Title: Nucleic acids encoding costimulatory molecule B7-4
Abstract: The invention provides isolated nucleic acids molecules, designated B7-4 nucleic acid molecules, which encode novel B7-4 polypeptides. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing B7-4 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a B7-4 gene has been introduced or disrupted. The invention still further provides isolated B7-4 proteins, fusion proteins, antigenic peptides and anti-B7-4 antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.
Patent Number: 6,936,704 Issued on 08/30/2005 to Freeman, et al.
| Inventors:
|
Freeman; Gordon (Brookline, MA);
Boussiotis; Vassiliki (Brookline, MA);
Chernova; Tatyana (Brighton, MA);
Malenkovich; Nelly (Boston, MA)
|
| Assignee:
|
Dana-Farber Cancer Institute, Inc. (Boston, MA)
|
| Appl. No.:
|
644934 |
| Filed:
|
August 23, 2000 |
| Current U.S. Class: |
536/23.5; 536/23.1; 536/23.4; 435/69.1; 435/455; 435/252.3; 435/320.1; 435/471; 435/326 |
| Intern'l Class: |
C07H 021/04; C12P 021/02; C12N 005/10; C12N 015/87; C12N 015/63 |
| Field of Search: |
536/235,231,234
435/455,252.3,320.1,471,326,691,255
|
References Cited [Referenced By]
U.S. Patent Documents
| 2002/0055139 | May., 2002 | Holtzman et al.
| |
| Foreign Patent Documents |
| WO 95/0340/8 | Feb., 1995 | WO.
| |
| WO 01/1455/7 | Mar., 2001 | WO.
| |
| WO 01/3972/2 | Jun., 2001 | WO.
| |
Other References
Code #'s 27-7975-01, 27-7609-01,27-7610-01, 27-7856-01, 27-7857-01, or 27-7858-01
in Pharmacia Biotech "BioDirectory" 1997 catalog, p. 44, Pharmacia Biotech Inc,
800 Centennial Ave., Piscataway, New Jersey 08855-1327.
Metzler et al. Nature Structural Biol. 1997; 4:527-531.
Stedman's Medical Dictionary, 24th Edition, 1982 Williams & Wilkins, Baltimore,
MD, p. 42.
Voet et al. In Biochemistry. John Wiley & Sons. 1990, vol. 1, pp. 126-128, and
p. 230.
Attwood Science 290(5491):471-473, Oct. 27, 2000.
Skolnick et al. Trends in Biotech., 18(1):34-39, 2000.
Coyle et al. Nature Immunol. 2:203-209 2001.
Dong, Haidong et al., GenBank Acession No. AF177937, Jan. 19, 2000.
Dong, Haidong et al., "B7-H1, a third member, a third of the B7 family, co-stimulates
T-cell proliferation and interleukin-10 secretion." Natural Medicine, (1999),
vol. 5, No. 12, pp.: 1365-1369.
Freeman, Gordon, J. et al., "Engagement of the PD-1 Immunoinhibitory Receptor
by a Novel B7 Family Member Leads to Negative Regulation of Lymphocyte Activation."
Journal Experimental Medicine, (1998), vol. 192 No. 7, pp.: 1027-1034.
Henry, Joelle et al., "Structure and evolution of the extended B7 family." Immunology
Today, (1999), vol. 20, No. 6, pp.: 285-288.
GenBank Acession No. AA292201, Apr. 21, 1997.
GenBank Acession No. AA399416, Apr. 29, 1997.
GenBank Acession No. Q13410, Nov. 1, 1997.
|
Primary Examiner: Gambel; Phillip
Assistant Examiner: Ouspenski; Ilia
Attorney, Agent or Firm: Lahive & Cockfield, LLP, Mandragouras, Esq.; Amy E., Williams, Esq.; Megan E.
Goverment Interests
GOVERNMENT FUNDING
Work described herein was supported under AI 39671, AI 44690, CA 94500 and AI
41584 awarded by the National Institutes of Health. The U.S. government therefore
may have certain rights in this invention.
Parent Case Text
RELATED APPLICATIONS
This application claims priority to U.S. provisional application Ser. No. 60/150,390,
filed on Aug. 23, 1999, incorporated herein in its entirety by this reference.
Claims
1. An isolated nucleic acid molecule comprising the nucleotide sequence set forth
in SEQ ID NO: 1.
2. An isolated nucleic acid molecule encoding a polypeptide comprising the amino
acid sequence set forth in SEQ ID NO: 2.
3. An isolated nucleic acid molecule comprising nucleotides 107-766 of SEQ ID
NO: 3.
4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding
amino acids 19-238 of SEQ ID NO: 4.
5. An isolated nucleic acid molecule comprising the nucleotide sequence set forth
in SEQ ID NO: 3.
6. The isolated nucleic acid molecule of claim 5 which also encodes a heterologous polypeptide.
7. A vector comprising the nucleic acid molecule of claim 5.
8. The vector of claim 7, which is an expression vector.
9. A host cell transfected with the expression vector of claim 8.
10. A method of producing a polypeptide encoded by the nucleic acid comprising
the nucleotide sequence set forth in SEQ ID NO: 3, comprising culturing the host
cell of claim 9 in an appropriate culture medium, to thereby produce the polypeptide.
11. An isolated nucleic acid molecule encoding a polypeptide comprising the amino
acid sequence set forth in SEQ ID NO: 4.
12. The isolated nucleic acid molecule of claim 11 which also encodes a heterologous polypeptide.
13. A vector comprising the nucleic acid molecule of claim 11.
14. The vector of claim 13, which is an expression vector.
15. A host cell transfected with the expression vector of claim 14.
16. A method of producing a polypeptide comprising the amino acid sequence set
forth in SEQ ID NO:4, comprising culturing the host cell of claim 15 in an appropriate
culture medium, to thereby produce the polypeptide.
17. An isolated nucleic acid molecule comprising a nucleotide sequence which
hybridizes to the complement of the nucleic acid molecule of SEQ ID NO:3 over its
full length under conditions of incubation at 45° C. in 6× sodium chloride/sodium
citrate (SSC), followed by washing in 0.2×SSC, 0.1% SDS, at 50-60° C.,
wherein the nucleic acid molecule encodes a polypeptide that costimulates T cell
proliferation in vitro when the polypeptide is present on a first surface and an
antigen or a polyclonal activator that transmits an activating signal via the T-cell
receptor is present on a second, different surface.
18. The isolated nucleic acid molecule of claim 17 which also encodes a heterologous polypeptide.
19. A vector comprising the nucleic acid molecule of claim 17.
20. The vector of claim 19, which is an expression vector.
21. A host cell transfected with the expression vector of claim 20.
22. A method of producing a polypeptide encoded by the nucleic acid of claim
17 in an appropriate culture medium, to thereby produce the polypeptide.
23. An isolated nucleic acid molecule comprising a nucleotide sequence which
hybridizes to the complement of the nucleic acid molecule of SEQ ID NO:1 over its
full length under conditions of incubation at 45° C. in 6× sodium chloride/sodium
citrate (SSC), followed by washing in 0.2×SSC, 0.1% SDS, at 50-60° C.,
wherein the nucleic acid molecule encodes a polypeptide that costimulates T cell
proliferation in vitro when the polypeptide is present on a first surface and an
antigen or a polyclonal activator that transmits an activating signal via the T-cell
receptor is present on a second, different surface.
24. The isolated nucleic acid molecule of claim 23 which also encodes a heterologous polypeptide.
25. A vector comprising the nucleic acid molecule of claim 23.
26. The vector of claim 25, which is an expression vector.
27. A host cell transfected with the expression vector of claim 26.
28. A method of producing a polypeptide encoded by the nucleic acid of claim
23 in an appropriate culture medium, to thereby produce the polypeptide.
29. An isolated nucleic acid molecule comprising a nucleotide sequence that is
at least 95% identical to the nucleotide sequence of SEQ ID NO: 3, and that encodes
a polypeptide that costimulates T cell proliferation in vitro when the polypeptide
is present on a first surface and an antigen or a polyclonal activator that transmits
an activating signal via the T-cell receptor is present on a second, different surface.
30. An isolated nucleic acid molecule comprising a nucleotide sequence encoding
a polypeptide comprising an amino acid sequence that is at least 95% identical
to the amino acid sequence of SEQ ID NO: 4, wherein the polypeptide costimulates
T cell proliferation in vitro when the polypeptide is present on a first surface
and an antigen or a polyclonal activator that transmits an activating signal via
the T-cell receptor is present on a second, different surface.
31. An isolated nucleic acid molecule which is a fragment of SEQ ID NO:1 or SEQ
ID NO:3, or the complement thereof, wherein the fragment consists of at least 15
contiguous nucleotides of a nucleotide sequence selected from the group consisting of:
(a) nucleotides 1-319 of SEQ ID NO: 1;
(b) nucleotides 53-922 of SEQ ID NO: 3;
(c) nucleotides 1-314 of SEQ ID NO: 3; and
(d) nucleotides 955-1285 of SEQ ID NO: 3.
32. An isolated nucleic acid molecule comprising a nucleotide sequence encoding
the amino acids 19-245 of SEQ ID NO:2.
33. The isolated nucleic acid molecule of claim 3 which also encodes a heterologous polypeptide.
34. A vector comprising the nucleic acid molecule of claim 3.
35. The vector of claim 34, which is an expression vector.
36. A host cell transfected with the expression vector of claim 35.
37. A method of producing a polypeptide encoded by the nucleic acid of claim
3 in an appropriate culture medium, to thereby produce the polypeptide.
38. The isolated nucleic acid molecule of claim 4 which also encodes a heterologous polypeptide.
39. A vector comprising the nucleic acid molecule of claim 4.
40. The vector of claim 39, which is an expression vector.
41. A host cell transfected with the expression vector of claim 40.
42. A method of producing a polypeptide encoded by the nucleic acid of claim
4 in an appropriate culture medium, to thereby produce the polypeptide.
43. An isolated nucleic acid molecule comprising nucleotides 107-733 of SEQ ID
NO: 3.
44. The isolated nucleic acid molecule of claim 43 which also encodes a heterologous polypeptide.
45. A vector comprising the nucleic acid molecule of claim 43.
46. The vector of claim 45, which is an expression vector.
47. A host cell transfected with the expression vector of claim 46.
48. A method of producing a polypeptide encoded by the nucleic acid of claim
43 in an appropriate culture medium, to thereby produce the polypeptide.
49. An isolated nucleic acid molecule comprising a nucleotide sequence encoding
amino acids 19-227 of SEQ ID NO: 4.
50. The isolated nucleic acid molecule of claim 49 which also encodes a heterologous polypeptide.
51. A vector comprising the nucleic acid molecule of claim 49.
52. The vector of claim 51, which is an expression vector.
53. A host cell transfected with the expression vector of claim 52.
54. A method of producing a polypeptide encoded by the nucleic acid of claim
49 in an appropriate culture medium, to thereby produce the polypeptide.
55. The isolated nucleic acid molecule of claim 1 which also encodes a heterologous polypeptide.
56. A vector comprising the nucleic acid molecule of claim 1.
57. The vector of claim 56, which is an expression vector.
58. A host cell transfected with the expression vector of claim 57.
59. A method of producing a polypeptide encoded by the nucleic acid of claim
43 in an appropriate culture medium, to thereby produce the polypeptide.
60. The isolated nucleic acid molecule of claim 2 which also encodes a heterologous polypeptide.
61. A vector comprising the nucleic acid molecule of claim 2.
62. The vector of claim 61, which is an expression vector.
63. A host cell transfected with the expression vector of claim 62.
64. A method of producing a polypeptide encoded by the nucleic acid of claim
2 in an appropriate culture medium, to thereby produce the polypeptide.
Description
BACKGROUND OF THE INVENTION
In order for T cells to respond to foreign proteins, two signals must be provided
by antigen-presenting cells (APCs) to resting T lymphocytes (Jenkins, M. and Schwartz,
R. (1987)
J. Exp. Med. 165, 302-319; Mueller, D. L., et al. (1990)
J.
Immunol 144, 3701-3709). The first signal, which confers specificity to the
immune response, is transduced via the T cell receptor (TCR) following recognition
of foreign antigenic peptide presented in the context of the major histocompatibility
complex (MHC). The second signal, termed costimulation, induces T cells to proliferate
and become functional (Lenschow et al. 1996
. Annu. Rev. Immunol. 14:233).
Costimulation is neither antigen-specific, nor MHC restricted and is thought to
be provided by one or more distinct cell surface molecules expressed by APCs (Jenkins,
M. K., et al. 1988
J. Immunol. 140, 3324-3330; Linsley, P. S., et al. 1991
J. Exp. Med. 173, 721-730; Gimmi, C. D., et al., 1991
Proc. Natl. Acad.
Sci. USA. 88, 6575-6579; Young, J. W., et al. 1992
J. Clin. Invest 90,
229-237; Koulova, L., et al. 1991
J. Exp. Med. 173, 759-762; Reiser, H.,
et al. 1992
Proc. Natl. Acad. Sci. USA. 89, 271-275; van-Seventer, G. A.,
et al. (1990)
J. Immunol. 144, 4579-4586; LaSalle, J. M., et al., 1991
J.
Immunol. 147, 774-80; Dustin, M. I., et al., 1989
J. Exp. Med 169, 503;
Armitage, R. J., et al. 1992
Nature 357, 80-82; Liu, Y., et al. 1992
J.
Exp. Med. 175, 437445).
The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs, are critical costimulatory
molecules (Freeman et al. 1991
. J. Exp. Med. 174:625; Freeman et al. 1989
J. Immunol. 143:2714; Azuma et al. 1993
Nature 366:76; Freeman et
al. 1993
. Science 262:909). B7-2 appears to play a predominant role during
primary immune responses, while B7-1, which is upregulated later in the course
of an immune response, may be important in prolonging primary T cell responses
or costimulating secondary T cell responses (Bluestone. 1995
. Immunity. 2:555).
One ligand to which B7-1 and B7-2 bind, CD28, is constitutively expressed on
resting T cells and increases in expression after activation. After signaling through
the T cell receptor, ligation of CD28 and transduction of a costimulatory signal
induces T cells to proliferate and secrete IL-2 (Linsley, P. S., et al. 1991
J.
Exp. Med. 173, 721-730; Gimmi, C. D., et al. 1991
Proc. Natl. Acad. Sci.
USA. 88, 6575-6579; June, C. H., et al. 1990
Immunol. Today 11, 211-6;
Harding, F. A., et al. 1992
Nature. 356, 607-609). A second ligand, termed
CTLA4 (CD152) is homologous to CD28 but is not expressed on resting T cells and
appears following T cell activation (Brunet, J. F., et al., 1987
Nature 328,
267-270). CTLA4 appears to be critical in negative regulation of T cell responses
(Waterhouse et al. 1995
. Science 270:985). Blockade of CTLA4 has been found
to remove inhibitory signals, while aggregation of CTLA4 has been found to provide
inhibitory signals that downregulate T cell responses (Allison and Krummel. 1995
.
Science 270:932). The B7 molecules have a higher affinity for CTLA4 than for
CD28 (Linsley, P. S., et al., 1991
J. Exp. Med 174, 561-569) and B7-1 and
B7-2 have been found to bind to distinct regions of the CTLA4 molecule and have
different kinetics of binding to CTLA4 (Linsley et al. 1994
. Immunity. 1:793).
A new molecule related to CD28 and CTLA4, ICOS, has been identified (Hutloff et
al. 1999
. Nature. 397:263; WO 98/38216), as has its ligand, which is a new
B7 family member (Aicher A. et al. (2000)
J. Immunol. 164:4689-96; Mages
H. W. et al. (2000)
Eur. J. Immunol. 30:1040-7; Brodie D. et al. (2000)
Curr. Biol. 10:333-6; Ling V. et al. (2000)
J. Immunol. 164:1653-7;
Yoshinaga S. K. et al. (1999)
Nature 402:827-32). If T cells are only stimulated
through the T cell receptor, without receiving an additional costimulatory signal,
they become nonresponsive, anergic, or die, resulting in downrnodulation of the
immune response.
The importance of the B7:CD28/CTLA4 costimulatory pathway has been demonstrated
in vitro and in several in vivo model systems. Blockade of this costimulatory pathway
results in the development of antigen specific tolerance in murine and humans systems
(Harding, F. A., et al. (1992)
Nature. 356, 607-609; Lenschow, D. J., et
al. (1992)
Science. 257, 789-792; Turka, L. A., et al. (1992)
Proc. Natl.
Acad. Sci. USA. 89, 11102-11105; Gimmi, C. D., et al. (1993)
Proc. Natl.
Acad. Sci USA 90, 6586-6590; Boussiotis, V., et al. (1993)
J. Exp. Med 178,
1753-1763). Conversely, expression of B7 by B7 negative murine tumor cells induces
T-cell mediated specific immunity accompanied by tumor rejection and long lasting
protection to tumor challenge (Chen, L., et al. (1992)
Cell 71, 1093-1102;
Townsend, S. E. and Allison, J. P. (1993)
Science 259, 368-370; Baskar,
S., et al. (1993)
Proc. Natl. Acad. Sci. 90, 5687-5690.). Therefore, manipulation
of the costimulatory pathways offers great potential to stimulate or suppress immune
responses in humans.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel nucleic
acid molecules and polypeptides encoded by such nucleic acid molecules, referred
to herein as the B7-4 family. Preferred B7-4 molecules include antigens on the
surface of professional antigen presenting cells (e.g., B lymphocytes, monocytes,
dendritic cells, Langerhan cells) and other antigen presenting cells (e.g., keratinocytes,
endothelial cells, astrocytes, fibroblasts, oligodendrocytes), costimulate T cell
proliferation and/or are bound by antibodies which recognize B7 members, e.g.,
anti-BB1 antibodies. The B7-4 nucleic acid and polypeptide molecules of the present
invention are useful, e.g., in modulating the immune response. Accordingly, in
one aspect, this invention provides isolated nucleic acid molecules encoding B7-4
polypeptides, as well as nucleic acid fragments suitable as primers or hybridization
probes for the detection of B7-4-encoding nucleic acids.
In one embodiment, a B7-4 nucleic acid molecule of the invention is at least
50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to a nucleotide
sequence (e.g., to the entire length of the nucleotide sequence) including SEQ
ID NO:1 or 3, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide
sequence shown SEQ ID NO:1 or 3, or a complement thereof. In another preferred
embodiment, an isolated nucleic acid molecule of the invention encodes the amino
acid sequence of a B7-4 polypeptide.
Another embodiment of the invention features nucleic acid molecules, preferably
the B7-4 nucleic acid molecules, which specifically detect the B7-4 nucleic acid
molecules relative to nucleic acid molecules encoding non- the B7-4 polypeptides.
For example, in one embodiment, such a nucleic acid molecule is at least 20, 30,
40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or
800 nucleotides in length and hybridizes under stringent conditions to a nucleic
acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1 or 3, or
a complement thereof.
In other preferred embodiments, the nucleic acid molecule encodes a naturally
occurring allelic variant of a human B7-4 polypeptide, wherein the nucleic acid
molecule hybridizes to a nucleic acid molecule which includes SEQ ID NO:1 or 3
under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule
which is antisense to a B7-4 nucleic acid molecule, e.g., the coding strand of
a B7-4 nucleic acid molecule.
Another aspect of the invention provides a vector comprising a B7-4 nucleic
acid molecule. In certain embodiments, the vector is a recombinant expression vector.
In another embodiment, the invention provides a host cell containing a vector of
the invention. The invention also provides a method for producing a polypeptide,
preferably a B7-4 polypeptide, by culturing in a suitable medium, a host cell,
e.g., a mammalian host cell such as a non-human mammalian cell, of the invention
containing a recombinant expression vector, such that the polypeptide is produced.
Another aspect of this invention features isolated or recombinant B7-4 polypeptides
and proteins. In one embodiment, the isolated polypeptide, is a human or murine
B7-4 polypeptide. In yet another embodiment, the isolated B7-4 polypeptide is a
soluble B7-4 polypeptide. In a further embodiment, the isolated B7-4 polypeptide,
is expressed on the surface of a cell, e.g., has a transmembrane domain.
In a further embodiment, the isolated B7-4 polypeptide plays a role in costimulating
the cytokine secretion and/or proliferation of activated T cells. In another embodiment,
the isolated B7-4 polypeptide is encoded by a nucleic acid molecule having a nucleotide
sequence which hybridizes under stringent hybridization conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3.
Another embodiment of the invention features an isolated polypeptide, preferably
a B7-4 polypeptide, which is encoded by a nucleic acid molecule having a nucleotide
sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or
more identity to a nucleotide sequence (e.g., to the entire length of the nucleotide
sequence) including SEQ ID NO:1 or 3 or a complement thereof.
Another embodiment of the invention features an isolated polypeptide, preferably
a B7-4 polypeptide, which is encoded by a nucleic acid molecule having a nucleotide
sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or
more identity to an amino acid sequence (e.g., to the entire length of the amino
acid sequence) including SEQ ID NO:2 or 4.
This invention further features an isolated B7-4 polypeptide which is encoded
by a nucleic acid molecule having a nucleotide sequence which hybridizes under
stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:1 or 3, or a complement thereof.
The polypeptides of the present invention can be operatively linked to a non-B7-4
polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
The invention further features antibodies, such as monoclonal or polyclonal antibodies,
that specifically bind polypeptides of the invention, preferably B7-4 polypeptides.
In addition, the B7-4 polypeptides, e.g., biologically active polypeptides, can
be incorporated into pharmaceutical compositions, which optionally include pharmaceutically
acceptable carriers.
In another aspect, the present invention provides a method for detecting the
presence
of a B7-4 nucleic acid molecule or polypeptide in a biological sample by contacting
the biological sample with an agent capable of detecting a B7-4 nucleic acid molecule
or polypeptide such that the presence of a B7-4 nucleic acid molecule or polypeptide
is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the
presence
of B7-4 activity in a biological sample by contacting the biological sample with
an agent capable of detecting an indicator of B7-4 polypeptide activity such that
the presence of the B7-4 polypeptide activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating B7-4 polypeptide
activity comprising contacting a cell capable of expressing B7-4 polypeptide with
an agent that modulates B7-4 activity such that the B7-4 activity in the cell is
modulated. In one embodiment, the agent inhibits B7-4 activity. In another embodiment,
the agent stimulates B7-4 activity. In one embodiment, the agent is an antibody
that binds, preferably specifically, to a B7-4 polypeptide. In another embodiment,
the agent modulates expression of the B7-4 by modulating transcription of a B7-4
gene or translation of a B7-4 mRNA. In yet another embodiment, the agent is a nucleic
acid molecule having a nucleotide sequence that is antisense to the coding strand
of a B7-4 mRNA or a B7-4 gene.
In one embodiment, the methods of the present invention are used to treat a subject
having a disorder (characterized by aberrant B7-4 polypeptide or nucleic acid expression
or activity) or a condition that would benefit from modulation, either up or downmodulation,
of a B7-4 molecule by administering an agent which is a B7-4 modulator to the subject.
In one embodiment, the B7-4 modulator is a B7-4 polypeptide. In another embodiment
the B7-4 modulator is a B7-4 nucleic acid molecule. In yet another embodiment,
the B7-4 modulator is a peptide, peptidomimetic, or other small molecule. In a
preferred embodiment, the disorder characterized by aberrant B7-4 polypeptide or
nucleic acid expression is an immune system disorder or condition that would benefit
from modulation of a B7-4 activity.
The present invention also provides a diagnostic assay for identifying the presence
or absence of a genetic alteration characterized by at least one of (i) aberrant
modification or mutation of a gene encoding a B7-4 polypeptide; (ii) mis-regulation
of the gene; and (iii) aberrant post-translational modification of a B7-4 polypeptide,
wherein a wild-type form of the gene encodes a polypeptide with a B7-4 activity.
In another aspect the invention provides a method for identifying a compound
that
binds to or modulates the activity of a B7-4 polypeptide. The method includes providing
an indicator composition comprising a B7-4 polypeptide having B7-4 activity, respectively,
contacting the indicator composition with a test compound, and determining the
effect of the test compound on B7-4 activity in the indicator composition to identify
a compound that modulates the activity of a B7-4 polypeptide.
In another aspect, the invention pertains to nonhuman transgenic animal that
contains
cells carrying a transgene encoding a B7-4 member polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the nucleotide sequence encoding a human secreted B7-4, B7-4S
(SEQ ID NO: 1).
FIG. 2 depicts the nucleotide sequence encoding a human B7-4, B7-4M (SEQ ID
NO: 3).
FIG. 3 depicts the amino acid sequence of human B7-4S (SEQ ID NO:2) and illustrates
the signal, IgV, IgC, and hydrophilic tail domains.
FIG. 4 depicts the amino acid sequence of human B7-4M (SEQ ID NO:4) and illustrates
the signal, IgV, IgC, and transmembrane and cytoplasmic domains.
FIG. 5 depicts the nucleotide sequence of murine B7-4 (SEQ ID NO: 10).
FIG. 6 depicts the amino acid sequence of murine B7-4 (SEQ ID NO: 11).
FIG. 7 depicts an alignment of the human B7-4M (SEQ ID NO: 4) and murine B7-4
(SEQ ID NO: 11) amino acid sequences. Identical residues are reiterated between
the two sequences.
FIG. 8 illustrates the results of FACS analysis of binding of CD28Ig, CTLA4-Ig,
and control Ig by B7-4M-transfected COS cells.
FIG. 9 illustrates the results FACS analysis of binding of IgG and murine ICOS-his
fusion protein by B7-4M-transfected COS cells.
FIG. 10 illustrates the results FACS analysis of binding of IgM, BB 1 and 133
antibodies to B7-4M-transfected COS cells.
FIG. 11 illustrates that COS cells transfected with B7-4M (292) can costimulate
T cell proliferation.
FIG. 12 illustrates that COS cells transfected with a B7-4M (292) can costimulate
T cell proliferation.
DETAILED DESCRIPTION OF THE INVENTION
In addition to the previously characterized B lymphocyte activation antigens,
e.g., B7-1 and B7-2, there are other antigens on the surface of antigen presenting
cells (e.g., B cells, monocytes, dendritic cells, Langerhan cells, keratinocytes,
endothelial cells, astrocytes, fibroblasts, oligodendrocytes) which costimulate
T cells. The present invention is based, at least in part, on the discovery of
novel molecules, referred to herein as B7-4 polypeptides which were isolated from
keratinocyte and placental cDNA libraries and which costimulate T cells. The ability
of a B7-4 polypeptide to costimulate activated T cells can be demonstrated using
techniques that are known in the art, e.g., as described in WO 96/40915 or U.S.
Pat. No. 5,580,756, the contents of which are incorporated herein by reference.
One embodiment of the invention features B7-4 nucleic acid molecules, preferably
human B7-4 molecules, which were identified based on amino acid sequence homology
to the B7 proteins. (Such families are described below).
B7-4 Nucleic Acid and Polypeptide Molecules
In one embodiment, the isolated nucleic acid molecules of the present invention
encode eukaryotic protein B7-4 polypeptides. The B7-4 family of molecules share
a number of conserved regions, including signal domains, IgV domains and the IgC
domains. These regions are both Ig superfamily member domains and are art recognized.
These domains correspond to structural units that have distinct folding patterns
called Ig folds. Ig folds are comprised of a sandwich of two β sheets, each
consisting of antiparallel β strands of 5-10 amino acids with a conserved
disulfide bond between the two sheets in most, but not all, domains. IgC domains
of Ig, TCR, and MHC molecules share the same types of sequence patterns and are
called the C1-set within the Ig superfamily. Other IgC domains fall within other
sets. IgV domains also share sequence patterns and are called V set domains. IgV
domains are longer than C-domains and form an additional pair of β strands.
Two novel human B7-4 molecules were identified. One form is a naturally occurring
B7-4 soluble polypeptide, i.e., having a short hydrophilic domain and no transmembrane
domain, and is referred to herein as B7-4S (shown in SEQ ID NO:2). One form is
a cell-associated polypeptide, i.e., having a transmembrane and cytoplasmic domain,
referred to herein as B7-4M (shown in SEQ ID NO:4).
B7-4 proteins comprise a signal sequence, and an IgV domain and an IgC domain.
The signal sequence of SEQ ID NO:
2 is shown from amino acids 1-18. The signal
sequence of SEQ ID NO:4 is shown from about amino acids 1-18. The IgV domain of
SEQ ID NO:2 is shown from about amino acids 19-134 and the IgV domain of SEQ ID
NO:4 is shown from about amino acids 19-134. The IgC domain of SEQ ID NO:2 is shown
from about amino acids 135-227 and the IgC domain of SEQ ID NO:4 is shown from
about amino acids 135-227. The hydrophilic tail of the B7-4 exemplified in SEQ
ID NO:2 comprises a hydrophilic tail shown from about amino acid 228-245. The B7-4
polypeptide exemplified in SEQ ID NO:4 comprises a transmembrane domain shown from
about amino acids 239-259 of SEQ ID NO:4 and a cytoplasmic domain shown from about
amino acids 260-290 of SEQ ID NO:4.
Murine B7-4 molecules were also identified. The munne cDNA sequence is presented
in Figure
5 and the mwine B7-4 amino acid sequence is presented in Figure
6. The present invention also pertains to these murine B7-4 molecules.
Various aspects of the invention are described in further detail in the following subsections:
I. Definitions
As used herein, the term "costimulate" with reference to activated T cells includes
the ability of a molecule to provide a second, non-T cell receptor mediated, signal
that induces proliferation or effector function, e.g., cytokine secretion, in a
T cell that has received a T cell-receptor-mediated signal, e.g., by interaction
with antigen or a polyclonal activator. Such a costimulatory signal can prevent
the induction of unresponsiveness to antigen, anergy, or cell death in the T cell.
The B7-4 protein and nucleic acid molecules, which comprise a family of molecules
having certain conserved structural and functional features. The term "family"
when referring to the protein and nucleic acid molecules of the invention is intended
to mean two or more proteins or nucleic acid molecules having a common structural
domain or motif and having sufficient amino acid or nucleotide sequence homology
as defined herein. Such family members can be naturally or non-naturally occurring
and can be from either the same or different species. For example, a family can
contain a first protein of human origin, as well as other, distinct proteins of
human origin or alternatively, can contain homologues of non-human origin. Members
of a family may also have common functional characteristics.
The B7-4 molecules described herein are members of the B7 family of molecules.
The term "B7 family" or "B7 molecules" as used herein includes costimulatory molecules
that share sequence homology with B7 polypeptides, e.g., with B7-1, B7-2, B7-3
(recognized by the antibody BB-1), and/or B7-4. For example, human B7-1 and B7-2
share approximately 26% amino acid sequence identity when compared using the BLAST
program at NCBI with the default parameters (Blosum62 matrix with gap penalties
set at existence 11 and extension 1.
Preferred B7 polypeptides are capable of providing costimulation to activated
T cells to thereby induce T cell proliferation and/or cytokine secretion or of
inhibiting costimulation of T cells, e.g., when present in soluble form. Preferred
B7 family members include B7-1, B7-2, and B7-4 and soluble fragments or derivatives
thereof. In one embodiment, B7 family members bind to CTLA4, CD28, ICOS, and/or
other ligands on immune cells and have the ability to inhibit or induce costimulation
of immune cells.
In addition, preferred B7 family members are bound by antibodies generated against
one or more other B7 family members, for example, the anti-BB1 antibody recognizes
B7-4 molecules.
As used herein, the term "activity" with respect to a B7-4 polypeptide includes
activities which are inherent in the structure of a B7-4 protein. The term "activity"
includes the ability to costimulate activated T cells and induce proliferation
and/or cytokine secretion. In addition, the term "activity" includes the ability
of a B7-4 polypeptide to bind its natural ligand.
As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA
or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes
a natural protein).
As used herein, an "antisense" nucleic acid comprises a nucleotide sequence which
is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary
to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA
sequence or complementary to the coding strand of a gene. Accordingly, an antisense
nucleic acid can hydrogen bond to a sense nucleic acid.
As used herein, the term "coding region" refers to regions of a nucleotide sequence
comprising codons which are translated into amino acid residues, whereas the term
"noncoding region" refers to regions of a nucleotide sequence that are not translated
into amino acids (e.g., 5′ and 3′ untranslated regions).
As used herein, the term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One type of vector
is a "plasmid", which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a viral vector,
wherein additional DNA segments may be ligated into the viral genome. Certain vectors
are capable of autonomous replication in a host cell into which they are introduced
(e.g., bacterial vectors having a bacterial origin of replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host cell, and thereby
are replicated along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors" or simply "expression
vectors". In general, expression vectors of utility in recombinant DNA techniques
are often in the form of plasmids. In the present specification, "plasmid" and
"vector" may be used interchangeably as the plasmid is the most commonly used form
of vector. However, the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses
and adeno-associated viruses), which serve equivalent functions.
As used herein, the term "host cell" is intended to refer to a cell into which
a nucleic acid of the invention, such as a recombinant expression vector of the
invention, has been introduced. The terms "host cell" and "recombinant host cell"
are used interchangeably herein. It should be understood that such terms refer
not only to the particular subject cell but to the progeny or potential progeny
of such a cell. Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny may not, in fact,
be identical to the parent cell, but are still included within the scope of the
term as used herein.
As used herein, a "transgenic animal" refers to a non-human animal, preferably
a mammal, more preferably a mouse, in which one or more of the cells of the animal
includes a "transgene". The term "transgene" refers to exogenous DNA which is integrated
into the genome of a cell from which a transgenic animal develops and which remains
in the genome of the mature animal, for example directing the expression of an
encoded gene product in one or more cell types or tissues of the transgenic animal.
As used herein, a "homologous recombinant animal" refers to a type of transgenic
non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous
gene has been altered by homologous recombination between the endogenous gene and
an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
As used herein, an "isolated protein" refers to a protein that is substantially
free of other proteins, cellular material and culture medium when isolated from
cells or produced by recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized.
As used herein, the term "antibody" is intended to include immunoglobulin molecules
and immunologically active portions of immunoglobulin molecules, i.e., molecules
that contain an antigen binding site which binds (immunoreacts with) an antigen,
such as Fab and F(ab′)
2 fragments, single chain antibodies, scFv,
Fd, or other fragments. Preferably, antibodies of the invention bind specifically
or substantially specifically to B7-4 molecules. The terms "monoclonal antibodies"
and "monoclonal antibody composition", as used herein, refer to a population of
antibody molecules that contain only one species of an antigen binding site capable
of immunoreacting with a particular epitope of an antigen, whereas the term "polyclonal
antibodies" and "polyclonal antibody composition" refer to a population of antibody
molecules that contain multiple species of antigen binding sites capable of interacting
with a particular antigen. A monoclonal antibody compositions thus typically display
a single binding affinity for a particular antigen with which it immunoreacts.
There is a known and definite correspondence between the amino acid sequence
of a particular protein and the nucleotide sequences that can code for the protein,
as defined by the genetic code (shown below). Likewise, there is a known and definite
correspondence between the nucleotide sequence of a particular nucleic acid molecule
and the amino acid sequence encoded by that nucleic acid molecule, as defined by
the genetic code.
| |
Alanine (Ala, A) |
GCA, GCC, GCG, GCT |
| |
Arginine (Arg, R) |
AGA, ACG, CGA, CGC, CGG, CGT |
| |
Asparagine (Asn, N) |
AAC, AAT |
| |
Aspartic acid (Asp, D) |
GAC, GAT |
| |
Cysteine (Cys, C) |
TGC, TGT |
| |
Glutamic acid (Glu, E) |
GAA, GAG |
| |
Glutamine (Gln, Q) |
CAA, CAG |
| |
Glycine (Gly, G) |
GGA, GGC, GGG, GGT |
| |
Histidine (His, H) |
CAC, CAT |
| |
Isoleucine (Ile, I) |
ATA, ATC, ATT |
| |
Leucine (Leu, L) |
CTA, CTC, CTG, CTT, TTA, TTG |
| |
Lysine (Lys, K) |
AAA, AAG |
| |
Methionine (Met, M) |
ATG |
| |
Phenylalanine (Phe, F) |
TTC, TTT |
| |
Proline (Pro, P) |
CCA, CCC, CCG, CCT |
| |
Serine (Ser, S) |
AGC, AGT, TCA, TCC, TCG, TCT |
| |
Threonine (Thr, T) |
ACA, ACC, ACG, ACT |
| |
Tryptophan (Trp, W) |
TGG |
| |
Tyrosine (Tyr, Y) |
TAC, TAT |
| |
Valine (Val, V) |
GTA, GTC, GTG, GTT |
| |
Termination signal (end) |
TAA, TAG, TGA |
| |
|
An important and well known feature of the genetic code is its redundancy, whereby,
for most of the amino acids used to make proteins, more than one coding nucleotide
triplet may be employed (illustrated above). Therefore, a number of different nucleotide
sequences may code for a given amino acid sequence. Such nucleotide sequences are
considered functionally equivalent since they result in the production of the same
amino acid sequence in all organisms (although certain organisms may translate
some sequences more efficiently than they do others). Moreover, occasionally, a
methylated variant of a purine or pyrimidine may be found in a given nucleotide
sequence. Such methylations do not affect the coding relationship between the trinucleotide
codon and the corresponding amino acid.
In view of the foregoing, the nucleotide sequence of a DNA or RNA molecule coding
for a B7-4 polypeptide of the invention (or any portion thereof) can be use to
derive the B7-4 amino acid sequence, using the genetic code to translate the DNA
or RNA molecule into an amino acid sequence. Likewise, for any B7-4-amino acid
sequence, corresponding nucleotide sequences that can encode B7-4 protein can be
deduced from the genetic code (which, because of its redundancy, will produce multiple
nucleic acid sequences for any given amino acid sequence). Thus, description and/or
disclosure herein of a B7-4 nucleotide sequence should be considered to also include
description and/or disclosure of the amino acid sequence encoded by the nucleotide
sequence. Similarly, description and/or disclosure of a B7-4 amino acid sequence
herein should be considered to also include description and/or disclosure of all
possible nucleotide sequences that can encode the amino acid sequence.
II. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that
encode B7-4 proteins or biologically active portions thereof, as well as nucleic
acid fragments sufficient for use as hybridization probes to identify B7-4-encoding
nucleic acids (e.g., B7-4 mRNA) and fragments for use as PCR primers for the amplification
or mutation of B7-4 nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and
RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide
analogs. The nucleic acid molecule can be single-stranded or double-stranded, but
preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from other nucleic
acid molecules which are present in the natural source of the nucleic acid. For
example, with regards to genomic DNA, the term "isolated" includes nucleic acid
molecules which are separated from the chromosome with which the genomic DNA is
naturally associated. Preferably, an "isolated" nucleic acid molecule is free of
sequences which naturally flank the nucleic acid (i.e., sequences located at the
5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid molecule is derived. For example, in various embodiments,
the isolated B7-4 nucleic acid molecule can contain less than about 5 kb, 4 kb,
3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank
the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid
is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule,
can be substantially free of other cellular material, or culture medium when produced
by recombinant techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. An "isolated" B7-4 nucleic acid molecule
may, however, be linked to other nucleotide sequences that do not normally flank
the B7-4 sequences in genomic DNA (e.g., the B7-4 nucleotide sequences may be linked
to vector sequences). In certain preferred embodiments, an "isolated" nucleic acid
molecule, such as a cDNA molecule, also may be free of other cellular material.
However, it is not necessary for the B7-4 nucleic acid molecule to be free of other
cellular material to be considered "isolated" (e.g., a B7-4 DNA molecule separated
from other mammalian DNA and inserted into a bacterial cell would still be considered
to be "isolated").
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO:1 or 3, or a portion thereof, can be
isolated using standard molecular biology techniques and the sequence information
provided herein. For example, using all or portion of the nucleic acid sequence
of SEQ ID NO:1 or 3, as a hybridization probe, B7-4 nucleic acid molecules can
be isolated using standard hybridization and cloning techniques (e.g., as described
in Sambrook, J., Fritsh, E. F., and Maniatis, T.
Molecular Cloning: A Laboratory
Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ
ID NO:1 or 3 can be isolated by the polymerase chain reaction (PCR) using synthetic
oligonucleotide primers designed based upon the sequence of SEQ ID NO:1 or 3, respectively.
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. The nucleic acid so amplified can be cloned
into an appropriate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to B7-4 nucleotide sequences can be prepared by
standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention
comprises the nucleotide sequence shown in SEQ ID NO:1 or 3.
In another preferred embodiment, an isolated nucleic acid molecule of the invention
comprises a nucleic acid molecule which is a complement of the nucleotide sequence
shown in SEQ ID NO:1 or 3, or a portion of any of these nucleotide sequences. A
nucleic acid molecule which is complementary to the nucleotide sequence shown in
SEQ ID NO:1 or 3, is one which is sufficiently complementary to the nucleotide
sequence shown in SEQ ID NO:1 or 3, respectively, such that it can hybridize to
the nucleotide sequence shown in SEQ ID NO:1 or 3, respectively, thereby forming
a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of the
present invention comprises a nucleotide sequence which is at least about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide
sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID
NO:1 or 3, or a portion of any of these nucleotide sequences.
Moreover, the nucleic acid molecule of the invention can comprise only
a portion of the nucleic acid sequence of SEQ ID NO:1 or 3, for example a fragment
which can be used as a probe or primer or a fragment encoding a biologically active
portion of a B7-4 protein. The nucleotide sequence determined from the cloning
of the B7-4 genes allows for the generation of probes and primers designed for
use in identifying and/or cloning other B7-4 family members, as well as B7-4 family
homologues from other species. The probe/primer typically comprises a substantially
purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide
sequence that hybridizes under stringent conditions to at least about 12 or 15,
preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65,
or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:1 or 3, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:1 or 3. In an exemplary
embodiment, a nucleic acid molecule of the present invention comprises a nucleotide
sequence which is at least 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800
nucleotides in length and hybridizes under stringent hybridization conditions to
a nucleic acid molecule of SEQ ID NO:1 or 3.
In another embodiment, a second nucleic acid molecule comprises at least about
500, 600, 700, 800, 900, or 1000 contiguous nucleotides of SEQ ID NO:1 or SEQ ID
NO: 3.
In one embodiment, a nucleic acid molecule of the invention, e.g., for use as
a probe, does not include the portion of SEQ ID NO:1 from about nucleotides 815
to about 850 of SEQ ID NO:1 or about nucleotides 320 to 856 of SEQ ID NO:1. In
another embodiment, a nucleic acid molecule of the invention does not include the
portion of SEQ ID NO:3 from about nucleotide 314 to about 734, or from about nucleotide
835 to about 860, or from about nucleotide 1085 to about 1104 or from about nucleotide
1286 to about 1536 of SEQ ID NO:3.
In one embodiment, a nucleic acid molecule of the invention comprises at least
about 500 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:3. In a preferred
embodiment, a nucleic acid molecule of the invention comprises at least about 600,
at least about 700, at east about 800, at least about 900 or at least about 950
contiguous nucleotides of SEQ ID NO:1 or about 1000 contiguous nucleotides of SEQ
ID NO:3. In another embodiment, a nucleic acid molecule of the invention comprises
at least about 1500 or 1550 nucleotides of SEQ ID NO:3.
Preferably, an isolated nucleic acid molecule of the invention comprises
at least a portion of the coding region of SEQ ID NO:1 (shown in nucleotides 59-793)
or SEQ ID NO:3 (shown in nucleotides 53-922). In another embodiment, a B7-4 nucleic
acid molecule comprises from about nucleotide 1 to about nucleotide 319 of SEQ
ID NO:1. In another embodiment, a B7-4 nucleic acid molecule comprises from about
nucleotide 855 to about nucleotide 968 of SEQ ID NO:1. In another embodiment, a
B7-4 nucleic acid molecule comprises from about nucleotide 1 to about nucleotide
314 of SEQ ID NO:3. In another embodiment, a B7-4 nucleic acid molecule comprises
from about nucleotide 955 to about nucleotide 1285 of SEQ ID NO:3. In another embodiment,
a B7-4 nucleic acid molecule comprises from about nucleotide 1535 to about nucleotide
1552 of SEQ ID NO:3.
In other embodiments, a nucleic acid molecule of the invention has at least 70%
identity, more preferably 80% identity, and even more preferably 90% identity with
a nucleic acid molecule comprising: at least about 500, at least about 600, at
least about 700, at east about 800, at least about 900 or at least about 1000 contiguous
nucleotides of SEQ ID NO:1 or SEQ ID NO:3.
Probes based on the B7-4 nucleotide sequences can be used to detect transcripts
or genomic sequences encoding the same or homologous proteins. In preferred embodiments,
the probe further comprises a label group attached thereto, e.g., the label group
can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
Such probes can be used as a part of a diagnostic test kit for identifying cells
or tissues which misexpress a B7-4 protein, such as by measuring a level of a B7-4-encoding
nucleic acid in a sample of cells from a subject e.g., detecting B7-4 mRNA levels
or determining whether a genomic B7-4 gene has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion of a B7-4 protein"
can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:1
or 3, which encodes a polypeptide having a B7-4 biological activity (the biological
activities of the B7-4 proteins are described herein), expressing the encoded portion
of the B7-4 protein (e.g., by recombinant expression in vitro) and assessing the
activity of the encoded portion of the B7-4 protein.
Nucleic acid molecules that differ from SEQ ID NO:1 or 3 due to degeneracy
of the genetic code, and thus encode the same a B7-4 member protein as that encoded
by SEQ ID NO:1 and 3, are encompassed by the invention. Accordingly, in another
embodiment, an isolated nucleic acid molecule of the invention has a nucleotide
sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2
or SEQ ID NO:4. In another embodiment, an isolated nucleic acid molecule of the
invention has a nucleotide sequence encoding a B7-4 protein.
In addition to the B7-4 nucleotide sequences shown in SEQ ID NO:1 or 3, it will
be appreciated by those skilled in the art that DNA sequence polymorphisms that
lead to changes in the amino acid sequences of the B7-4 proteins may exist within
a population (e.g., the human population). Such genetic polymorphism in the B7-4
genes may exist among individuals within a population due to natural allelic variation.
As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules
which include an open reading frame encoding a B7-4 protein, preferably a mammalian
B7-4 protein, and can further include non-coding regulatory sequences, and introns.
Such natural allelic variations include both functional and non-functional B7-4
proteins and can typically result in 1-5% variance in the nucleotide sequence of
a B7-4 gene. Any and all such nucleotide variations and resulting amino acid polymorphisms
in B7-4 genes that are the result of natural allelic variation and that do not
alter the functional activity of a B7-4 protein are intended to be within the scope
of the invention.
Moreover, nucleic acid molecules encoding other B7-4 family members and,
thus, which have a nucleotide sequence which differs from the B7-4 family sequences
of SEQ ID NO:1 or 3 are intended to be within the scope of the invention. For example,
another B7-4 cDNA can be identified based on the nucleotide sequence of human B7-4.
Moreover, nucleic acid molecules encoding B7-4 proteins from different species,
and thus which have a nucleotide sequence which differs from the B7-4 sequences
of SEQ ID NO:1 or 3 are intended to be within the scope of the invention. For example,
a mouse B7-4 cDNA can be identified based on the nucleotide sequence of a human
B7-4 molecule.
Nucleic acid molecules corresponding to natural allelic variants and homologues
of the B7-4 cDNAs of the invention can be isolated based on their homology to the
B7-4 nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion
thereof, as a hybridization probe according to standard hybridization techniques.
For example, a B7-4 DNA can be isolated from a human genomic DNA library using
all or portion of SEQ ID NO:1 or 3 as a hybridization probe and standard hybridization
techniques (e.g., as described in Sambrook, J., et. al.
Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or a portion of
a B7-4 gene can be isolated by the polymerase chain reaction using oligonucleotide
primers designed based upon the sequence of SEQ ID NO:1 or 3. For example, mRNA
can be isolated from cells (e.g., by the guanidinium-thiocyanate extraction procedure
of Chirgwin et al. (1979)
Biochemistry 18: 5294-5299) and cDNA can be prepared
using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available
from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku
America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for PCR
amplification can be designed based upon the nucleotide sequence shown in SEQ ID
NO:1 or 3. A nucleic acid molecule of the invention can be amplified using cDNA
or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers
according to standard PCR amplification techniques. The nucleic acid so amplified
can be cloned into an appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to a B7-4 nucleotide sequence can be
prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In another embodiment, an isolated nucleic acid molecule of the invention is
at
least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent
conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO:1 or 3. In other embodiment, the nucleic acid molecule is at least 30, 50,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides in length.
As used herein, the term "hybridizes under stringent conditions" is intended to
describe conditions for hybridization and washing under which nucleotide sequences
at least 30%, 40%, 50%, or 60% homologous to each other typically remain hybridized
to each other. Preferably, the conditions are such that sequences at least about
70%, more preferably at least about 80%, even more preferably at least about 85%
or 90% homologous to each other typically remain hybridized to each other. Such
stringent conditions are known to those skilled in the art and can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions
are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°
C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.
Preferably, an isolated nucleic acid molecule of the invention that hybridizes
under stringent conditions to the sequence of SEQ ID NO:1 or 3 corresponds to a
naturally-occurring nucleic acid molecule.
As used herein, a "naturally-occurring" nucleic
