeptide receptor-selective
binding agents (such as antibodies and derivatives thereof), small
molecules, and antisense oligonucleotides, any of which can be used
for treating one or more disease or disorder, including those
disclosed herein.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIGS. 1A-1B illustrate the nucleotide sequence of the human
B7RP-2 gene (SEQ ID NO: 1) and the deduced amino acid sequence of
human B7RP-2 polypeptide (SEQ ID NO: 2);
[0051] FIGS. 2A-2B illustrate the nucleotide sequence of the murine
B7RP-2 gene (SEQ ID NO: 3) and the deduced amino acid sequence of
the murine B7RP-2 polypeptide (SEQ ID NO: 4);
[0052] FIGS. 3A-3C illustrate the nucleotide sequence of the rat
B7RP-2 gene (SEQ ID NO: 5) and the deduced amino acid sequence of
rat B7RP-2 polypeptide (SEQ ID NO: 6);
[0053] FIGS. 4A-4B illustrate the amino acid sequence alignment of
human B7RP-2 polypeptide (huB7RP-2; SEQ ID NO: 2), murine B7RP-2
polypeptide (muB7RP-2; SEQ ID NO: 4), and rat B7RP-2 polypeptide
(raB7RP-2; SEQ ID NO: 6), which was prepared using the ClustalW
algorithm. The sequences were aligned using the application
MacVector 7.1.1 (Accelrys, Cambridge, UK; http://www.accelrys.com)
at the default settings. Conserved residues are boxed;
[0054] FIG. 5 illustrates the locations of several conserved
domains possessed by human B7RP-2 polypeptide (SEQ ID NO: 2),
murine B7RP-2 polypeptide (SEQ ID NO: 4), and rat B7RP-2
polypeptide (SEQ ID NO: 6), as indicated following a BLAST analysis
of the amino acid sequences against the Conserved Domain
Database;
[0055] FIGS. 6A-6B illustrate the amino acid sequence alignment of
human butyrophilin, subfamily 1, member A1 (hu_BTN1A1; SEQ ID NO:
7; GenBank Accession No. NP.sub.--001723), bovine butyrophilin
precursor (bo_BTN; SEQ ID NO: 8; GenBank Accession No. P18892),
murine butyrophilin (mu_BTN; SEQ ID NO: 9; GenBank Accession No.
NP.sub.--038511), human butyrophilin, subfamily 2, member A1
(hu_BTN2A1; SEQ ID NO: 10; Accession No. NP.sub.--008980), human
butyrophilin-like protein (hu_BT3.2; SEQ ID NO: 11; GenBank
Accession No. ACC02652), human butyrophilin, subfamily 3, member A2
(hu_BTN3A2; SEQ ID NO: 12; GenBank Accession No. NP.sub.--008978),
Grus americana B-G-like protein (gr_BG2; SEQ ID NO: 13; GenBank
Accession No. AF033107), and human B7RP-2 polypeptide (hu_B7RP-2;
SEQ ID NO: 2);
[0056] FIG. 7 illustrates the expression of B7RP-2 mRNA as detected
by Northern blot analysis of osteoblast cells following treatment
with dexamethasone, vitamin C, and
.quadrature.-glycerophosphate;
[0057] FIG. 8 illustrates the expression of B7RP-2 mRNA in an E18.5
mouse embryo as detected by in situ hybridization;
[0058] FIG. 9 illustrates the inhibition of bone mineralization by
B7RP-2 polypeptide. Cells were either treated with no polypeptide
(plate A), 10 .mu.g/ml of an IgG1 isotype control (plate B), 1
.mu.g/ml of soluble B7RP-2 polypeptide (plate C) or 10 .mu.g/ml
(plate D) of soluble B7RP-2 polypeptide;
[0059] FIGS. 10A-10C illustrate the effect of B7RP-2 on T-cell
proliferation, interleukin-2 production, and interferon-.gamma.
production;
[0060] FIG. 11 illustrates the wild type murine B7RP-2 locus,
targeting vector, and mutated B7RP-2 allele for generating
B7RP-2-/-mice. (B, Bgl II; H, Hind III; X, Xba I);
[0061] FIG. 12 depicts a Southern analysis of genomic DNA from F2
progeny verifying the disruption of the B7RP-2 gene;
[0062] FIG. 13 depicts a flow cytometric analysis of B7RP-2 protein
expression in MEF cells of the indicated genotypes;
[0063] FIG. 14 shows total and lymphocyte cell counts in the
bronchoalveolar lavage (BAL) fluid from the B7RP-2-/- and +/+mice
having cytokine-induced airway inflammation;
[0064] FIG. 15 depicts hematoxylin and exosin staining of lung
sections from the B7RP-2 -/- and +/+mice having cytokine-induced
airway inflammation;
[0065] FIG. 16 depicts flow cytometric analysis of
lung-infiltrating T-cells based on lung infiltrates pooled from
B7RP-2-/- and +/+mice having cytokine-induced airway inflammation
(four mice in each group);
[0066] FIG. 17 depicts LCMV-induced footpad swelling of B7RP-2-/-
and +/+mice;
[0067] FIGS. 18A-18B depict the disease course for experimental
autoimmune encephalomyelitis (EAE) in B7RP-2-/- and +/+mice as
determined by the average clinical score of all mice in a group
(FIG. 16A), and the time of EAE onset compared among littermates
(FIG. 16B); Ci Circles in the same row represent individual mice
from the same litter; results shown are a summary of four
independent experiments;
[0068] FIG. 19 illustrates the CTL response to LCMV in splenocytes
from B7RP-2-/- and +/+mice harvested 8 days post-infection, and a
memory CTL response to LCMV in splenocytes from B7RP-2-/- and
+/+mice harvested 30 days post-infection and restimulated for 5
days in vitro;
[0069] FIG. 20 illustrates the CTL response and CTL memory response
to influenza virus in splenocytes harvested from B7RP-2-/- and
+/+mice.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All references cited in this application are
expressly incorporated by reference herein.
[0071] Definitions
[0072] The terms "B7RP-2 gene" or "B7RP-2 nucleic acid molecule" or
"B7RP-2 polynucleotide" refer to a nucleic acid molecule comprising
or consisting of a nucleotide sequence as set forth in any of SEQ
ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, a nucleotide sequence
encoding the polypeptide as set forth in any of SEQ ID NO: 2, SEQ
ID NO: 4, or SEQ ID NO: 6, and nucleic acid molecules as defined
herein.
[0073] The term "B7RP-2 polypeptide allelic variant" refers to one
of several possible naturally occurring alternate forms of a gene
occupying a given locus on a chromosome of an organism or a
population of organisms.
[0074] The term "B7RP-2 polypeptide splice variant" refers to a
nucleic acid molecule, usually RNA, which is generated by
alternative processing of intron sequences in an RNA transcript of
B7RP-2 polypeptide amino acid sequence as set forth in any of SEQ
ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
[0075] The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule of the invention that (1) has been separated
from at least about 50 percent of proteins, lipids, carbohydrates,
or other materials with which it is naturally found when total
nucleic acid is isolated from the source cells, (2) is not linked
to all or a portion of a polynucleotide to which the "isolated
nucleic acid molecule" is linked in nature, (3) is operably linked
to a polynucleotide which it is not linked to in nature, or (4)
does not occur in nature as part of a larger polynucleotide
sequence. Preferably, the isolated nucleic acid molecule of the
present invention is substantially free from any other
contaminating nucleic acid molecule(s) or other contaminants that
are found in its natural environment that would interfere with its
use in polypeptide production or its therapeutic, diagnostic,
prophylactic or research use.
[0076] The term "nucleic acid sequence" or "nucleic acid molecule"
refers to a DNA or RNA sequence. The term encompasses molecules
formed from any of the known base analogs of DNA and RNA such as,
but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,
N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiou- racil,
beta-D-mannosylqueosine, 5'-methoxycarbonyl-methyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0077] The term "vector" is used to refer to any molecule (e.g.,
nucleic acid, plasmid, or virus) used to transfer coding
information to a host cell.
[0078] The term "expression vector" refers to a vector that is
suitable for transformation of a host cell and contains nucleic
acid sequences that direct and/or control the expression of
inserted heterologous nucleic acid sequences. Expression includes,
but is not limited to, processes such as transcription,
translation, and RNA splicing, if introns are present.
[0079] The term "operably linked" is used herein to refer to an
arrangement of flanking sequences wherein the flanking sequences so
described are configured or assembled so as to perform their usual
function. Thus, a flanking sequence operably linked to a coding
sequence may be capable of effecting the replication, transcription
and/or translation of the coding sequence. For example, a coding
sequence is operably linked to a promoter when the promoter is
capable of directing transcription of that coding sequence. A
flanking sequence need not be contiguous with the coding sequence,
so long as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0080] The term "host cell" is used to refer to a cell which has
been transformed, or is capable of being transformed with a nucleic
acid sequence and then of expressing a selected gene of interest.
The term includes the progeny of the parent cell, whether or not
the progeny is identical in morphology or in genetic make-up to the
original parent, so long as the selected gene is present.
[0081] The term "B7RP-2 polypeptide" refers to a polypeptide
comprising the amino acid sequence of any of SEQ ID NO: 2, SEQ ID
NO: 4, or SEQ ID NO: 6 and related polypeptides. Related
polypeptides include B7RP-2 polypeptide fragments, B7RP-2
polypeptide orthologs, B7RP-2 polypeptide variants, and B7RP-2
polypeptide derivatives, which possess at least one activity of the
polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or
SEQ ID NO: 6. B7RP-2 polypeptides may be mature polypeptides, as
defined herein, and may or may not have an amino-terminal
methionine residue, depending on the method by which they are
prepared.
[0082] The term "B7RP-2 polypeptide fragment" refers to a
polypeptide that comprises a truncation at the amino-terminus (with
or without a leader sequence) and/or a truncation at the
carboxyl-terminus of the polypeptide as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. The term "B7RP-2 polypeptide
fragment" also refers to amino-terminal and/or carboxyl-terminal
truncations of B7RP-2 polypeptide orthologs, B7RP-2 polypeptide
derivatives, or B7RP-2 polypeptide variants, or to amino-terminal
and/or carboxyl-terminal truncations of the polypeptides encoded by
B7RP-2 polypeptide allelic variants or B7RP-2 polypeptide splice
variants. B7RP-2 polypeptide fragments may result from alternative
RNA splicing or from in vivo protease activity. Membrane-bound
forms of a B7RP-2 polypeptide are also contemplated by the present
invention. In preferred embodiments, truncations and/or deletions
comprise about 10 amino acids, or about 20 amino acids, or about 50
amino acids, or about 75 amino acids, or about 100 amino acids, or
more than about 100 amino acids. The polypeptide fragments so
produced will comprise about 25 contiguous amino acids, or about 50
amino acids, or about 75 amino acids, or about 100 amino acids, or
about 150 amino acids, or more than about 150 amino acids. Such
B7RP-2 polypeptide fragments may optionally comprise an
amino-terminal methionine residue. It will be appreciated that such
fragments can be used, for example, to generate antibodies to
B7RP-2 polypeptides.
[0083] The term "B7RP-2 polypeptide ortholog" refers to a
polypeptide from another species that corresponds to B7RP-2
polypeptide amino acid sequence as set forth in any of SEQ ID NO:
2, SEQ ID NO: 4, or SEQ ID NO: 6. For example, mouse and human
B7RP-2 polypeptides are considered orthologs of each other.
[0084] The term "B7RP-2 polypeptide variants" refers to B7RP-2
polypeptides comprising amino acid sequences having one or more
amino acid sequence substitutions, deletions (such as internal
deletions and/or B7RP-2 polypeptide fragments), and/or additions
(such as internal additions and/or B7RP-2 fusion polypeptides) as
compared to the B7RP-2 polypeptide amino acid sequence set forth in
any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 (with or without
a leader sequence). Variants may be naturally occurring (e.g.,
B7RP-2 polypeptide allelic variants, B7RP-2 polypeptide orthologs,
and B7RP-2 polypeptide splice variants) or artificially
constructed. Such B7RP-2 polypeptide variants may be prepared from
the corresponding nucleic acid molecules having a DNA sequence that
varies accordingly from the DNA sequence as set forth in any of SEQ
ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In preferred embodiments,
the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or
from 1 to 15, or from 1 to 20 or from 1 to 25, or from 1 to 50, or
from 1 to 75, or from 1 to 100, or more than 100 amino acid
substitutions, insertions, additions and/or deletions, wherein the
substitutions may be conservative, or non-conservative, or any
combination thereof.
[0085] The term "B7RP-2 polypeptide derivatives" refers to the
polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or
SEQ ID NO: 6, B7RP-2 polypeptide fragments, B7RP-2 polypeptide
orthologs, or B7RP-2 polypeptide variants, as defined herein, that
have been chemically modified. The term "B7RP-2 polypeptide
derivatives" also refers to the polypeptides encoded by B7RP-2
polypeptide allelic variants or B7RP-2 polypeptide splice variants,
as defined herein, that have been chemically modified.
[0086] The term "mature B7RP-2 polypeptide" refers to a B7RP-2
polypeptide lacking a leader sequence. A mature B7RP-2 polypeptide
may also include other modifications such as proteolytic processing
of the amino-terminus (with or without a leader sequence) and/or
the carboxyl-terminus, cleavage of a smaller polypeptide from a
larger precursor, N-linked and/or O-linked glycosylation, and the
like.
[0087] The term "B7RP-2 fusion polypeptide" refers to a fusion of
one or more amino acids (such as a heterologous protein or peptide)
at the amino- or carboxyl-terminus of the polypeptide as set forth
in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, B7RP-2
polypeptide fragments, B7RP-2 polypeptide orthologs, B7RP-2
polypeptide variants, or B7RP-2 derivatives, as defined herein. The
term "B7RP-2 fusion polypeptide" also refers to a fusion of one or
more amino acids at the amino- or carboxyl-terminus of the
polypeptide encoded by B7RP-2 polypeptide allelic variants or
B7RP-2 polypeptide splice variants, as defined herein.
[0088] The term "biologically active B7RP-2 polypeptides" refers to
B7RP-2 polypeptides having at least one activity characteristic of
the polypeptide comprising the amino acid sequence of any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In addition, a B7RP-2
polypeptide may be active as an immunogen; that is, the B7RP-2
polypeptide contains at least one epitope to which antibodies may
be raised.
[0089] The term "isolated polypeptide" refers to a polypeptide of
the present invention that (1) has been separated from at least
about 50 percent of polynucleotides, lipids, carbohydrates, or
other materials with which it is naturally found when isolated from
the source cell, (2) is not linked (by covalent or noncovalent
interaction) to all or a portion of a polypeptide to which the
"isolated polypeptide" is linked in nature, (3) is operably linked
(by covalent or noncovalent interaction) to a polypeptide with
which it is not linked in nature, or (4) does not occur in nature.
Preferably, the isolated polypeptide is substantially free from any
other contaminating polypeptides or other contaminants that are
found in its natural environment that would interfere with its
therapeutic, diagnostic, prophylactic or research use.
[0090] The term "identity," as known in the art, refers to a
relationship between the sequences of two or more polypeptide
molecules or two or more nucleic acid molecules, as determined by
comparing the sequences. In the art, "identity" also means the
degree of sequence relatedness between nucleic acid molecules or
polypeptides, as the case may be, as determined by the match
between strings of two or more nucleotide or two or more amino acid
sequences. "Identity" measures the percent of identical matches
between the smaller of two or more sequences with gap alignments
(if any) addressed by a particular mathematical model or computer
program (i.e., "algorithms").
[0091] The term "similarity" is a related concept, but in contrast
to "identity," "similarity" refers to a measure of relatedness
which includes both identical matches and conservative substitution
matches. If two polypeptide sequences have, for example, 10/20
identical amino acids, and the remainder are all non-conservative
substitutions, then the percent identity and similarity would both
be 50%. If in the same example, there are five more positions where
there are conservative substitutions, then the percent identity
remains 50%, but the percent similarity would be 75% (15/20).
Therefore, in cases where there are conservative substitutions, the
percent similarity between two polypeptides will be higher than the
percent identity between those two polypeptides.
[0092] The term "naturally occurring" or "native" when used in
connection with biological materials such as nucleic acid
molecules, polypeptides, host cells, and the like, refers to
materials which are found in nature and are not manipulated by man.
Similarly, "non-naturally occurring" or "non-native" as used herein
refers to a material that is not found in nature or that has been
structurally modified or synthesized by man.
[0093] The terms "effective amount" and "therapeutically effective
amount" each refer to the amount of a B7RP-2 polypeptide or B7RP-2
nucleic acid molecule used to support an observable level of one or
more biological activities of the B7RP-2 polypeptides as set forth
herein.
[0094] The term "pharmaceutically acceptable carrier" or
"physiologically acceptable carrier" as used herein refers to one
or more formulation materials suitable for accomplishing or
enhancing the delivery of the B7RP-2 polypeptide, B7RP-2 nucleic
acid molecule, or B7RP-2 selective binding agent as a
pharmaceutical composition.
[0095] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antibody, and additionally capable of being used in an animal
to produce antibodies capable of binding to an epitope of that
antigen. An antigen may have one or more epitopes.
[0096] The term "selective binding agent" refers to a molecule or
molecules having specificity for a B7RP-2 polypeptide. As used
herein, the terms, "specific" and "specificity" refer to the
ability of the selective binding agents to bind to human B7RP-2
polypeptides and not to bind to human non-B7RP-2 polypeptides. It
will be appreciated, however, that the selective binding agents may
also bind orthologs of the polypeptide as set forth in any of SEQ
ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, that is, interspecies
versions thereof, such as mouse and rat B7RP-2 polypeptides.
[0097] The term "transduction" is used to refer to the transfer of
genes from one bacterium to another, usually by a phage.
"Transduction" also refers to the acquisition and transfer of
eukaryotic cellular sequences by retroviruses.
[0098] The term "transfection" is used to refer to the uptake of
foreign or exogenous DNA by a cell, and a cell has been
"transfected" when the exogenous DNA has been introduced inside the
cell membrane. A number of transfection techniques are well known
in the art and are disclosed herein. See, e.g., Graham et al.,
1973, Virology 52:456; Sambrook et al., Molecular Cloning, A
Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et
al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu
et al., 1981, Gene 13:197. Such techniques can be used to introduce
one or more exogenous DNA moieties into suitable host cells.
[0099] The term "transformation" as used herein refers to a change
in a cell's genetic characteristics, and a cell has been
transformed when it has been modified to contain a new DNA. For
example, a cell is transformed where it is genetically modified
from its native state. Following transfection or transduction, the
transforming DNA may recombine with that of the cell by physically
integrating into a chromosome of the cell, may be maintained
transiently as an episomal element without being replicated, or may
replicate independently as a plasmid. A cell is considered to have
been stably transformed when the DNA is replicated with the
division of the cell.
[0100] Relatedness of Nucleic Acid Molecules and/or
Polypeptides
[0101] It is understood that related nucleic acid molecules include
allelic or splice variants of the nucleic acid molecule of any of
SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, and include sequences
which are complementary to any of the above nucleotide sequences.
Related nucleic acid molecules also include a nucleotide sequence
encoding a polypeptide comprising or consisting essentially of a
substitution, modification, addition and/or deletion of one or more
amino acid residues compared to the polypeptide in any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. Such related B7RP-2
polypeptides may comprise, for example, an addition and/or a
deletion of one or more N-linked or O-linked glycosylation sites or
an addition and/or a deletion of one or more cysteine residues.
[0102] Related nucleic acid molecules also include fragments of
B7RP-2 nucleic acid molecules which encode a polypeptide of at
least about 25 contiguous amino acids, or about 50 amino acids, or
about 75 amino acids, or about 100 amino acids, or about 150 amino
acids, or more than about 150 amino acid residues of the B7RP-2
polypeptide of any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
6.
[0103] In addition, related B7RP-2 nucleic acid molecules also
include those molecules which comprise nucleotide sequences which
hybridize under moderately or highly stringent conditions as
defined herein with the fully complementary sequence of the B7RP-2
nucleic acid molecule of any of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ
ID NO: 5, or of a molecule encoding a polypeptide, which
polypeptide comprises the amino acid sequence as shown in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or of a nucleic acid
fragment as defined herein, or of a nucleic acid fragment encoding
a polypeptide as defined herein. Hybridization probes may be
prepared using the B7RP-2 sequences provided herein to screen cDNA,
genomic or synthetic DNA libraries for related sequences. Regions
of the DNA and/or amino acid sequence of B7RP-2 polypeptide that
exhibit significant identity to known sequences are readily
determined using sequence alignment algorithms as described herein
and those regions may be used to design probes for screening.
[0104] The term "highly stringent conditions" refers to those
conditions that are designed to permit hybridization of DNA strands
whose sequences are highly complementary, and to exclude
hybridization of significantly mismatched DNAs. Hybridization
stringency is principally determined by temperature, ionic
strength, and the concentration of denaturing agents such as
formamide. Examples of "highly stringent conditions" for
hybridization and washing are 0.015 M sodium chloride, 0.0015 M
sodium citrate at 65-68.degree. C. or 0.015 M sodium chloride,
0.0015 M sodium citrate, and 50% formamide at 42.degree. C. See
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et
al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL
Press Limited).
[0105] More stringent conditions (such as higher temperature, lower
ionic strength, higher formamide, or other denaturing agent) may
also be used-however, the rate of hybridization will be affected.
Other agents may be included in the hybridization and washing
buffers for the purpose of reducing non-specific and/or background
hybridization. Examples are 0.1% bovine serum albumin, 0.1%
polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium
dodecylsulfate, NaDodSO.sub.4, (SDS), ficoll, Denhardt's solution,
sonicated salmon sperm DNA (or another non-complementary DNA), and
dextran sulfate, although other suitable agents can also be used.
The concentration and types of these additives can be changed
without substantially affecting the stringency of the hybridization
conditions. Hybridization experiments are usually carried out at pH
6.8-7.4; however, at typical ionic strength conditions, the rate of
hybridization is nearly independent of pH. See Anderson et al.,
Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press
Limited).
[0106] Factors affecting the stability of DNA duplex include base
composition, length, and degree of base pair mismatch.
Hybridization conditions can be adjusted by one skilled in the art
in order to accommodate these variables and allow DNAs of different
sequence relatedness to form hybrids. The melting temperature of a
perfectly matched DNA duplex can be estimated by the following
equation:
T.sub.m(.degree. C)=81.5+16.6(log[Na+])+0.41(% G+C)-600/N-0.72(%
formamide)
[0107] where N is the length of the duplex formed, [Na+] is the
molar concentration of the sodium ion in the hybridization or
washing solution, % G+C is the percentage of (guanine+cytosine)
bases in the hybrid. For imperfectly matched hybrids, the melting
temperature is reduced by approximately 1.degree. C. for each 1%
mismatch.
[0108] The term "moderately stringent conditions" refers to
conditions under which a DNA duplex with a greater degree of base
pair mismatching than could occur under "highly stringent
conditions" is able to form. Examples of typical "moderately
stringent conditions" are 0.015 M sodium chloride, 0.0015 M sodium
citrate at 50-65.degree. C. or 0.015 M sodium chloride, 0.0015 M
sodium citrate, and 20% formamide at 37-50.degree. C. By way of
example, "moderately stringent conditions" of 50.degree. C. in
0.015 M sodium ion will allow about a 21% mismatch.
[0109] It will be appreciated by those skilled in the art that
there is no absolute distinction between "highly stringent
conditions" and "moderately stringent conditions." For example, at
0.015 M sodium ion (no formamide), the melting temperature of
perfectly matched long DNA is about 71.degree. C. With a wash at
65.degree. C. (at the same ionic strength), this would allow for
approximately a 6% mismatch. To capture more distantly related
sequences, one skilled in the art can simply lower the temperature
or raise the ionic strength.
[0110] A good estimate of the melting temperature in 1M NaCl* for
oligonucleotide probes up to about 20 nt is given by:
Tm=2.degree. C. per A-T base pair+4.degree. C. per G-C base
pair
[0111] *The sodium ion concentration in 6.times.salt sodium citrate
(SSC) is 1M. See Suggs et al., Developmental Biology Using Purified
Genes 683 (Brown and Fox, eds., 1981).
[0112] High stringency washing conditions for oligonucleotides are
usually at a temperature of 0-5.degree. C. below the Tm of the
oligonucleotide in 6.times.SSC, 0.1% SDS.
[0113] In another embodiment, related nucleic acid molecules
comprise or consist of a nucleotide sequence that is at least about
70 percent identical to the nucleotide sequence as shown in any of
SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or comprise or consist
essentially of a nucleotide sequence encoding a polypeptide that is
at least about 70 percent identical to the polypeptide as set forth
in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In preferred
embodiments, the nucleotide sequences are about 75 percent, or
about 80 percent, or about 85 percent, or about 90 percent, or
about 95, 96, 97, 98, or 99 percent identical to the nucleotide
sequence as shown in any of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID
NO: 5, or the nucleotide sequences encode a polypeptide that is
about 75 percent, or about 80 percent, or about 85 percent, or
about 90 percent, or about 95, 96, 97, 98, or 99 percent identical
to the polypeptide sequence as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6. Related nucleic acid molecules
encode polypeptides possessing at least one activity of the
polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6.
[0114] Differences in the nucleic acid sequence may result in
conservative and/or non-conservative modifications of the amino
acid sequence relative to the amino acid sequence of any of SEQ ID
NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
[0115] Conservative modifications to the amino acid sequence of any
of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 (and the
corresponding modifications to the encoding nucleotides) will
produce a polypeptide having functional and chemical
characteristics similar to those of B7RP-2 polypeptides. In
contrast, substantial modifications in the functional and/or
chemical characteristics of B7RP-2 polypeptides may be accomplished
by selecting substitutions in the amino acid sequence of any of SEQ
ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 that differ significantly
in their effect on maintaining (a) the structure of the molecular
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
[0116] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
nonnative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis."
[0117] Conservative amino acid substitutions also encompass
non-naturally occurring amino acid residues that are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics, and other
reversed or inverted forms of amino acid moieties.
[0118] Naturally occurring residues may be divided into classes
based on common side chain properties:
[0119] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0120] 2) neutral hydrophilic: Cys, Ser, Thr;
[0121] 3) acidic: Asp, Glu;
[0122] 4) basic: Asn, Gln, His, Lys, Arg;
[0123] 5) residues that influence chain orientation: Gly, Pro;
and
[0124] 6) aromatic: Trp, Tyr, Phe.
[0125] For example, non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the human B7RP-2 polypeptide that are homologous with
non-human B7RP-2 polypeptides, or into the non-homologous regions
of the molecule.
[0126] In making such changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic
index on the basis of its hydrophobicity and charge
characteristics. The hydropathic indices are: isoleucine (+4.5);
valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine
(-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);
lysine (-3.9); and arginine (-4.5).
[0127] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte et al., 1982, J. Mol. Biol.
157:105-31). It is known that certain amino acids may be
substituted for other amino acids having a similar hydropathic
index or score and still retain a similar biological activity. In
making changes based upon the hydropathic index, the substitution
of amino acids whose hydropathic indices are within .+-.2 is
preferred, those which are within .+-.1 are particularly preferred,
and those within .+-.0.5 are even more particularly preferred.
[0128] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functionally
equivalent protein or peptide thereby created is intended for use
in immunological embodiments, as in the present case. The greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property
of the protein.
[0129] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, the substitution of amino acids whose
hydrophilicity values are within .+-.2 is preferred, those which
are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred. One may also identify
epitopes from primary amino acid sequences on the basis of
hydrophilicity. These regions are also referred to as "epitopic
core regions."
[0130] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
B7RP-2 polypeptide, or to increase or decrease the affinity of the
B7RP-2 polypeptides described herein. Exemplary amino acid
substitutions are set forth in Table I.
1TABLE I Amino Acid Substitutions Original Residues Exemplary
Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg
Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn
Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile
Leu, Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile
Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn
Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly
Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,
Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine
[0131] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 4, or SEQ ID NO: 6 using well-known techniques. For
identifying suitable areas of the molecule that may be changed
without destroying biological activity, one skilled in the art may
target areas not believed to be important for activity. For
example, when similar polypeptides with similar activities from the
same species or from other species are known, one skilled in the
art may compare the amino acid sequence of a B7RP-2 polypeptide to
such similar polypeptides. With such a comparison, one can identify
residues and portions of the molecules that are conserved among
similar polypeptides. It will be appreciated that changes in areas
of the B7RP-2 molecule that are not conserved relative to such
similar polypeptides would be less likely to adversely affect the
biological activity and/or structure of a B7RP-2 polypeptide. One
skilled in the art would also know that, even in relatively
conserved regions, one may substitute chemically similar amino
acids for the naturally occurring residues while retaining activity
(conservative amino acid residue substitutions). Therefore, even
areas that may be important for biological activity or for
structure may be subject to conservative amino acid substitutions
without destroying the biological activity or without adversely
affecting the polypeptide structure.
[0132] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a B7RP-2 polypeptide that correspond to amino acid
residues that are important for activity or structure in similar
polypeptides. One skilled in the art may opt for chemically similar
amino acid substitutions for such predicted important amino acid
residues of B7RP-2 polypeptides.
[0133] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of B7RP-2 polypeptide with respect to its three
dimensional structure. One skilled in the art may choose not to
make radical changes to amino acid residues predicted to be on the
surface of the protein, since such residues may be involved in
important interactions with other molecules. Moreover, one skilled
in the art may generate test variants containing a single amino
acid substitution at each amino acid residue. The variants could be
screened using activity assays known to those with skill in the
art. Such variants could be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change would
be avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided
either alone or in combination with other mutations.
[0134] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult, 1996, Curr. Opin.
Biotechnol. 7:422-27; Chou et al., 1974, Biochemistry 13:222-45;
Chou et al., 1974, Biochemistry 113:211-22; Chou et al., 1978, Adv.
Enzymol. Relat. Areas Mol. Biol. 47:45-48; Chou et al, 1978, Ann.
Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys. J.
26:367-84. Moreover, computer programs are currently available to
assist with predicting secondary structure. One method of
predicting secondary structure is based upon homology modeling. For
example, two polypeptides or proteins which have a sequence
identity of greater than 30%, or similarity greater than 40%, often
have similar structural topologies. The recent growth of the
protein structural database (PDB) has provided enhanced
predictability of secondary structure, including the potential
number of folds within the structure of a polypeptide or protein.
See Holm et al., 1999, Nucleic Acids Res. 27:244-47. It has been
suggested that there are a limited number of folds in a given
polypeptide or protein and that once a critical number of
structures have been resolved, structural prediction will become
dramatically more accurate (Brenner et al., 1997, Curr. Opin.
Struct. Biol. 7:369-76).
[0135] Additional methods of predicting secondary structure include
"threading" (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl
et al., 1996, Structure 4:15-19), "profile analysis" (Bowie et al.,
1991, Science, 253:164-70; Gribskov et al., 1990, Methods Enzymol.
183:146-59; Gribskov et al., 1987, Proc. Nat. Acad. Sci. U.S.A.
84:4355-58), and "evolutionary linkage" (See Holm et al., supra,
and Brenner et al., supra).
[0136] Preferred B7RP-2 polypeptide variants include glycosylation
variants wherein the number and/or type of glycosylation sites have
been altered compared to the amino acid sequence set forth in any
of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In one embodiment,
B7RP-2 polypeptide variants comprise a greater or a lesser number
of N-linked glycosylation sites than the amino acid sequence set
forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. An
N-linked glycosylation site is characterized by the sequence:
Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated
as X may be any amino acid residue except proline. The substitution
of amino acid residues to create this sequence provides a potential
new site for the addition of an N-linked carbohydrate chain.
Alternatively, substitutions that eliminate this sequence will
remove an existing N-linked carbohydrate chain. Also provided is a
rearrangement of N-linked carbohydrate chains wherein one or more
N-linked glycosylation sites (typically those that are naturally
occurring) are eliminated and one or more new N-linked sites are
created. Additional preferred B7RP-2 variants include cysteine
variants, wherein one or more cysteine residues are deleted or
substituted with another amino acid (e.g., serine) as compared to
the amino acid sequence set forth in any of SEQ ID NO: 2, SEQ ID
NO: 4, or SEQ ID NO: 6. Cysteine variants are useful when B7RP-2
polypeptides must be refolded into a biologically active
conformation such as after the isolation of insoluble inclusion
bodies. Cysteine variants generally have fewer cysteine residues
than the native protein, and typically have an even number to
minimize interactions resulting from unpaired cysteines.
[0137] In other embodiments, related nucleic acid molecules
comprise or consist of a nucleotide sequence encoding a polypeptide
as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6
with at least one amino acid insertion and wherein the polypeptide
has an activity of the polypeptide set forth in any of SEQ ID NO:
2, SEQ ID NO: 4, or SEQ ID NO: 6, or a nucleotide sequence encoding
a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or
SEQ ID NO: 6 with at least one amino acid deletion and wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. Related nucleic acid
molecules also comprise or consist of a nucleotide sequence
encoding a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID
NO: 4, or SEQ ID NO: 6 wherein the polypeptide has a carboxyl-
and/or amino-terminal truncation and further wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. Related nucleic acid
molecules also comprise or consist of a nucleotide sequence
encoding a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID
NO: 4, or SEQ ID NO: 6 with at least one modification selected from
the group consisting of amino acid substitutions, amino acid
insertions, amino acid deletions, carboxyl-terminal truncations,
and amino-terminal truncations and wherein the polypeptide has an
activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ
ID NO: 4, or SEQ ID NO: 6.
[0138] In addition, the polypeptide comprising the amino acid
sequence of any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or
other B7RP-2 polypeptide, may be fused to a homologous polypeptide
to form a homodimer or to a heterologous polypeptide to form a
heterodimer. Heterologous peptides and polypeptides include, but
are not limited to: an epitope to allow for the detection and/or
isolation of a B7RP-2 fusion polypeptide; a transmembrane receptor
protein or a portion thereof, such as an extracellular domain or a
transmembrane and intracellular domain; a ligand or a portion
thereof which binds to a transmembrane receptor protein; an enzyme
or portion thereof which is catalytically active; a polypeptide or
peptide which promotes oligomerization, such as a leucine zipper
domain; a polypeptide or peptide which increases stability, such as
an immunoglobulin constant region; and a polypeptide which has a
therapeutic activity different from the polypeptide comprising the
amino acid sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO:
4, or SEQ ID NO: 6, or other B7RP-2 polypeptide.
[0139] Fusions can be made either at the amino-terminus or at the
carboxyl-terminus of the polypeptide comprising the amino acid
sequence set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID
NO: 6, or other B7RP-2 polypeptide. Fusions may be direct with no
linker or adapter molecule or may be through a linker or adapter
molecule. A linker or adapter molecule may be one or more amino
acid residues, typically from about 20 to about 50 amino acid
residues. A linker or adapter molecule may also be designed with a
cleavage site for a DNA restriction endonuclease or for a protease
to allow for the separation of the fused moieties. It will be
appreciated that once constructed, the fusion polypeptides can be
derivatized according to the methods described herein.
[0140] In a further embodiment of the invention, the polypeptide
comprising the amino acid sequence of any of SEQ ID NO: 2, SEQ ID
NO: 4, or SEQ ID NO: 6, or other B7RP-2 polypeptide, is fused to
one or more domains of an Fc region of human IgG. Antibodies
comprise two functionally independent parts, a variable domain
known as "Fab," that binds an antigen, and a constant domain known
as "Fc," that is involved in effector functions such as complement
activation and attack by phagocytic cells. An Fc has a long serum
half-life, whereas an Fab is short-lived. Capon et al., 1989,
Nature 337:525-31. When constructed together with a therapeutic
protein, an Fc domain can provide longer half-life or incorporate
such functions as Fc receptor binding, protein A binding,
complement fixation, and perhaps even placental transfer. Id. Table
II summarizes the use of certain Fc fusions known in the art.
2TABLE II Fc Fusion with Therapeutic Proteins Form of Fc Fusion
partner Therapeutic implications Reference IgG1 N-terminus of
Hodgkin's disease; U.S. Pat. No. CD30-L anaplastic lymphoma:
5,480,981 T-cell leukemia Murine IL-10 anti-inflammatory; Zheng et
al., Fc.gamma.2a transplant rejection 1995, J. Immunol.
154:5590-600 IgG1 TNF receptor septic shock Fisher et al., 1996, N.
Engl. J. Med. 334:1697- 1702; Van Zee et al., 1996, J. Immunol.
156:2221-30 IgG, IgA, TNF receptor inflammation, U.S. Pat. No. IgM,
or IgE autoimmune disorders 5,808,029 (excluding the first domain)
IgG1 CD4 receptor AIDS Capon et al., 1989, Nature 337:525-31 IgG1,
IgG3 N-terminus anti-cancer, antiviral Harvill et al., of IL-2
1995, Immunotech. 1:95-105 IgG1 C-terminus of osteoarthritis; WO
97/23614 OPG bone density IgG1 N-terminus of anti-obesity PCT/US
leptin 97/23183, filed Dec. 11, 1997 Human Ig CTLA-4 autoimmune
disorders Linsley, 1991, C.gamma.1 J. Exp. Med., 174:561-69
[0141] In one example, a human IgG hinge, CH2, and CH3 region may
be fused at either the amino-terminus or carboxyl-terminus of the
B7RP-2 polypeptides using methods known to the skilled artisan. In
another example, a human IgG hinge, CH2, and CH3 region may be
fused at either the amino-terminus or carboxyl-terminus of a B7RP-2
polypeptide fragment (e.g., the predicted extracellular portion of
B7RP-2 polypeptide).
[0142] The resulting B7RP-2 fusion polypeptide may be purified by
use of a Protein A affinity column. Peptides and proteins fused to
an Fc region have been found to exhibit a substantially greater
half-life in vivo than the unfused counterpart. Also, a fusion to
an Fc region allows for dimerization/multimerization of the fusion
polypeptide. The Fc region may be a naturally occurring Fc region,
or may be altered to improve certain qualities, such as therapeutic
qualities, circulation time, or reduced aggregation.
[0143] Identity and similarity of related nucleic acid molecules
and polypeptides are readily calculated by known methods. Such
methods include, but are not limited to those described in
Computational Molecular Biology (A. M. Lesk, ed., Oxford University
Press 1988); Biocomputing: Informatics and Genome Projects (D. W.
Smith, ed., Academic Press 1993); Computer Analysis of Sequence
Data (Part 1, A. M. Griffin and H. G. Griffin, eds., Humana Press
1994); G. von Heijne, Sequence Analysis in Molecular Biology
(Academic Press 1987); Sequence Analysis Primer (M. Gribskov and J.
Devereux, eds., M. Stockton Press 1991); and Carillo et al., 1988,
SIAM J. Applied Math., 48:1073.
[0144] Preferred methods to determine identity and/or similarity
are designed to give the largest match between the sequences
tested. Methods to determine identity and similarity are described
in publicly available computer programs. Preferred computer program
methods to determine identity and similarity between two sequences
include, but are not limited to, the GCG program package, including
GAP (Devereux et al., 1984, Nucleic Acids Res. 12:387; Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP,
BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol.
215:403-10). The BLASTX program is publicly available from the
National Center for Biotechnology Information (NCBI) and other
sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda,
Md.); Altschul et al., 1990, supra). The well-known Smith Waterman
algorithm may also be used to determine identity.
[0145] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, in a preferred
embodiment, the selected alignment method (GAP program) will result
in an alignment that spans at least 50 contiguous amino acids of
the claimed polypeptide.
[0146] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span," as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal; the "average diagonal" is the average of the diagonal of
the comparison matrix being used; the "diagonal" is the score or
number assigned to each perfect amino acid match by the particular
comparison matrix) and a gap extension penalty (which is usually
0.1.times. the gap opening penalty), as well as a comparison matrix
such as PAM 250 or BLOSUM 62 are used in conjunction with the
algorithm. A standard comparison matrix is also used by the
algorithm (see Dayhoff et al., 5 Atlas of Protein Sequence and
Structure (Supp. 3 1978)(PAM250 comparison matrix); Henikoff et
al., 1992, Proc. Natl. Acad. Sci USA 89:10915-19 (BLOSUM 62
comparison matrix)).
[0147] Preferred parameters for polypeptide sequence comparison
include the following:
[0148] Algorithm: Needleman and Wunsch, 1970, J. Mol. Biol.
48:443-53;
[0149] Comparison matrix: BLOSUM 62 (Henikoff et al., supra);
[0150] Gap Penalty: 12
[0151] Gap Length Penalty: 4
[0152] Threshold of Similarity: 0
[0153] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for
polypeptide comparisons (along with no penalty for end gaps) using
the GAP algorithm.
[0154] Preferred parameters for nucleic acid molecule sequence
comparison include the following:
[0155] Algorithm: Needleman and Wunsch, supra;
[0156] Comparison matrix: matches=+10, mismatch=0
[0157] Gap Penalty: 50
[0158] Gap Length Penalty: 3
[0159] The GAP program is also useful with the above parameters.
The aforementioned parameters are the default parameters for
nucleic acid molecule comparisons.
[0160] Other exemplary algorithms, gap opening penalties, gap
extension penalties, comparison matrices, and thresholds of
similarity may be used, including those set forth in the Program
Manual, Wisconsin Package, Version 9, September, 1997. The
particular choices to be made will be apparent to those of skill in
the art and will depend on the specific comparison to be made, such
as DNA-to-DNA, protein-to-protein, protein-to-DNA; and
additionally, whether the comparison is between given pairs of
sequences (in which case GAP or BestFit are generally preferred) or
between one sequence and a large database of sequences (in which
case FASTA or BLASTA are preferred).
[0161] Nucleic Acid Molecules
[0162] The nucleic acid molecules encoding a polypeptide comprising
the amino acid sequence of a B7RP-2 polypeptide can readily be
obtained in a variety of ways including, without limitation,
chemical synthesis, cDNA or genomic library screening, expression
library screening, and/or PCR amplification of cDNA.
[0163] Recombinant DNA methods used herein are generally those set
forth in Sambrook et al., Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, 1989) and/or Current
Protocols in Molecular Biology (Ausubel et al., eds., Green
Publishers Inc. and Wiley and Sons 1994). The invention provides
for nucleic acid molecules as described herein and methods for
obtaining such molecules.
[0164] Where a gene encoding the amino acid sequence of a B7RP-2
polypeptide has been identified from one species, all or a portion
of that gene may be used as a probe to identify orthologs or
related genes from the same species. The probes or primers may be
used to screen cDNA libraries from various tissue sources believed
to express the B7RP-2 polypeptide. In addition, part or all of a
nucleic acid molecule having the sequence as set forth in any of
SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 may be used to screen a
genomic library to identify and isolate a gene encoding the amino
acid sequence of a B7RP-2 polypeptide. Typically, conditions of
moderate or high stringency will be employed for screening to
minimize the number of false positives obtained from the
screening.
[0165] Nucleic acid molecules encoding the amino acid sequence of
B7RP-2 polypeptides may also be identified by expression cloning
which employs the detection of positive clones based upon a
property of the expressed protein. Typically, nucleic acid
libraries are screened by the binding an antibody or other binding
partner (e.g., receptor or ligand) to cloned proteins that are
expressed and displayed on a host cell surface. The antibody or
binding partner is modified with a detectable label to identify
those cells expressing the desired clone.
[0166] Recombinant expression techniques conducted in accordance
with the descriptions set forth below may be followed to produce
these polynucleotides and to express the encoded polypeptides. For
example, by inserting a nucleic acid sequence that encodes the
amino acid sequence of a B7RP-2 polypeptide into an appropriate
vector, one skilled in the art can readily produce large quantities
of the desired nucleotide sequence. The sequences can then be used
to generate detection probes or amplification primers.
Alternatively, a polynucleotide encoding the amino acid sequence of
a B7RP-2 polypeptide can be inserted into an expression vector. By
introducing the expression vector into an appropriate host, the
encoded B7RP-2 polypeptide may be produced in large amounts.
[0167] Another method for obtaining a suitable nucleic acid
sequence is the polymerase chain reaction (PCR). In this method,
cDNA is prepared from poly(A)+RNA or total RNA using the enzyme
reverse transcriptase. Two primers, typically complementary to two
separate regions of cDNA encoding the amino acid sequence of a
B7RP-2 polypeptide, are then added to the cDNA along with a
polymerase such as Taq polymerase, and the polymerase amplifies the
cDNA region between the two primers.
[0168] Another means of preparing a nucleic acid molecule encoding
the amino acid sequence of a B7RP-2 polypeptide is chemical
synthesis using methods well known to the skilled artisan such as
those described by Engels et al., 1989, Angew. Chem. Intl. Ed.
28:716-34. These methods include, inter alia, the phosphotriester,
phosphoramidite, and H-phosphonate methods for nucleic acid
synthesis. A preferred method for such chemical synthesis is
polymer-supported synthesis using standard phosphoramidite
chemistry. Typically, the DNA encoding the amino acid sequence of a
B7RP-2 polypeptide will be several hundred nucleotides in length.
Nucleic acids larger than about 100 nucleotides can be synthesized
as several fragments using these methods. The fragments can then be
ligated together to form the full-length nucleotide sequence of a
B7RP-2 gene. Usually, the DNA fragment encoding the amino-terminus
of the polypeptide will have an ATG, which encodes a methionine
residue. This methionine may or may not be present on the mature
form of the B7RP-2 polypeptide, depending on whether the
polypeptide produced in the host cell is designed to be secreted
from that cell. Other methods known to the skilled artisan may be
used as well.
[0169] In certain embodiments, nucleic acid variants contain codons
which have been altered for optimal expression of a B7RP-2
polypeptide in a given host cell. Particular codon alterations will
depend upon the B7RP-2 polypeptide and host cell selected for
expression. Such "codon optimization" can be carried out by a
variety of methods, for example, by selecting codons which are
preferred for use in highly expressed genes in a given host cell.
Computer algorithms which incorporate codon frequency tables such
as "Eco_high.Cod" for codon preference of highly expressed
bacterial genes may be used and are provided by the University of
Wisconsin Package Version 9.0 (Genetics Computer Group, Madison,
Wis.). Other useful codon frequency tables include
"Celegans_high.cod," "Celegans_low.cod," "Drosophila_high.cod,"
"Human_high.cod," "Maize_high.cod," and "Yeast_high.cod."
[0170] In some cases, it may be desirable to prepare nucleic acid
molecules encoding B7RP-2 polypeptide variants. Nucleic acid
molecules encoding variants may be produced using site directed
mutagenesis, PCR amplification, or other appropriate methods, where
the primer(s) have the desired point mutations (see Sambrook et
al., supra, and Ausubel et al., supra, for descriptions of
mutagenesis techniques). Chemical synthesis using methods described
by Engels et al., supra, may also be used to prepare such variants.
Other methods known to the skilled artisan may be used as well.
[0171] Vectors and Host Cells
[0172] A nucleic acid molecule encoding the amino acid sequence of
a B7RP-2 polypeptide is inserted into an appropriate expression
vector using standard ligation techniques. The vector is typically
selected to be functional in the particular host cell employed
(i.e., the vector is compatible with the host cell machinery such
that amplification of the gene and/or expression of the gene can
occur). A nucleic acid molecule encoding the amino acid sequence of
a B7RP-2 polypeptide may be amplified/expressed in prokaryotic,
yeast, insect (baculovirus systems) and/or eukaryotic host cells.
Selection of the host cell will depend in part on whether a B7RP-2
polypeptide is to be post-translationally modified (e.g.,
glycosylated and/or phosphorylated). If so, yeast, insect, or
mammalian host cells are preferable. For a review of expression
vectors, see Meth. Enz., vol. 185 (D. V. Goeddel, ed., Academic
Press 1990).
[0173] Typically, expression vectors used in any of the host cells
will contain sequences for plasmid maintenance and for cloning and
expression of exogenous nucleotide sequences. Such sequences,
collectively referred to as "flanking sequences" in certain
embodiments will typically include one or more of the following
nucleotide sequences: a promoter, one or more enhancer sequences,
an origin of replication, a transcriptional termination sequence, a
complete intron sequence containing a donor and acceptor splice
site, a sequence encoding a leader sequence for polypeptide
secretion, a ribosome binding site, a polyadenylation sequence, a
polylinker region for inserting the nucleic acid encoding the
polypeptide to be expressed, and a selectable marker element. Each
of these sequences is discussed below.
[0174] Optionally, the vector may contain a "tag"-encoding
sequence, i.e., an oligonucleotide molecule located at the 5' or 3'
end of the B7RP-2 polypeptide coding sequence; the oligonucleotide
sequence encodes polyllis (such as hexalis), or another "tag" such
as FLAG, HA (hemaglutinin influenza virus), or myc for which
commercially available antibodies exist. This tag is typically
fused to the polypeptide upon expression of the polypeptide, and
can serve as a means for affinity purification of the B7RP-2
polypeptide from the host cell. Affinity purification can be
accomplished, for example, by column chromatography using
antibodies against the tag as an affinity matrix. Optionally, the
tag can subsequently be removed from the purified B7RP-2
polypeptide by various means such as using certain peptidases for
cleavage.
[0175] Flanking sequences may be homologous (i.e., from the same
species and/or strain as the host cell), heterologous (i.e., from a
species other than the host cell species or strain), hybrid (i.e.,
a combination of flanking sequences from more than one source), or
synthetic, or the flanking sequences may be native sequences which
normally function to regulate B7RP-2 polypeptide expression. As
such, the source of a flanking sequence may be any prokaryotic or
eukaryotic organism, any vertebrate or invertebrate organism, or
any plant, provided that the flanking sequence is functional in,
and can be activated by, host cell machinery.
[0176] Flanking sequences useful in the vectors of this invention
may be obtained by any of several methods well known in the art.
Typically, flanking sequences useful herein--other than the B7RP-2
gene flanking sequences--will have been previously identified by
mapping and/or by restriction endonuclease digestion and can thus
be isolated from the proper tissue source using the appropriate
restriction endonucleases. In some cases, the full nucleotide
sequence of a flanking sequence may be known. Here, the flanking
sequence may be synthesized using the methods described herein for
nucleic acid synthesis or cloning.
[0177] Where all or only a portion of the flanking sequence is
known, it may be obtained using PCR and/or by screening a genomic
library with a suitable oligonucleotide and/or flanking sequence
fragment from the same or another species. Where the flanking
sequence is not known, a fragment of DNA containing a flanking
sequence may be isolated from a larger piece of DNA that may
contain, for example, a coding sequence or even another gene or
genes. Isolation may be accomplished by restriction endonuclease
digestion to produce the proper DNA fragment followed by isolation
using agarose gel purification, Qiagen.RTM. column chromatography
(Chatsworth, Calif.), or other methods known to the skilled
artisan. The selection of suitable enzymes to accomplish this
purpose will be readily apparent to one of ordinary skill in the
art.
[0178] An origin of replication is typically a part of those
prokaryotic expression vectors purchased commercially, and the
origin aids in the amplification of the vector in a host cell.
Amplification of the vector to a certain copy number can, in some
cases, be important for the optimal expression of a B7RP-2
polypeptide. If the vector of choice does not contain an origin of
replication site, one may be chemically synthesized based on a
known sequence, and ligated into the vector. For example, the
origin of replication from the plasmid pBR322 (New England Biolabs,
Beverly, Mass.) is suitable for most gram-negative bacteria and
various origins (e.g., SV40, polyoma, adenovirus, vesicular
stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are
useful for cloning vectors in mammalian cells. Generally, the
origin of replication component is not needed for mammalian
expression vectors (for example, the SV40 origin is often used only
because it contains the early promoter).
[0179] A transcription termination sequence is typically located 3'
of the end of a polypeptide coding region and serves to terminate
transcription. Usually, a transcription termination sequence in
prokaryotic cells is a G-C rich fragment followed by a poly-T
sequence. While the sequence is easily cloned from a library or
even purchased commercially as part of a vector, it can also be
readily synthesized using methods for nucleic acid synthesis such
as those described herein.
[0180] A selectable marker gene element encodes a protein necessary
for the survival and growth of a host cell grown in a selective
culture medium. Typical selection marker genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, tetracycline, or kanamycin for prokaryotic host cells;
(b) complement auxotrophic deficiencies of the cell; or (c) supply
critical nutrients not available from complex media. Preferred
selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, and the tetracycline resistance gene. A
neomycin resistance gene may also be used for selection in
prokaryotic and eukaryotic host cells.
[0181] Other selection genes may be used to amplify the gene that
will be expressed. Amplification is the process wherein genes that
are in greater demand for the production of a protein critical for
growth are reiterated in tandem within the chromosomes of
successive generations of recombinant cells. Examples of suitable
selectable markers for mammalian cells include dihydrofolate
reductase (DHFR) and thymidine kinase. The mammalian cell
transformants are placed under selection pressure wherein only the
transformants are uniquely adapted to survive by virtue of the
selection gene present in the vector. Selection pressure is imposed
by culturing the transformed cells under conditions in which the
concentration of selection agent in the medium is successively
changed, thereby leading to the amplification of both the selection
gene and the DNA that encodes a B7RP-2 polypeptide. As a result,
increased quantities of B7RP-2 polypeptide are synthesized from the
amplified DNA.
[0182] A ribosome binding site is usually necessary for translation
initiation of mRNA and is characterized by a Shine-Dalgarno
sequence (prokaryotes) or a Kozak sequence (eukaryotes). The
element is typically located 3' to the promoter and 5' to the
coding sequence of a B7RP-2 polypeptide to be expressed. The
Shine-Dalgarno sequence is varied but is typically a polypurine
(i.e., having a high A-G content). Many Shine-Dalgarno sequences
have been identified, each of which can be readily synthesized
using methods set forth herein and used in a prokaryotic
vector.
[0183] A leader, or signal, sequence may be used to direct a B7RP-2
polypeptide out of the host cell. Typically, a nucleotide sequence
encoding the signal sequence is positioned in the coding region of
a B7RP-2 nucleic acid molecule, or directly at the 5' end of a
B7RP-2 polypeptide coding region. Many signal sequences have been
identified, and any of those that are functional in the selected
host cell may be used in conjunction with a B7RP-2 nucleic acid
molecule. Therefore, a signal sequence may be homologous (naturally
occurring) or heterologous to the B7RP-2 nucleic acid molecule.
Additionally, a signal sequence may be chemically synthesized using
methods described herein. In most cases, the secretion of a B7RP-2
polypeptide from the host cell via the presence of a signal peptide
will result in the removal of the signal peptide from the secreted
B7RP-2 polypeptide. The signal sequence may be a component of the
vector, or it may be a part of a B7RP-2 nucleic acid molecule that
is inserted into the vector.
[0184] Included within the scope of this invention is the use of
either a nucleotide sequence encoding a native B7RP-2 polypeptide
signal sequence joined to a B7RP-2 polypeptide coding region or a
nucleotide sequence encoding a heterologous signal sequence joined
to a B7RP-2 polypeptide coding region. The heterologous signal
sequence selected should be one that is recognized and processed,
i.e., cleaved by a signal peptidase, by the host cell. For
prokaryotic host cells that do not recognize and process the native
B7RP-2 polypeptide signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, or
heat-stable enterotoxin II leaders. For yeast secretion, the native
B7RP-2 polypeptide signal sequence may be substituted by the yeast
invertase, alpha factor, or acid phosphatase leaders. In mammalian
cell expression the native signal sequence is satisfactory,
although other mammalian signal sequences may be suitable.
[0185] In some cases, such as where glycosylation is desired in a
eukaryotic host cell expression system, one may manipulate the
various presequences to improve glycosylation or yield. For
example, one may alter the peptidase cleavage site of a particular
signal peptide, or add pro-sequences, which also may affect
glycosylation. The final protein product may have, in the -1
position (relative to the first amino acid of the mature protein)
one or more additional amino acids incident to expression, which
may not have been totally removed. For example, the final protein
product may have one or two amino acid residues found in the
peptidase cleavage site, attached to the amino-terminus.
Alternatively, use of some enzyme cleavage sites may result in a
slightly truncated form of the desired B7RP-2 polypeptide, if the
enzyme cuts at such area within the mature polypeptide.
[0186] In many cases, transcription of a nucleic acid molecule is
increased by the presence of one or more introns in the vector;
this is particularly true where a polypeptide is produced in
eukaryotic host cells, especially mammalian host cells. The introns
used may be naturally occurring within the B7RP-2 gene especially
where the gene used is a full-length genomic sequence or a fragment
thereof. Where the intron is not naturally occurring within the
gene (as for most cDNAs), the intron may be obtained from another
source. The position of the intron with respect to flanking
sequences and the B7RP-2 gene is generally important, as the intron
must be transcribed to be effective. Thus, when a B7RP-2 cDNA
molecule is being transcribed, the preferred position for the
intron is 3' to the transcription start site and 5' to the poly-A
transcription termination sequence. Preferably, the intron or
introns will be located on one side or the other (i.e., 5' or 3')
of the cDNA such that it does not interrupt the coding sequence.
Any intron from any source, including viral, prokaryotic and
eukaryotic (plant or animal) organisms, may be used to practice
this invention, provided that it is compatible with the host cell
into which it is inserted. Also included herein are synthetic
introns. Optionally, more than one intron may be used in the
vector.
[0187] The expression and cloning vectors of the present invention
will typically contain a promoter that is recognized by the host
organism and operably linked to the molecule encoding the B7RP-2
polypeptide. Promoters are untranscribed sequences located upstream
(i.e., 5') to the start codon of a structural gene (generally
within about 100 to 1000 bp) that control the transcription of the
structural gene. Promoters are conventionally grouped into one of
two classes: inducible promoters and constitutive promoters.
Inducible promoters initiate increased levels of transcription from
DNA under their control in response to some change in culture
conditions, such as the presence or absence of a nutrient or a
change in temperature. Constitutive promoters, on the other hand,
initiate continual gene product production; that is, there is
little or no control over gene expression. A large number of
promoters, recognized by a variety of potential host cells, are
well known. A suitable promoter is operably linked to the DNA
encoding B7RP-2 polypeptide by removing the promoter from the
source DNA by restriction enzyme digestion and inserting the
desired promoter sequence into the vector. The native B7RP-2
promoter sequence may be used to direct amplification and/or
expression of a B7RP-2 nucleic acid molecule. A heterologous
promoter is preferred, however, if it permits greater transcription
and higher yields of the expressed protein as compared to the
native promoter, and if it is compatible with the host cell system
that has been selected for use.
[0188] Promoters suitable for use with prokaryotic hosts include
the beta-lactamase and lactose promoter systems; alkaline
phosphatase; a tryptophan (trp) promoter system; and hybrid
promoters such as the tac promoter. Other known bacterial promoters
are also suitable. Their sequences have been published, thereby
enabling one skilled in the art to ligate them to the desired DNA
sequence, using linkers or adapters as needed to supply any useful
restriction sites.
[0189] Suitable promoters for use with yeast hosts are also well
known in the art. Yeast enhancers are advantageously used with
yeast promoters. Suitable promoters for use with mammalian host
cells are well known and include, but are not limited to, those
obtained from the genomes of viruses such as polyoma virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B
virus and most preferably Simian Virus 40 (SV40). Other suitable
mammalian promoters include heterologous mammalian promoters, for
example, heat-shock promoters and the actin promoter.
[0190] Additional promoters which may be of interest in controlling
B7RP-2 gene expression include, but are not limited to: the SV40
early promoter region (Bemoist and Chambon, 1981, Nature
290:304-10); the CMV promoter; the promoter contained in the 3'
long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980,
Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et
al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-45); the
regulatory sequences of the metallothionine gene (Brinster et al.,
1982, Nature 296:39-42); prokaryotic expression vectors such as the
beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl.
Acad. Sci. U.S.A., 75:3727-31); or the tac promoter (DeBoer et al.,
1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Also of interest
are the following animal transcriptional control regions, which
exhibit tissue specificity and have been utilized in transgenic
animals: the elastase I gene control region which is active in
pancreatic acinar cells (Swift et al., 1984, Cell 38:639-46; Ornitz
et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409
(1986); MacDonald, 1987, Hepatology 7:425-515); the insulin gene
control region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315:115-22); the immunoglobulin gene control region
which is active in lymphoid cells (Grosschedl et al., 1984, Cell
38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et
al., 1987, Mol. Cell. Biol., 7:1436-44); the mouse mammary tumor
virus control region which is active in testicular, breast,
lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95); the
albumin gene control region which is active in liver (Pinkert et
al., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein gene
control region which is active in liver (Krumlauf et al., 1985,
Mol. Cell. Biol., 5:1639-48; Hammer et al., 1987, Science
235:53-58); the alpha 1-antitrypsin gene control region which is
active in the liver (Kelsey et al., 1987, Genes and Devel.
1:161-71); the beta-globin gene control region which is active in
myeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et
al., 1986, Cell 46:89-94); the myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2
gene control region which is active in skeletal muscle (Sani, 1985,
Nature 314:283-86); and the gonadotropic releasing hormone gene
control region which is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-78).
[0191] An enhancer sequence may be inserted into the vector to
increase the transcription of a DNA encoding a B7RP-2 polypeptide
of the present invention by higher eukaryotes. Enhancers are
cis-acting elements of DNA, usually about 10-300 bp in length, that
act on the promoter to increase transcription. Enhancers are
relatively orientation and position independent. They have been
found 5' and 3' to the transcription unit. Several enhancer
sequences available from mammalian genes are known (e.g., globin,
elastase, albumin, alpha-feto-protein and insulin). Typically,
however, an enhancer from a virus will be used. The SV40 enhancer,
the cytomegalovirus early promoter enhancer, the polyoma enhancer,
and adenovirus enhancers are exemplary enhancing elements for the
activation of eukaryotic promoters. While an enhancer may be
spliced into the vector at a position 5' or 3' to a B7RP-2 nucleic
acid molecule, it is typically located at a site 5' from the
promoter.
[0192] Expression vectors of the invention may be constructed from
a starting vector such as a commercially available vector. Such
vectors may or may not contain all of the desired flanking
sequences. Where one or more of the flanking sequences described
herein are not already present in the vector, they may be
individually obtained and ligated into the vector. Methods used for
obtaining each of the flanking sequences are well known to one
skilled in the art.
[0193] Preferred vectors for practicing this invention are those
which are compatible with bacterial, insect, and mammalian host
cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1
(Invitrogen, San Diego, Calif.), pBSII (Stratagene, La Jolla,
Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,
Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL
(BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO 90/14363) and
pFastBacDual (Gibco-BRL, Grand Island, N.Y.).
[0194] Additional suitable vectors include, but are not limited to,
cosmids, plasmids, or modified viruses, but it will be appreciated
that the vector system must be compatible with the selected host
cell. Such vectors include, but are not limited to plasmids such as
Bluescript.RTM. plasmid derivatives (a high copy number ColE1-based
phagemid, Stratagene Cloning Systems, La Jolla Calif.), PCR cloning
plasmids designed for cloning Taq-amplified PCR products (e.g.,
TOPO.TM. TA Cloning.RTM. Kit, PCR2.1.RTM. plasmid derivatives,
Invitrogen, Carlsbad, Calif.), and mammalian, yeast or virus
vectors such as a baculovirus expression system (pBacPAK plasmid
derivatives, Clontech, Palo Alto, Calif.).
[0195] After the vector has been constructed and a nucleic acid
molecule encoding a B7RP-2 polypeptide has been inserted into the
proper site of the vector, the completed vector may be inserted
into a suitable host cell for amplification and/or polypeptide
expression. The transformation of an expression vector for a B7RP-2
polypeptide into a selected host cell may be accomplished by well
known methods including methods such as transfection, infection,
calcium chloride, electroporation, microinjection, lipofection,
DEAE-dextran method, or other known techniques. The method selected
will in part be a function of the type of host cell to be used.
These methods and other suitable methods are well known to the
skilled artisan, and are set forth, for example, in Sambrook et
al., supra.
[0196] Host cells may be prokaryotic host cells (such as E. coli)
or eukaryotic host cells (such as a yeast, insect, or vertebrate
cell). The host cell, when cultured under appropriate conditions,
synthesizes a B7RP-2 polypeptide which can subsequently be
collected from the culture medium (if the host cell secretes it
into the medium) or directly from the host cell producing it (if it
is not secreted). The selection of an appropriate host cell will
depend upon various factors, such as desired expression levels,
polypeptide modifications that are desirable or necessary for
activity (such as glycosylation or phosphorylation) and ease of
folding into a biologically active molecule.
[0197] A number of suitable host cells are known in the art and
many are available from the American Type Culture Collection
(ATCC), Manassas, Va. Examples include, but are not limited to,
mammalian cells, such as Chinese hamster ovary cells (CHO), CHO
DHFR(-) cells (Urlaub et al., 1980, Proc. Natl. Acad. Sci. U.S.A.
97:4216-20), human embryonic kidney (HEK) 293 or 293T cells, or 3T3
cells. The selection of suitable mammalian host cells and methods
for transformation, culture, amplification, screening, product
production, and purification are known in the art. Other suitable
mammalian cell lines, are the monkey COS-1 and COS-7 cell lines,
and the CV-1 cell line. Further exemplary mammalian host cells
include primate cell lines and rodent cell lines, including
transformed cell lines. Normal diploid cells, cell strains derived
from in vitro culture of primary tissue, as well as primary
explants, are also suitable. Candidate cells may be genotypically
deficient in the selection gene, or may contain a dominantly acting
selection gene. Other suitable mammalian cell lines include but are
not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929
cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK
hamster cell lines. Each of these cell lines is known by and
available to those skilled in the art of protein expression.
[0198] Similarly useful as host cells suitable for the present
invention are bacterial cells. For example, the various strains of
E. coli (e.g., HB101, DH5.alpha., DH10, and MC1061) are well-known
as host cells in the field of biotechnology. Various strains of B.
subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp.,
and the like may also be employed in this method.
[0199] Many strains of yeast cells known to those skilled in the
art are also available as host cells for the expression of the
polypeptides of the present invention. Preferred yeast cells
include, for example, Saccharomyces cerivisae and Pichia
pastoris.
[0200] Additionally, where desired, insect cell systems may be
utilized in the methods of the present invention. Such systems are
described, for example, in Kitts et al., 1993, Biotechniques,
14:810-17; Lucklow, 1993, Curr. Opin. Biotechnol. 4:564-72; and
Lucklow et al., 1993, J. Virol., 67:4566-79. Preferred insect cells
are Sf-9 and Hi5 (Invitrogen).
[0201] One may also use transgenic animals to express glycosylated
B7RP-2 polypeptides. For example, one may use a transgenic
milk-producing animal (a cow or goat, for example) and obtain the
present glycosylated polypeptide in the animal milk. One may also
use plants to produce B7RP-2 polypeptides, however, in general, the
glycosylation occurring in plants is different from that produced
in mammalian cells, and may result in a glycosylated product which
is not suitable for human therapeutic use.
[0202] Polypeptide Production
[0203] Host cells comprising a B7RP-2 polypeptide expression vector
may be cultured using standard media well known to the skilled
artisan. The media will usually contain all nutrients necessary for
the growth and survival of the cells. Suitable media for culturing
E. coli cells include, for example, Luria Broth (LB) and/or
Terrific Broth (TB). Suitable media for culturing eukaryotic cells
include Roswell Park Memorial Institute medium 1640 (RPMI 1640),
Minimal Essential Medium (MEM) and/or Dulbecco's Modified Eagle
Medium (DMEM), all of which may be supplemented with serum and/or
growth factors as necessary for the particular cell line being
cultured. A suitable medium for insect cultures is Grace's medium
supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal
calf serum as necessary.
[0204] Typically, an antibiotic or other compound useful for
selective growth of transfected or transformed cells is added as a
supplement to the media. The compound to be used will be dictated
by the selectable marker element present on the plasmid with which
the host cell was transformed. For example, where the selectable
marker element is kanamycin resistance, the compound added to the
culture medium will be kanamycin. Other compounds for selective
growth include ampicillin, tetracycline, and neomycin.
[0205] The amount of a B7RP-2 polypeptide produced by a host cell
can be evaluated using standard methods known in the art. Such
methods include, without limitation, Western blot analysis,
SDS-polyacrylamide gel electrophoresis, non-denaturing gel
electrophoresis, High Performance Liquid Chromatography (HPLC)
separation, immunoprecipitation, and/or activity assays such as DNA
binding gel shift assays.
[0206] If a B7RP-2 polypeptide has been designed to be secreted
from the host cells, the majority of polypeptide may be found in
the cell culture medium. If however, the B7RP-2 polypeptide is not
secreted from the host cells, it will be present in the cytoplasm
and/or the nucleus (for eukaryotic host cells) or in the cytosol
(for gram-negative bacteria host cells).
[0207] For a B7RP-2 polypeptide situated in the host cell cytoplasm
and/or nucleus (for eukaryotic host cells) or in the cytosol (for
bacterial host cells), the intracellular material (including
inclusion bodies for gram-negative bacteria) can be extracted from
the host cell using any standard technique known to the skilled
artisan. For example, the host cells can be lysed to release the
contents of the periplasm/cytoplasm by French press,
homogenization, and/or sonication followed by centrifugation.
[0208] If a B7RP-2 polypeptide has formed inclusion bodies in the
cytosol, the inclusion bodies can often bind to the inner and/or
outer cellular membranes and thus will be found primarily in the
pellet material after centrifugation. The pellet material can then
be treated at pH extremes or with a chaotropic agent such as a
detergent, guanidine, guanidine derivatives, urea, or urea
derivatives in the presence of a reducing agent such as
dithiothreitol at alkaline pH or tris carboxyethyl phosphine at
acid pH to release, break apart, and solubilize the inclusion
bodies. The solubilized B7RP-2 polypeptide can then be analyzed
using gel electrophoresis, immunoprecipitation, or the like. If it
is desired to isolate the B7RP-2 polypeptide, isolation may be
accomplished using standard methods such as those described herein
and in Marston et al., 1990, Meth. Enz., 182:264-75.
[0209] In some cases, a B7RP-2 polypeptide may not be biologically
active upon isolation. Various methods for "refolding" or
converting the polypeptide to its tertiary structure and generating
disulfide linkages can be used to restore biological activity. Such
methods include exposing the solubilized polypeptide to a pH
usually above 7 and in the presence of a particular concentration
of a chaotrope. The selection of chaotrope is very similar to the
choices used for inclusion body solubilization, but usually the
chaotrope is used at a lower concentration and is not necessarily
the same as chaotropes used for the solubilization. In most cases
the refolding/oxidation solution will also contain a reducing agent
or the reducing agent plus its oxidized form in a specific ratio to
generate a particular redox potential allowing for disulfide
shuffling to occur in the formation of the protein's cysteine
bridges. Some of the commonly used redox couples include
cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric
chloride, dithiothreitol(DTT)/dithiane DTT, and
2-2-mercaptoethanol(bME)/dithio-b(ME). In many instances, a
cosolvent may be used or may be needed to increase the efficiency
of the refolding, and the more common reagents used for this
purpose include glycerol, polyethylene glycol of various molecular
weights, arginine and the like.
[0210] If inclusion bodies are not formed to a significant degree
upon expression of a B7RP-2 polypeptide, then the polypeptide will
be found primarily in the supernatant after centrifugation of the
cell homogenate. The polypeptide may be further isolated from the
supernatant using methods such as those described herein.
[0211] The purification of a B7RP-2 polypeptide from solution can
be accomplished using a variety of techniques. If the polypeptide
has been synthesized such that it contains a tag such as
Hexahistidine (B7RP-2 polypeptide/hexaHis) or other small peptide
such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc
(Invitrogen, Carlsbad, Calif.) at either its carboxyl- or
amino-terminus, it may be purified in a one-step process by passing
the solution through an affinity column where the column matrix has
a high affinity for the tag.
[0212] For example, polyhistidine binds with great affinity and
specificity to nickel. Thus, an affinity column of nickel (such as
the Qiagen.RTM. nickel columns) can be used for purification of
B7RP-2 polypeptide/polyHis. See, e.g., Current Protocols in
Molecular Biology .sctn. 10.11.8 (Ausubel et al., eds., Green
Publishers Inc. and Wiley and Sons 1993).
[0213] Additionally, B7RP-2 polypeptides may be purified through
the use of a monoclonal antibody that is capable of specifically
recognizing and binding to a B7RP-2 polypeptide.
[0214] Other suitable procedures for purification include, without
limitation, affinity chromatography, immunoaffinity chromatography,
ion exchange chromatography, molecular sieve chromatography, HPLC,
electrophoresis (including native gel electrophoresis) followed by
gel elution, and preparative isoelectric focusing ("Isoprime"
machine/technique, Hoefer Scientific, San Francisco, Calif.). In
some cases, two or more purification techniques may be combined to
achieve increased purity.
[0215] B7RP-2 polypeptides may also be prepared by chemical
synthesis methods (such as solid phase peptide synthesis) using
techniques known in the art such as those set forth by Merrifield
et al., 1963, J. Am. Chem. Soc. 85:2149; Houghten et al., 1985,
Proc Natl Acad. Sci. USA 82:5132; and Stewart and Young, Solid
Phase Peptide Synthesis (Pierce Chemical Co. 1984). Such
polypeptides may be synthesized with or without a methionine on the
amino-terminus. Chemically synthesized B7RP-2 polypeptides may be
oxidized using methods set forth in these references to form
disulfide bridges. Chemically synthesized B7RP-2 polypeptides are
expected to have comparable biological activity to the
corresponding B7RP-2 polypeptides produced recombinantly or
purified from natural sources, and thus may be used interchangeably
with a recombinant or natural B7RP-2 polypeptide.
[0216] Another means of obtaining B7RP-2 polypeptide is via
purification from biological samples such as source tissues and/or
fluids in which the B7RP-2 polypeptide is naturally found. Such
purification can be conducted using methods for protein
purification as described herein. The presence of the B7RP-2
polypeptide during purification may be monitored, for example,
using an antibody prepared against recombinantly produced B7RP-2
polypeptide or peptide fragments thereof.
[0217] A number of additional methods for producing nucleic acids
and polypeptides are known in the art, and the methods can be used
to produce polypeptides having specificity for B7RP-2 polypeptide.
See, e.g., Roberts et al., 1997, Proc. Natl. Acad. Sci. U.S.A.
94:12297-303, which describes the production of fusion proteins
between an mRNA and its encoded peptide. See also, Roberts, 1999,
Curr. Opin. Chem. Biol. 3:268-73. Additionally, U.S. Pat. No.
5,824,469 describes methods for obtaining oligonucleotides capable
of carrying out a specific biological function. The procedure
involves generating a heterogeneous pool of oligonucleotides, each
having a 5' randomized sequence, a central preselected sequence,
and a 3' randomized sequence. The resulting heterogeneous pool is
introduced into a population of cells that do not exhibit the
desired biological function. Subpopulations of the cells are then
screened for those that exhibit a predetermined biological
function. From that subpopulation, oligonucleotides capable of
carrying out the desired biological function are isolated.
[0218] U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and
5,817,483 describe processes for producing peptides or
polypeptides. This is done by producing stochastic genes or
fragments thereof, and then introducing these genes into host cells
which produce one or more proteins encoded by the stochastic genes.
The host cells are then screened to identify those clones producing
peptides or polypeptides having the desired activity.
[0219] Another method for producing peptides or polypeptides is
described in PCT/US98/20094 (WO99/15650) filed by Athersys, Inc.
Known as "Random Activation of Gene Expression for Gene Discovery"
(RAGE-GD), the process involves the activation of endogenous gene
expression or over-expression of a gene by in situ recombination
methods. For example, expression of an endogenous gene is activated
or increased by integrating a regulatory sequence into the target
cell which is capable of activating expression of the gene by
non-homologous or illegitimate recombination. The target DNA is
first subjected to radiation, and a genetic promoter inserted. The
promoter eventually locates a break at the front of a gene,
initiating transcription of the gene. This results in expression of
the desired peptide or polypeptide.
[0220] It will be appreciated that these methods can also be used
to create comprehensive B7RP-2 polypeptide expression libraries,
which can subsequently be used for high throughput phenotypic
screening in a variety of assays, such as biochemical assays,
cellular assays, and whole organism assays (e.g., plant, mouse,
etc.).
[0221] Synthesis
[0222] It will be appreciated by those skilled in the art that the
nucleic acid and polypeptide molecules described herein may be
produced by recombinant and other means.
[0223] Selective Binding Agents
[0224] The term "selective binding agent" refers to a molecule that
has specificity for one or more B7RP-2 polypeptides. Suitable
selective binding agents include, but are not limited to,
antibodies and derivatives thereof, polypeptides, and small
molecules. Suitable selective binding agents may be prepared using
methods known in the art. An exemplary B7RP-2 polypeptide selective
binding agent of the present invention is capable of binding a
certain portion of the B7RP-2 polypeptide thereby inhibiting the
binding of the polypeptide to a B7RP-2 polypeptide receptor.
[0225] Selective binding agents such as antibodies and antibody
fragments that bind B7RP-2 polypeptides are within the scope of the
present invention. The antibodies may be polyclonal including
monospecific polyclonal; monoclonal (MAbs); recombinant; chimeric;
humanized, such as complementarity-determining region
(CDR)-grafted; human; single chain; and/or bispecific; as well as
fragments; variants; or derivatives thereof. Antibody fragments
include those portions of the antibody that bind to an epitope on
the B7RP-2 polypeptide. Examples of such fragments include Fab and
F(ab') fragments generated by enzymatic cleavage of full-length
antibodies. Other binding fragments include those generated by
recombinant DNA techniques, such as the expression of recombinant
plasmids containing nucleic acid sequences encoding antibody
variable regions.
[0226] Polyclonal antibodies directed toward a B7RP-2 polypeptide
generally are produced in animals (e.g., rabbits or mice) by means
of multiple subcutaneous or intraperitoneal injections of B7RP-2
polypeptide and an adjuvant. It may be useful to conjugate a B7RP-2
polypeptide to a carrier protein that is immunogenic in the species
to be immunized, such as keyhole limpet hemocyanin, serum, albumin,
bovine thyroglobulin, or soybean trypsin inhibitor. Also,
aggregating agents such as alum are used to enhance the immune
response. After immunization, the animals are bled and the serum is
assayed for anti-B7RP-2 antibody titer.
[0227] Monoclonal antibodies directed toward B7RP-2 polypeptides
are produced using any method that provides for the production of
antibody molecules by continuous cell lines in culture. Examples of
suitable methods for preparing monoclonal antibodies include the
hybridoma methods of Kohler et al., 1975, Nature 256:495-97 and the
human B-cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001;
Brodeur et al., Monoclonal Antibody Production Techniques and
Applications 51-63 (Marcel Dekker, Inc., 1987). Also provided by
the invention are hybridoma cell lines that produce monoclonal
antibodies reactive with B7RP-2 polypeptides.
[0228] Monoclonal antibodies of the invention may be modified for
use as therapeutics. One embodiment is a "chimeric" antibody in
which a portion of the heavy (H) and/or light (L) chain is
identical with or homologous to a corresponding sequence in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is/are identical with or homologous to a corresponding
sequence in antibodies derived from another species or belonging to
another antibody class or subclass. Also included are fragments of
such antibodies, so long as they exhibit the desired biological
activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985, Proc.
Natl. Acad. Sci. 81:6851-55.
[0229] In another embodiment, a monoclonal antibody of the
invention is a "humanized" antibody. Methods for humanizing
non-human antibodies are well known in the art. See U.S. Pat. Nos.
5,585,089 and 5,693,762. Generally, a humanized antibody has one or
more amino acid residues introduced into it from a source that is
non-human. Humanization can be performed, for example, using
methods described in the art (Jones et al., 1986, Nature
321:522-25; Riechmann et al., 1998, Nature 332:323-27; Verhoeyen et
al., 1988, Science 239:1534-36), by substituting at least a portion
of a rodent complementarity-determining region for the
corresponding regions of a human antibody.
[0230] Also encompassed by the invention are human antibodies that
bind B7RP-2 polypeptides. Using transgenic animals (e.g., mice)
that are capable of producing a repertoire of human antibodies in
the absence of endogenous immunoglobulin production such antibodies
are produced by immunization with a B7RP-2 polypeptide antigen
(i.e., having at least 6 contiguous amino acids), optionally
conjugated to a carrier. See, e.g., Jakobovits et al., 1993, Proc.
Natl. Acad. Sci. 90:2551-55; Jakobovits et al., 1993, Nature
362:255-58; Bruggermann et al., 1993, Year in Immuno. 7:33. In one
method, such transgenic animals are produced by incapacitating the
endogenous loci encoding the heavy and light immunoglobulin chains
therein, and inserting loci encoding human heavy and light chain
proteins into the genome thereof. Partially modified animals, that
is those having less than the full complement of modifications, are
then cross-bred to obtain an animal having all of the desired
immune system modifications. When administered an immunogen, these
transgenic animals produce antibodies with human (rather than,
e.g., murine) amino acid sequences, including variable regions
which are immunospecific for these antigens. See PCT App. Nos.
PCT/US96/05928 and PCT/US93/06926. Additional methods are described
in U.S. Pat. No. 5,545,807, PCT App. Nos. PCT/US91/245 and
PCT/GB89/01207, and in European Patent Nos. 546073B1 and 546073A1.
Human antibodies can also be produced by the expression of
recombinant DNA in host cells or by expression in hybridoma cells
as described herein.
[0231] In an alternative embodiment, human antibodies can also be
produced from phage-display libraries (Hoogenboom et al., 1991, J.
Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581).
These processes mimic immune selection through the display of
antibody repertoires on the surface of filamentous bacteriophage,
and subsequent selection of phage by their binding to an antigen of
choice. One such technique is described in PCT App. No.
PCT/US98/17364, which describes the isolation of high affinity and
functional agonistic antibodies for MPL- and msk-receptors using
such an approach.
[0232] Chimeric, CDR grafted, and humanized antibodies are
typically produced by recombinant methods. Nucleic acids encoding
the antibodies are introduced into host cells and expressed using
materials and procedures described herein. In a preferred
embodiment, the antibodies are produced in mammalian host cells,
such as CHO cells. Monoclonal (e.g., human) antibodies may be
produced by the expression of recombinant DNA in host cells or by
expression in hybridoma cells as described herein.
[0233] The anti-B7RP-2 antibodies of the invention may be employed
in any known assay method, such as competitive binding assays,
direct and indirect sandwich assays, and immunoprecipitation assays
(Sola, Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC
Press, Inc., 1987)) for the detection and quantitation of B7RP-2
polypeptides. The antibodies will bind B7RP-2 polypeptides with an
affinity that is appropriate for the assay method being
employed.
[0234] For diagnostic applications, in certain embodiments,
anti-B7RP-2 antibodies may be labeled with a detectable moiety. The
detectable moiety can be any one that is capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32P, .sup.35S, .sup.125, .sup.99Tc, .sup.111In, or
.sup.67Ga; a fluorescent or chemiluminescent compound, such as
fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme,
such as alkaline phosphatase, .beta.-galactosidase, or horseradish
peroxidase (Bayer, et al., 1990, Meth. Enz. 184:138-63).
[0235] Competitive binding assays rely on the ability of a labeled
standard (e.g., a B7RP-2 polypeptide, or an immunologically
reactive portion thereof) to compete with the test sample analyte
(an B7RP-2 polypeptide) for binding with a limited amount of
anti-B7RP-2 antibody. The amount of a B7RP-2 polypeptide in the
test sample is inversely proportional to the amount of standard
that becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies typically are
insolubilized before or after the competition, so that the standard
and analyte that are bound to the antibodies may conveniently be
separated from the standard and analyte which remain unbound.
[0236] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected and/or quantitated. In a
sandwich assay, the test sample analyte is typically bound by a
first antibody which is immobilized on a solid support, and
thereafter a second antibody binds to the analyte, thus forming an
insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110.
The second antibody may itself be labeled with a detectable moiety
(direct sandwich assays) or may be measured using an
anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assays). For example, one type of
sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in
which case the detectable moiety is an enzyme.
[0237] The selective binding agents, including anti-B7RP-2
antibodies, are also useful for in vivo imaging. An antibody
labeled with a detectable moiety may be administered to an animal,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host assayed. The antibody may be
labeled with any moiety that is detectable in an animal, whether by
nuclear magnetic resonance, radiology, or other detection means
known in the art.
[0238] Selective binding agents of the invention, including
antibodies, may be used as therapeutics. These therapeutic agents
are generally agonists or antagonists, in that they either enhance
or reduce, respectively, at least one of the biological activities
of a B7RP-2 polypeptide. In one embodiment, antagonist antibodies
of the invention are antibodies or binding fragments thereof which
are capable of specifically binding to a B7RP-2 polypeptide and
which are capable of inhibiting or eliminating the functional
activity of a B7RP-2 polypeptide in vivo or in vitro. In preferred
embodiments, the selective binding agent, e.g., an antagonist
antibody, will inhibit the functional activity of a B7RP-2
polypeptide by at least about 50%, and preferably by at least about
80%. In another embodiment, the selective binding agent may be an
anti-B7RP-2 polypeptide antibody that is capable of interacting
with a B7RP-2 polypeptide binding partner (a ligand or receptor)
thereby inhibiting or eliminating B7RP-2 polypeptide activity in
vitro or in vivo. Selective binding agents, including agonist and
antagonist anti-B7RP-2 polypeptide antibodies, are identified by
screening assays that are well known in the art.
[0239] The invention also relates to a kit comprising B7RP-2
selective binding agents (such as antibodies) and other reagents
useful for detecting B7RP-2 polypeptide levels in biological
samples. Such reagents may include a detectable label, blocking
serum, positive and negative control samples, and detection
reagents.
[0240] Microarrays
[0241] It will be appreciated that DNA microarray technology can be
utilized in accordance with the present invention. DNA microarrays
are miniature, high-density arrays of nucleic acids positioned on a
solid support, such as glass. Each cell or element within the array
contains numerous copies of a single nucleic acid species that acts
as a target for hybridization with a complementary nucleic acid
sequence (e.g., mRNA). In expression profiling using DNA microarray
technology, mRNA is first extracted from a cell or tissue sample
and then converted enzymatically to fluorescently labeled cDNA.
This material is hybridized to the microarray and unbound cDNA is
removed by washing. The expression of discrete genes represented on
the array is then visualized by quantitating the amount of labeled
cDNA that is specifically bound to each target nucleic acid
molecule. In this way, the expression of thousands of genes can be
quantitated in a high throughput, parallel manner from a single
sample of biological material.
[0242] This high throughput expression profiling has a broad range
of applications with respect to the B7RP-2 molecules of the
invention, including, but not limited to: the identification and
validation of B7RP-2 disease-related genes as targets for
therapeutics; molecular toxicology of related B7RP-2 molecules and
inhibitors thereof; stratification of populations and generation of
surrogate markers for clinical trials; and enhancing related B7RP-2
polypeptide small molecule drug discovery by aiding in the
identification of selective compounds in high throughput
screens.
[0243] Chemical Derivatives
[0244] Chemically modified derivatives of B7RP-2 polypeptides may
be prepared by one skilled in the art, given the disclosures
described herein. B7RP-2 polypeptide derivatives are modified in a
manner that is different--either in the type or location of the
molecules naturally attached to the polypeptide. Derivatives may
include molecules formed by the deletion of one or more
naturally-attached chemical groups. The polypeptide comprising the
amino acid sequence of any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID
NO: 6, or other B7RP-2 polypeptide, may be modified by the covalent
attachment of one or more polymers. For example, the polymer
selected is typically water-soluble so that the protein to which it
is attached does not precipitate in an aqueous environment, such as
a physiological environment. Included within the scope of suitable
polymers is a mixture of polymers. Preferably, for therapeutic use
of the end-product preparation, the polymer will be
pharmaceutically acceptable.
[0245] The polymers each may be of any molecular weight and may be
branched or unbranched. The polymers each typically have an average
molecular weight of between about 2 kDa to about 100 kDa (the term
"about" indicating that in preparations of a water-soluble polymer,
some molecules will weigh more, some less, than the stated
molecular weight). The average molecular weight of each polymer is
preferably between about 5 kDa and about 50 kDa, more preferably
between about 12 kDa and about 40 kDa and most preferably between
about 20 kDa and about 35 kDa.
[0246] Suitable water-soluble polymers or mixtures thereof include,
but are not limited to, N-linked or O-linked carbohydrates, sugars,
phosphates, polyethylene glycol (PEG) (including the forms of PEG
that have been used to derivatize proteins, including
mono-(C.sub.1-C.sub.10), alkoxy-, or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular
weight dextran of, for example, about 6 kD), cellulose, or other
carbohydrate based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, propylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g.,
glycerol), and polyvinyl alcohol. Also encompassed by the present
invention are bifunctional crosslinking molecules which may be used
to prepare covalently attached B7RP-2 polypeptide multimers.
[0247] In general, chemical derivatization may be performed under
any suitable condition used to react a protein with an activated
polymer molecule. Methods for preparing chemical derivatives of
polypeptides will generally comprise the steps of: (a) reacting the
polypeptide with the activated polymer molecule (such as a reactive
ester or aldehyde derivative of the polymer molecule) under
conditions whereby the polypeptide comprising the amino acid
sequence of any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or
other B7RP-2 polypeptide, becomes attached to one or more polymer
molecules, and (b) obtaining the reaction products. The optimal
reaction conditions will be determnined based on known parameters
and the desired result. For example, the larger the ratio of
polymer molecules to protein, the greater the percentage of
attached polymer molecule. In one embodiment, the B7RP-2
polypeptide derivative may have a single polymer molecule moiety at
the amino-terminus. See, e.g., U.S. Pat. No. 5,234,784.
[0248] The pegylation of a polypeptide may be specifically carried
out using any of the pegylation reactions known in the art. Such
reactions are described, for example, in the following references:
Francis et al., 1992, Focus on Growth Factors 3:4-10; European
Patent Nos. 0154316 and 0401384; and U.S. Pat. No. 4,179,337. For
example, pegylation may be carried out via an acylation reaction or
an alkylation reaction with a reactive polyethylene glycol molecule
(or an analogous reactive water-soluble polymer) as described
herein. For the acylation reactions, a selected polymer should have
a single reactive ester group. For reductive alkylation, a selected
polymer should have a single reactive aldehyde group. A reactive
aldehyde is, for example, polyethylene glycol propionaldehyde,
which is water stable, or mono C.sub.1-C.sub.10 alkoxy or aryloxy
derivatives thereof (see U.S. Pat. No. 5,252,714).
[0249] In another embodiment, B7RP-2 polypeptides may be chemically
coupled to biotin. The biotin/B7RP-2 polypeptide molecules are then
allowed to bind to avidin, resulting in tetravalent
avidin/biotin/B7RP-2 polypeptide molecules. B7RP-2 polypeptides may
also be covalently coupled to dinitrophenol (DNP) or trinitrophenol
(TNP) and the resulting conjugates precipitated with anti-DNP or
anti-TNP-IgM to form decameric conjugates with a valency of 10.
[0250] Generally, conditions that may be alleviated or modulated by
the administration of the present B7RP-2 polypeptide derivatives
include those described herein for B7RP-2 polypeptides. However,
the B7RP-2 polypeptide derivatives disclosed herein may have
additional activities, enhanced or reduced biological activity, or
other characteristics, such as increased or decreased half-life, as
compared to the non-derivatized molecules.
[0251] Genetically Engineered Non-human Animals
[0252] Additionally included within the scope of the present
invention are non-human animals such as mice, rats, or other
rodents; rabbits, goats, sheep, or other farm animals, in which the
genes encoding native B7RP-2 polypeptide have been disrupted (i.e.,
"knocked out") such that the level of expression of B7RP-2
polypeptide is significantly decreased or completely abolished.
Such animals may be prepared using techniques and methods such as
those described in U.S. Pat. No. 5,557,032.
[0253] The present invention further includes non-human animals
such as mice, rats, or other rodents; rabbits, goats, sheep, or
other farm animals, in which either the native form of a B7RP-2
gene for that animal or a heterologous B7RP-2 gene is
over-expressed by the animal, thereby creating a "transgenic"
animal. Such transgenic animals may be prepared using well known
methods such as those described in U.S. Pat. No 5,489,743 and PCT
Pub. No. WO 94/28122.
[0254] The present invention further includes non-human animals in
which the promoter for one or more of the B7RP-2 polypeptides of
the present invention is either activated or inactivated (e.g., by
using homologous recombination methods) to alter the level of
expression of one or more of the native B7RP-2 polypeptides.
[0255] These non-human animals may be used for drug candidate
screening. In such screening, the impact of a drug candidate on the
animal may be measured. For example, drug candidates may decrease
or increase the expression of the B7RP-2 gene. In certain
embodiments, the amount of B7RP-2 polypeptide that is produced may
be measured after the exposure of the animal to the drug candidate.
Additionally, in certain embodiments, one may detect the actual
impact of the drug candidate on the animal. For example,
over-expression of a particular gene may result in, or be
associated with, a disease or pathological condition. In such
cases, one may test a drug candidate's ability to decrease
expression of the gene or its ability to prevent or inhibit a
pathological condition. In other examples, the production of a
particular metabolic product such as a fragment of a polypeptide,
may result in, or be associated with, a disease or pathological
condition. In such cases, one may test a drug candidate's ability
to decrease the production of such a metabolic product or its
ability to prevent or inhibit a pathological condition.
[0256] Assaying for Other Modulators of B7RP-2 Polypeptide
Activity
[0257] In some situations, it may be desirable to identify
molecules that are modulators, i.e., agonists or antagonists, of
the activity of B7RP-2 polypeptide. Natural or synthetic molecules
that modulate B7RP-2 polypeptide may be identified using one or
more screening assays, such as those described herein. Such
molecules may be administered either in an ex vivo manner or in an
in vivo manner by injection, or by oral delivery, implantation
device, or the like.
[0258] "Test molecule" refers to a molecule that is under
evaluation for the ability to modulate (i.e., increase or decrease)
the activity of a B7RP-2 polypeptide. Most commonly, a test
molecule will interact directly with a B7RP-2 polypeptide. However,
it is also contemplated that a test molecule may also modulate
B7RP-2 polypeptide activity indirectly, such as by affecting B7RP-2
gene expression, or by binding to a B7RP-2 polypeptide binding
partner (e.g., receptor or ligand). In one embodiment, a test
molecule will bind to a B7RP-2 polypeptide with an affinity
constant of at least about 10.sup.-6 M, preferably about 10.sup.-8
M, more preferably about 10.sup.-9 M, and even more preferably
about 10.sup.-10 M.
[0259] Methods for identifying compounds that interact with B7RP-2
polypeptides are encompassed by the present invention. In certain
embodiments, a B7RP-2 polypeptide is incubated with a test molecule
under conditions that permit the interaction of the test molecule
with a B7RP-2 polypeptide, and the extent of the interaction is
measured. The test molecule can be screened in a substantially
purified form or in a crude mixture.
[0260] In certain embodiments, a B7RP-2 polypeptide agonist or
antagonist may be a protein, peptide, carbohydrate, lipid, or small
molecular weight molecule that interacts with B7RP-2 polypeptide to
regulate its activity. Molecules which regulate B7RP-2 polypeptide
expression include nucleic acids which are complementary to nucleic
acids encoding a B7RP-2 polypeptide, or are complementary to
nucleic acids sequences which direct or control the expression of
B7RP-2 polypeptide, and which act as anti-sense regulators of
expression.
[0261] Once a test molecule has been identified as interacting with
a B7RP-2 polypeptide, the molecule may be further evaluated for its
ability to increase or decrease B7RP-2 polypeptide activity. The
measurement of the interaction of a test molecule with B7RP-2
polypeptide may be carried out in several formats, including
cell-based binding assays, membrane binding assays, solution-phase
assays, and immunoassays. In general, a test molecule is incubated
with a B7RP-2 polypeptide for a specified period of time, and
B7RP-2 polypeptide activity is determined by one or more assays for
measuring biological activity.
[0262] The interaction of test molecules with B7RP-2 polypeptides
may also be assayed directly using polyclonal or monoclonal
antibodies in an immunoassay. Alternatively, modified forms of
B7RP-2 polypeptides containing epitope tags as described herein may
be used in solution and immunoassays.
[0263] In the event that B7RP-2 polypeptides display biological
activity through an interaction with a binding partner (e.g., a
receptor or a ligand), a variety of in vitro assays may be used to
measure the binding of a B7RP-2 polypeptide to the corresponding
binding partner (such as a selective binding agent, receptor, or
ligand). These assays may be used to screen test molecules for
their ability to increase or decrease the rate and/or the extent of
binding of a B7RP-2 polypeptide to its binding partner. In one
assay, a B7RP-2 polypeptide is immobilized in the wells of a
microtiter plate. Radiolabeled B7RP-2 polypeptide binding partner
(for example, iodinated B7RP-2 polypeptide binding partner) and a
test molecule can then be added either one at a time (in either
order) or simultaneously to the wells. After incubation, the wells
can be washed and counted for radioactivity, using a scintillation
counter, to determine the extent to which the binding partner bound
to the B7RP-2 polypeptide. Typically, a molecule will be tested
over a range of concentrations, and a series of control wells
lacking one or more elements of the test assays can be used for
accuracy in the evaluation of the results. An alternative to this
method involves reversing the "positions" of the proteins, i.e.,
immobilizing B7RP-2 polypeptide binding partner to the microtiter
plate wells, incubating with the test molecule and radiolabeled
B7RP-2 polypeptide, and determining the extent of B7RP-2
polypeptide binding. See, e.g., Current Protocols in Molecular
Biology, chap. 18 (Ausubel et al., eds., Green Publishers Inc. and
Wiley and Sons 1995).
[0264] As an alternative to radiolabeling, a B7RP-2 polypeptide or
its binding partner may be conjugated to biotin, and the presence
of biotinylated protein can then be detected using streptavidin
linked to an enzyme, such as horse radish peroxidase (HRP) or
alkaline phosphatase (AP), which can be detected colorometrically,
or by fluorescent tagging of streptavidin. An antibody directed to
a B7RP-2 polypeptide or to a B7RP-2 polypeptide binding partner,
and which is conjugated to biotin, may also be used for purposes of
detection following incubation of the complex with enzyme-linked
streptavidin linked to AP or HRP.
[0265] A B7RP-2 polypeptide or a B7RP-2 polypeptide binding partner
can also be immobilized by attachment to agarose beads, acrylic
beads, or other types of such inert solid phase substrates. The
substrate-protein complex can be placed in a solution containing
the complementary protein and the test compound. After incubation,
the beads can be precipitated by centrifugation, and the amount of
binding between a B7RP-2 polypeptide and its binding partner can be
assessed using the methods described herein. Alternatively, the
substrate-protein complex can be immobilized in a column with the
test molecule and complementary protein passing through the column.
The formation of a complex between a B7RP-2 polypeptide and its
binding partner can then be assessed using any of the techniques
described herein (e.g., radiolabelling or antibody binding).
[0266] Another in vitro assay that is useful for identifying a test
molecule which increases or decreases the formation of a complex
between a B7RP-2 polypeptide binding protein and a B7RP-2
polypeptide binding partner is a surface plasmon resonance detector
system such as the BIAcore assay system (Pharmacia, Piscataway,
N.J.). The BIAcore system is utilized as specified by the
manufacturer. This assay essentially involves the covalent binding
of either B7RP-2 polypeptide or a B7RP-2 polypeptide binding
partner to a dextran-coated sensor chip that is located in a
detector. The test compound and the other complementary protein can
then be injected, either simultaneously or sequentially, into the
chamber containing the sensor chip. The amount of complementary
protein that binds can be assessed based on the change in molecular
mass that is physically associated with the dextran-coated side of
the sensor chip, with the change in molecular mass being measured
by the detector system.
[0267] In some cases, it may be desirable to evaluate two or more
test compounds together for their ability to increase or decrease
the formation of a complex between a B7RP-2 polypeptide and a
B7RP-2 polypeptide binding partner. In these cases, the assays set
forth herein can be readily modified by adding such additional test
compound(s) either simultaneously with, or subsequent to, the first
test compound. The remainder of the steps in the assay are as set
forth herein.
[0268] In vitro assays such as those described herein may be used
advantageously to screen large numbers of compounds for an effect
on the formation of a complex between a B7RP-2 polypeptide and
B7RP-2 polypeptide binding partner. The assays may be automated to
screen compounds generated in phage display, synthetic peptide, and
chemical synthesis libraries.
[0269] Compounds which increase or decrease the formation of a
complex between a B7RP-2 polypeptide and a B7RP-2 polypeptide
binding partner may also be screened in cell culture using cells
and cell lines expressing either B7RP-2 polypeptide or B7RP-2
polypeptide binding partner. Cells and cell lines may be obtained
from any mammal, but preferably will be from human or other
primate, canine, or rodent sources. The binding of a B7RP-2
polypeptide to cells expressing B7RP-2 polypeptide binding partner
at the surface is evaluated in the presence or absence of test
molecules, and the extent of binding may be determined by, for
example, flow cytometry using a biotinylated antibody to a B7RP-2
polypeptide binding partner. Cell culture assays can be used
advantageously to further evaluate compounds that score positive in
protein binding assays described herein.
[0270] Cell cultures can also be used to screen the impact of a
drug candidate. For example, drug candidates may decrease or
increase the expression of the B7RP-2 gene. In certain embodiments,
the amount of B7RP-2 polypeptide or a B7RP-2 polypeptide fragment
that is produced may be measured after exposure of the cell culture
to the drug candidate. In certain embodiments, one may detect the
actual impact of the drug candidate on the cell culture. For
example, the over-expression of a particular gene may have a
particular impact on the cell culture. In such cases, one may test
a drug candidate's ability to increase or decrease the expression
of the gene or its ability to prevent or inhibit a particular
impact on the cell culture. In other examples, the production of a
particular metabolic product such as a fragment of a polypeptide,
may result in, or be associated with, a disease or pathological
condition. In such cases, one may test a drug candidate's ability
to decrease the production of such a metabolic product in a cell
culture.
[0271] Internalizing Proteins
[0272] The tat protein sequence (from HIV) can be used to
internalize proteins into a cell. See, e.g., Falwell et al., 1994,
Proc. Natl. Acad. Sci. U.S.A. 91:664-68. For example, an 11 amino
acid sequence (Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 14) of the HIV tat
protein (termed the "protein transduction domain," or TAT PDT) has
been described as mediating delivery across the cytoplasmic
membrane and the nuclear membrane of a cell. See Schwarze et al.,
1999, Science 285:1569-72; and Nagahara et al., 1998, Nat. Med.
4:1449-52. In these procedures, FITC-constructs (FITC-labeled
G-G-G-G-Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 15), which penetrate
tissues following intraperitoneal administration, are prepared, and
the binding of such constructs to cells is detected by
fluorescence-activated cell sorting (FACS) analysis. Cells treated
with a tat-.beta.-gal fusion protein will demonstrate .beta.-gal
activity. Following injection, expression of such a construct can
be detected in a number of tissues, including liver, kidney, lung,
heart, and brain tissue. It is believed that such constructs
undergo some degree of unfolding in order to enter the cell, and as
such, may require a refolding following entry into the cell.
[0273] It will thus be appreciated that the tat protein sequence
may be used to internalize a desired polypeptide into a cell. For
example, using the tat protein sequence, a B7RP-2 antagonist (such
as an anti-B7RP-2 selective binding agent, small molecule, soluble
receptor, or antisense oligonucleotide) can be administered
intracellularly to inhibit the activity of a B7RP-2 molecule. As
used herein, the term "B7RP-2 molecule" refers to both B7RP-2
nucleic acid molecules and B7RP-2 polypeptides as defined herein.
Where desired, the B7RP-2 protein itself may also be internally
administered to a cell using these procedures. See also, Straus,
1999, Science 285:1466-67.
[0274] Cell Source Identification Using B7RP-2 Polypeptide
[0275] In accordance with certain embodiments of the invention, it
may be useful to be able to determine the source of a certain cell
type associated with a B7RP-2 polypeptide. For example, it may be
useful to determine the origin of a disease or pathological
condition as an aid in selecting an appropriate therapy. In certain
embodiments, nucleic acids encoding a B7RP-2 polypeptide can be
used as a probe to identify cells described herein by screening the
nucleic acids of the cells with such a probe. In other embodiments,
one may use anti-B7RP-2 polypeptide antibodies to test for the
presence of B7RP-2 polypeptide in cells, and thus, determine if
such cells are of the types described herein.
[0276] B7RP-2 Polypeptide Compositions and Administration
[0277] Therapeutic compositions are within the scope of the present
invention. Such B7RP-2 polypeptide pharmaceutical compositions may
comprise a therapeutically effective amount of a B7RP-2 polypeptide
or a B7RP-2 nucleic acid molecule in admixture with a
pharmaceutically or physiologically acceptable formulation agent
selected for suitability with the mode of administration.
Pharmaceutical compositions may comprise a therapeutically
effective amount of one or more B7RP-2 polypeptide selective
binding agents in admixture with a pharmaceutically or
physiologically acceptable formulation agent selected for
suitability with the mode of administration.
[0278] Acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed.
[0279] The pharmaceutical composition may contain formulation
materials for modifying, maintaining, or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption,
or penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine, or lysine), antimicrobials,
antioxidants (such as ascorbic acid, sodium sulfite, or sodium
hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, or other organic acids), bulking agents (such
as mannitol or glycine), chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,
disaccharides, and other carbohydrates (such as glucose, mannose,
or dextrins), proteins (such as serum albumin, gelatin, or
immunoglobulins), coloring, flavoring and diluting agents,
emulsifying agents, hydrophilic polymers (such as
polyvinylpyrrolidone), low molecular weight polypeptides,
salt-forming countelions (such as sodium), preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, B7RP-2orhexidine,
sorbic acid, or hydrogen peroxide), solvents (such as glycerin,
propylene glycol, or polyethylene glycol), sugar alcohols (such as
mannitol or sorbitol), suspending agents, surfactants or wetting
agents (such as pluronics; PEG; sorbitan esters; polysorbates such
as polysorbate 20 or polysorbate 80; triton; tromethamine;
lecithin; cholesterol or tyloxapal), stability enhancing agents
(such as sucrose or sorbitol), tonicity enhancing agents (such as
alkali metal halides--preferably sodium or potassium chloride--or
mannitol sorbitol), delivery vehicles, diluents, excipients and/or
pharmaceutical adjuvants. See Remington's Pharmaceutical Sciences
(18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990.
[0280] The optimal pharmaceutical composition will be determined by
a skilled artisan depending upon, for example, the intended route
of administration, delivery format, and desired dosage. See, e.g.,
Remington's Pharmaceutical Sciences, supra. Such compositions may
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the B7RP-2 molecule.
[0281] The primary vehicle or carrier in a pharmaceutical
composition may be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier for injection may be water,
physiological saline solution, or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may
further include sorbitol or a suitable substitute. In one
embodiment of the present invention, B7RP-2 polypeptide
compositions may be prepared for storage by mixing the selected
composition having the desired degree of purity with optional
formulation agents (Remington's Pharmaceutical Sciences, supra) in
the form of a lyophilized cake or an aqueous solution. Further, the
B7RP-2 polypeptide product may be formulated as a lyophilizate
using appropriate excipients such as sucrose.
[0282] The B7RP-2 polypeptide pharmaceutical compositions can be
selected for parenteral delivery. Alternatively, the compositions
may be selected for inhalation or for delivery through the
digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the
art.
[0283] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at a slightly lower pH, typically within a pH range of from about 5
to about 8.
[0284] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be in the
form of a pyrogen-free, parenterally acceptable, aqueous solution
comprising the desired B7RP-2 molecule in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which a B7RP-2 molecule is
formulated as a sterile, isotonic solution, properly preserved. Yet
another preparation can involve the formulation of the desired
molecule with an agent, such as injectable microspheres,
bio-erodible particles, polymeric compounds (such as polylactic
acid or polyglycolic acid), beads, or liposomes, that provides for
the controlled or sustained release of the product which may then
be delivered via a depot injection. Hyaluronic acid may also be
used, and this may have the effect of promoting sustained duration
in the circulation. Other suitable means for the introduction of
the desired molecule include implantable drug delivery devices.
[0285] In one embodiment, a pharmaceutical composition may be
formulated for inhalation. For example, B7RP-2 polypeptide may be
formulated as a dry powder for inhalation. B7RP-2 polypeptide or
nucleic acid molecule inhalation solutions may also be formulated
with a propellant for aerosol delivery. In yet another embodiment,
solutions may be nebulized. Pulmonary administration is further
described in PCT Pub. No. WO 94/20069, which describes the
pulmonary delivery of chemically modified proteins.
[0286] It is also contemplated that certain formulations may be
administered orally. In one embodiment of the present invention,
B7RP-2 polypeptides that are administered in this fashion can be
formulated with or without those carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. For
example, a capsule may be designed to release the active portion of
the formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. Additional agents can be included to facilitate
absorption of the B7RP-2 polypeptide. Diluents, flavorings, low
melting point waxes, vegetable oils, lubricants, suspending agents,
tablet disintegrating agents, and binders may also be employed.
[0287] Another pharmaceutical composition may involve an effective
quantity of B7RP-2 polypeptides in a mixture with non-toxic
excipients that are suitable for the manufacture of tablets. By
dissolving the tablets in sterile water, or another appropriate
vehicle, solutions can be prepared in unit-dose form. Suitable
excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or
calcium phosphate; or binding agents, such as starch, gelatin, or
acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
[0288] Additional B7RP-2 polypeptide pharmaceutical compositions
will be evident to those skilled in the art, including formulations
involving B7RP-2 polypeptides in sustained- or controlled-delivery
formulations. Techniques for formulating a variety of other
sustained- or controlled-delivery means, such as liposome carriers,
bio-erodible microparticles or porous beads and depot injections,
are also known to those skilled in the art. See, e.g.,
PCT/US93/00829, which describes the controlled release of porous
polymeric microparticles for the delivery of pharmaceutical
compositions.
[0289] Additional examples of sustained-release preparations
include semipermeable polymer matrices in the form of shaped
articles, e.g. films, or microcapsules. Sustained release matrices
may include polyesters, hydrogels, polylactides (U.S. Pat. No.
3,773,919 and European Patent No. 058481), copolymers of L-glutamic
acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers
22:547-56), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981,
J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech.
12:98-105), ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (European Patent No. 133988).
Sustained-release compositions may also include liposomes, which
can be prepared by any of several methods known in the art. See,
e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92;
and European Patent Nos. 036676, 088046, and 143949.
[0290] The B7RP-2 pharmaceutical composition to be used for in vivo
administration typically must be sterile. This may be accomplished
by filtration through sterile filtration membranes. Where the
composition is lyophilized, sterilization using this method may be
conducted either prior to, or following, lyophilization and
reconstitution. The composition for parenteral administration may
be stored in lyophilized form or in a solution. In addition,
parenteral compositions generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0291] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0292] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
may each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0293] The effective amount of a B7RP-2 pharmaceutical composition
to be employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment will
thus vary depending, in part, upon the molecule delivered, the
indication for which the B7RP-2 molecule is being used, the route
of administration, and the size (body weight, body surface, or
organ size) and condition (the age and general health) of the
patient. Accordingly, the clinician may titer the dosage and modify
the route of administration to obtain the optimal therapeutic
effect. A typical dosage may range from about 0.1 .mu.g/kg to up to
about 100 mg/kg or more, depending on the factors mentioned above.
In other embodiments, the dosage may range from 0.1 .mu.g/kg up to
about 100 mg/kg; or 1 .mu.g/kg up to about 100 mg/kg; or 5 .mu.g/kg
up to about 100 mg/kg.
[0294] The frequency of dosing will depend upon the pharmacokinetic
parameters of the B7RP-2 molecule in the formulation being used.
Typically, a clinician will administer the composition until a
dosage is reached that achieves the desired effect. The composition
may therefore be administered as a single dose, as two or more
doses (which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. Appropriate dosages may be ascertained through use of
appropriate dose-response data.
[0295] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g., orally; through
injection by intravenous, intraperitoneal, intracerebral
(intraparenchymal), intracerebroventricular, intramuscular,
intraocular, intraarterial, intraportal, or intralesional routes;
by sustained release systems; or by implantation devices. Where
desired, the compositions may be administered by bolus injection or
continuously by infusion, or by implantation device.
[0296] Alternatively or additionally, the composition may be
administered locally via implantation of a membrane, sponge, or
other appropriate material onto which the desired molecule has been
absorbed or encapsulated. Where an implantation device is used, the
device may be implanted into any suitable tissue or organ, and
delivery of the desired molecule may be via diffusion,
timed-release bolus, or continuous administration.
[0297] In some cases, it may be desirable to use B7RP-2 polypeptide
pharmaceutical compositions in an ex vivo manner. In such
instances, cells, tissues, or organs that have been removed from
the patient are exposed to B7RP-2 polypeptide pharmaceutical
compositions after which the cells, tissues, or organs are
subsequently implanted back into the patient.
[0298] In other cases, a B7RP-2 polypeptide can be delivered by
implanting certain cells that have been genetically engineered,
using methods such as those described herein, to express and
secrete the B7RP-2 polypeptide. Such cells may be animal or human
cells, and may be autologous, heterologous, or xenogeneic.
Optionally, the cells may be immortalized. In order to decrease the
chance of an immunological response, the cells may be encapsulated
to avoid infiltration of surrounding tissues. The encapsulation
materials are typically biocompatible, semi-permeable polymeric
enclosures or membranes that allow the release of the protein
product(s) but prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissues.
[0299] As discussed herein, it may be desirable to treat isolated
cell populations (such as stem cells, lymphocytes, red blood cells,
chondrocytes, neurons, and the like) with one or more B7RP-2
polypeptides. This can be accomplished by exposing the isolated
cells to the polypeptide directly, where it is in a form that is
permeable to the cell membrane.
[0300] Additional embodiments of the present invention relate to
cells and methods (e.g., homologous recombination and/or other
recombinant production methods) for both the in vitro production of
therapeutic polypeptides and for the production and delivery of
therapeutic polypeptides by gene therapy or cell therapy.
Homologous and other recombination methods may be used to modify a
cell that contains a normally transcriptionally-silent B7RP-2 gene,
or an under-expressed gene, and thereby produce a cell which
expresses therapeutically efficacious amounts of B7RP-2
polypeptides.
[0301] Homologous recombination is a technique originally developed
for targeting genes to induce or correct mutations in
transcriptionally active genes. Kucherlapati, 1989, Prog. in Nucl.
Acid Res. & Mol. Biol. 36:301. The basic technique was
developed as a method for introducing specific mutations into
specific regions of the mammalian genome (Thomas et al., 1986, Cell
44:419-28; Thomas and Capecchi, 1987, Cell 51:503-12; Doetschman et
al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8583-87) or to correct
specific mutations within defective genes (Doetschman et al., 1987,
Nature 330:576-78). Exemplary homologous recombination techniques
are described in U.S. Pat. No. 5,272,071; European Patent Nos.
9193051 and 505500; PCT/US90/07642, and PCT Pub No. WO
91/09955).
[0302] Through homologous recombination, the DNA sequence to be
inserted into the genome can be directed to a specific region of
the gene of interest by attaching it to targeting DNA. The
targeting DNA is a nucleotide sequence that is complementary
(homologous) to a region of the genomic DNA. Small pieces of
targeting DNA that are complementary to a specific region of the
genome are put in contact with the parental strand during the DNA
replication process. It is a general property of DNA that has been
inserted into a cell to hybridize, and therefore, recombine with
other pieces of endogenous DNA through shared homologous regions.
If this complementary strand is attached to an oligonucleotide that
contains a mutation or a different sequence or an additional
nucleotide, it too is incorporated into the newly synthesized
strand as a result of the recombination. As a result of the
proofreading function, it is possible for the new sequence of DNA
to serve as the template. Thus, the transferred DNA is incorporated
into the genome.
[0303] Attached to these pieces of targeting DNA are regions of DNA
that may interact with or control the expression of a B7RP-2
polypeptide, e.g., flanking sequences. For example, a
promoter/enhancer element, a suppressor, or an exogenous
transcription modulatory element is inserted in the genome of the
intended host cell in proximity and orientation sufficient to
influence the transcription of DNA encoding the desired B7RP-2
polypeptide. The control element controls a portion of the DNA
present in the host cell genome. Thus, the expression of the
desired B7RP-2 polypeptide may be achieved not by transfection of
DNA that encodes the B7RP-2 gene itself, but rather by the use of
targeting DNA (containing regions of homology with the endogenous
gene of interest) coupled with DNA regulatory segments that provide
the endogenous gene sequence with recognizable signals for
transcription of a B7RP-2 gene.
[0304] In an exemplary method, the expression of a desired targeted
gene in a cell (i.e., a desired endogenous cellular gene) is
altered via homologous recombination into the cellular genome at a
preselected site, by the introduction of DNA which includes at
least a regulatory sequence, an exon, and a splice donor site.
These components are introduced into the chromosomal (genomic) DNA
in such a manner that this, in effect, results in the production of
a new transcription unit (in which the regulatory sequence, the
exon, and the splice donor site present in the DNA construct are
operatively linked to the endogenous gene). As a result of the
introduction of these components into the chromosomal DNA, the
expression of the desired endogenous gene is altered.
[0305] Altered gene expression, as described herein, encompasses
activating (or causing to be expressed) a gene which is normally
silent (unexpressed) in the cell as obtained, as well as increasing
the expression of a gene which is not expressed at physiologically
significant levels in the cell as obtained. The embodiments further
encompass changing the pattern of regulation or induction such that
it is different from the pattern of regulation or induction that
occurs in the cell as obtained, and reducing (including
eliminating) the expression of a gene which is expressed in the
cell as obtained.
[0306] One method by which homologous recombination can be used to
increase, or cause, B7RP-2 polypeptide production from a cell's
endogenous B7RP-2 gene involves first using homologous
recombination to place a recombination sequence from a
site-specific recombination system (e.g., Cre/loxP, FLP/FRT)
(Sauer, 1994, Curr. Opin. Biotechnol., 5:521-27; Sauer, 1993,
Methods Enzymol., 225:890-900) upstream of (i.e., 5' to) the cell's
endogenous genomic B7RP-2 polypeptide coding region. A plasmid
containing a recombination site homologous to the site that was
placed just upstream of the genomic B7RP-2 polypeptide coding
region is introduced into the modified cell line along with the
appropriate recombinase enzyme. This recombinase causes the plasmid
to integrate, via the plasmid's recombination site, into the
recombination site located just upstream of the genomic B7RP-2
polypeptide coding region in the cell line (Baubonis and Sauer,
1993, Nucleic Acids Res. 21:2025-29; O'Gorman et al., 1991, Science
251:1351-55). Any flanking sequences known to increase
transcription (e.g., enhancer/promoter, intron, translational
enhancer), if properly positioned in this plasmid, would integrate
in such a manner as to create a new or modified transcriptional
unit resulting in de novo or increased B7RP-2 polypeptide
production from the cell's endogenous B7RP-2 gene.
[0307] A further method to use the cell line in which the site
specific recombination sequence had been placed just upstream of
the cell's endogenous genomic B7RP-2 polypeptide coding region is
to use homologous recombination to introduce a second recombination
site elsewhere in the cell line's genome. The appropriate
recombinase enzyme is then introduced into the
two-recombination-site cell line, causing a recombination event
(deletion, inversion, and translocation) (Sauer, 1994, Curr. Opin.
Biotechnol., 5:521-27; Sauer, 1993, Methods Enzymol., 225:890-900)
that would create a new or modified transcriptional unit resulting
in de novo or increased B7RP-2 polypeptide production from the
cell's endogenous B7RP-2 gene.
[0308] An additional approach for increasing, or causing, the
expression of B7RP-2 polypeptide from a cell's endogenous B7RP-2
gene involves increasing, or causing, the expression of a gene or
genes (e.g., transcription factors) and/or decreasing the
expression of a gene or genes (e.g., transcriptional repressors) in
a manner which results in de novo or increased B7RP-2 polypeptide
production from the cell's endogenous B7RP-2 gene. This method
includes the introduction of a non-naturally occurring polypeptide
(e.g., a polypeptide comprising a site specific DNA binding domain
fused to a transcriptional factor domain) into the cell such that
de novo or increased B7RP-2 polypeptide production from the cell's
endogenous B7RP-2 gene results.
[0309] The present invention further relates to DNA constructs
useful in the method of altering expression of a target gene. In
certain embodiments, the exemplary DNA constructs comprise: (a) one
or more targeting sequences, (b) a regulatory sequence, (c) an
exon, and (d) an unpaired splice-donor site. The targeting sequence
in the DNA construct directs the integration of elements (a)-(d)
into a target gene in a cell such that the elements (b)-(d) are
operatively linked to sequences of the endogenous target gene. In
another embodiment, the DNA constructs comprise: (a) one or more
targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a
splice-donor site, (e) an intron, and (f) a splice-acceptor site,
wherein the targeting sequence directs the integration of elements
(a)-(f) such that the elements of (b)-(f) are operatively linked to
the endogenous gene. The targeting sequence is homologous to the
preselected site in the cellular chromosomal DNA with which
homologous recombination is to occur. In the construct, the exon is
generally 3' of the regulatory sequence and the splice-donor site
is 3' of the exon.
[0310] If the sequence of a particular gene is known, such as the
nucleic acid sequence of B7RP-2 polypeptide presented herein, a
piece of DNA that is complementary to a selected region of the gene
can be synthesized or otherwise obtained, such as by appropriate
restriction of the native DNA at specific recognition sites
bounding the region of interest. This piece serves as a targeting
sequence upon insertion into the cell and will hybridize to its
homologous region within the genome. If this hybridization occurs
during DNA replication, this piece of DNA, and any additional
sequence attached thereto, will act as an Okazaki fragment and will
be incorporated into the newly synthesized daughter strand of DNA.
The present invention, therefore, includes nucleotides encoding a
B7RP-2 polypeptide, which nucleotides may be used as targeting
sequences.
[0311] B7RP-2 polypeptide cell therapy, e.g., the implantation of
cells producing B7RP-2 polypeptides, is also contemplated. This
embodiment involves implanting cells capable of synthesizing and
secreting a biologically active form of B7RP-2 polypeptide. Such
B7RP-2 polypeptide-producing cells can be cells that are natural
producers of B7RP-2 polypeptides or may be recombinant cells whose
ability to produce B7RP-2 polypeptides has been augmented by
transformation with a gene encoding the desired B7RP-2 polypeptide
or with a gene augmenting the expression of B7RP-2 polypeptide.
Such a modification may be accomplished by means of a vector
suitable for delivering the gene as well as promoting its
expression and secretion. In order to minimize a potential
immunological reaction in patients being administered a B7RP-2
polypeptide, as may occur with the administration of a polypeptide
of a foreign species, it is preferred that the natural cells
producing B7RP-2 polypeptide be of human origin and produce human
B7RP-2 polypeptide. Likewise, it is preferred that the recombinant
cells producing B7RP-2 polypeptide be transformed with an
expression vector containing a gene encoding a human B7RP-2
polypeptide.
[0312] Implanted cells may be encapsulated to avoid the
infiltration of surrounding 3 0 tissue. Human or non-human animal
cells may be implanted in patients in biocompatible, semipermeable
polymeric enclosures or membranes that allow the release of B7RP-2
polypeptide, but that prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissue. Alternatively, the patient's own cells,
transformed to produce B7RP-2 polypeptides ex vivo, may be
implanted directly into the patient without such encapsulation.
[0313] Techniques for the encapsulation of living cells are known
in the art, and the preparation of the encapsulated cells and their
implantation in patients may be routinely accomplished. For
example, Baetge et al. (PCT Pub. No. WO 95/05452 and
PCT/US94/09299) describe membrane capsules containing genetically
engineered cells for the effective delivery of biologically active
molecules. The capsules are biocompatible and are easily
retrievable. The capsules encapsulate cells transfected with
recombinant DNA molecules comprising DNA sequences coding for
biologically active molecules operatively linked to promoters that
are not subject to down-regulation in vivo upon implantation into a
mammalian host. The devices provide for the delivery of the
molecules from living cells to specific sites within a recipient.
In addition, see U.S. Pat. Nos. 4,892,538; 5,011,472; and
5,106,627. A system for encapsulating living cells is described in
PCT Pub. No. WO 91/10425 (Aebischer et al.). See also, PCT Pub. No.
WO 91/10470 (Aebischer et al.); Winn et al., 1991, Exper. Neurol.
113:322-29; Aebischer et al., 1991, Exper. Neurol. 111:269-75; and
Tresco et al., 1992, ASAIO 38:17-23.
[0314] In vivo and in vitro gene therapy delivery of B7RP-2
polypeptides is also envisioned. One example of a gene therapy
technique is to use the B7RP-2 gene (either genomic DNA, cDNA,
and/or synthetic DNA) encoding a B7RP-2 polypeptide which may be
operably linked to a constitutive or inducible promoter to form a
"gene therapy DNA construct." The promoter may be homologous or
heterologous to the endogenous B7RP-2 gene, provided that it is
active in the cell or tissue type into which the construct will be
inserted. Other components of the gene therapy DNA construct may
optionally include DNA molecules designed for site-specific
integration (e.g., endogenous sequences useful for homologous
recombination), tissue-specific promoters, enhancers or silencers,
DNA molecules capable of providing a selective advantage over the
parent cell, DNA molecules useful as labels to identify transformed
cells, negative selection systems, cell specific binding agents
(as, for example, for cell targeting), cell-specific
internalization factors, transcription factors enhancing expression
from a vector, and factors enabling vector production.
[0315] A gene therapy DNA construct can then be introduced into
cells (either ex vivo or in vivo) using viral or non-viral vectors.
One means for introducing the gene therapy DNA construct is by
means of viral vectors as described herein. Certain vectors, such
as retroviral vectors, will deliver the DNA construct to the
chromosomal DNA of the cells, and the gene can integrate into the
chromosomal DNA. Other vectors will function as episomes, and the
gene therapy DNA construct will remain in the cytoplasm.
[0316] In yet other embodiments, regulatory elements can be
included for the controlled expression of the B7RP-2 gene in the
target cell. Such elements are turned on in response to an
appropriate effector. In this way, a therapeutic polypeptide can be
expressed when desired. One conventional control means involves the
use of small molecule dimerizers or rapalogs to dimerize chimeric
proteins which contain a small molecule-binding domain and a domain
capable of initiating a biological process, such as a DNA-binding
protein or transcriptional activation protein (see PCT Pub. Nos. WO
96/41865, WO 97/31898, and WO 97/31899). The dimerization of the
proteins can be used to initiate transcription of the
transgene.
[0317] An alternative regulation technology uses a method of
storing proteins expressed from the gene of interest inside the
cell as an aggregate or cluster. The gene of interest is expressed
as a fusion protein that includes a conditional aggregation domain
that results in the retention of the aggregated protein in the
endoplasmic reticulum. The stored proteins are stable and inactive
inside the cell. The proteins can be released, however, by
administering a drug (e.g., small molecule ligand) that removes the
conditional aggregation domain and thereby specifically breaks
apart the aggregates or clusters so that the proteins may be
secreted from the cell. See Aridor et al., 2000, Science 287:816-17
and Rivera et al., 2000, Science 287:826-30.
[0318] Other suitable control means or gene switches include, but
are not limited to, the systems described herein. Mifepristone
(RU486) is used as a progesterone antagonist. The binding of a
modified progesterone receptor ligand-binding domain to the
progesterone antagonist activates transcription by forming a dimer
of two transcription factors that then pass into the nucleus to
bind DNA. The ligand-binding domain is modified to eliminate the
ability of the receptor to bind to the natural ligand. The modified
steroid hormone receptor system is further described in U.S. Pat.
No. 5,364,791 and PCT Pub. Nos. WO 96/40911 and WO 97/10337.
[0319] Yet another control system uses ecdysone (a fruit fly
steroid hormone) which binds to and activates an ecdysone receptor
(cytoplasmic receptor). The receptor then translocates to the
nucleus to bind a specific DNA response element (promoter from
ecdysone-responsive gene). The ecdysone receptor includes a
transactivation domain, DNA-binding domain, and ligand-binding
domain to initiate transcription. The ecdysone system is further
described in U.S. Pat. No. 5,514,578 and PCT Pub. Nos. WO 97/38117,
WO 96/37609, and WO 93/03162.
[0320] Another control means uses a positive
tetracycline-controllable transactivator. This system involves a
mutated tet repressor protein DNA-binding domain (mutated tet R-4
amino acid changes which resulted in a reverse
tetracycline-regulated transactivator protein, i.e., it binds to a
tet operator in the presence of tetracycline) linked to a
polypeptide which activates transcription. Such systems are
described in U.S. Pat. Nos. 5,464,758, 5,650,298, and
5,654,168.
[0321] Additional expression control systems and nucleic acid
constructs are described in U.S. Pat. Nos. 5,741,679 and 5,834,186,
to Innovir Laboratories Inc.
[0322] In vivo gene therapy may be accomplished by introducing the
gene encoding B7RP-2 polypeptide into cells via local injection of
a B7RP-2 nucleic acid molecule or by other appropriate viral or
non-viral delivery vectors. Hefti 1994, Neurobiology 25:1418-35.
For example, a nucleic acid molecule encoding a B7RP-2 polypeptide
may be contained in an adeno-associated virus (AAV) vector for
delivery to the targeted cells (see, e.g., Johnson, PCT Pub. No. WO
95/34670; PCT App. No. PCT/US95/07178). The recombinant AAV genome
typically contains AAV inverted terminal repeats flanking a DNA
sequence encoding a B7RP-2 polypeptide operably linked to
functional promoter and polyadenylation sequences.
[0323] Alternative suitable viral vectors include, but are not
limited to, retrovirus, adenovirus, herpes simplex virus,
lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus,
alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma
virus vectors. U.S. Pat. No. 5,672,344 describes an in vivo
viral-mediated gene transfer system involving a recombinant
neurotrophic HSV-1 vector. U.S. Pat. No. 5,399,346 provides
examples of a process for providing a patient with a therapeutic
protein by the delivery of human cells which have been treated in
vitro to insert a DNA segment encoding a therapeutic protein.
Additional methods and materials for the practice of gene therapy
techniques are described in U.S. Pat. Nos. 5,631,236 (involving
adenoviral vectors), 5,672,510 (involving retroviral vectors),
5,635,399 (involving retroviral vectors expressing cytokines).
[0324] Nonviral delivery methods include, but are not limited to,
liposome-mediated transfer, naked DNA delivery (direct injection),
receptor-mediated transfer (ligand-DNA complex), electroporation,
calcium phosphate precipitation, and microparticle bombardment
(e.g., gene gun). Gene therapy materials and methods may also
include inducible promoters, tissue-specific enhancer-promoters,
DNA sequences designed for site-specific integration, DNA sequences
capable of providing a selective advantage over the parent cell,
labels to identify transformed cells, negative selection systems
and expression control systems (safety measures), cell-specific
binding agents (for cell targeting), cell-specific internalization
factors, and transcription factors to enhance expression by a
vector as well as methods of vector manufacture. Such additional
methods and materials for the practice of gene therapy techniques
are described in U.S. Pat. Nos. 4,970,154 (involving
electroporation techniques), 5,679,559 (describing a
lipoprotein-containing system for gene delivery), 5,676,954
(involving liposome carriers), 5,593,875 (describing methods for
calcium phosphate transfection), and 4,945,050 (describing a
process wherein biologically active particles are propelled at
cells at a speed whereby the particles penetrate the surface of the
cells and become incorporated into the interior of the cells), and
PCT Pub. No. WO 96/40958 (involving nuclear ligands).
[0325] It is also contemplated that B7RP-2 gene therapy or cell
therapy can further include the delivery of one or more additional
polypeptide(s) in the same or a different cell(s). Such cells may
be separately introduced into the patient, or the cells may be
contained in a single implantable device, such as the encapsulating
membrane described above, or the cells may be separately modified
by means of viral vectors.
[0326] A means to increase endogenous B7RP-2 polypeptide expression
in a cell via gene therapy is to insert one or more enhancer
elements into the B7RP-2 polypeptide promoter, where the enhancer
elements can serve to increase transcriptional activity of the
B7RP-2 gene. The enhancer elements used will be selected based on
the tissue in which one desires to activate the gene--enhancer
elements known to confer promoter activation in that tissue will be
selected. For example, if a gene encoding a B7RP-2 polypeptide is
to be "turned on" in T-cells, the lck promoter enhancer element may
be used. Here, the functional portion of the transcriptional
element to be added may be inserted into a fragment of DNA
containing the B7RP-2 polypeptide promoter (and optionally,
inserted into a vector and/or 5' and/or 3' flanking sequences)
using standard cloning techniques. This construct, known as a
"homologous recombination construct," can then be introduced into
the desired cells either ex vivo or in vivo.
[0327] Gene therapy also can be used to decrease B7RP-2 polypeptide
expression by modifying the nucleotide sequence of the endogenous
promoter. Such modification is typically accomplished via
homologous recombination methods. For example, a DNA molecule
containing all or a portion of the promoter of the B7RP-2 gene
selected for inactivation can be engineered to remove and/or
replace pieces of the promoter that regulate transcription. For
example, the TATA box and/or the binding site of a transcriptional
activator of the promoter may be deleted using standard molecular
biology techniques; such deletion can inhibit promoter activity
thereby repressing the transcription of the corresponding B7RP-2
gene. The deletion of the TATA box or the transcription activator
binding site in the promoter may be accomplished by generating a
DNA construct comprising all or the relevant portion of the B7RP-2
polypeptide promoter (from the same or a related species as the
B7RP-2 gene to be regulated) in which one or more of the TATA box
and/or transcriptional activator binding site nucleotides are
mutated via substitution, deletion and/or insertion of one or more
nucleotides. As a result, the TATA box and/or activator binding
site has decreased activity or is rendered completely inactive.
This construct, which also will typically contain at least about
500 bases of DNA that correspond to the native (endogenous) 5' and
3' DNA sequences adjacent to the promoter segment that has been
modified, may be introduced into the appropriate cells (either ex
vivo or in vivo) either directly or via a viral vector as described
herein. Typically, the integration of the construct into the
genomic DNA of the cells will be via homologous recombination,
where the 5' and 3' DNA sequences in the promoter construct can
serve to help integrate the modified promoter region via
hybridization to the endogenous chromosomal DNA.
[0328] Therapeutic Uses
[0329] B7RP-2 nucleic acid molecules, polypeptides, and agonists
and antagonists thereof can be used to treat, diagnose, ameliorate,
or prevent a number of diseases, disorders, or conditions,
including those recited herein.
[0330] B7RP-2 polypeptide agonists and antagonists include those
molecules which regulate B7RP-2 polypeptide activity and either
increase or decrease at least one activity of the mature form of
the B7RP-2 polypeptide. Agonists or antagonists may be co-factors,
such as a protein, peptide, carbohydrate, lipid, or small molecular
weight molecule, which interact with B7RP-2 polypeptide and thereby
regulate its activity. Potential polypeptide agonists or
antagonists include antibodies that react with either soluble or
membrane-bound forms of B7RP-2 polypeptides that comprise part or
all of the extracellular domains of the said proteins. Molecules
that regulate B7RP-2 polypeptide expression typically include
nucleic acids encoding B7RP-2 polypeptide that can act as
anti-sense regulators of expression.
[0331] Bone tissue consists of a matrix of collagenous and
noncollagenous proteins, minerals (largely calcium and
phosphorous), and cells. Three types of cells are involved in the
dynamic process by which bone is continually formed and resorbed:
osteocytes, osteoblasts, and osteoclasts. Osteoblasts promote
formation of bone tissue whereas osteoclasts are associated with
resorption. Resorption, or the dissolution of bone matrix and
mineral, is a fast and efficient process compared to bone formation
and can release large amounts of mineral from bone. Osteoclasts are
involved in the regulation of the normal remodeling of skeletal
tissue and in resorption induced by hormones. For instance,
resorption is stimulated by the secretion of parathyroid hormone in
response to decreasing concentrations of calcium ion in
extracellular fluids. In contrast, inhibition of resorption is the
principal function of calcitonin. In addition, metabolites of
vitamin D alter the responsiveness of bone to parathyroid hormone
and calcitonin.
[0332] Following skeletal maturity, the amount of bone in the
skeleton reflects the balance (or imbalance) of bone formation and
bone resorption. Peak bone mass occurs after skeletal maturity
prior to the fourth decade. Between the fourth and fifth decades,
the equilibrium shifts and bone resorption dominates. The
inevitable decrease in bone mass with advancing years starts
earlier in females than males and is distinctly accelerated after
menopause in some females (principally those of Caucasian and Asian
descent).
[0333] Osteopenia is a condition relating generally to any decrease
in bone mass to below normal levels. Such a condition may arise
from a decrease in the rate of bone synthesis or an increase in the
rate of bone destruction or both. The most common form of
osteopenia is primary osteoporosis, also referred to as
postmenopausal and senile osteoporosis. This form of osteoporosis
is a consequence of the universal loss of bone with age and is
usually a result of increase in bone resorption with a normal rate
of bone formation. About 25 to 30 percent of all white females in
the United States develop symptomatic osteoporosis. A direct
relationship exists between osteoporosis and the incidence of hip,
femoral, neck, and inter-trochanteric fracture in women 45 years
and older. Elderly males develop symptomatic osteoporosis between
the ages of 50 and 70, but the disease primarily affects
females.
[0334] The cause of postmenopausal and senile osteoporosis is
unknown. Several factors have been identified which may contribute
to the condition. They include alteration in hormone levels
accompanying aging, and inadequate calcium consumption attributed
to decreased intestinal absorption of calcium and other minerals.
Treatments have usually included hormone therapy or dietary
supplements in an attempt to retard the process. To date, however,
an effective treatment for bone loss does not exist.
[0335] The B7RP-2 nucleic acid molecules, polypeptides, and
agonists and antagonists of the present invention may be used to
treat, diagnose, ameliorate, or prevent diseases and disorders of
the bones, such as diseases or disorders characterized by a net
bone loss (such as osteopenia or osteolysis). For example, B7RP-2
polypeptides may be used to suppress the rate of bone resorption.
In this manner, an individual may be treated with B7RP-2
polypeptides in order to reduce the rate of bone resorption where
the resorption rate is above normal or to reduce bone resorption to
below normal levels in order to compensate for below normal levels
of bone formation.
[0336] Conditions which may be treatable with the B7RP-2 nucleic
acid molecules, polypeptides, and agonists and antagonists of the
present invention include the following: osteoporosis, such as
primary osteoporosis, endocrine osteoporosis (hyperthyroidism,
hyperparathryoidism, Cushing's syndrome, and acromegaly),
hereditary and congenital forms of osteoporosis (osteogenesis
imperfecta, homocystinuria, Menkes' syndrome, and Riley-Day
syndrome), and osteoporosis due to immobilization of extremities;
Paget's disease of bone (osteitis deformans) in adults and
juveniles; osteomyelitis, or an infectious lesion in bone, leading
to bone loss; hypercalcemia resulting from solid tumors (breast,
lung, and kidney) and hematologic malignacies (multiple myeloma,
lymphoma, and leukemia), idiopathic hypercalcemia, and
hypercalcemia associated with hyperthryoidism and renal function
disorders; osteopenia following surgery, induced by steroid
administration, and associated with disorders of the small and
large intestine and with chronic hepatic and renal diseases;
osteonecrosis, or bone cell death, associated with traumatic injury
or nontraumatic necrosis associated with Gaucher's disease, sickle
cell anemia, systemic lupus erythematosus, rheumatoid arthritis,
periodontal disease, osteolytic metastasis, and other conditions.
Other bone diseases and disorders are encompassed within the scope
of the invention.
[0337] The B7RP-2 nucleic acid molecules, polypeptides, and
agonists and antagonists of the present invention may be used to
treat, diagnose, ameliorate, or prevent diseases associated with
T-cell function (e.g., functioning as a T-cell receptor decoy). For
example, antibodies, soluble proteins comprising extracellular
domains, or other regulators of B7RP-2 polypeptide that result in
prolonged or enhanced T-cell activation can be used to increase the
immune response to tumors.
[0338] The B7RP-2 nucleic acid molecules, polypeptides, and
agonists and antagonists of the present invention may be used in
the treatment of autoimmune disease, graft survival, immune cell
activation for inhibiting tumor cell growth, T-cell dependent
B-cell mediated diseases, and cancer gene immunotherapy. In one
embodiment, agonists or antagonists of B7RP-2 polypeptide function,
soluble B7RP-2 polypeptides, or B7RP-2 polypeptide derivatives may
be beneficial to alleviate symptoms in diseases with chronic immune
cell dysfunction. Autoimmune diseases, such as systemic lupus
erythematosis, rheumatoid arthritis, osteoarthritis, immune
thrombocytopenic purpura (ITP), and psoriasis, may be treated with
agonists or antagonists of B7RP-2 polypeptide function, soluble
B7RP-2 polypeptides, or B7RP-2 polypeptide derivatives. In
addition, chronic inflammatory diseases, such as inflammatory bowel
disease (Crohn's disease and ulcerative colitis), Grave's disease,
Hashimoto's thyroiditis, and diabetes mellitis, may also be treated
with agonists or antagonists of B7RP-2 polypeptide function,
soluble B7RP-2 polypeptides, or B7RP-2 polypeptide derivatives.
[0339] Agonists or antagonists of B7RP-2 polypeptide function,
soluble B7RP-2 polypeptides, or B7RP-2 polypeptide derivatives may
be used as immunosuppressive agents for bone marrow and organ
transplantation and may be used to prolong graft survival. Such
agonists or antagonists of B7RP-2 polypeptide function, soluble
B7RP-2 polypeptides, or B7RP-2 polypeptide derivatives may provide
significant advantages over existing treatments. Bone marrow and
organ transplantation therapy must contend with T-cell mediated
rejection of the foreign cells or tissue by the host. Present
therapeutic regimens for inhibiting T-cell mediated rejection
involve treatment with the drugs cyclosporine or FK506. While drugs
are effective, patients suffer from serious side effects, including
hepatotoxicity, nephrotoxicity, and neurotoxicity. The target for
the cyclosporin/FK506 class of therapeutics is calcineurin, a
phosphatase with ubiquitous expression. Agonists or antagonists of
B7RP-2 polypeptide function, soluble B7RP-2 polypeptides, or B7RP-2
polypeptide derivatives may lack the severe side effects observed
with the use of the present immunotherapeutic agents. Agonists or
antagonists of B7RP-2 polypeptide function, soluble B7RP-2
polypeptides, or B7RP-2 polypeptide derivatives may be used as
immunosuppressive agents for autoimmune disorders, such as
rheumatoid arthritis, osteoarthritis psoriasis, multiple sclerosis,
diabetes, and systemic lupus erythematosus. Agonists or antagonists
of B7RP-2 polypeptide function, soluble B7RP-2 polypeptides, or
B7RP-2 polypeptide derivatives may also be used to alleviate toxic
shock syndrome, inflammatory bowel disease, allosensitization due
to blood transfusions, T-cell dependent B-cell mediated diseases,
and the treatment of graft versus host disease.
[0340] Gene therapy using B7RP-2 genes of the invention may be used
in cancer immunotherapy. B7RP-2 genes introduced into cancer cells
can transform them into antigen presenting cells that can be
recognized by the T-cells of the immune system when introduced back
into an animal. Recognition of the transfected tumor cells by the
T-cells results in the eradication of tumors expressing and tumors
not expressing the B7RP-2 gene. This immunotherapy approach may be
used for various leukemias, sarcomas, melanomas, adenocarcinomas,
breast carcinomas, prostate tumors, lung carcinomas, colon
carcinomas, and other tumors. This invention encompasses using the
B7RP-2 gene in a similar manner to enhance T-cell activation in
response to variety of tumors.
[0341] For instance, many vaccines act by eliciting an effective
and specific antibody response. Some vaccines, especially those
against intestinal microorganisms (e.g., Hepatitis A virus and
Salmonella), elicit a short-lived antibody response. It is
desirable to potentiate and prolong this response in order to
increase the effectiveness of the vaccine. Therefore, soluble
B7RP-2 polypeptides may serve as vaccine adjuvants.
[0342] Conversely, since B7RP-2 may have negative immune regulatory
functions, inhibition of B7RP-2 activity using antibodies, small
molecules, peptibodies, or other antagonists of B7RP-2 function may
result in immune enhancement and anti-tumor activity.
[0343] Anti-viral responses may also be enhanced by activators or
agonists of the B7RP-2 polypeptide pathway. The enhancement of
cellular immune functions by B7RP-2 polypeptide antagonists may
also be beneficial in eliminating virus-infected cells. In a
complementary fashion, B7RP-2 polypeptide antagonists may also have
effects on humoral immune functions that may enhance antibody
mediated responses and that may function to help clear free virus
from the body.
[0344] Conversely, there are a number of clinical conditions that
would be ameliorated by the inhibition of antibody production.
Hypersensitivity is a normally beneficial immune response that is
exaggerated or inappropriate, and leads to inflammatory reactions
and tissue damage. Hypersensitivity reactions that are
antibody-mediated may be particularly susceptible to antagonism by
agonists of B7RP-2 polypeptide function, soluble B7RP-2
polypeptides, or B7RP-2 polypeptide derivatives. Allergies, hay
fever, asthma, and acute edema cause type I hypersensitivity
reactions, and these reactions may be suppressed by agonists of
B7RP-2 polypeptide function, soluble B7RP-2 polypeptides, or B7RP-2
polypeptide derivatives.
[0345] Diseases that cause antibody-mediated hypersensitivity
reactions, including systemic lupus erythematosis, arthritis
(rheumatoid arthritis, reactive arthritis, and psoriatic
arthritis), nephropathies (glomerulo-nephritis, membranous,
mesangiocapillary, focal segmental, focal necrotizing, crescentic,
and proliferative tubulopathies), skin disorders (pemphigus,
pemphigoid, and erythema nodosum), endocrinopathies (thyroiditis,
Grave's, Hashimoto's, insulin-dependent diabetes mellitus), various
pneumopathies (especially extrinsic alveolitis), various
vasculopathies, coeliac disease, with aberrant production of IgA,
many anemias and thrombocytopenias, Guillain-Barre Syndrome, and
myasthenia gravis, may be treated with agonists of B7RP-2
polypeptide function, soluble B7RP-2 polypeptides, or B7RP-2
polypeptide derivatives.
[0346] In addition, lymphoproliferative disorders, such as multiple
myeloma, Waldenstrom's macroglobulinemia, and crioglobulinemias,
may be inhibited by agonists of B7RP-2 polypeptide function,
soluble B7RP-2 polypeptides, or B7RP-2 polypeptide derivatives.
Finally, graft versus host disease, an "artificial" immune
disorder, may benefit from the inhibition of antibody production by
agonists of B7RP-2 polypeptide function, soluble B7RP-2
polypeptides, or B7RP-2 polypeptide derivatives.
[0347] Agonists or antagonists of B7RP-2 polypeptide function may
be used (simultaneously or sequentially) in combination with one or
more cytokines, growth factors, antibiotics, anti-inflammatories,
and/or chemotherapeutic agents as is appropriate for the condition
being treated.
[0348] Other diseases caused by or mediated by undesirable levels
of B7RP-2 polypeptides are encompassed within the scope of the
invention. Undesirable levels include excessive levels of B7RP-2
polypeptides and sub-normal levels of B7RP-2 polypeptides.
[0349] Uses of B7RP-2 Nucleic Acids and Polypeptides
[0350] Nucleic acid molecules of the invention (including those
that do not themselves encode biologically active polypeptides) may
be used to map the locations of the B7RP-2 gene and related genes
on chromosomes. Mapping may be done by techniques known in the art,
such as PCR amplification and in situ hybridization.
[0351] B7RP-2 nucleic acid molecules (including those that do not
themselves encode biologically active polypeptides), may be useful
as hybridization probes in diagnostic assays to test, either
qualitatively or quantitatively, for the presence of a B7RP-2
nucleic acid molecule in mammalian tissue or bodily fluid
samples.
[0352] Other methods may also be employed where it is desirable to
inhibit the activity of one or more B7RP-2 polypeptides. Such
inhibition may be effected by nucleic acid molecules that are
complementary to and hybridize to expression control sequences
(triple helix formation) or to B7RP-2 mRNA. For example, antisense
DNA or RNA molecules, which have a sequence that is complementary
to at least a portion of a B7RP-2 gene can be introduced into the
cell. Anti-sense probes may be designed by available techniques
using the sequence of the B7RP-2 gene disclosed herein. Typically,
each such antisense molecule will be complementary to the start
site (5' end) of each selected B7RP-2 gene. When the antisense
molecule then hybridizes to the corresponding B7RP-2 mRNA,
translation of this mRNA is prevented or reduced. Anti-sense
inhibitors provide information relating to the decrease or absence
of a B7RP-2 polypeptide in a cell or organism.
[0353] Alternatively, gene therapy may be employed to create a
dominant-negative inhibitor of one or more B7RP-2 polypeptides. In
this situation, the DNA encoding a mutant polypeptide of each
selected B7RP-2 polypeptide can be prepared and introduced into the
cells of a patient using either viral or non-viral methods as
described herein. Each such mutant is typically designed to compete
with endogenous polypeptide in its biological role.
[0354] In addition, a B7RP-2 polypeptide, whether biologically
active or not, may be used as an immunogen, that is, the
polypeptide contains at least one epitope to which antibodies may
be raised. Selective binding agents that bind to a B7RP-2
polypeptide (as described herein) may be used for in vivo and in
vitro diagnostic purposes, including, but not limited to, use in
labeled form to detect the presence of B7RP-2 polypeptide in a body
fluid or cell sample. The antibodies may also be used to prevent,
treat, or diagnose a number of diseases and disorders, including
those recited herein. The antibodies may bind to a B7RP-2
polypeptide so as to diminish or block at least one activity
characteristic of a B7RP-2 polypeptide, or may bind to a
polypeptide to increase at least one activity characteristic of a
B7RP-2 polypeptide (including by increasing the pharmacokinetics of
the B7RP-2 polypeptide).
[0355] The B7RP-2 polypeptides of the present invention can be used
to clone B7RP-2 polypeptide receptors, using an expression cloning
strategy. Radiolabeled (.sup.125Iodine) B7RP-2 polypeptide or
affinity/activity-tagged B7RP-2 polypeptide (such as an Fc fusion
or an alkaline phosphatase fusion) can be used in binding assays to
identify a cell type or cell line or tissue that expresses B7RP-2
polypeptide receptors. RNA isolated from such cells or tissues can
be converted to cDNA, cloned into a mammalian expression vector,
and transfected into mammalian cells (such as COS or 293 cells) to
create an expression library. A radiolabeled or tagged B7RP-2
polypeptide can then be used as an affinity ligand to identify and
isolate from this library the subset of cells that express the
B7RP-2 polypeptide receptors on their surface. DNA can then be
isolated from these cells and transfected into mammalian cells to
create a secondary expression library in which the fraction of
cells expressing B7RP-2 polypeptide receptors is many-fold higher
than in the original library. This enrichment process can be
repeated iteratively until a single recombinant clone containing a
B7RP-2 polypeptide receptor is isolated. Isolation of the B7RP-2
polypeptide receptors is useful for identifying or developing novel
agonists and antagonists of the B7RP-2 polypeptide signaling
pathway. Such agonists and antagonists include soluble B7RP-2
polypeptide receptors, anti-B7RP-2 polypeptide receptor antibodies,
small molecules, or antisense oligonucleotides, and they may be
used for treating, preventing, or diagnosing one or more of the
diseases or disorders described herein.
[0356] The murine and human B7RP-2 nucleic acids of the present
invention are also useful tools for isolating the corresponding
chromosomal B7RP-2 polypeptide genes. For example, mouse
chromosomal DNA containing B7RP-2 sequences can be used to
construct knockout mice, thereby permitting an examination of the
in vivo role for B7RP-2 polypeptide. The human B7RP-2 genomic DNA
can be used to identify heritable tissue-degenerating diseases.
[0357] The following examples are intended for illustration
purposes only, and should not be construed as limiting the scope of
the invention in any way.
EXAMPLE 1
[0358] Cloning of the Human B7RP-2 Polypeptide Gene
[0359] Generally, materials and methods as described in Sambrook et
al. supra were used to clone and analyze the gene encoding human
B7RP-2 polypeptide.
[0360] A search of a proprietary database was performed using the
rat B7RP-2 ortholog (SEQ ID NO: 5) as the query sequence. A 342 bp
clone was identifying as containing nucleic acid sequences encoding
a portion of human B7RP-2 polypeptide. This sequence was expanded
to 792 bp (SEQ ID NO: 16) by examination of related contigs.
[0361] Due to the high level of B7RP-2 expression detected in bone
calveria by in situ hybridization, cDNA sequences encoding human
B7RP-2 polypeptide were isolated in amplification reactions
performed using a human bone calveria cDNA library and the
amplimers 2245-71 (5'-C-A-A-C-G-A-G-C-A-G-G-G-C-T-T-G-T-T-T-G-3';
SEQ ID NO: 17) and 2245-72
(5'-G-G-T-C-T-G-T-G-T-A-T-C-G-C-A-T-C-C-T-T-T-G-G-3'; SEQ ID NO:
18). A PCR product of the expected size was isolated and subcloned
into the Topo II vector. The resulting ligation reactions were used
to transform competent bacteria and the clones obtained in this
manner were then analyzed by sequencing.
[0362] Both 5'RACE and 3'RACE reactions were performed in order to
generate the full-length cDNA sequence for human B7RP-2
polypeptide. To isolate cDNA sequences corresponding to the 3' end
of the cDNA sequence, 3'RACE was performed using a human fetal
calveria cDNA library in the pSPORT1 vector and the primers 2245-72
and 1071-80 (5'-T-G-C-A-G-G-T-A-C-C-G-G-T-C-C-G-G-A-A-T-3'; SEQ ID
NO: 19). Nested PCR was performed using a portion of the 3'RACE
amplification product and the primers 2279-24
(5'-T-G-T-C-A-G-A-G-C-A-G-G-A-T-G-C-A-T-C-T-G-T-3'; SEQ ID NO: 20)
and 1071-80. A PCR product of 800 bp was isolated and subcloned
into the Topo II vector. The resulting ligation reactions were used
to transform competent bacteria and the clones obtained in this
manner were then analyzed by sequencing.
[0363] To isolate cDNA sequences corresponding to the 5' end of the
cDNA sequence, 5'RACE was performed using the human fetal calveria
cDNA library described above and the primers 2279-22
(5'-T-G-C-A-T-T-G-C-C-T-T- -G-T-G-C-C-A-G-C-A-G-G-T-3'; SEQ ID NO:
21) and 1071-80. Nested PCR was performed using a portion of the
5'RACE amplification product and the primers 2279-21
(5'-C-T-G-T-C-A-G-C-T-G-C-C-A-G-A-T-G-A-G-G-T-T-G-3'; SEQ ID NO:
22) and 1071-80. A PCR product of 400 bp was isolated and subcloned
into the Topo II vector. The resulting ligation reactions were used
to transform competent bacteria and the clones obtained in this
manner were then analyzed by sequencing.
[0364] To isolate the full-length cDNA sequence encoding B7RP-2
polypeptide, amplification reactions were performed using the human
fetal calveria cDNA library described above and the primers 2318-34
(5'-G-C-G-T-C-C-C-T-G-A-G-T-C-C-C-A-G-A-G-3'; SEQ ID NO: 23) and
2318-35 (5'-G-T-G-T-A-T-C-G-C-A-T-C-C-T-T-T-G-G-A-G-A-A-G-3'; SEQ
ID NO: 24). A PCR product of approximately 1.6 kb was isolated and
subcloned into the Topo II vector. The resulting ligation reactions
were used to transform competent bacteria and the clones obtained
in this manner were then analyzed by sequencing. Sequence analysis
indicated that the human B7RP-2 gene comprises a 948 bp open
reading frame encoding a protein of 316 amino acids (FIGS.
1A-1B).
[0365] Isolation of the cDNA sequences encoding murine and rat
B7RP-2 polypeptide, indicate that both the murine B7RP-2 gene
(FIGS. 2A-2B) and the rat B7RP-2 gene (FIGS. 3A-3C) also comprise
open reading frames of 948 bp, each encoding a protein of 316 amino
acids. The amino acid sequences for human B7RP-2 polypeptide (SEQ
ID NO: 2), murine B7RP-2 polypeptide (SEQ ID NO: 4), and rat B7RP-2
polypeptide (SEQ ID NO: 6) were aligned using the ClustalW
algorithm (Thompson et al., 1994, Nucleic Acids Res. 22:4673-80).
The ClustalW alignment of the human, murine, and rat B7RP-2
orthologs (FIGS. 4A-4B) suggests that human B7RP-2 polypeptide will
tolerate nonconservative amino acid substitutions at a number of
positions (see SEQ ID NO: 32), and further, that conservative amino
acid substitutions may be made at several other positions in the
human B7RP-2 amino acid sequence (e.g., at positions 20, 29, 101,
120, 184, 260, 261, 291, and 306). A BLAST analysis of the human,
murine, and rat B7RP-2 orthologs against the Conserved Domain
Database (a collection of functional and structural domains derived
primarily from the Smart and Pfam databases) indicated that the
three proteins also share at least two conserved protein domains,
namely an immunoglobulin V-type domain and an immunoglobulin C-type
or C-2 type domain (FIG. 5).
[0366] Sequence analysis also revealed that B7RP-2 polypeptide
shares homology with the B7 family of proteins. FIGS. 6A-6B
illustrate the amino acid sequence alignment of human butyrophilin,
subfamily 1, member A1 (hu_BTN1A1; SEQ ID NO: 7), bovine
butyrophilin precursor (bo_BTN; SEQ ID NO: 8), murine butyrophilin
(mu_BTN; SEQ ID NO: 9), human butyrophilin, subfamily 2, member A1
(hu_BTN2A1; SEQ ID NO: 10), human butyrophilin-like protein
(hu_BT3.2; SEQ ID NO: 11), human butyrophilin, subfamily 3, member
A2 (hu_BTN3A2; SEQ ID NO: 12), Grus americana B-G-like protein
(gr_BG2; SEQ ID NO: 13), and human B7RP-2 polypeptide (hu_B7RP-2;
SEQ ID NO: 2).
[0367] The predicted protein product of the B7RP-2 gene is related
to the B7 family of proteins. These proteins are members of the
immunoglobulin superfamily and function as regulators of the T-cell
mediated immune response. Members of the B7 family of proteins are
Type-1 membrane proteins with a small cytoplasmic domain and
extracellular regions that contain immunoglobulin V (variable) and
C (constant) domains. The known members of the B7 family include
CD80 (B7-1), CD86 (B7-2), B7RP-1, and B7-H1. B7-1 and B7-2 interact
with CD28 and CTLA-4 and are mediators of the T-cell costimulatory
pathway. B7RP-1 binds to a distinct receptor (ICOS; inducible
co-stimulator) and is also a stimulator of T-cell proliferation.
B7-H1 also co-stimulates T-cell proliferation, but does not bind
CD28, CTLA-4, or ICOS. The protein sequences of this family are
poorly conserved and consequently, are difficult to distinguish
from other related molecules using computational methods,
especially when only a portion of the full-length coding region
sequence is compared. Other proteins exhibiting sequence homology
to the B7 family include the butyrophilins and PRO352. Still more
distantly related are the myelin oligodendrocyte proteins
(MOGs).
[0368] Baker et al. (PCT Publication No. WO 99/46281) dislocse a
nucleic acid sequence of 1998 bp (SEQ ID NO: 25) encoding a
polypeptide of 316 amino acids (SEQ ID NO: 26) that they designate
as PRO352. Chapoval et al, 2001, Nat. Immun. 2:269-274, disclose a
nucleic acid sequence of 951 bp (SEQ ID NO: 27) encoding a
polypeptide of 316 amino acids (SEQ ID NO: 28) which they designate
B7-H3. The nucleic acid sequence disclosed by Chapoval et al. is
identical to the nucleic acid sequence that encodes B7RP-2
polypeptide.
EXAMPLE 2
[0369] B7RP-2 mRNA Expression
[0370] The kinetics of B7RP-2 mRNA expression during bone formation
was examined in osteoblast cells following treatment with
dexamethasone, vitamin C, and .quadrature.-glycerophosphate.
Osteoblasts were isolated from rat bone marrow and cultured in
.alpha.-Minimal Essential Media containing 10% fetal calf serum, 3
ng/ml .beta.-FGF, 50 .quadrature.M .beta.-mercaptoethanol, and
antibiotics. Dexamethasone (10 nM) and vitamin C (50
.quadrature.g/ml) were added to the media when the cells reached
confluency, and the media was then renewed every other day until
day 14. At day 8, .beta.-glycerophosphate (50 .quadrature.g/ml) was
also added. Total RNA was prepared at days 2, 3, 4, 6, 8, 10, 12,
and 14 and analysed by Northern blot analysis using the full-length
rat B7RP-2 cDNA sequence as a probe. Each lane was loaded with an
equal amount of RNA (20 .quadrature.g/lane) as assessed by 28S and
18S rRNA. The increase in the expression of B7RP-2 mRNA following
the addition of dexamethasone, vitamin C, and
.beta.-glycerophosphate (FIG. 7) indicates that B7RP-2 polypeptide
might be involved in osteoblast growth or differentiation.
[0371] The expression of B7RP-2 mRNA was localized by in situ
hybridization. Normal mouse embryos (E18.5) were fixed in 4%
paraformaldehyde, embedded in paraffin, and sectioned at 5 .mu.m.
Prior to hybridization, sections were permeabilized with 0.2 M HCl,
digested with Proteinase K, and acetylated with triethanolamine and
acetic anhydride. Sections were hybridized overnight at 55.degree.
C. with a .sup.33P-labeled riboprobe corresponding to 5' and 3'
sequences of the mouse B7RP-2 protein coding sequence. Following
hybridization, sections were treated with RNaseA to digest
unhybridized probe, and then washed with a series of buffers
containing decreasing salt concentrations to a high stringency of
0.1.times.SSC at 55.degree. C. Sections were then immersed in NTB2
emulsion (Kodak, Rochester, N.Y.), exposed for 2-3 weeks at
4.degree. C., developed, and counterstained with hematoxilyn and
eosin. Sections were examined with darkfield and transmitted light
illumination to allow simultaneous evaluation of tissue morphology
and hybridization signal. B7RP-2 mRNA expression was detected in
the developing bones of an E18.5 mouse embryo (FIG. 8).
EXAMPLE 3
[0372] Production of B7RP-2 Polypeptides
[0373] A. Expression of B7RP-2 Polypeptides in Bacteria
[0374] PCR is used to amplify template DNA sequences encoding a
B7RP-2 polypeptide using primers corresponding to the 5' and 3'
ends of the sequence. The amplified DNA products may be modified to
contain restriction enzyme sites to allow for insertion into
expression vectors. PCR products are gel purified and inserted into
expression vectors using standard recombinant DNA methodology. An
exemplary vector, such as pAMG21 (ATCC no. 98113) containing the
lux promoter and a gene encoding kanamycin resistance is digested
with Bam FH and Nde I for directional cloning of inserted DNA. The
ligated mixture is transformed into an E. coli host strain by
electroporation and transformants are selected for kanamycin
resistance. Plasmid DNA from selected colonies is isolated and
subjected to DNA sequencing to confirm the presence of the
insert.
[0375] Transformed host cells are incubated in 2.times.YT medium
containing 30 .mu.g/mL kanamycin at 30.degree. C. prior to
induction. Gene expression is induced by the addition of
N-(3-oxohexanoyl)-dl-homose- rine lactone to a final concentration
of 30 ng/mL followed by incubation at either 30.degree. C. or
37.degree. C. for six hours. The expression of B7RP-2 polypeptide
is evaluated by centrifugation of the culture, resuspension and
lysis of the bacterial pellets, and analysis of host cell proteins
by SDS-polyacrylamide gel electrophoresis.
[0376] Inclusion bodies containing B7RP-2 polypeptide are purified
as follows. Bacterial cells are pelleted by centrifugation and
resuspended in water. The cell suspension is lysed by sonication
and pelleted by centrifugation at 195,000.times.g for 5 to 10
minutes. The supernatant is discarded, and the pellet is washed and
transferred to a homogenizer. The pellet is homogenized in 5 mL of
a Percoll solution (75% liquid Percoll and 0.15 M NaCl) until
uniformly suspended and then diluted and centrifuged at
21,600.times.g for 30 minutes. Gradient fractions containing the
inclusion bodies are recovered and pooled. The isolated inclusion
bodies are analyzed by SDS-PAGE.
[0377] A single band on an SDS polyacrylamide gel corresponding to
E. coli-produced B7RP-2 polypeptide is excised from the gel, and
the N-terminal amino acid sequence is determined essentially as
described by Matsudaira et al., 1987, J. Biol. Chem. 262:10-35.
[0378] B. Expression of B7RP-2 Polypeptide in Mammalian Cells
[0379] PCR is used to amplify template DNA sequences encoding a
B7RP-2 polypeptide using primers corresponding to the 5' and 3'
ends of the sequence. The amplified DNA products may be modified to
contain restriction enzyme sites to allow for insertion into
expression vectors. PCR products are gel purified and inserted into
expression vectors using standard recombinant DNA methodology. An
exemplary expression vector, pCEP4 (Invitrogen, Carlsbad, Calif.),
that contains an Epstein-Barr virus origin of replication, may be
used for the expression of B7RP-2 polypeptides in 293-EBNA-1 cells.
Amplified and gel purified PCR products are ligated into pCEP4
vector and introduced into 293-EBNA cells by lipofection. The
transfected cells are selected in 100 .mu.g/mL hygromycin and the
resulting drug-resistant cultures are grown to confluence. The
cells are then cultured in serum-free media for 72 hours. The
conditioned media is removed and B7RP-2 polypeptide expression is
analyzed by SDS-PAGE.
[0380] B7RP-2 polypeptide expression may be detected by silver
staining. Alternatively, B7RP-2 polypeptide is produced as a fusion
protein with an epitope tag, such as an IgG constant domain or a
FLAG epitope, which may be detected by Western blot analysis using
antibodies to the peptide tag.
[0381] B7RP-2 polypeptides may be excised from an
SDS-polyacrylamide gel, or B7RP-2 fusion proteins are purified by
affinity chromatography to the epitope tag, and subjected to
N-terminal amino acid sequence analysis as described herein.
[0382] C. Expression and Purification of B7RP-2 Polypeptide in
Mammalian Cells
[0383] B7RP-2 polypeptide expression constructs are introduced into
293 EBNA or CHO cells using either a lipofection or calcium
phosphate protocol.
[0384] To conduct functional studies on the B7RP-2 polypeptides
that are produced, large quantities of conditioned media are
generated from a pool of hygromycin selected 293 EBNA clones. The
cells are cultured in 500 cm Nunc Triple Flasks to 80% confluence
before switching to serum free media a week prior to harvesting the
media. Conditioned media is harvested and frozen at -20.degree. C.
until purification.
[0385] Conditioned media is purified by affinity chromatography as
described below. The media is thawed and then passed through a 0.2
.mu.m filter. A Protein G column is equilibrated with PBS at pH
7.0, and then loaded with the filtered media. The column is washed
with PBS until the absorbance at A.sub.280 reaches a baseline.
B7RP-2 polypeptide is eluted from the column with 0.1 M Glycine-HCl
at pH 2.7 and immediately neutralized with 1 M Tris-HCl at pH 8.5.
Fractions containing B7RP-2 polypeptide are pooled, dialyzed in
PBS, and stored at -70.degree. C.
[0386] For Factor Xa cleavage of the human B7RP-2 polypeptide-Fc
fusion polypeptide, affinity chromatography-purified protein is
dialyzed in 50 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl.sub.2 at pH 8.0.
The restriction protease Factor Xa is added to the dialyzed protein
at 1/100 (w/w) and the sample digested overnight at room
temperature.
EXAMPLE 4
[0387] In Vitro Characterization of B7RP-2 Polypeptides
[0388] The inhibitory activity of B7RP-2 mRNA expression during
bone mineralization was examined in calvarial cells following
treatment with dexamethasone, vitamin C, and
.beta.-glycerophosphate. Calvarial cells were isolated from
neonatal mice (CD1 strain) and cultured in .alpha.-Minimal
Essential Media containing 10% fetal calf serum, 50 .quadrature.M
.beta.-mercaptoethanol, and antibiotics. Dexamethasone (10 nM) and
vitamin C (50 .quadrature.g/ml) were added to the media when the
cells reached confluency, and the media was then renewed every
other day until day 14. At day 12, .beta.-glycerophosphate (10 mM)
was also added. At day 14, the degree of bone mineralization was
determined by von Kossa staining. Soluble B7RP-2 polypeptide was
found to inhibit nodule formation and mineralization in a dose
dependent manner in vitro (FIG. 9), indicating that B7RP-2
polypeptide might be involved in the regulation of bone
formation.
[0389] Recombinant protein comprising the two extracellular Ig
domains of B7RP-2 fused in-frame to the Fc portion of human IgG1
(B7RP-2-Fc) was synthesized. Lymph node cells from C57BL/6 mice
were depleted of B220.sup.+ cells using magnetic beads (Dynal,
Oslo, Norway). Purified lymph node T-cells were activated using
plate-bound anti-CD3 (0.1 .mu.g/ml) plus 10 .mu.g/ml of plate-bound
human IgG1 (isotype control), B7RP-2-Fc, or B7-2-Fc (positive
control) in U-bottom 96-well plates. T-cell proliferation was
assayed by pulsing the cells with .sup.3H-thymidine during the last
8 hours of a 72-hour incubation period. The B7RP-2-Fc inhibited
T-cell proliferation up to 5-fold compared to controls (FIG. 10A).
Interleukin-2 (IL-2) (FIG. 10B) and interferon-.gamma.
(IFN-.gamma.) (FIG. 10C) production in the culture supernatants was
measured using ELISA. IL-2 and IFN-.gamma. production were
similarly reduced, most likely because of the decrease in T-cell
proliferation (FIGS. 10B and 10C). These results indicate that
B7RP-2 inhibits TCR-mediated T-cell proliferation in vitro.
EXAMPLE 5
[0390] In vivo Characterization of B7RP-2
[0391] A. Generation of B7RP-2-/-Mice
[0392] The in vivo role of B7RP-2 was examined by generating B7RP-2
deficient mice. A murine B7RP-2 genomic clone was isolated from a
129/J phage library using the full-length rat B7RP-2 cDNA as a
probe. A targeting vector was designed to replace the exon encoding
the second Ig domain of B7RP-2 with a neomycin resistance cassette
(FIG. 11). A .about.3.2 kb genomic sequence encompassing the exon
encoding the second Ig domain of B7RP-2 (filled rectangle in FIG.
11) was replaced by the PGK promoter-driven neomycin resistance
gene (Neo). The diphtheria toxin A gene (DT-A) was used for
negative selection. The targeting vector was introduced into E14
embryonic stem (ES) cells (129/Ola) by direct micoinjection, and ES
cell clones were screened by PCR to identify clones that had
undergone homologous recombination.
[0393] Selected ES clones were verified by Southern blot analysis.
For Southern blotting, Bgl II-digested genomic DNA from B7RP-2+/+,
+/- and -/-mice was hybridized to the 5' flanking probe shown in
FIG. 11. C57BL/6 blastocysts were injected with B7RP-2+/-ES cells
and implanted in pseudopregnant female mice. The resulting chimeric
mice were bred with C57BL/6 mice to obtain heterozygous F1 progeny,
which were interbred to generate B7RP-2-/-mice. B7RP-2-/-mice
derived from two independent ES clones showed the same
phenotypes.
[0394] Disruption of the B7RP-2 gene was confirmed by Southern
analysis of genomic DNA from F2 progeny (FIG. 12). Anti-B7RP-2
antibodies were used to verify that the mice were not producing
B7RP-2 protein. Rabbit polyclonal antibody was raised against rat
B7RP-2-Fc protein. The antiserum was purified through a protein A
column and anti-Fc antibodies were removed using an Fc affinity
column. The flow cytometric analysis of mouse embryonic fibroblasts
(MEF) from the B7RP-2-/-mice confirmed the absence of B7RP-2
protein (FIG. 13). MEF were stained with anti-B7RP-2 rabbit IgG and
FITC-conjugated goat anti-rabbit IgG.
[0395] B7RP-2-/-mice were obtained at the expected Mendelian ratio,
and were found to be of normal size, maturation, and fertility. T-,
B-, and NK-cell populations in the bone marrow, thymus, lymph node,
spleen, and peripheral blood were normal in B7RP-2-/-mice.
C57BL/6.times.129/Ola F2 and F3 animals were used for subsequent
analysis.
[0396] B. T-Cell Response In B7RP-2-/-Mice
[0397] The B7RP-2-/-mice were used to determine if the absence of
B7RP-2 protein contributes to heightened T-cell-mediated
hypersensitivity. Airway inflammation models driven by either Th1
or Th2 cells, and a footpad-swelling model that reflects cytotoxic
T lymphocyte (CTL) response to lymphocytic choriomeningitis virus
(LCMV) infection, were used to examine T-cell response. Cytokine
(Th1- or Th2-polarizing) microenvironments were established in the
airways of B7RP-2+/+ and B7RP-2-/-mice by transient,
adenovirus-mediated intranasal expression of GM-CSF plus IL-12
(Th1) or GM-CSF alone (Th2). Intranasal expression of
replication-deficient adenovirus carrying the appropriate cytokine
cDNA was initiated on day (-)1. The +/+ and -/-mice were exposed to
ovalbumin (OVA) aerosol (1% wt/vol in 0.9% saline) for 20 minutes
on 10 consecutive days (days 0-9) to cause airway inflammation. On
day 11, the mice were killed and immune cell populations in the
bronchoalveolar lavage (BAL) fluids were differentially stained and
counted. The remaining lung tissue was processed for histologic
examination by hematoxylin and eosin (H&E) staining. In some
cases, lung cells were released by collagenase treatment and
analyzed by flow cytometry. Ex vivo splenocytes were cultured in
the absence or presence of 400 .mu.g/ml OVA for 5 days prior to
determination of cytokine production by ELISA (R&D
Systems).
[0398] After OVA sensitization under Th1 conditions, B7RP-2-/-mice
had 2.5-fold more infiltrating immune cells in the airway compared
to B7RP-2+/+ (FIG. 14; Total). Lymphocyte (FIG. 14; Lymphocytes),
macrophage, and neutrophil subsets were increased to a similar
degree. Histological examination of lung sections confirmed
infiltration of increased severity in the absence of B7RP-2 (FIG.
15; Th1). The portion of activated (CD69.sup.+) cells in both
CD4.sup.+ and CD8.sup.+ T-cell populations in lung infiltrates of
B7RP-2-/-mice compared to controls (59.5% and 47.2%, respectively,
among CD4.sup.+ T-cells; 76.7% and 65.9% among CD8.sup.+ T-cells;
FIG. 16).
[0399] Splenocytes harvested from B7RP-2-/-mice sensitized under
Th1 conditions produced about 60% more IFN-.gamma. when
restimulated in vitro with OVA compared to B7RP-2+/+splenocytes.
Under Th2 conditions, however, similar numbers of lung-infiltrating
immune cells were found in BAL fluids and lung sections from
B7RP-2+/+ and B7RP-2-/-mice (FIGS. 14 and 15; Th2). Eosinophilia, a
hallmark of Th2-driven airway inflammation, was also comparable
(FIG. 15; Th2; bottom panel), and OVA-stimulated B7RP-2+/+ and
-/-splenocytes consistently produced similar levels of IL-4, IL-s,
and IL-13. Thus, in situations of Th1- but not Th2-driven airway
inflammation, B7RP-2-/-mice develop more severe disease than
control mice and display augmented T-cell activation.
[0400] The role of B7RP-2 in a CTL-mediated hypersensitivity
reaction was examined by injecting LCMV into the footpads of
B7RP-2+/+ and -/-mice. The extent and kinetics of footpad swelling
were indistinguishable between genotypes (FIG. 17). These data
indicate that B7RP-2 is involved in downregulating Th1-mediated
responses, but not in Th2- or CTL-mediated hypersensitivity
reactions. cl EXAMPLE 6
[0401] Role of B7RP-2 in Experimental Autoimmune Encephalomyelitis
(EAE)
[0402] The role of B7RP-2 was also investigated in experimental
autoimmune encephalomyelitis (EAE), another Th1-driven disease
model. Induction and clinical scoring of EAE was performed as
follows. EAE was induced by immunizing mice with the peptide
antigen representing amino acids 35-55 of myelin oligodendrocyte
glycoprotein (MOG) M-E-V-G-W-Y-R-S-P-F-S-R-V-V-- H-L-Y-R-N-G-K (SEQ
ID NO: 29). B7RP-2+/+ and -/-littermates (8-12 weeks) were injected
subcutaneously (s.c.) on day 0 and day 7 with 300 .mu.g MOG 35-55
peptide emulsified in CFA (Sigma) plus 500 .mu.g Mycobacterium
tuberculosis. The mice were injected intraperitoneally (i.p.) with
500 ng pertussis toxin (List Biological, Campbell, Calif.) on day 0
and day 2. EAE clinical scores were determined daily as follows
(with a gradation of 0.5 for intermediate levels): 0, no clinical
signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limb
paralysis; 4, hind and fore limb paralysis; 5, death.
[0403] The average day of disease onset (the first day when the
clinical score was higher than 1) was earlier in B7RP-2-/-mice (day
16.1; n =16) than in B7RP-2 +/+mice (day 18.4; n=14) (FIG. 18A), a
trend clarified when littermates were compared (FIG. 18B). Despite
the earlier onset, B7RP-2-/-mice had the same clinical scores as
B7RP-2+/+mice by the late stages of the disease (FIG. 18A). The
rates of disease incidence (14/16 in+/+ and 16/18 in-/-) or
mortality (1/14 in+/+ vs. 3/16 in-/-) were also equivalent. The
earlier onset of EAE in the absence of B7RP-2 provides support for
the hypothesis that B7RP-2 negatively regulates Th1-driven immune
responses.
EXAMPLE7
[0404] Cytotoxic T lymphocytes (CTL) Response in B7RP-2-/-Mice
[0405] A. Lymphocytic Choriomeningitis Virus (LCMV)-Specific CTL
Response
[0406] The effect of B7RP-2 on the anti-viral cytotoxic T
lymphocytes (CTL) responses to lymphocytic choriomeningitis virus
(LCMV) or influenza virus was also examined in vivo. For primary
CTL responses, B7RP-2-/- and +/+mice were injected intravenously
(i.v.) with 2000 pfu LCMV (Armstrong strain). At day 8
post-infection, splenocytes were harvested and ex vivo CTL activity
was measured by a standard .sup.51Cr release assay using
.sup.51Cr-labeled EL4 cells pulsed with LCMV glycoprotein peptide
p33 (K-A-V-Y-N-F-A-T-M; SEQ ID NO: 30). The splenocytes from
B7RP-2-/-mice on day 8 post-infection with LCMV showed the same
level of ex vivo CTL activity as those from B7RP-2+/+mice (FIG. 19;
Primary).
[0407] The CTL memory response in B7RP-2-/- and +/+mice was
examined using the footpad swelling assays. For footpad swelling,
3000 pfu LCMV (Armstrong strain) was injected into the hind footpad
of B7RP-2-/- and +/+mice and the footpad thickness was measured
with calipers. To measure memory CTL activity, the mice that were
used for the footpad swelling experiments were killed at day 30 and
the splenocytes were harvested. The harvested splenocytes were
restimulated in vitro by culturing the cells for 5 days in the
presence of 1 .mu.M p33 peptide and rat splenocyte ConA culture
supernatant. Cytotoxicity was measured as above. The levels of CTL
activity detected in the splenocytes harvested from the
B7RP-2-/-mice were comparable to those harvested from the B7RP
+/+mice (FIG. 19; Memory). These experiments indicate that normal
primary and memory CTL responses against LCMV can be mounted in the
absence of B7RP-2.
[0408] B. Influenza Virus Nucleoprotein (NP)-Specific CTL
Response
[0409] To rule out the possibility that the strong antigenic
stimulation associated with LCMV infection masked a need for
costimulatory signals, CTL responses to influenza virus were
examined. B7RP-2-/- and +/+mice were infected intraperitoneally
(i.p.) with 200 hemaglutinin units (HAU) of influenza A
HKx31(H3N1). Expansion of influenza nucleoprotein (NP)-specific CTL
was monitored by flow cytometric analysis of splenocytes stained on
day 7 and day 21 post-infection with anti-CD8 mAb (ebioscience, San
Diego, Calif.) and the tetramer H-2D.sup.b/NP366-374
(A-S-N-E-N-M-E-T-M; SEQ ID NO: 31) (NIAID MHC Tetramer Core
Facility, Atlanta, Ga.). The expansion of NP366-374/H-2Db-specific
CTLs during primary and secondary influenza virus infections was
indistinguishable in B7RP-2+/+ and -/-mice (FIG. 20). For memory
CTL responses, B7RP-2+/+ and -/-mice (four mice/group) were
infected with HKx31 and re-infected 4 weeks later with the
serologically distinct influenza virus A/PR/8/34 (H1N1), which
shares the same NP gene with HKx31. Splenocytes were harvested and
analyzed by tetramer staining 7 days after re-infection. There were
no differences in the number of IFN-.gamma. producing cells among
CD8.sup.+ T-cells and the cytotoxicity of splenocytes restimulated
in vitro.
EXAMPLE 8
[0410] Production of Anti-B7RP-2 Polypeptide Antibodies
[0411] Antibodies to B7RP-2 polypeptides may be obtained by
immunization with purified protein or with B7RP-2 peptides produced
by biological or chemical synthesis. Suitable procedures for
generating antibodies include those described in Hudson and Bay,
Practical Immunology (2nd ed., Blackwell Scientific
Publications).
[0412] In one procedure for the production of antibodies, animals
(typically mice or rabbits) are injected with a B7RP-2 antigen
(such as a B7RP-2 polypeptide), and those with sufficient serum
titer levels as determined by ELISA are selected for hybridoma
production. Spleens of immunized animals are collected and prepared
as single cell suspensions from which splenocytes are recovered.
The splenocytes are fused to mouse myeloma cells (such as
Sp2/0-Ag14 cells), are first incubated in DMEM with 200 U/mL
penicillin, 200 .mu.g/mL streptomycin sulfate, and 4 mM glutamine,
and are then incubated in HAT selection medium (hypoxanthine,
aminopterin, and thymidine). After selection, the tissue culture
supernatants are taken from each fusion well and tested for
anti-B7RP-2 antibody production by ELISA.
[0413] Alternative procedures for obtaining anti-B7RP-2 antibodies
may also be employed, such as the immunization of transgenic mice
harboring human Ig loci for production of human antibodies, and the
screening of synthetic antibody libraries, such as those generated
by mutagenesis of an antibody variable domain.
EXAMPLE 9
[0414] Expression of B7RP-2 Polypeptide in Transgenic Mice
[0415] To assess the biological activity of B7RP-2 polypeptide, a
construct encoding a B7RP-2 polypeptide/Fc fusion protein under the
control of a liver specific ApoE promoter is prepared. The delivery
of this construct is expected to cause pathological changes that
are informative as to the function of B7RP-2 polypeptide.
Similarly, a construct containing the full-length B7RP-2
polypeptide under the control of the beta actin promoter is
prepared. The delivery of this construct is expected to result in
ubiquitous expression.
[0416] To generate these constructs, PCR is used to amplify
template DNA sequences encoding a B7RP-2 polypeptide using primers
that correspond to the 5' and 3' ends of the desired sequence and
which incorporate restriction enzyme sites to permit insertion of
the amplified product into an expression vector. Following
amplification, PCR products are gel purified, digested with the
appropriate restriction enzymes, and ligated into an expression
vector using standard recombinant DNA techniques. For example,
amplified B7RP-2 polypeptide sequences can be cloned into an
expression vector under the control of the human .beta.-actin
promoter as described by Graham et al., 1997, Nature Genetics,
17:272-74 and Ray et al., 1991, Genes Dev. 5:2265-73.
[0417] Following ligation, reaction mixtures are used to transform
an E. coli host strain by electroporation and transformants are
selected for drug resistance. Plasmid DNA from selected colonies is
isolated and subjected to DNA sequencing to confirm the presence of
an appropriate insert and absence of mutation. The B7RP-2
polypeptide expression vector is purified through two rounds of
CsCl density gradient centrifugation, cleaved with a suitable
restriction enzyme, and the linearized fragment containing the
B7RP-2 polypeptide transgene is purified by gel electrophoresis.
The purified fragment is resuspended in 5 mM Tris, pH 7.4, and 0.2
mM EDTA at a concentration of 2 mg/mL.
[0418] Single-cell embryos from BDF1.times.BDF1 bred mice are
injected as described (PCT Pub. No. WO 97/23614). Embryos are
cultured overnight in a CO.sub.2 incubator and 15-20 two-cell
embryos are transferred to the oviducts of a pseudopregnant CD1
female mice. Offspring obtained from the implantation of
microinjected embryos are screened by PCR amplification of the
integrated transgene in genomic DNA samples as follows. Ear pieces
are digested in 20 mL ear buffer (20 mM Tris, pH 8.0, 10 mM EDTA,
0.5% SDS, and 500 mg/mL proteinase K) at 55.degree. C. overnight.
The sample is then diluted with 200 mL of TE, and 2 mL of the ear
sample is used in a PCR reaction using appropriate primers.
[0419] At 8 weeks of age, transgenic founder animals and control
animals are sacrificed for necropsy and pathological analysis.
Portions of spleen are removed and total cellular RNA isolated from
the spleens using the Total RNA Extraction Kit (Qiagen) and
transgene expression determined by RT-PCR. RNA recovered from
spleens is converted to cDNA using the SuperScrip.TM.
Preamplification System (Gibco-BRL) as follows. A suitable primer,
located in the expression vector sequence and 3' to the B7RP-2
polypeptide transgene, is used to prime cDNA synthesis from the
transgene transcripts. Ten mg of total spleen RNA from transgenic
founders and controls is incubated with 1 mM of primer for 10
minutes at 70.degree. C. and placed on ice. The reaction is then
supplemented with 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM
MgCl.sub.2, 10 mM of each dNTP, 0.1 mM DTT, and 200 U of
SuperScript II reverse transcriptase. Following incubation for 50
minutes at 42.degree. C., the reaction is stopped by heating for 15
minutes at 72.degree. C. and digested with 2U of RNase H for 20
minutes at 37.degree. C. Samples are then amplified by PCR using
primers specific for B7RP-2 polypeptide.
EXAMPLE 10
[0420] Biological Activity of B7RP-2 Polypeptide in Transgenic
Mice
[0421] Prior to euthanasia, transgenic animals are weighed,
anesthetized by isofluorane and blood drawn by cardiac puncture.
The samples are subjected to hematology and serum chemistry
analysis. Radiography is performed after terminal exsanguination.
Upon gross dissection, major visceral organs are subject to weight
analysis.
[0422] Following gross dissection, tissues (i.e., liver, spleen,
pancreas, stomach, the entire gastrointestinal tract, kidney,
reproductive organs, skin and mammary glands, bone, brain, heart,
lung, thymus, trachea, esophagus, thyroid, adrenals, urinary
bladder, lymph nodes and skeletal muscle) are removed and fixed in
10% buffered Zn-Formalin for histological examination. After
fixation, the tissues are processed into paraffin blocks, and 3 mm
sections are obtained. All sections are stained with hematoxylin
and exosin, and are then subjected to histological analysis.
[0423] The spleen, lymph node, and Peyer's patches of both the
transgenic and the control mice are subjected to immunohistology
analysis with B-cell and T-cell specific antibodies as follows. The
formalin fixed paraffin embedded sections are deparaffinized and
hydrated in deionized water. The sections are quenched with 3%
hydrogen peroxide, blocked with Protein Block (Lipshaw, Pittsburgh,
Pa.), and incubated in rat monoclonal anti-mouse B220 and CD3
(Harlan, Indianapolis, Ind.). Antibody binding is detected by
biotinylated rabbit anti-rat immunoglobulins and peroxidase
conjugated streptavidin (BioGenex, San Ramon, Calif.) with DAB as a
chromagen (BioTek, Santa Barbara, Calif.). Sections are
counterstained with hematoxylin.
[0424] After necropsy, MLN and sections of spleen and thymus from
transgenic animals and control littermates are removed. Single cell
suspensions are prepared by gently grinding the tissues with the
flat end of a syringe against the bottom of a 100 mm nylon cell
strainer (Becton Dickinson, Franklin Lakes, N.J.). Cells are washed
twice, counted, and approximately 1.times.10.sup.6 cells from each
tissue are then incubated for 10 minutes with 0.5 .mu.g
CD16/32(Fc.gamma.III/II) Fc block in a 20 .mu.L volume. Samples are
then stained for 30 minutes at 2-8.degree. C. in a 100 .mu.L volume
of PBS (lacking Ca.sup.+ and Mg.sup.+), 0.1% bovine serum albumin,
and 0.01% sodium azide with 0.5 .mu.g antibody of FITC or
PE-conjugated monoclonal antibodies against CD90.2 (Thy-1.2), CD45R
(B220), CD11b (Mac-1), Gr-1, CD4, or CD8 (PharMingen, San Diego,
Calif.). Following antibody binding, the cells are washed and then
analyzed by flow cytometry on a FACScan (Becton Dickinson).
[0425] While the present invention has been described in terms of
the preferred embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations that come within the scope of the invention as claimed.
Sequence CWU 1
1
32 1 1337 DNA Homo sapiens CDS (135)..(1082) 1 ccgggtcgac
ccacgcgtcc ggcggcggcg actgagccag gctgggccgc gtccctgagt 60
cccagagtcg gcgcggcgcg gcaggggcag ccttccacca cggggagccc agctgtcagc
120 cgcctcacag gaag atg ctg cgt cgg cgg ggc agc cct ggc atg ggt gtg
170 Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val 1 5 10 cat gtg
ggt gca gcc ctg gga gca ctg tgg ttc tgc ctc aca gga gcc 218 His Val
Gly Ala Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala 15 20 25
ctg gag gtc cag gtc cct gaa gac cca gtg gtg gca ctg gtg ggc acc 266
Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr 30
35 40 gat gcc acc ctg tgc tgc tcc ttc tcc cct gag cct ggc ttc agc
ctg 314 Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser
Leu 45 50 55 60 gca cag ctc aac ctc atc tgg cag ctg aca gat acc aaa
cag ctg gtg 362 Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys
Gln Leu Val 65 70 75 cac agc ttt gct gag ggc cag gac cag ggc agc
gcc tat gcc aac cgc 410 His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser
Ala Tyr Ala Asn Arg 80 85 90 acg gcc ctc ttc ccg gac ctg ctg gca
caa ggc aat gca tcc ctg agg 458 Thr Ala Leu Phe Pro Asp Leu Leu Ala
Gln Gly Asn Ala Ser Leu Arg 95 100 105 ctg cag cgc gtg cgt gtg gcg
gac gag ggc agc ttc acc tgc ttc gtg 506 Leu Gln Arg Val Arg Val Ala
Asp Glu Gly Ser Phe Thr Cys Phe Val 110 115 120 agc atc cgg gat ttc
ggc agc gct gcc gtc agc ctg cag gtg gcc gct 554 Ser Ile Arg Asp Phe
Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala 125 130 135 140 ccc tac
tcg aag ccc agc atg acc ctg gag ccc aac aag gac ctg cgg 602 Pro Tyr
Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg 145 150 155
cca ggg gac acg gtg acc atc acg tgc tcc agc tac cgg ggc tac cct 650
Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro 160
165 170 gag gct gag gtg ttc tgg cag gat ggg cag ggt gtg ccc ctg act
ggc 698 Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr
Gly 175 180 185 aac gtg acc acg tcg cag atg gcc aac gag cag ggc ttg
ttt gat gtg 746 Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu
Phe Asp Val 190 195 200 cac agc gtc ctg cgg gtg gtg ctg ggt gcg aat
ggc acc tac agc tgc 794 His Ser Val Leu Arg Val Val Leu Gly Ala Asn
Gly Thr Tyr Ser Cys 205 210 215 220 ctg gtg cgc aac ccc gtg ctg cag
cag gat gcg cac ggc tct gtc acc 842 Leu Val Arg Asn Pro Val Leu Gln
Gln Asp Ala His Gly Ser Val Thr 225 230 235 atc aca ggg cag cct atg
aca ttc ccc cca gag gcc ctg tgg gtg acc 890 Ile Thr Gly Gln Pro Met
Thr Phe Pro Pro Glu Ala Leu Trp Val Thr 240 245 250 gtg ggg ctg tct
gtc tgt ctc att gca ctg ctg gtg gcc ctg gct ttc 938 Val Gly Leu Ser
Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe 255 260 265 gtg tgc
tgg aga aag atc aaa cag agc tgt gag gag gag aat gca gga 986 Val Cys
Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly 270 275 280
gct gag gac cag gat ggg gag gga gaa ggc tcc aag aca gcc ctg cag
1034 Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu
Gln 285 290 295 300 cct ctg aaa cac tct gac agc aaa gaa gat gat gga
caa gaa ata gcc 1082 Pro Leu Lys His Ser Asp Ser Lys Glu Asp Asp
Gly Gln Glu Ile Ala 305 310 315 tgaccatgag gaccagggag ctgctacccc
tccctacagc tcctaccctc tggctgcaat 1142 ggggctgcac tgtgagccct
gcccccaaca gatgcatcct gctctgacag gtgggctcct 1202 tctccaaagg
atgcgataca cagaccactg tgcagcctta tttctccaat ggacatgatt 1262
cccaagtcat cctgctgcct tttttcttat agacacaatg aacagaccac ccacaacctt
1322 agttctctaa gtcat 1337 2 316 PRT Homo sapiens 2 Met Leu Arg Arg
Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10 15 Ala Leu
Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30
Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35
40 45 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu
Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His
Ser Phe Ala 65 70 75 80 Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn
Arg Thr Ala Leu Phe 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala
Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Ala Asp Glu Gly Ser
Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala
Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met
Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160
Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val 165
170 175 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr
Thr 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His
Ser Val Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser
Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Gly
Ser Val Thr Ile Thr Gly Gln 225 230 235 240 Pro Met Thr Phe Pro Pro
Glu Ala Leu Trp Val Thr Val Gly Leu Ser 245 250 255 Val Cys Leu Ile
Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg 260 265 270 Lys Ile
Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln 275 280 285
Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro Leu Lys His 290
295 300 Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315
3 1095 DNA Mus musculus CDS (73)..(1020) 3 tggtaccgag ctcggatcca
ctagtaacgg ccgccagtgt gctggaattc gcccttgctg 60 tctcacagga ag atg
ctt cga gga tgg ggt ggc ccc agt gtg ggt gtg tgt 111 Met Leu Arg Gly
Trp Gly Gly Pro Ser Val Gly Val Cys 1 5 10 gtg cgc aca gca ctg ggg
gtg ctg tgc ctc tgc ctc aca gga gct gtg 159 Val Arg Thr Ala Leu Gly
Val Leu Cys Leu Cys Leu Thr Gly Ala Val 15 20 25 gaa gtc cag gtc
tct gaa gac ccc gtg gtg gcc ctg gtg gac acg gat 207 Glu Val Gln Val
Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp 30 35 40 45 gcc acc
cta cgc tgc tcc ttt tcc cca gag cct ggc ttc agt ctg gca 255 Ala Thr
Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala 50 55 60
cag ctc aac ctc atc tgg cag ctg aca gac acc aaa cag ctg gtg cac 303
Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His 65
70 75 agc ttc acg gag ggc cgg gac caa ggc agt gcc tac tcc aac cgc
aca 351 Ser Phe Thr Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg
Thr 80 85 90 gcg ctc ttc cct gac ctg ttg gtg caa ggc aat gcg tcc
ttg agg ctg 399 Ala Leu Phe Pro Asp Leu Leu Val Gln Gly Asn Ala Ser
Leu Arg Leu 95 100 105 cag cgc gtc cga gta acc gac gag ggc agc tac
acc tgc ttt gtg agc 447 Gln Arg Val Arg Val Thr Asp Glu Gly Ser Tyr
Thr Cys Phe Val Ser 110 115 120 125 atc cag gac ttt gac agc gct gct
gtt agc ctg cag gtg gcc gcc ccc 495 Ile Gln Asp Phe Asp Ser Ala Ala
Val Ser Leu Gln Val Ala Ala Pro 130 135 140 tac tcg aag ccc agc atg
acc ctg gag ccc aac aag gac cta cgt cca 543 Tyr Ser Lys Pro Ser Met
Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro 145 150 155 ggg aac atg gtg
acc atc acg tgc tct agc tac cag ggc tat ccg gag 591 Gly Asn Met Val
Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu 160 165 170 gcc gag
gtg ttc tgg aag gat gga cag gga gtg ccc ttg act ggc aat 639 Ala Glu
Val Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Asn 175 180 185
gtg acc aca tcc cag atg gcc aac gag cgg ggc ttg ttc gat gtt cac 687
Val Thr Thr Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val His 190
195 200 205 agc gtg ctg agg gtg gtg ctg ggt gct aac ggc acc tac agc
tgc ctg 735 Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser
Cys Leu 210 215 220 gta cgc aac ccg gtg ttg cag caa gat gct cac ggc
tca gtc acc atc 783 Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly
Ser Val Thr Ile 225 230 235 aca ggg cag ccc ctg aca ttc ccc cct gag
gct ctg tgg gta acc gtg 831 Thr Gly Gln Pro Leu Thr Phe Pro Pro Glu
Ala Leu Trp Val Thr Val 240 245 250 ggg ctc tct gtc tgt ctt gtg gta
cta ctg gtg gcc ctg gct ttc gtg 879 Gly Leu Ser Val Cys Leu Val Val
Leu Leu Val Ala Leu Ala Phe Val 255 260 265 tgc tgg aga aag atc aag
cag agc tgc gag gag gag aat gca ggt gcc 927 Cys Trp Arg Lys Ile Lys
Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala 270 275 280 285 gag gac cag
gat gga gat gga gaa gga tcc aag aca gct cta cgg cct 975 Glu Asp Gln
Asp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu Arg Pro 290 295 300 ctg
aaa ccc tct gaa aac aaa gaa gat gac gga caa gaa att gct 1020 Leu
Lys Pro Ser Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315
tgattgggag ctgctgcaag ggcgaattct gcagatatcc atcacactgg cggccgctcg
1080 agcatgcatc tagag 1095 4 316 PRT Mus musculus 4 Met Leu Arg Gly
Trp Gly Gly Pro Ser Val Gly Val Cys Val Arg Thr 1 5 10 15 Ala Leu
Gly Val Leu Cys Leu Cys Leu Thr Gly Ala Val Glu Val Gln 20 25 30
Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp Ala Thr Leu 35
40 45 Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu
Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His
Ser Phe Thr 65 70 75 80 Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn
Arg Thr Ala Leu Phe 85 90 95 Pro Asp Leu Leu Val Gln Gly Asn Ala
Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Thr Asp Glu Gly Ser
Tyr Thr Cys Phe Val Ser Ile Gln Asp 115 120 125 Phe Asp Ser Ala Ala
Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met
Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asn Met 145 150 155 160
Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165
170 175 Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr
Thr 180 185 190 Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val His
Ser Val Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser
Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Gly
Ser Val Thr Ile Thr Gly Gln 225 230 235 240 Pro Leu Thr Phe Pro Pro
Glu Ala Leu Trp Val Thr Val Gly Leu Ser 245 250 255 Val Cys Leu Val
Val Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg 260 265 270 Lys Ile
Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln 275 280 285
Asp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu Arg Pro Leu Lys Pro 290
295 300 Ser Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315
5 2797 DNA Rattus norvegicus CDS (137)..(1084) 5 cctcgcggct
gctctaccga cggtggcggc gattgtgctg cgccccgccg cgtccccgag 60
tcccgggagt cggcgcggcg cggcaggagc agccatccgc cacggagagt ccagctgtca
120 gctgtctcac aggaag atg ctt cga gga tgg ggt ggc ccc agt gtg ggt
gtg 172 Met Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val 1 5 10 tct
atg ggc acg gca ctg gga gtg ttg tgc ctc tgc ctt aca gga gct 220 Ser
Met Gly Thr Ala Leu Gly Val Leu Cys Leu Cys Leu Thr Gly Ala 15 20
25 gtg gag gtc caa gtc tct gaa gac cct gtg gtg gcc cta gtg gat acg
268 Val Glu Val Gln Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr
30 35 40 gat gcc acc cta cgc tgc tcc ttc tcc cca gag cct ggc ttc
agc ctg 316 Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe
Ser Leu 45 50 55 60 aga cag ctc aac ctc atc tgg cag ctg aca gac acc
aaa cag ctg gtg 364 Arg Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr
Lys Gln Leu Val 65 70 75 cac agc ttc act gag ggc cgg gac caa ggc
agt gcc tat gcc aac cgc 412 His Ser Phe Thr Glu Gly Arg Asp Gln Gly
Ser Ala Tyr Ala Asn Arg 80 85 90 acg gcg ctc ttc cct gac ttg ttg
gtg cag ggc aat gca tcc ctg agg 460 Thr Ala Leu Phe Pro Asp Leu Leu
Val Gln Gly Asn Ala Ser Leu Arg 95 100 105 ctg cag cgt gtc cga gta
acc gac gag ggc agc tac acc tgc ttt gtg 508 Leu Gln Arg Val Arg Val
Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val 110 115 120 agc atc cag gac
ttt gac agc gct gct gtt agc ctg cag gtg gcc gcc 556 Ser Ile Gln Asp
Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala 125 130 135 140 ccc
tac tca aag ccc agc atg acc ctg gag ccc aac aag gac ctg cgt 604 Pro
Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg 145 150
155 cca ggg gac atg gtg acc atc acg tgc tcc agc tac cag ggc tat ccc
652 Pro Gly Asp Met Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro
160 165 170 gag gct gag gtg ttc tgg aag gac gga cag gga ttg ccc ttg
act ggc 700 Glu Ala Glu Val Phe Trp Lys Asp Gly Gln Gly Leu Pro Leu
Thr Gly 175 180 185 aat gtg acc aca tcc cag atg gcc aac gag cgg ggc
ctg ttc gat gtt 748 Asn Val Thr Thr Ser Gln Met Ala Asn Glu Arg Gly
Leu Phe Asp Val 190 195 200 cac agt gtg ctg agg gtg gtg ctg ggt gct
aat ggc acc tac agc tgc 796 His Ser Val Leu Arg Val Val Leu Gly Ala
Asn Gly Thr Tyr Ser Cys 205 210 215 220 ctg gtc cgc aac ccg gtg ttg
cag caa gat gct cat ggc tcg gtc acc 844 Leu Val Arg Asn Pro Val Leu
Gln Gln Asp Ala His Gly Ser Val Thr 225 230 235 atc aca ggg cag ccc
atg aca ttc ccc cct gag gct cta tgg gtg act 892 Ile Thr Gly Gln Pro
Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr 240 245 250 gtg ggg ctc
tct gtc tgt ctt gtg ata ctg ctg gtg gcc ctg gcc ttc 940 Val Gly Leu
Ser Val Cys Leu Val Ile Leu Leu Val Ala Leu Ala Phe 255 260 265 gtg
tgc tgg aga aag atc aag cag agc tgt gaa gag gag aat gca ggt 988 Val
Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly 270 275
280 gct gag gac cag gat ggg gat gga gaa gga tcc aag aca gct ctt cgg
1036 Ala Glu Asp Gln Asp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu
Arg 285 290 295 300 cct ctg aaa cac tct gaa aac aaa gaa gat gac gga
caa gaa ata gct 1084 Pro Leu Lys His Ser Glu Asn Lys Glu Asp Asp
Gly Gln Glu Ile Ala 305 310 315 tgactggaag ctgctgccct tccctggtgg
gggggcccac cctctggctg tattgagcct 1144 caatgtgagc cctgccccca
atgaatgggt tttgttccac agatctaccc attctttaga 1204 ggacgtggtt
tacaggctac ccacagcctt attttcccaa tggacttaat tcccatcatc 1264
ctgaagcctc ctttctccag tgacacgata cacgaaccat cctgcggcct tatttcttac
1324 ggactcgaca caaagagttc tccacctcag tgtccctcca gagtcatccg
gtggccttgt 1384 gatactacac ggaccttcct tctgccttac tttaatagat
atacacaaac catccccatg 1444 tccttgtgcc tccaaagcca tgcagagact
gtattactgc tactattctc caaggcacat 1504 gctattcaga tgaacccctg
ccttattcct ctgaagacag atgcttagtt acctcttggt 1564 tctttctccc
atggccctga catatcttag tcacccatca acgatgggat cccatctctc 1624
agcaagtcct caacctgact ccctgccctc atctggccct ggctttggtt ttctccctcc
1684 ctaagtgaga tggggcacac tccccatcca cacacatggg tcacagctgt
gcgtgctgga 1744 tcgcgtacat acttgccttg catggtctcc tctggctgcc
ctgggctgtg ccctttctcg 1804 cctcaggaag caggtgctgg tcggcctggt
tctcagggcc cctcagggag tcagccttca 1864 accctgtgct tcccgtgttg
gaaatctttg ttacttttcc tttcttagta aattaacatc 1924 tgttgaacaa
ccacagcgtc caacaggact ttcacagacc ctgccagcta gattaaataa 1984
tgatacagaa gtttattaat tattttaaag cttaggtttt tttgccggga ggtatcccaa
2044 atactctatc ccgactaatc ctggcactat gtcccaccac atggccaggc
ctacctctgc 2104 tccactctga atcatccacc tctgtgtccg ccgacaaatc
tcccatgatt cagttcttct 2164 cccagcgtcc ctatctctgc ccggaagtac
gacctttgac ttcctgacca actattggcc 2224 gtcaactctt tgttaaaggt
gatcagatat aattttgcct taggcacgtg aggaagaaac 2284 atatttataa
aatacgagac cagagatggg ccatggaaat aacaccagat tctgacagcc 2344
tttagccctc tgctggtaca aattaacaat tgaatatata gagacacacc ttcacacagt
2404 gcaccccaac aacaggggtg agcattgtgc tgggtactag ggtcctgctg
aaatcagaga 2464 ccttaactcc agctggggaa tggccttgct ccctgctgtg
cccacagctt ccaacactgt 2524 ccctgacccc agggtagggg tggaaacctg
gagaaggcac agccccttac cataccttga 2584 gaactgggta tttttcagag
tctatatgtg tgcactggaa ggcaggtggc cacagccatg 2644 cagacctggg
tagggtcaga agcctatgcc acgctgggac ctcctcaaca gctgaagtct 2704
gaggacaaga agggccttct tactgtggtg ctattctgga gctggggtat atacctggct
2764 tgtctctgac agccctggct tttggcagaa ctt 2797 6 316 PRT Rattus
norvegicus 6 Met Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val Ser
Met Gly Thr 1 5 10 15 Ala Leu Gly Val Leu Cys Leu Cys Leu Thr Gly
Ala Val Glu Val Gln 20 25 30 Val Ser Glu Asp Pro Val Val Ala Leu
Val Asp Thr Asp Ala Thr Leu 35 40 45 Arg Cys Ser Phe Ser Pro Glu
Pro Gly Phe Ser Leu Arg Gln Leu Asn 50 55 60 Leu Ile Trp Gln Leu
Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr 65 70 75 80 Glu Gly Arg
Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 Pro
Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105
110 Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val Ser Ile Gln Asp
115 120 125 Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr
Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg
Pro Gly Asp Met 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Gln
Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Lys Asp Gly Gln Gly
Leu Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met Ala Asn
Glu Arg Gly Leu Phe Asp Val His Ser Val Leu 195 200 205 Arg Val Val
Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 Pro
Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln 225 230
235 240 Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu
Ser 245 250 255 Val Cys Leu Val Ile Leu Leu Val Ala Leu Ala Phe Val
Cys Trp Arg 260 265 270 Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala
Gly Ala Glu Asp Gln 275 280 285 Asp Gly Asp Gly Glu Gly Ser Lys Thr
Ala Leu Arg Pro Leu Lys His 290 295 300 Ser Glu Asn Lys Glu Asp Asp
Gly Gln Glu Ile Ala 305 310 315 7 526 PRT Homo sapiens 7 Met Ala
Val Phe Pro Ser Ser Gly Leu Pro Arg Cys Leu Leu Thr Leu 1 5 10 15
Ile Leu Leu Gln Leu Pro Lys Leu Asp Ser Ala Pro Phe Asp Val Ile 20
25 30 Gly Pro Pro Glu Pro Ile Leu Ala Val Val Gly Glu Asp Ala Glu
Leu 35 40 45 Pro Cys Arg Leu Ser Pro Asn Ala Ser Ala Glu His Leu
Glu Leu Arg 50 55 60 Trp Phe Arg Lys Lys Val Ser Pro Ala Val Leu
Val His Arg Asp Gly 65 70 75 80 Arg Glu Gln Glu Ala Glu Gln Met Pro
Glu Tyr Arg Gly Arg Ala Thr 85 90 95 Leu Val Gln Asp Gly Ile Ala
Lys Gly Arg Val Ala Leu Arg Ile Arg 100 105 110 Gly Val Arg Val Ser
Asp Asp Gly Glu Tyr Thr Cys Phe Phe Arg Glu 115 120 125 Asp Gly Ser
Tyr Glu Glu Ala Leu Val His Leu Lys Val Ala Ala Leu 130 135 140 Gly
Ser Asp Pro His Ile Ser Met Gln Val Gln Glu Asn Gly Glu Ile 145 150
155 160 Cys Leu Glu Cys Thr Ser Val Gly Trp Tyr Pro Glu Pro Gln Val
Gln 165 170 175 Trp Arg Thr Ser Lys Gly Glu Lys Phe Pro Ser Thr Ser
Glu Ser Arg 180 185 190 Asn Pro Asp Glu Glu Gly Leu Phe Thr Val Ala
Ala Ser Val Ile Ile 195 200 205 Arg Asp Thr Ser Thr Lys Asn Val Ser
Cys Tyr Ile Gln Asn Leu Leu 210 215 220 Leu Gly Gln Glu Lys Lys Val
Glu Ile Ser Ile Pro Ala Ser Ser Leu 225 230 235 240 Pro Arg Leu Thr
Pro Trp Ile Val Ala Val Ala Val Ile Leu Met Val 245 250 255 Leu Gly
Leu Leu Thr Ile Gly Ser Ile Phe Phe Thr Trp Arg Leu Tyr 260 265 270
Asn Glu Arg Pro Arg Glu Arg Arg Asn Glu Phe Ser Ser Lys Glu Arg 275
280 285 Leu Leu Glu Glu Leu Lys Trp Lys Lys Ala Thr Leu His Ala Val
Asp 290 295 300 Val Thr Leu Asp Pro Asp Thr Ala His Pro His Leu Phe
Leu Tyr Glu 305 310 315 320 Asp Ser Lys Ser Val Arg Leu Glu Asp Ser
Arg Gln Lys Leu Pro Glu 325 330 335 Lys Thr Glu Arg Phe Asp Ser Trp
Pro Cys Val Leu Gly Arg Glu Thr 340 345 350 Phe Thr Ser Gly Arg His
Tyr Trp Glu Val Glu Val Gly Asp Arg Thr 355 360 365 Asp Trp Ala Ile
Gly Val Cys Arg Glu Asn Val Met Lys Lys Gly Phe 370 375 380 Asp Pro
Met Thr Pro Glu Asn Gly Phe Trp Ala Val Glu Leu Tyr Gly 385 390 395
400 Asn Gly Tyr Trp Ala Leu Thr Pro Leu Arg Thr Pro Leu Pro Leu Ala
405 410 415 Gly Pro Pro Arg Arg Val Gly Ile Phe Leu Asp Tyr Glu Ser
Gly Asp 420 425 430 Ile Ser Phe Tyr Asn Met Asn Asp Gly Ser Asp Ile
Tyr Thr Phe Ser 435 440 445 Asn Val Thr Phe Ser Gly Pro Leu Arg Pro
Phe Phe Cys Leu Trp Ser 450 455 460 Ser Gly Lys Lys Pro Leu Thr Ile
Cys Pro Ile Ala Asp Gly Pro Glu 465 470 475 480 Arg Val Thr Val Ile
Ala Asn Ala Gln Asp Leu Ser Lys Glu Ile Pro 485 490 495 Leu Ser Pro
Met Gly Glu Glu Ser Ala Pro Arg Asp Ala Asp Thr Leu 500 505 510 His
Ser Lys Leu Ile Pro Thr Gln Pro Ser Gln Gly Ala Pro 515 520 525 8
526 PRT Bos taurus 8 Met Ala Val Phe Pro Asn Ser Cys Leu Ala Gly
Cys Leu Leu Ile Phe 1 5 10 15 Ile Leu Leu Gln Leu Pro Lys Leu Asp
Ser Ala Pro Phe Asp Val Ile 20 25 30 Gly Pro Gln Glu Pro Ile Leu
Ala Val Val Gly Glu Asp Ala Glu Leu 35 40 45 Pro Cys Arg Leu Ser
Pro Asn Val Ser Ala Lys Gly Met Glu Leu Arg 50 55 60 Trp Phe Arg
Glu Lys Val Ser Pro Ala Val Phe Val Ser Arg Glu Gly 65 70 75 80 Gln
Glu Gln Glu Gly Glu Glu Met Ala Glu Tyr Arg Gly Arg Val Ser 85 90
95 Leu Val Glu Asp His Ile Ala Glu Gly Ser Val Ala Val Arg Ile Gln
100 105 110 Glu Val Lys Ala Ser Asp Asp Gly Glu Tyr Arg Cys Phe Phe
Arg Gln 115 120 125 Asp Glu Asn Tyr Glu Glu Ala Ile Val His Leu Lys
Val Ala Ala Leu 130 135 140 Gly Ser Asp Pro His Ile Ser Met Lys Val
Gln Glu Ser Gly Glu Ile 145 150 155 160 Gln Leu Glu Cys Thr Ser Val
Gly Trp Tyr Pro Glu Pro Gln Val Gln 165 170 175 Trp Arg Thr His Arg
Gly Glu Glu Phe Pro Ser Met Ser Glu Ser Arg 180 185 190 Asn Pro Asp
Glu Glu Gly Leu Phe Thr Val Arg Ala Ser Val Ile Ile 195 200 205 Arg
Asp Ser Ser Met Lys Asn Val Ser Cys Cys Ile Arg Asn Leu Leu 210 215
220 Leu Gly Gln Glu Lys Glu Val Glu Val Ser Ile Pro Ala Ser Phe Phe
225 230 235 240 Pro Arg Leu Thr Pro Trp Met Val Ala Val Ala Val Ile
Leu Val Val 245 250 255 Leu Gly Leu Leu Thr Ile Gly Ser Ile Phe Phe
Thr Trp Arg Leu Tyr 260 265 270 Lys Glu Arg Ser Arg Gln Arg Arg Asn
Glu Phe Ser Ser Lys Glu Lys 275 280 285 Leu Leu Glu Glu Leu Lys Trp
Lys Arg Ala Thr Leu His Ala Val Asp 290 295 300 Val Thr Leu Asp Pro
Asp Thr Ala His Pro His Leu Phe Leu Tyr Glu 305 310 315 320 Asp Ser
Lys Ser Val Arg Leu Glu Asp Ser Arg Gln Lys Leu Pro Glu 325 330 335
Lys Pro Glu Arg Phe Asp Ser Trp Pro Cys Val Met Gly Arg Glu Ala 340
345 350 Phe Thr Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Asp Arg
Thr 355 360 365 Asp Trp Ala Ile Gly Val Cys Arg Glu Asn Val Met Lys
Lys Gly Phe 370 375 380 Asp Pro Met Thr Pro Glu Asn Gly Phe Trp Ala
Val Glu Leu Tyr Gly 385 390 395 400 Asn Gly Tyr Trp Ala Leu Thr Pro
Leu Arg Thr Pro Leu Pro Leu Ala 405 410 415 Gly Pro Pro Arg Arg Val
Gly Val Phe Leu Asp Tyr Glu Ser Gly Asp 420 425 430 Ile Phe Phe Tyr
Asn Met Thr Asp Gly Ser His Ile Tyr Thr Phe Ser 435 440 445 Lys Ala
Ser Phe Ser Gly Pro Leu Arg Pro Phe Phe Cys Leu Trp Ser 450 455 460
Cys Gly Lys Lys Pro Leu Thr Ile Cys Pro Val Thr Asp Gly Leu Glu 465
470 475 480 Gly Val Met Val Val Ala Asp Ala Lys Asp Ile Ser Lys Glu
Ile Pro 485 490 495 Leu Ser Pro Met Gly Glu Asp Ser Ala Ser Gly Asp
Ile Glu Thr Leu 500 505 510 His Ser Lys Leu Ile Pro Leu Gln Pro Ser
Gln Gly Val Pro 515 520 525 9 524 PRT Mus musculus 9 Met Ala Val
Pro Thr Asn Ser Cys Leu Leu Val Cys Leu Leu Thr Leu 1 5 10 15 Thr
Val Leu Gln Leu Pro Thr Leu Asp Ser Ala Ala Pro Phe Asp Val 20 25
30 Thr Ala Pro Gln Glu Pro Val Leu Ala Leu Val Gly Ser Asp Ala Glu
35 40 45 Leu Thr Cys Gly Phe Ser Pro Asn Ala Ser Ser Glu Tyr Met
Glu Leu 50 55 60 Leu Trp Phe Arg Gln Thr Arg Ser Thr Ala Val Leu
Leu Tyr Arg Asp 65 70 75 80 Gly Gln Glu Gln Glu Gly Gln Gln Met Thr
Glu Tyr Arg Gly Arg Ala 85 90 95 Thr Leu Ala Thr Ala Gly Leu Leu
Asp Gly Arg Ala Thr Leu Leu Ile 100 105 110 Arg Asp Val Arg Val Ser
Asp Gln Gly Glu Tyr Arg Cys Leu Phe Lys 115 120 125 Asp Asn Asp Asp
Phe Glu Glu Ala Ala Val Tyr Leu Lys Val Ala Ala 130 135 140 Val Gly
Ser Asp Pro Gln Ile Ser Met Thr Val Gln Glu Asn Gly Glu 145 150 155
160 Met Glu Leu Glu Cys Thr Ser Ser Gly Trp Tyr Pro Glu Pro Gln Val
165 170 175 Gln Trp Arg Thr Gly Asn Arg Glu Met Leu Pro Ser Thr Ser
Glu Ser 180 185 190 Lys Lys His Asn Glu Glu Gly Leu Phe Thr Val Ala
Val Ser Met Met 195 200 205 Ile Arg Asp Ser Ser Ile Lys Asn Met Ser
Cys Cys Ile Gln Asn Ile 210 215 220 Leu Leu Gly Gln Gly Lys Glu Val
Glu Ile Ser Leu Pro Ala Pro Phe 225 230 235 240 Val Pro Arg Leu Thr
Pro Trp Ile Val Ala Val Ala Ile Ile Leu Leu 245 250 255 Ala Leu Gly
Phe Leu Thr Ile Gly Ser Ile Phe Phe Thr Trp Lys Leu 260 265 270 Tyr
Lys Glu Arg Ser Ser Leu Arg Lys Lys Glu Phe Gly Ser Lys Glu 275 280
285 Arg Leu Leu Glu Glu Leu Arg Cys Lys Lys Thr Val Leu His Glu Val
290 295 300 Asp Val Thr Leu Asp Pro Asp Thr Ala His Pro His Leu Phe
Leu Tyr 305 310 315 320 Glu Asp Ser Lys Ser Val Arg Leu Glu Asp Ser
Arg Gln Ile Leu Pro 325 330 335 Asp Arg Pro Glu Arg Phe Asp Ser Trp
Pro Cys Val Leu Gly Arg Glu 340 345 350 Thr Phe Thr Ser Gly Arg His
Tyr Trp Glu Val Glu Val Gly Asp Arg 355 360 365 Thr Asp Trp Ala Ile
Gly Val Cys Arg Glu Asn Val Val Lys Lys Gly 370 375 380 Phe Asp Pro
Met Thr Pro Asp Asn Gly Phe Trp Ala Val Glu Leu Tyr 385 390 395 400
Gly Asn Gly Tyr Trp Ala Leu Thr Pro Leu Arg Thr Ser Leu Arg Leu 405
410 415 Ala Gly Pro Pro Arg Arg Val Gly Val Phe Leu Asp Tyr Asp Ala
Gly 420 425 430 Asp Ile Ser Phe Tyr Asn Met Ser Asn Gly Ser Leu Ile
Tyr Thr Phe 435 440 445 Pro Ser Ile Ser Phe Ser Gly Pro Leu Arg Pro
Phe Phe Cys Leu Trp 450 455 460 Ser Cys Gly Lys Lys Pro Leu Thr Ile
Cys Ser Thr Ala Asn Gly Pro 465 470 475 480 Glu Lys Val Thr Val Ile
Ala Asn Val Gln Asp Asp Ile Pro Leu Ser 485 490 495 Pro Leu Gly Glu
Gly Cys Thr Ser Gly Asp Lys Asp Thr Leu His Ser 500 505 510 Lys Leu
Ile Pro Phe Ser Pro Ser Gln Ala Ala Pro 515 520 10 527 PRT Homo
sapiens 10 Met Glu Ser Ala Ala Ala Leu His Phe Ser Arg Pro Ala Ser
Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Leu Cys Ala Leu Val Ser Ala
Gln Phe Ile Val 20 25 30 Val Gly Pro Thr Asp Pro Ile Leu Ala Thr
Val Gly Glu Asn Thr Thr 35 40 45 Leu Arg Cys His Leu Ser Pro Glu
Lys Asn Ala Glu Asp Met Glu Val 50 55 60 Arg Trp Phe Arg Ser Gln
Phe Ser Pro Ala Val Phe Val Tyr Lys Gly 65 70 75 80 Gly Arg Glu Arg
Thr Glu Glu Gln Met Glu Glu Tyr Arg Gly Arg Thr 85 90 95 Thr Phe
Val Ser Lys Asp Ile Ser Arg Gly Ser Val Ala Leu Val Ile 100 105 110
His Asn Ile Thr Ala Gln Glu Asn Gly Thr Tyr Arg Cys Tyr Phe Gln 115
120 125 Glu Gly Arg Ser Tyr Asp Glu Ala Ile Leu His Leu Val Val Ala
Gly 130 135 140 Leu Gly Ser Lys Pro Leu Ile Ser Met Arg Gly His Glu
Asp Gly Gly 145 150 155 160 Ile Arg Leu Glu Cys Ile Ser Arg Gly Trp
Tyr Pro Lys Pro Leu Thr 165 170 175 Val Trp Arg Asp Pro Tyr Gly Gly
Val Ala Pro Ala Leu Lys Glu Val 180 185 190 Ser Met Pro Asp Ala Asp
Gly Leu Phe Met Val Thr Thr Ala Val Ile 195 200 205 Ile Arg Asp Lys
Ser Val Arg Asn Met Ser Cys Ser Ile Asn Asn Thr 210 215 220 Leu Leu
Gly Gln Lys Lys Glu Ser Val Ile Phe Ile Pro Glu Ser Phe 225 230 235
240 Met Pro Ser Val Ser Pro Cys Ala Val Ala Leu Pro Ile Ile Val Val
245 250 255 Ile Leu Met Ile Pro Ile Ala Val Cys Ile Tyr Trp Ile Asn
Lys Leu 260 265 270 Gln Lys Glu Lys Lys Ile Leu Ser Gly Glu Lys Glu
Phe Glu Arg Glu 275 280 285 Thr Arg Glu Ile Ala Leu Lys Glu Leu Glu
Lys Glu Arg Val Gln Lys 290 295 300 Glu Glu Glu Leu Gln Val Lys Glu
Lys Leu Gln Glu Glu Leu Arg Trp 305 310 315 320 Arg Arg Thr Phe Leu
His Ala Val Asp Val Val Leu Asp Pro Asp Thr 325 330 335 Ala His Pro
Asp Leu Phe Leu Ser Glu Asp Arg Arg Ser Val Arg Arg 340 345 350 Cys
Pro Phe Arg His Leu Gly Glu Ser Val Pro Asp Asn Pro Glu Arg 355 360
365 Phe Asp Ser Gln Pro Cys Val Leu Gly Arg Glu Ser Phe Ala Ser Gly
370 375 380 Lys His Tyr Trp Glu Val Glu Val Glu
Asn Val Ile Glu Trp Thr Val 385 390 395 400 Gly Val Cys Arg Asp Ser
Val Glu Arg Lys Gly Glu Val Leu Leu Ile 405 410 415 Pro Gln Asn Gly
Phe Trp Thr Leu Glu Met His Lys Gly Gln Tyr Arg 420 425 430 Ala Val
Ser Ser Pro Asp Arg Ile Leu Pro Leu Lys Glu Ser Leu Cys 435 440 445
Arg Val Gly Val Phe Leu Asp Tyr Glu Ala Gly Asp Val Ser Phe Tyr 450
455 460 Asn Met Arg Asp Arg Ser His Ile Tyr Thr Cys Pro Arg Ser Ala
Phe 465 470 475 480 Ser Val Pro Val Arg Pro Phe Phe Arg Leu Gly Cys
Glu Asp Ser Pro 485 490 495 Ile Phe Ile Cys Pro Ala Leu Thr Gly Ala
Asn Gly Val Thr Val Pro 500 505 510 Glu Glu Gly Leu Thr Leu His Arg
Val Gly Thr His Gln Ser Leu 515 520 525 11 359 PRT Homo sapiens 11
Met Lys Met Ala Ser Ser Leu Ala Phe Leu Leu Leu Asn Phe His Val 1 5
10 15 Ser Leu Leu Leu Val Gln Leu Leu Thr Pro Cys Ser Ala Gln Phe
Ser 20 25 30 Val Leu Gly Pro Ser Gly Pro Ile Leu Ala Met Val Gly
Glu Asp Ala 35 40 45 Asp Leu Pro Cys His Leu Phe Pro Thr Met Ser
Ala Glu Thr Met Glu 50 55 60 Leu Lys Trp Val Ser Ser Ser Leu Arg
Gln Val Val Asn Val Tyr Ala 65 70 75 80 Asp Gly Lys Glu Val Glu Asp
Arg Gln Ser Ala Pro Tyr Arg Gly Arg 85 90 95 Thr Ser Ile Leu Arg
Asp Gly Ile Thr Ala Gly Lys Ala Ala Leu Arg 100 105 110 Ile His Asn
Val Thr Ala Ser Asp Ser Gly Lys Tyr Leu Cys Tyr Phe 115 120 125 Gln
Asp Gly Asp Phe Tyr Glu Lys Ala Leu Val Glu Leu Lys Val Ala 130 135
140 Ala Leu Gly Ser Asn Leu His Val Glu Val Lys Gly Tyr Glu Asp Gly
145 150 155 160 Gly Ile His Leu Glu Cys Arg Ser Thr Gly Trp Tyr Pro
Gln Pro Gln 165 170 175 Ile Gln Trp Ser Asn Ala Lys Gly Glu Asn Ile
Pro Ala Val Glu Ala 180 185 190 Pro Val Val Ala Asp Gly Val Gly Leu
Tyr Glu Val Ala Ala Ser Val 195 200 205 Ile Met Arg Gly Gly Ser Gly
Glu Gly Val Ser Cys Ile Ile Arg Asn 210 215 220 Ser Leu Leu Gly Leu
Glu Lys Thr Ala Ser Ile Ser Ile Ala Asp Pro 225 230 235 240 Phe Phe
Arg Ser Ala Gln Pro Trp Ile Ala Ala Leu Ala Gly Thr Leu 245 250 255
Pro Ile Leu Leu Leu Leu Leu Ala Gly Ala Ser Tyr Phe Leu Trp Arg 260
265 270 Gln Gln Lys Glu Ile Thr Ala Leu Ser Ser Glu Ile Glu Ser Glu
Gln 275 280 285 Glu Met Lys Glu Met Gly Tyr Ala Ala Thr Glu Arg Glu
Ile Ser Leu 290 295 300 Arg Glu Ser Leu Gln Glu Glu Leu Lys Arg Lys
Lys Ile Gln Tyr Leu 305 310 315 320 Thr Arg Gly Glu Glu Ser Ser Ser
Asp Thr Asn Lys Ser Ala Leu Met 325 330 335 Leu Lys Trp Lys Lys Ala
Leu Leu Lys Pro Gly Glu Glu Met Leu Gln 340 345 350 Met Arg Leu His
Leu Val Lys 355 12 319 PRT Homo sapiens 12 Met Lys Met Ala Ser Ser
Leu Ala Phe Leu Leu Leu Asn Phe His Val 1 5 10 15 Ser Leu Leu Leu
Val Gln Leu Leu Thr Pro Cys Ser Ala Gln Phe Ser 20 25 30 Val Leu
Gly Pro Ser Gly Pro Ile Leu Ala Met Val Gly Glu Asp Ala 35 40 45
Asp Leu Pro Cys His Leu Phe Pro Thr Met Ser Ala Glu Thr Met Glu 50
55 60 Leu Lys Trp Val Ser Ser Ser Leu Arg Gln Val Val Asn Val Tyr
Ala 65 70 75 80 Asp Gly Lys Glu Val Glu Asp Arg Gln Ser Ala Pro Tyr
Arg Gly Arg 85 90 95 Thr Ser Ile Leu Arg Asp Gly Ile Thr Ala Gly
Lys Ala Ala Leu Arg 100 105 110 Ile His Asn Val Thr Ala Ser Asp Ser
Gly Lys Tyr Leu Cys Tyr Phe 115 120 125 Gln Asp Gly Asp Phe Tyr Glu
Lys Ala Leu Val Glu Leu Lys Val Ala 130 135 140 Ala Leu Gly Ser Asn
Leu His Val Glu Val Lys Gly Tyr Glu Asp Gly 145 150 155 160 Gly Ile
His Leu Glu Cys Arg Ser Thr Gly Trp Tyr Pro Gln Pro Gln 165 170 175
Ile Gln Trp Ser Asn Ala Lys Gly Glu Asn Ile Pro Ala Val Glu Ala 180
185 190 Pro Val Val Ala Asp Gly Val Gly Leu Tyr Glu Val Ala Ala Ser
Val 195 200 205 Ile Met Arg Gly Gly Ser Gly Glu Gly Val Ser Cys Ile
Ile Arg Asn 210 215 220 Ser Leu Leu Gly Leu Glu Lys Thr Ala Ser Ile
Ser Ile Ala Asp Pro 225 230 235 240 Phe Phe Arg Ser Ala Gln Pro Trp
Ile Ala Ala Leu Ala Gly Thr Leu 245 250 255 Pro Ile Leu Leu Leu Leu
Leu Ala Gly Ala Ser Tyr Phe Leu Trp Arg 260 265 270 Gln Gln Lys Glu
Ile Thr Ala Leu Ser Ser Glu Ile Glu Ser Glu Gln 275 280 285 Glu Met
Lys Glu Met Gly Tyr Ala Ala Thr Glu Arg Glu Ile Ser Leu 290 295 300
Arg Glu Ser Leu Gln Glu Glu Leu Lys Arg Lys Lys Ser Ser Thr 305 310
315 13 280 PRT Grus americana 13 Met Gln Met Trp Leu Pro Ala Ser
Pro Arg Gly Leu Leu Ser Tyr Leu 1 5 10 15 Val Thr Leu His Val Leu
Arg Leu Gly Ser Ala Asn Phe Ser Val Val 20 25 30 Gly Pro Gly His
Pro Leu Arg Val Thr Val Gly Gln Asp Val Met Leu 35 40 45 Pro Cys
His Leu Ser Pro Ser Met Glu Ala Arg Ser Leu Asp Ile Arg 50 55 60
Trp Ile Arg His Gln Val Ser Glu Ile Val His Arg Tyr Arg Asn Gly 65
70 75 80 Glu Asp Leu Tyr Gly Asp Gln Met Glu Glu Tyr Val Gly Arg
Thr Glu 85 90 95 Leu Val Arg Asp Gly Leu Ser Arg Gly Arg Leu Asp
Leu Arg Ile Ser 100 105 110 Gly Leu Arg Pro Ser Asp Asp Gly Gln Tyr
Val Cys Thr Val Arg Asp 115 120 125 Gly Ser Ser Tyr Gly Glu Ala Thr
Val Asp Leu Glu Val Ser Ala Thr 130 135 140 Gly Ser Gly Pro Gln Leu
Ser Leu Glu Ala Tyr Glu Asp Gly Gly Ile 145 150 155 160 Arg Val Val
Cys Arg Ser Ala Gly Trp Tyr Pro Arg Pro Glu Val Leu 165 170 175 Trp
Lys Asp Pro Gly Gly Gln His Leu Pro Ser Val Ser Gln Arg Tyr 180 185
190 Ser Phe Asp Glu Arg Gly Leu Phe Asp Thr Glu Asp Val Ile Ile Val
195 200 205 Thr Asp Gly Asn Arg Asp Gly Lys Trp Ser Cys Val Val Arg
Asn Ser 210 215 220 His Leu Asn Gln Glu Gln Glu Thr Ser Leu His Ile
Ser Ala Pro Phe 225 230 235 240 Phe His Asn Ala Arg Pro Trp Met Val
Gly Val Gln Val Leu Leu Val 245 250 255 Leu Ser Gly Val Leu Leu Gly
Leu Gly Ala Tyr Leu Trp Arg Arg Lys 260 265 270 Val Leu Gln Ser Arg
Glu Leu Glu 275 280 14 11 PRT Human immunodeficiency virus type 1
14 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 15 15 PRT
Artificial Sequence Description of Artificial Sequence
internalizing domain derived from HIV tat protein 15 Gly Gly Gly
Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 15 16 792
DNA Homo sapiens unsure (125) "n" is a, g, c, or t 16 tctggcagga
tgggcagggt gtgcccatga ctggcaacgt gaccacgtcg cagatggcca 60
acgagcaggg cttgtttgat gtgcacagcg tcctgcgggt ggtgctgggt gcgaatggca
120 ctacngcntg cctgttncgc aaccccgtgc tgcagcagga tgcgcacggc
tctgtcacca 180 tcacagggca gcctatgaca ttccccccag aggccctgtg
gtgaccgtgg ggctgtctgt 240 ctgtctcatt gcactgctgg tggccctggc
tttcgtgtgc tggagaaaga tcaaacagag 300 ctgtgaggag gagaatgcag
gagctgagga ccaggatggg gagggagaag gctccaagac 360 agccctgcag
cctctgaaac actctgacag caaagaagat gatggacaag aaatagcctg 420
accatgagga ccagggagct gctacccctc cctacagctc ctaccctctg gctgcaatgg
480 ggctgcactg tgagccctgc ccccaacaga tgcatcctgc tctgacaggt
gggctccttc 540 tccaaaggat gcgatacaca gaccactgtg cagccttatt
tctccaatgg acatgattcc 600 caagtcatcc tgctgccttt tttcttatag
acacaatgaa cagaccaccc acaaccttga 660 gttctgtaaa gtcatcctgg
cctgctggcc tntattttca cagttacata catttnttta 720 gggggacaca
gttacattga accacattta accacctttt tnttttccag ttgttgcgtg 780
gggaccnttt gg 792 17 20 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide; PCR primer 2245-71 17
caacgagcag ggcttgtttg 20 18 23 DNA Artificial Sequence Description
of Artificial Sequence oligonucleotide; PCR primer 2245-72 18
ggtctgtgta tcgcatcctt tgg 23 19 20 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide; PCR primer
1071-80 19 tgcaggtacc ggtccggaat 20 20 22 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide; PCR primer
2279-24 20 tgtcagagca ggatgcatct gt 22 21 23 DNA Artificial
Sequence Description of Artificial Sequence oligonucleotide; PCR
primer 2279-22 21 tgcattgcct tgtgccagca ggt 23 22 23 DNA Artificial
Sequence Description of Artificial Sequence oligonucleotide; PCR
primer 2279-21 22 ctgtcagctg ccagatgagg ttg 23 23 19 DNA Artificial
Sequence Description of Artificial Sequence oligonucleotide; PCR
primer 2318-34 23 gcgtccctga gtcccagag 19 24 23 DNA Artificial
Sequence Description of Artificial Sequenceoligonucleotide; PCR
primer 2318-35 24 gtgtatcgca tcctttggag aag 23 25 1998 DNA Homo
sapiens CDS (152)..(1099) UNSURE (233) "Xaa" is Ser or Ala 25
cgggccgccc ccggccccca ttcgggccgg gcctcgctgc ggcggcgact gagccaggct
60 gggccgcgtc cctgagtccc agagtcggcg cggcgcggca ggggcagcct
tccaccacgg 120 ggagcccagc tgtcagccgc ctcacaggaa g atg ctg cgt cgg
cgg ggc agc 172 Met Leu Arg Arg Arg Gly Ser 1 5 cct ggc atg ggt gtg
cat gtg ggt gca gcc ctg gga gca ctg tgg ttc 220 Pro Gly Met Gly Val
His Val Gly Ala Ala Leu Gly Ala Leu Trp Phe 10 15 20 tgc ctc aca
gga gcc ctg gag gtc cag gtc cct gaa gac cca gtg gtg 268 Cys Leu Thr
Gly Ala Leu Glu Val Gln Val Pro Glu Asp Pro Val Val 25 30 35 gca
ctg gtg ggc acc gat gcc acc ctg tgc tgc tcc ttc tcc cct gag 316 Ala
Leu Val Gly Thr Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu 40 45
50 55 cct ggc ttc agc ctg gca cag ctc aac ctc atc tgg cag ctg aca
gat 364 Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr
Asp 60 65 70 acc aaa cag ctg gtg cac agc ttt gct gag ggc cag gac
cag ggc agc 412 Thr Lys Gln Leu Val His Ser Phe Ala Glu Gly Gln Asp
Gln Gly Ser 75 80 85 gcc tat gcc aac cgc acg gcc ctc ttc ccg gac
ctg ctg gca cag ggc 460 Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp
Leu Leu Ala Gln Gly 90 95 100 aac gca tcc ctg agg ctg cag cgc gtg
cgt gtg gcg gac gag ggc agc 508 Asn Ala Ser Leu Arg Leu Gln Arg Val
Arg Val Ala Asp Glu Gly Ser 105 110 115 ttc acc tgc ttc gtg agc atc
cgg gat ttc ggc agc gct gcc gtc agc 556 Phe Thr Cys Phe Val Ser Ile
Arg Asp Phe Gly Ser Ala Ala Val Ser 120 125 130 135 ctg cag gtg gcc
gct ccc tac tcg aag ccc agc atg acc ctg gag ccc 604 Leu Gln Val Ala
Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro 140 145 150 aac aag
gac ctg cgg cca ggg gac acg gtg acc atc acg tgc tcc agc 652 Asn Lys
Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser 155 160 165
tac cag ggc tac cct gag gct gag gtg ttc tgg cag gat ggg cag ggt 700
Tyr Gln Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly 170
175 180 gtg ccc ctg act ggc aac gtg acc acg tcg cag atg gcc aac gag
cag 748 Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu
Gln 185 190 195 ggc ttg ttt gat gtg cac agc gtc ctg cgg gtg gtg ctg
ggt gcg aat 796 Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu
Gly Ala Asn 200 205 210 215 ggc acc tac agc tgc ctg gtg cgc aac ccc
gtg ctg cag cag gat gcg 844 Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro
Val Leu Gln Gln Asp Ala 220 225 230 cac rgc tct gtc acc atc aca ggg
cag cct atg aca ttc ccc cca gag 892 His Xaa Ser Val Thr Ile Thr Gly
Gln Pro Met Thr Phe Pro Pro Glu 235 240 245 gcc ctg tgg gtg acc gtg
ggg ctg tct gtc tgt ctc att gca ctg ctg 940 Ala Leu Trp Val Thr Val
Gly Leu Ser Val Cys Leu Ile Ala Leu Leu 250 255 260 gtg gcc ctg gct
ttc gtg tgc tgg aga aag atc aaa cag agc tgt gag 988 Val Ala Leu Ala
Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu 265 270 275 gag gag
aat gca gga gct gag gac cag gat ggg gag gga gaa ggc tcc 1036 Glu
Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser 280 285
290 295 aag aca gcc ctg cag cct ctg aaa cac tct gac agc aaa gaa gat
gat 1084 Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu
Asp Asp 300 305 310 gga caa gaa ata gcc tgaccatgag gaccagggag
ctgctacccc tccctacagc 1139 Gly Gln Glu Ile Ala 315 tcctaccctc
tggctgcaat ggggctgcac tgtgagccct gcccccaaca gatgcatcct 1199
gctctgacag gtgggctcct tctccaaagg atgcgataca cagaccactg tgcagcctta
1259 tttctccaat ggacatgatt cccaagtcat cctgctgcct tttttcttat
agacacaatg 1319 aacagaccac ccacaacctt agttctctaa gtcatcctgc
ctgctgcctt atttcacagt 1379 acatacattt cttagggaca cagtacactg
accacatcac caccctcttc ttccagtgct 1439 gcgtggacca tctggctgcc
ttttttctcc aaaagatgca atattcagac tgactgaccc 1499 cctgccttat
ttcaccaaag acacgatgca tagtcacccc ggccttgttt ctccaatggc 1559
cgtgatacac tagtgatcat gttcagccct gcttccacct gcatagaatc ttttcttctc
1619 agacagggac agtgcggcct caacatctcc tggagtctag aagctgtttc
ctttcccctc 1679 cttcctccct gccccaagtg aagacagggc agggccagga
atgctttggg gacaccgagg 1739 ggactgcccc ccacccccac catggtgcta
ttctggggct ggggcagtct tttcctggct 1799 tgcctctggc cagctcctgg
cctctggtag agtgagactt cagacgttct gatgccttcc 1859 ggatgtcatc
tctccctgcc ccaggaatgg aagatgtgag gacttctaat ttaaatgtgg 1919
gactcggagg gattttgtaa actgggggta tattttgggg aaaataaatg tctttgtaaa
1979 aaaaaaaaaa aaaaaaaaa 1998 26 316 PRT Homo sapiens UNSURE (233)
"Xaa" is Ser or Ala 26 Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly
Val His Val Gly Ala 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu
Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val Val
Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe Ser
Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp
Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80 Glu
Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90
95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val
100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile
Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala
Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp
Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser Ser
Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly
Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met
Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu 195 200 205 Arg
Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215
220 Pro Val Leu Gln
Gln Asp Ala His Xaa Ser Val Thr Ile Thr Gly Gln 225 230 235 240 Pro
Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser 245 250
255 Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg
260 265 270 Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu
Asp Gln 275 280 285 Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln
Pro Leu Lys His 290 295 300 Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu
Ile Ala 305 310 315 27 951 DNA Homo sapiens CDS (1)..(948) 27 atg
ctg cgt cgg cgg ggc agc cct ggc atg ggt gtg cat gtg ggt gca 48 Met
Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10
15 gcc ctg gga gca ctg tgg ttc tgc ctc aca gga gcc ctg gag gtc cag
96 Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln
20 25 30 gtc cct gaa gac cca gtg gtg gca ctg gtg ggc acc gat gcc
acc ctg 144 Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala
Thr Leu 35 40 45 tgc tgc tcc ttc tcc cct gag cct ggc ttc agc ctg
gca cag ctc aac 192 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu
Ala Gln Leu Asn 50 55 60 ctc atc tgg cag ctg aca gat acc aaa cag
ctg gtg cac agc ttt gct 240 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln
Leu Val His Ser Phe Ala 65 70 75 80 gag ggc cag gac cag ggc agc gcc
tat gcc aac cgc acg gcc ctc ttc 288 Glu Gly Gln Asp Gln Gly Ser Ala
Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 ccg gac ctg ctg gca cag
ggc aac gca tcc ctg agg ctg cag cgc gtg 336 Pro Asp Leu Leu Ala Gln
Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 cgt gtg gcg gac
gag ggc agc ttc acc tgc ttc gtg agc atc cgg gat 384 Arg Val Ala Asp
Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 ttc ggc
agc gct gcc gtc agc ctg cag gtg gcc gct ccc tac tcg aag 432 Phe Gly
Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140
ccc agc atg acc ctg gag ccc aac aag gac ctg cgg cca ggg gac acg 480
Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145
150 155 160 gtg acc atc acg tgc tcc agc tac cgg ggc tac cct gag gct
gag gtg 528 Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala
Glu Val 165 170 175 ttc tgg cag gat ggg cag ggt gtg ccc ctg act ggc
aac gtg acc acg 576 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly
Asn Val Thr Thr 180 185 190 tcg cag atg gcc aac gag cag ggc ttg ttt
gat gtg cac agc gtc ctg 624 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe
Asp Val His Ser Val Leu 195 200 205 cgg gtg gtg ctg ggt gcg aat ggc
acc tac agc tgc ctg gtg cgc aac 672 Arg Val Val Leu Gly Ala Asn Gly
Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 ccc gtg ctg cag cag gat
gcg cac ggc tct gtc acc atc aca ggg cag 720 Pro Val Leu Gln Gln Asp
Ala His Gly Ser Val Thr Ile Thr Gly Gln 225 230 235 240 cct atg aca
ttc ccc cca gag gcc ctg tgg gtg acc gtg ggg ctg tct 768 Pro Met Thr
Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser 245 250 255 gtc
tgt ctc att gca ctg ctg gtg gcc ctg gct ttc gtg tgc tgg aga 816 Val
Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg 260 265
270 aag atc aaa cag agc tgt gag gag gag aat gca gga gct gag gac cag
864 Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln
275 280 285 gat ggg gag gga gaa ggc tcc aag aca gcc ctg cag cct ctg
aaa cac 912 Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro Leu
Lys His 290 295 300 tct gac agc aaa gaa gat gat gga caa gaa ata gcc
tga 951 Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315
28 316 PRT Homo sapiens 28 Met Leu Arg Arg Arg Gly Ser Pro Gly Met
Gly Val His Val Gly Ala 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys
Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val
Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe
Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile
Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80
Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85
90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg
Val 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser
Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala
Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys
Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser
Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp
Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln
Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu 195 200 205
Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210
215 220 Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly
Gln 225 230 235 240 Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr
Val Gly Leu Ser 245 250 255 Val Cys Leu Ile Ala Leu Leu Val Ala Leu
Ala Phe Val Cys Trp Arg 260 265 270 Lys Ile Lys Gln Ser Cys Glu Glu
Glu Asn Ala Gly Ala Glu Asp Gln 275 280 285 Asp Gly Glu Gly Glu Gly
Ser Lys Thr Ala Leu Gln Pro Leu Lys His 290 295 300 Ser Asp Ser Lys
Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315 29 21 PRT Artificial
Sequence Description of Artificial Sequence Myelin oligodendrocyte
glycoprotein peptide antigen 29 Met Glu Val Gly Trp Tyr Arg Ser Pro
Phe Ser Arg Val Val His Lys 1 5 10 15 Tyr Arg Asn Gly Lys 20 30 9
PRT Artificial Sequence Description of Artificial Sequence LCMV
glycoprotein peptide p33 30 Lys Ala Val Tyr Asn Phe Ala Thr Met 1 5
31 9 PRT Artificial Sequence Description of Artificial Sequence
tetramer H-2Db/NP366-374 31 Ala Ser Asn Glu Asn Met Glu Thr Met 1 5
32 316 PRT Artificial Sequence Description of Artificial Sequence
artificial B7RP-2 amino acid sequence 32 Met Leu Arg Xaa Xaa Gly
Xaa Pro Xaa Xaa Gly Val Xaa Xaa Xaa Xaa 1 5 10 15 Ala Leu Gly Ala
Leu Xaa Xaa Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Xaa
Glu Asp Pro Val Val Ala Leu Val Xaa Thr Asp Ala Thr Leu 35 40 45
Xaa Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50
55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe
Xaa 65 70 75 80 Glu Gly Xaa Asp Gln Gly Ser Ala Tyr Xaa Asn Arg Thr
Ala Leu Phe 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu
Arg Leu Gln Arg Val 100 105 110 Arg Val Xaa Asp Glu Gly Ser Phe Thr
Cys Phe Val Ser Ile Xaa Asp 115 120 125 Phe Xaa Ser Ala Ala Val Ser
Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu
Glu Pro Asn Lys Asp Leu Arg Pro Gly Xaa Xaa 145 150 155 160 Val Thr
Ile Thr Cys Ser Ser Tyr Xaa Gly Tyr Pro Glu Ala Glu Val 165 170 175
Phe Trp Xaa Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180
185 190 Ser Gln Met Ala Asn Glu Xaa Gly Leu Phe Asp Val His Ser Val
Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu
Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Gly Ser Val
Thr Ile Thr Gly Gln 225 230 235 240 Pro Xaa Thr Phe Pro Pro Glu Ala
Leu Trp Val Thr Val Gly Leu Ser 245 250 255 Val Cys Leu Ile Ala Leu
Leu Val Ala Leu Ala Phe Val Cys Trp Arg 260 265 270 Lys Ile Lys Gln
Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln 275 280 285 Asp Gly
Glu Gly Glu Gly Ser Lys Thr Ala Leu Xaa Pro Leu Lys Xaa 290 295 300
Ser Asp Xaa Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315
* * * * *
References