U.S. patent application number 10/890789 was filed with the patent office on 2005-03-17 for b7-h1, a novel immunoregulatory molecule.
This patent application is currently assigned to Mayo Foundation for Medical Education and Research. Invention is credited to Chen, Lieping.
Application Number | 20050059051 10/890789 |
Document ID | / |
Family ID | 33098055 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059051 |
Kind Code |
A1 |
Chen, Lieping |
March 17, 2005 |
B7-H1, a novel immunoregulatory molecule
Abstract
The invention provides novel polypeptides useful for
co-stimulating T cells, isolated nucleic acid molecules encoding
them, vectors containing the nucleic acid molecules, and cells
containing the vectors. Also included are methods of making and
using these co-stimulatory polypeptides.
Inventors: |
Chen, Lieping; (Sparks
Glencoe, MD) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
CITIGROUP CENTER 52ND FLOOR
153 EAST 53RD STREET
NEW YORK
NY
10022-4611
US
|
Assignee: |
Mayo Foundation for Medical
Education and Research
Rochester
MN
|
Family ID: |
33098055 |
Appl. No.: |
10/890789 |
Filed: |
July 14, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10890789 |
Jul 14, 2004 |
|
|
|
09451291 |
Nov 30, 1999 |
|
|
|
6803192 |
|
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/351; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 2035/124 20130101; C07K 14/47 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00; C07K 014/52 |
Claims
What is claimed is:
1. An isolated DNA comprising: (a) a nucleic acid sequence that
encodes a polypeptide with the ability to co-stimulate a T cell,
wherein the polypeptide is an amino acid sequence consisting of SEQ
ID NO:1; or (b) the complement of the nucleic acid sequence.
2. The DNA of claim 1, wherein the nucleic acid sequence is a
nucleotide sequence consisting of SEQ ID NO:2.
3. A vector comprising the DNA of claim 1.
4. The vector of claim 3, wherein the nucleic acid sequence is
operably linked to a regulatory element which allows expression of
said nucleic acid sequence in a cell.
5. A cell comprising the vector of claim 3.
6. A cell comprising the vector of claim 4.
7. A method of producing a polypeptide that co-stimulates a T cell,
the method comprising culturing the cell of claim 6 and purifying
the polypeptide from the culture.
8. An isolated DNA comprising: (a) a nucleic acid sequence that
encodes a polypeptide consisting of (i) SEQ ID NO:1 but lacking
amino acid residues 1-22 of SEQ ID NO:1; or (b) the complement of
the nucleic acid sequence.
9. A vector comprising the DNA of claim 8.
10. The vector of claim 9, wherein the nucleic acid sequence is
operably linked to a regulatory element which allows expression of
said nucleic acid sequence in a cell.
11. A cell comprising the vector of claim 9.
12. A cell comprising the vector of claim 10.
13. A method of producing a polypeptide that co-stimulates a T
cell, the method comprising culturing the cell of claim 12 and
purifying the polypeptide from the culture.
14. A method of co-stimulating a T cell, the method comprising
contacting the T cell with a polypeptide encoded by a DNA
comprising: (a) a nucleic acid sequence that encodes a polypeptide
with the ability to co-stimulate a T cell, wherein the nucleic acid
sequence hybridizes under stringent conditions to the complement of
a sequence that encodes a polypeptide with an amino acid sequence
with SEQ ID NO:1 or SEQ ID NO:3; or (b) the complement of the
nucleic acid sequence.
15. The method of claim 14, wherein the contacting comprises
culturing the polypeptide with the T cell in vitro.
16. The method of claim 14, wherein the T cell is in a mammal.
17. The method of claim 16, wherein the contacting comprises
administering the polypeptide to the mammal.
18. The method of claim 16, wherein the contacting comprises
administering a nucleic acid encoding the polypeptide to the
mammal.
19. The method of claim 16, comprising: (a) providing a recombinant
cell which is the progeny of a cell obtained from the mammal and
has been transfected or transformed ex vivo with a nucleic acid
encoding the polypeptide so that the cell expresses the
polypeptide; and (b) administering the cell to the mammal.
20. The method of claim 19, wherein the cell is an antigen
presenting cell (APC) and the cell expresses the polypeptide on its
surface.
21. The method of claim 20, wherein, prior to the administering,
the APC is pulsed with an antigen or an antigenic peptide.
22. The method of claim 16, wherein the mammal is suspected of
having an immunodeficiency disease.
23. The method of claim 16, wherein the mammal is suspected of
having an inflammatory condition.
24. The method of claim 16, wherein the mammal is suspected of
having an autoimmune disease.
25. A method of identifying a compound that inhibits an immune
response, the method comprising: (a) providing a test compound; (b)
culturing, together, the compound, and a polypeptide, a T cell, and
a T cell activating stimulus; and (c) determining whether the test
compound inhibits the response of the T cell to the stimulus, as an
indication that the test compound inhibits an immune response,
wherein the polypeptide is encoded by a DNA comprising: (i) a
nucleic acid sequence that encodes a polypeptide with the ability
to co-stimulate a T cell, wherein the nucleic acid sequence
hybridizes under stringent conditions to the complement of a
sequence that encodes a polypeptide with an amino acid sequence
with SEQ ID NO:1 or SEQ ID NO:3; or (ii) the complement of the
nucleic acid sequence.
26. The method of claim 25, wherein the stimulus is an antibody
that binds to a T cell receptor or a CD3 polypeptide.
27. The method of claim 25, wherein the stimulus is an alloantigen
or an antigenic peptide bound to a major histocompatibility complex
(MHC) molecule on the surface of an antigen presenting cell
(APC).
28. The method of claim 27, wherein the APC is transfected or
transformed with a nucleic acid encoding the polypeptide and the
polypeptide is expressed on the surface of the. APC.
29. A method of identifying a compound that enhances an immune
response, the method comprising: (a) providing a test compound; (b)
culturing, together, the compound, a polypeptide, a T cell, and a T
cell activating stimulus; and (c) determining whether the test
compound enhances the response of the T cell to the antigen, as an
indication that the test compound enhances an immune response,
wherein the polypeptide is encoded by a DNA comprising: (i) a
nucleic acid sequence that encodes a polypeptide with the ability
to co-stimulate a T cell, wherein the nucleic acid sequence
hybridizes under stringent conditions to the complement of a
sequence that encodes a polypeptide with an amino acid sequence
with SEQ ID NO:1 or SEQ ID NO:3; or (ii) the complement of the
nucleic acid sequence.
30. The method of claim 29, wherein the stimulus is an antibody
that binds to a T cell receptor or a CD3 polypeptide.
31. The method of claim 29, wherein the stimulus is an alloantigen
or an antigenic peptide bound to a MHC molecule on the surface of
an APC.
32. The method of claim 31, wherein the APC is transfected or
transformed with a nucleic acid encoding the polypeptide and the
polypeptide is expressed on the surface of the APC.
33. An antibody that binds specifically to a polypeptide encoded by
a DNA comprising: (i) a nucleic acid sequence that encodes a
polypeptide with the ability to co-stimulate a T cell, wherein the
nucleic acid sequence hybridizes under stringent conditions to the
complement of a sequence that encodes a polypeptide with an amino
acid sequence with SEQ ID NO:1 or SEQ ID NO:3; or (ii) the
complement of the nucleic acid sequence.
34. The antibody of claim 33, wherein the antibody is a monoclonal
antibody.
35. The antibody of claim 33, wherein the antibody binds to the
polypeptide with SEQ ID NO:1.
36. A fusion protein comprising a first domain joined to at least
one additional domain, wherein the first domain comprises a
polypeptide encoded by a DNA comprising: (i) a nucleic acid
sequence that encodes a polypeptide with the ability to
co-stimulate a T cell, wherein the nucleic acid sequence hybridizes
under stringent conditions to the complement of a sequence that
encodes a polypeptide with an amino acid sequence with SEQ ID NO:1
or SEQ ID NO:3; or (ii) the complement of the nucleic acid
sequence.
37. The fusion protein of claim 36, wherein the at least one
additional domain comprises the constant region of an
immunoglobulin heavy chain or a fragment thereof.
38. A nucleic acid molecule encoding the fusion protein of claim
36.
39. A vector comprising the nucleic acid molecule of claim 38.
40. The vector of claim 39, wherein the nucleic acid molecule is
operably linked to a regulatory element which allows expression of
the nucleic acid molecule in a cell.
41. A cell comprising the vector of claim 40.
42. A method of producing a fusion protein, the method comprising
culturing the cell of claim 41 and purifying the fusion protein
from the culture.
Description
BACKGROUND OF THE INVENTION
[0001] The invention is generally in the field of immunoregulation,
and specifically T cell response regulation.
[0002] Mammalian T lymphocytes recognize antigenic peptides bound
to major histocompatibility complex (MHC) molecules on the surface
of antigen presenting cells (APC). The antigenic peptides are
generated by proteolytic degradation of protein antigens within the
APC. The interaction of the T cells with the APC and the subsequent
response of the T cells are qualitatively and quantitatively
regulated by interactions between cell surface receptors on the T
cells with both soluble mediators and ligands on the surface of
APC.
SUMMARY OF THE INVENTION
[0003] The invention is based on the cloning of human and mouse
cDNA molecules encoding novel homologous molecules that
co-stimulate the T cell responses of both species and the
functional characterization of the polypeptides that the cDNA
molecules encode. The human polypeptide is designated hB7-H1 and
the mouse polypeptide mB7-H1. Text that refers to B7-H1 without
specifying human versus mouse is pertinent to both forms of B7-H1.
The invention features DNA molecules encoding the hB7-H1, mB7-H1
polypeptides, functional fragments of the polypeptides, and fusion
proteins containing the polypeptides or functional fragments of
them, hB7-H1 and mB7-H1 and functional fragments of both, vectors
containing the DNA molecules, and cells containing the vectors.
Also included in the invention are antibodies that bind to the
B7-H1 polypeptides. The invention features in vitro, in vivo, and
ex vivo methods of co-stimulating T cell responses, methods of
screening for compounds that inhibit or enhance T cell responses,
and methods for producing the above polypeptides and fusion
proteins.
[0004] Specifically the invention features an isolated DNA
including: (a) a nucleic acid sequence that (i) encodes a B7-H1
polypeptide with the ability to co-stimulate a T cell, and (ii)
hybridizes under stringent conditions to the complement of a
sequence that encodes a polypeptide with an amino acid sequence
with SEQ ID NO:1 or SEQ ID NO:3; or (b) a complement of this
nucleic acid sequence. The nucleic acid sequence included in the
isolated DNA will be at least 10 bp, 15 bp, 25 bp, 50 bp, 75 bp,
100 bp, 125 bp, 150 bp, 175 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400
bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750, bp 800 bp,
850 bp, or 870 bp long. The nucleic acid sequence can encode a
B7-H1 polypeptide that includes an amino sequence with SEQ ID NO:1
or SEQ ID NO:3 or it can have a nucleotide sequences with SEQ ID
NO:2 or SEQ ID NO:4. The nucleic acid sequence can also encode
functional fragments of these B7-H1 polypeptides.
[0005] The invention also embodies an isolated B7-H1 polypeptide
encoded by a DNA that includes a nucleic acid sequence that (i)
encodes a polypeptide with the ability to co-stimulate a T cell and
(ii) hybridizes under stringent conditions to the complement of a
sequence that encodes a polypeptide with an amino acid sequence
with SEQ ID NO:1 or SEQ ID NO:3. The B7-H1 polypeptide can include
an amino sequence of amino acid residue 23 to amino acid residue
290 of SEQ ID NO:1 or SEQ ID NO:3. The invention also encompasses
B7-H1 polypeptides that include an amino acid sequence with SEQ ID
NO:1 or SEQ ID NO:3, or either of these amino acid sequences but
differing solely by one or more conservative substitutions. The
polypeptides of the invention include fusion proteins containing a
first domain and at least one additional domain. The first domain
can be any of the B7-H1 polypeptides described above or a
functional fragment of any of these polypeptides. The at least one
additional domain can be a heterologous targeting or leader
sequence, an amino acid sequence that facilitates purification,
detection, or solubility of the fusion protein. The second domain
can be, for example, all or part of an immunoglobulin (Ig) heavy
chain constant region. Also included are isolated nucleic acid
molecules encoding the fusion proteins.
[0006] The invention features vectors containing any of the DNAs of
the invention and nucleic acid molecules encoding the fusion
proteins of the invention. The vectors can be expression vectors in
which the nucleic acid coding sequence or molecule is operably
linked to a regulatory element which allows expression of the
nucleic acid sequence or molecule in a cell. Also included in the
invention are cells ( e.g., mammalian, insect, yeast, fungal, or
bacterial cells) containing any of the vectors of the
invention.
[0007] Another embodiment of the invention is a method of
co-stimulating a T cell that involves contacting the T cell with
any of the B7-H1 polypeptides of the invention, functional
fragments thereof, or fusion proteins of the invention; these 3
classes of molecule are, for convenience, designated "B7-H1
agents". The contacting can be by culturing any of these B7-H1
agents with the T cell in vitro. Alternatively, the T cell can be
in a mammal and the contacting can be, for example, by
administering any of the B7-H1 agents to the mammal or
administering a nucleic acid encoding the B7-H1 agent to the
mammal. In addition, the method can be an ex vivo procedure that
involves providing a recombinant cell which is the progeny of a
cell obtained from the mammal and has been transfected or
transformed ex vivo with a nucleic acid encoding any of the B7-H1
agents so that the cell expresses the B7-H1 agent; and
administering the cell to the mammal. In this ex vivo procedure,
the cell can be an antigen presenting cell (APC) that expresses the
B7-H1 agent on its surface. Furthermore, prior to administering to
the mammal, the APC can be pulsed with an antigen or an antigenic
peptide. In any of the above methods, the mammal can be suspected
of having, for example, an immunodeficiency disease, an
inflammatory condition, or an autoimmune disease.
[0008] The invention includes a method of identifying a compound
that inhibits an immune response. The method involves: providing a
test compound; culturing, together, the compound, one or more B7-H1
agents, a T cell, and a T cell activating stimulus; and determining
whether the test compound inhibits the response of the T cell to
the stimulus, as an indication that the test compound inhibits an
immune response. The invention also embodies a method of
identifying a compound that enhances an immune response. The method
involves: providing a test compound; culturing, together, the
compound, one or more of B7-H1 agents, a T cell, and a T cell
activating stimulus; and determining whether the test compound
enhances the response of the T cell to the stimulus, as an
indication that the test compound enhances an immune response. In
both these methods, the stimulus can be, for example, an antibody
that binds to a T cell receptor or a CD3 polypeptide.
Alternatively, the stimulus can be an alloantigen or an antigenic
peptide bound to a major histocompatibility complex (MHC) molecule
on the surface of an antigen presenting cell (APC). The APC can be
transfected or transformed with a nucleic acid encoding the B7-H1
agent and the B7-H1 agent can be expressed on the surface of the
APC.
[0009] The invention also features an antibody (e.g., a polyclonal
or a monoclonal antibody) that binds to any of the B7-H1
polypeptides of the invention, e.g., the polypeptide with SEQ ID
NO:1 or SEQ ID NO:3.
[0010] The invention also features a method of producing any of the
B7-H1 polypeptides of the invention, functional fragments thereof,
or fusion proteins of the invention. The method involves culturing
a cell of the invention and purifying the relevant B7-H1 protein
from the culture.
[0011] "Polypeptide" and "protein" are used interchangeably and
mean any peptide-linked chain of amino acids, regardless of length
or post-translational modification. The invention also features
B7-H1 polypeptides with conservative substitutions. Conservative
substitutions typically include substitutions within the following
groups: glycine and alanine; valine, isoleucine, and leucine;
aspartic acid and glutamic acid; asparagine, glutamine, serine and
threonine; lysine, histidine and arginine; and phenylalanine and
tyrosine.
[0012] The term "isolated" polypeptide or peptide fragment as used
herein refers to a polypeptide or a peptide fragment which either
has no naturally-occurring counterpart (e.g., a peptidomimetic), or
has been separated or purified from components which naturally
accompany it, e.g., in tissues such as pancreas, liver, spleen,
ovary, testis, muscle, joint tissue, neural tissue,
gastrointestinal tissue, or body fluids such as blood, serum, or
urine. Typically, the polypeptide or peptide fragment is considered
"isolated" when it is at least 70%, by dry weight, free from the
proteins and naturally-occurring organic molecules with which it is
naturally associated. Preferably, a preparation of a polypeptide
(or peptide fragment thereof) of the invention is at least 80%,
more preferably at least 90%, and most preferably at least 99%, by
dry weight, the polypeptide (or the peptide fragment thereof),
respectively, of the invention. Thus, for example, a preparation of
polypeptide x is at least 80%, more preferably at least 90%, and
most preferably at least 99%, by dry weight, polypeptide x. Since a
polypeptide that is chemically synthesized is, by its nature,
separated from the components that naturally accompany it, the
synthetic polypeptide or nucleic acid is "isolated."
[0013] An isolated polypeptide (or peptide fragment) of the
invention can be obtained, for example, by extraction from a
natural source (e.g., from human tissues or bodily fluids); by
expression of a recombinant nucleic acid encoding the peptide; or
by chemical synthesis. A peptide that is produced in a cellular
system different from the source from which it naturally originates
is "isolated," because it will be separated from components which
naturally accompany it. The extent of isolation or purity can be
measured by any appropriate method, e.g., column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis.
[0014] An "isolated DNA" means DNA free of the genes that flank the
gene of interest in the genome of the organism in which the gene of
interest naturally occurs. The term therefore includes a
recombinant DNA incorporated into a vector, into an autonomously
replicating plasmid or virus, or into the genomic DNA of a
prokaryote or eukaryote. It also includes a separate molecule such
as: a cDNA where the corresponding genomic DNA has introns and
therefore a different sequence; a genomic fragment; a fragment
produced by polymerase chain reaction (PCR); a restriction
fragment; a DNA encoding a non-naturally occurring protein, fusion
protein, or fragment of a given protein; or a nucleic acid which is
a degenerate variant of a naturally occurring nucleic acid. In
addition, it includes a recombinant nucleotide sequence that is
part of a hybrid gene, i.e., a gene encoding a fusion protein. Also
included is a recombinant DNA that includes a portion of SEQ ID
NO:2, SEQ ID NO:4, or SEQ ID NO:5.
[0015] As used herein, a polypeptide that "co-stimulates" a T cell
is a polypeptide that, upon interaction with a cell-surface
molecule on the T cell, enhances the response of the T cell. The T
cell response that results from the interaction will be greater
than the response in the absence of the polypeptide. The response
of the T cell in the absence of the co-stimulatory polypeptide can
be no response or it can be a response significantly lower than in
the presence of the co-stimulatory polypeptide. It is understood
that the response of the T cell can an effector, helper, or
suppressive response.
[0016] As used herein, an "activating stimulus" is a molecule that
delivers an activating signal to a T cell, preferably through the
antigen specific T cell receptor (TCR). The activating stimulus can
be sufficient to elicit a detectable response in the T cell.
Alternatively, the T cell may require co-stimulation (e.g., by a
B7-H1 polypeptide) in order to respond detectably to the activating
stimulus. Examples of activating stimuli include, without
limitation, antibodies that bind to the TCR or to a polypeptide of
the CD3 complex that is physically associated with the TCR on the T
cell surface, alloantigens, or an antigenic peptide bound to a MHC
molecule.
[0017] As used herein, a "fragment" of a B7-H1 polypeptide is a
fragment of the polypeptide that is shorter than the full-length
polypeptide. Generally, fragments will be five or more amino acids
in length. An antigenic fragment has the ability to be recognized
and bound by an antibody.
[0018] As used herein, a "functional fragment" of a B7-H1
polypeptide is a fragment of the polypeptide that is shorter than
the full-length polypeptide and has the ability to co-stimulate a T
cell. Methods of establishing whether a fragment of an B7-H1
molecule is functional are known in the art. For example, fragments
of interest can be made by either recombinant, synthetic, or
proteolytic digestive methods. Such fragments can then be isolated
and tested for their ability to co-stimulate T cells by procedures
described herein.
[0019] As used herein, "operably linked" means incorporated into a
genetic construct so that expression control sequences effectively
control expression of a coding sequence of interest.
[0020] As used herein, the term "antibody" refers not only to whole
antibody molecules, but also to antigen-binding fragments, e.g.,
Fab, F(ab').sub.2, Fv, and single chain Fv fragments. Also included
are chimeric antibodies.
[0021] As used herein, an antibody that "binds specifically" to an
isolated B7-H1 polypeptide encoded by a DNA that includes a nucleic
acid sequence that (i) encodes a polypeptide with the ability to
co-stimulate a T cell and (ii) hybridizes under stringent
conditions to the complement of a sequence that encodes a
polypeptide with an amino acid sequence with SEQ ID NO:1 or SEQ ID
NO:3, is an antibody that does not bind to B7-1 or B7-2
polypeptides.
[0022] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. In case
of conflict, the present document, including definitions, will
control. Preferred methods and materials are described below,
although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention. All publications, patent applications, patents
and other references mentioned herein are incorporated by reference
in their entirety. The materials, methods, and examples disclosed
herein are illustrative only and not intended to be limiting,
[0023] Other features and advantages of the invention, e.g.,
enhancing immune responses in mammalian subjects, will be apparent
from the following description, from the drawings and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a depiction of the nucleotide sequence of a cDNA
fragment (SEQ ID NO:5) that includes the coding sequence
(nucleotides 72-870 of SEQ ID NO:5) (SEQ ID NO:2) of hB7-H1.
[0025] FIG. 2a is a depiction of the amino acid sequence of hB7-H1
(SEQ ID NO:1).
[0026] FIG. 2b is a depiction of the amino acid sequences of the
extracellular domains of hB7-H1, B7-1, and B7-2 aligned for maximum
homology. Identical amino acid residues are shaded in bold and
conserved residues are boxed.
[0027] FIG. 3 is a photograph of a Northern blot showing expression
of hB7-H1 mRNA in various human tissues.
[0028] FIG. 4 is a series of two-dimensional fluorescence flow
cytometry histograms showing cell surface expression of hB7-H1 on
resting and activated CD3+ T cells, CD19+ B cells, and CD14+
monocytes.
[0029] FIG. 5a is a series of fluorescence flow cytometry
histograms showing binding of CTLA-4Ig, ICOSIg, and antibody
specific for hB7-H1 to 293 cells transfected with either a control
vector (Mock/293 cells) or a vector containing a cDNA insert
encoding hB7-H1 (B7-H1/293 cells), or Raji cells.
[0030] FIG. 5b is a series of fluorescence flow cytometry
histograms showing the binding of mB7-H1Ig and antibody to CD28 to
Jurkat cells.
[0031] FIG. 6a is a line graph showing the ability of immobilized
hB7-H1 to co-stimulate the proliferative response of human T cells
to immobilized antibody specific for human CD3.
[0032] FIG. 6b is a line graph showing the ability of soluble
hB7-H1 to co-stimulate the proliferative response of human T cells
to irradiated allogeneic PBMC.
[0033] FIGS. 7a-7d are a series of line graphs showing the ability
of hB7-H1Ig, B-7Ig, or antibody specific for CD28 to co-stimulate
the production by human T cells responding to immobilized antibody
specific for CD3 of IL-10 (FIG. 7a), IFN-.gamma. (FIG. 7b), IL-2
(FIG. 7c), IL-4 (FIG. 7d). FIG. 7d is a line graph showing the
ability of various concentrations of hB7-H1 to co-stimulate the
production by human T cells responding to immobilized antibody
specific for CD3 of IL-10.
[0034] FIG. 8a is a bar graph showing the ability of antibody
specific for IL-2 to inhibit the proliferation of human T cells
induced by antibody specific for human CD3 and co-stimulated by COS
cells transfected with and expressing either hB7-H1 or B7-1.
[0035] FIG. 8b is a bar graph showing the ability of antibody
specific for IL-2 to inhibit the production of IL-10 by human T
cells stimulated by immobilized antibody specific for human CD3 and
co-stimulated by either hB7-H1Ig or B7-1Ig.
[0036] FIG. 9a is a series of two-dimensional fluorescence flow
cytometry profiles showing the relative proportion of T cells in
the early (annexin V-positive, propidium iodide (PI)-negative) and
late (annexin V-positive, PI-positive) apoptosis following
activation by immobilized antibody specific for human CD3 and
co-stimulation with either control Ig or hB7-H1Ig.
[0037] FIG. 9b is series of fluorescence flow cytometry profiles
showing expression of Fas and FasL on human T cells following
activation by immobilized antibody specific for human CD3 and
co-stimulation with either control Ig or hB7-H1Ig.
[0038] FIG. 10 is a depiction of the nucleotide sequence of cDNA
encoding mB7-H1 (SEQ ID NO:4).
[0039] FIG. 11 is a depiction of the amino acid sequence of mB7-H1
(SEQ ID NO:3).
[0040] FIG. 12a and FIG. 12b are fluorescence flow cytometry
histograms showing lack of surface expression of murine B7-1 (FIG.
12a) and mB7-H1 (FIG. 12b) on P815 cells transfected with a control
expression vector.
[0041] FIG. 13a and FIG. 13b are fluorescence flow cytometry
histograms showing surface expression of murine B7-1 (FIG. 13a) and
lack of surface expression of mB7-H1 (FIG. 13b) on P815 cells
transfected with an expression vector containing a nucleic acid
sequence encoding murine B7-1.
[0042] FIG. 14a and FIG. 14b are fluorescence flow cytometry
histograms showing lack of surface expression of murine B7-1 (FIG.
14a) and surface expression of mB7-H1 (FIG. 14b) on P815 cells
transfected with an expression vector containing a nucleic acid
sequence encoding mB7-H1.
[0043] FIG. 15a and FIG. 15b are line graphs showing the growth
rate of P815 tumors in DBA/2 mice injected subcutaneously with P815
cells transfected with a control expression vector (FIG. 15a) or an
expression vector containing a nucleic acid sequence encoding
mB7-H1.
DETAILED DESCRIPTION
[0044] Using PCR primers with sequences derived from an expressed
sequence tag (EST) that had significant homology to human B7-1 and
B7-2 and a human cDNA library as a source of template, cDNA
sequences corresponding to regions of a transcript 5' and 3' of the
EST were identified. A cDNA molecule (SEQ ID NO:5) that included a
open reading frame (orf) (SEQ ID NO:2) encoding a novel B7-related
molecule was then generated using PCR primers with sequences
derived from the 3' and 5' ends and cloned.
[0045] Translation of the cDNA sequence indicated that the
polypeptide (SEQ ID NO:1) that it encoded (hB7-H1) is a type I
transmembrane protein of 290 amino acids containing an
immunoglobulin (Ig) V-like domain, Ig C-like domain, a
transmembrane domain and a cytoplasmic domain of 30 amino acids.
Northern blot analysis showed strong expression of the gene
encoding hB7-H1 in heart, skeletal muscle, placenta, and lung, and
weak expression in thymus, spleen, kidney, and liver. Expression
was undetectable in brain, colon, small intestine, and peripheral
blood mononuclear cells (PBMC).
[0046] Using an antiserum produced by immunization of mice with a
recombinantly produced fusion protein that included the hB7-H1
protein, expression by fluorescence flow cytometry indicated
negligible expression on resting T and B cells. On the other hand,
about 16% of CD 14+ monocytes constitutively expressed the molecule
on their surface. Activation of T cells increased expression such
that about 30% expressed cell-surface hB7-H1. Activation resulted
in about 90% of monocytes expressing hB7-1H, but only about 6% of B
cells expressed it after activation.
[0047] Transfection of 293 cells resulted in an hB7-H1 expressing
cell line (hB7-H1/293) which was used for binding experiments.
These experiments and others with a CD28 expressing cell line
indicated that neither CTLA4, ICOS, nor CD28 were receptors for
hB7-H1.
[0048] In vitro experiments with isolated human T cells and the
hB7-H1-containing fusion protein indicated that hB7-H1 had no
direct activity on T cells, it enhanced ("co-stimulated") T cell
proliferative responses induced by both antibody specific for human
CD3 and MHC alloantigens. This co-stimulatory activity was
significantly more potent when the hB7-H1 was immobilized in the
plastic tissue culture wells used for the cultures than when it as
in solution. Similar experiments indicated that hB7-H1 had a
dramatic and selective enhancing effect on the production of
interleukin (IL)-10 induced by T cell activation. Moreover this
IL-10 enhancing activity appeared to be dependent on at least low
amounts of IL-2. Analysis of T cells activated by anti-CD3 antibody
and hB7-H1Ig indicated that hB7-H1 enhances apoptosis and
expression of Fas and FasL
[0049] In addition, using a strategy essentially the same as that
used to clone hB7-H1 cDNA, a cDNA molecule containing an orf
encoding mouse B7-H1 (mB7-H1) was cloned, the nucleotide sequence
of the orf (SEQ ID NO:4) was obtained, and the amino acid sequence
of the encoded sequence (SEQ ID NO:3) was derived. mB7-H1 is
exactly the same length (290 amino acids) as hB7-H1 and has the
same domain structure.
[0050] B7-H1 can be useful as an augmenter of immune responses both
in vivo and in vitro. Furthermore, in light of (a) its ability to
selectively enhance IL-10 production, (b) its ability to enhance
apoptosis, and (c) is expression in placenta and lung, both organs
normally protected from unneeded cellular-mediated immune and
inflammatory responses, B7-H1 can be useful in controlling
pathologic cell-mediated conditions (e.g., those induced by
infectious agents such Mycobacterium tuberculosis or M leprae) or
other pathologic cell-mediated responses such as those involved in
autoimmune diseases (e.g., rheumatoid arthritis (RA), multiple
sclerosis (MS), or insulin-dependent diabetes mellitus (IDDM)).
[0051] B7-H1 Nucleic Acid Molecules
[0052] The B7-H1 nucleic acid molecules of the invention can be
cDNA, genomic DNA, synthetic DNA, or RNA, and can be
double-stranded or single-stranded (i.e., either a sense or an
antisense strand). Segments of these molecules are also considered
within the scope of the invention, and can be produced by, for
example, the polymerase chain reaction (PCR) or generated by
treatment with one or more restriction endonucleases. A ribonucleic
acid (RNA) molecule can be produced by in vitro transcription.
Preferably, the nucleic acid molecules encode polypeptides that,
regardless of length, are soluble under normal physiological
conditions the membrane forms would not be soluble.
[0053] The nucleic acid molecules of the invention can contain
naturally occurring sequences, or sequences that differ from those
that occur naturally, but, due to the degeneracy of the genetic
code, encode the same polypeptide (for example, the polypeptides
with SEQ ID NOS:1 and 3). In addition, these nucleic acid molecules
are not limited to coding sequences, e.g., they can include some or
all of the non-coding sequences that lie upstream or downstream
from a coding sequence. They include, for example, the nucleic acid
molecule with SEQ ID NO:5.
[0054] The nucleic acid molecules of the invention can be
synthesized (for example, by phosphoramidite-based synthesis) or
obtained from a biological cell, such as the cell of a mammal.
Thus, the nucleic acids can be those of a human, non-human primate
(e.g., monkey) mouse, rat, guinea pig, cow, sheep, horse, pig,
rabbit, dog, or cat.
[0055] In addition, the isolated nucleic acid molecules of the
invention encompass segments that are not found as such in the
natural state. Thus, the invention encompasses recombinant nucleic
acid molecules, (for example, isolated nucleic acid molecules
encoding hB7-H1 or mB7-H1) incorporated into a vector (for example,
a plasmid or viral vector) or into the genome of a heterologous
cell (or the genome of a homologous cell, at a position other than
the natural chromosomal location). Recombinant nucleic acid
molecules and uses therefor are discussed further below.
[0056] Certain nucleic acid molecules of the invention are
antisense molecules or are transcribed into antisense molecules.
These can be used, for example, to down-regulate translation of
B7-H1 mRNA within a cell.
[0057] Techniques associated with detection or regulation of genes
are well known to skilled artisans and such techniques can be used
to diagnose and/or treat disorders associated with aberrant B7-H1
expression. Nucleic acid molecules of the invention are discussed
further below in the context of their therapeutic utility.
[0058] A B7-H1 family gene or protein can be identified based on
its similarity to the relevant B7-H1 gene or protein, respectively.
For example, the identification can be based on sequence identity.
The invention features isolated nucleic acid molecules which are at
least 50% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to: (a) a
nucleic acid molecule that encodes the polypeptide of SEQ ID NO:1
or 3; (b) the nucleotide sequence of SEQ ID NO:2 or 4; or (c) a
nucleic acid molecule which includes a segment of at least 30
(e.g., at least 50, 60, 100, 125, 150, 175, 200, 250, 300, 325,
350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, or 865)
nucleotides of SEQ ID NO:2 or SEQ ID NO:4.
[0059] The determination of percent identity between two sequences
is accomplished using the mathematical algorithm of Karlin and
Altschul, Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an
algorithm is incorporated into the BLASTN and BLASTP programs of
Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST nucleotide
searches are performed with the BLASTN program, score=100,
wordlength=12 to obtain nucleotide sequences homologous to
B7-H1-encoding nucleic acids. BLAST protein searches are performed
with the BLASTP program, score=50, wordlength=3 to obtain amino
acid sequences homologous to B7-H1. To obtain gapped alignments for
comparative purposes, Gapped BLAST is utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) are used (See
http://www.ncbi.nlm.nih.gov).
[0060] Hybridization can also be used as a measure of homology
between two nucleic acid sequences. A B7-H1-encoding nucleic acid
sequence, or a portion thereof, can be used as hybridization probe
according to standard hybridization techniques. The hybridization
of a B7-H1 probe to DNA from a test source (e.g., a mammalian cell)
is an indication of the presence of B7-H1 DNA in the test source.
Hybridization conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y., 6.3.1-6.3.6, 1991. Moderate hybridization
conditions are defined as equivalent to hybridization in 2.times.
sodium chloride/sodium citrate (SSC) at 30.degree. C., followed by
one or more washes in 1.times.SSC, 0.1% SDS at 50-60.degree. C.
Highly stringent conditions are defined as equivalent to
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 50-65.degree. C.
[0061] The invention also encompasses: (a) vectors that contain any
of the foregoing B7-H 1-related coding sequences and/or their
complements (that is, "antisense" sequence); (b) expression vectors
that contain any of the foregoing B7-H1-related coding sequences
operatively associated with any transcriptional/translational
regulatory elements (examples of which are given below) necessary
to direct expression of the coding sequences; (c) expression
vectors containing, in addition to sequences encoding a B7-H1
polypeptide, nucleic acid sequences that are unrelated to nucleic
acid sequences encoding B7-H1, such as molecules encoding a
reporter, marker, or a signal peptide, e.g., fused to B7-H1; and
(d) genetically engineered host cells that contain any of the
foregoing expression vectors and thereby express the nucleic acid
molecules of the invention.
[0062] Recombinant nucleic acid molecules can contain a sequence
encoding hB7-H1 or mB7-H1, or B7-H1 having an heterologous signal
sequence. The full length B7-H1 polypeptide, a domain of B7-H1, or
a fragment thereof may be fused to additional polypeptides, as
described below. Similarly, the nucleic acid molecules of the
invention can encode the mature form of B7-H1 or a form that
includes an exogenous polypeptide which facilitates secretion.
[0063] The transcriptional/translational regulatory elements
referred to above and which are further described below, include,
but are not limited to, inducible and non-inducible promoters,
enhancers, operators and other elements, which are known to those
skilled in the art, and which drive or otherwise regulate gene
expression. Such regulatory elements include but are not limited to
the cytomegalovirus hCMV immediate early gene, the early or late
promoters of SV40 adenovirus, the lac system, the trp system, the
TAC system, the TRC system, the major operator and promoter regions
of phage A, the control regions of fd coat protein, the promoter
for 3-phosphoglycerate kinase, the promoters of acid phosphatase,
and the promoters of the yeast .alpha.-mating factors.
[0064] Similarly, the nucleic acid can form part of a hybrid gene
encoding additional polypeptide sequences, for example, sequences
that function as a marker or reporter. Examples of marker or
reporter genes include .beta.-lactamase, chloramphenicol
acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
phosphotransferase (neo.sub.r, G418.sup.r), dihydrofolate reductase
(DHFR), hygromycin-B-phosphotransfer- ase (HPH), thymidine kinase
(TK), lacZ (encoding .beta.-galactosidase), and xanthine guanine
phosphoribosyltransferase (XGPRT). As with many of the standard
procedures associated with the practice of the invention, skilled
artisans will be aware of additional useful reagents, for example,
additional sequences that can serve the function of a marker or
reporter. Generally, the hybrid polypeptide will include a first
portion and a second portion; the first portion being a B7-H1
polypeptide and the second portion being, for example, the reporter
described above or an Ig constant region or part of an Ig constant
region, e.g., the CH2 and CH3 domains of IgG2a.
[0065] The expression systems that may be used for purposes of the
invention include, but are not limited to, microorganisms such as
bacteria (for example, E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA
expression vectors containing the nucleic acid molecules of the
invention; yeast (for example, Saccharomyces and Pichia)
transformed with recombinant yeast expression vectors containing
the nucleic acid molecules of the invention (preferably containing
the nucleic acid sequence encoding B7-H1 (contained within SEQ ID
NOS:1 or 3); insect cell systems infected with recombinant virus
expression vectors (for example, baculovirus) containing the
nucleic acid molecules of the invention; plant cell systems
infected with recombinant virus expression vectors (for example,
cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or
transformed with recombinant plasmid expression vectors (for
example, Ti plasmid) containing B7-H1 nucleotide sequences; or
mammalian cell systems (for example, COS, CHO, BHK, 293, VERO,
HeLa, MDCK, WI38, and NIH 3T3 cells) harboring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells (for example, the metallothionein promoter) or
from mammalian viruses (for example, the adenovirus late promoter
and the vaccinia virus 7.5K promoter). Also useful as host cells
are primary or secondary cells obtained directly from a mammal,
transfected with a plasmid vector or infected with a viral
vector.
[0066] Polypeptides and Polypeptide Fragments
[0067] The polypeptides of the invention include hB7-H1, mB7-H1,
and functional fragments of these polypeptides. The polypeptides
embraced by the invention also include fusion proteins which
contain either full-length B7-H1 or a functional fragment of it
fused to unrelated amino acid sequence. The unrelated sequences can
be additional functional domains or signal peptides. Signal
peptides are described in greater detail and exemplified below.
[0068] The polypeptides can be purified from natural sources (e.g.,
blood, serum plasma, tissues or cells such as T cells or any cell
that naturally produces B7-H1). Smaller peptides (less than 50
amino acids long) can also be conveniently synthesized by standard
chemical means. In addition, both polypeptides and peptides can be
produced by standard in vitro recombinant DNA techniques and in
vivo recombination/genetic recombination (e.g., transgenesis),
using the nucleotide sequences encoding the appropriate
polypeptides or peptides. Methods well known to those skilled in
the art can be used to construct expression vectors containing
relevant coding sequences and appropriate
transcriptional/translational control signals. See, for example,
the techniques described in Sambrook et al., Molecular Cloning: A
Laboratory Manual (2nd Ed.) [Cold Spring Harbor Laboratory, N.Y.,
1989], and Ausubel et al., Current Protocols in Molecular Biology,
[Green Publishing Associates and Wiley Interscience, N.Y.,
1989].
[0069] Polypeptides and fragments of the invention also include
those described above, but modified for in vivo use by the
addition, the amino- and/or carboxyl-terminal ends, of a blocking
agent to facilitate survival of the relevant polypeptide in vivo.
This can be useful in those situations in which the peptide termini
tend to be degraded by proteases prior to cellular uptake. Such
blocking agents can include, without limitation, additional related
or unrelated peptide sequences that can be attached to the amino
and/or carboxyl terminal residues of the peptide to be
administered. This can be done either chemically during the
synthesis of the peptide or by recombinant DNA technology by
methods familiar to artisans of average skill.
[0070] Alternatively, blocking agents such as pyroglutamic acid or
other molecules known in the art can be attached to the amino
and/or carboxyl terminal residues, or the amino group at the amino
terminus or carboxyl group at the carboxyl terminus can be replaced
with a different moiety. Likewise, the peptides can be covalently
or noncovalently coupled to pharmaceutically acceptable "carrier"
proteins prior to administration.
[0071] Also of interest are peptidomimetic compounds that are
designed based upon the amino acid sequences of the functional
peptide fragments. Peptidomimetic compounds are synthetic compounds
having a three-dimensional conformation (i.e., a "peptide motif")
that is substantially the same as the three-dimensional
conformation of a selected peptide. The peptide motif provides the
peptidomimetic compound with the ability to co-stimulate T cells in
a manner qualitatively identical to that of the B7-H1 functional
peptide fragment from which the peptidomimetic was derived.
Peptidomimetic compounds can have additional characteristics that
enhance their therapeutic utility, such as increased cell
permeability and prolonged biological half-life.
[0072] The peptidomimetics typically have a backbone that is
partially or completely non-peptide, but with side groups that are
identical to the side groups of the amino acid residues that occur
in the peptide on which the peptidomimetic is based. Several types
of chemical bonds, e.g., ester, thioester, thioamide, retroamide,
reduced carbonyl, dimethylene and ketomethylene bonds, are known in
the art to be generally useful substitutes for peptide bonds in the
construction of protease-resistant peptidomimetics.
[0073] Methods of Therapy
[0074] The methods of the invention involve contacting a T cell
with a B7-H1 molecule of the invention, or a functional fragment
thereof, in order to co-stimulate the T cell. The contacting can
occur before, during, or after activation of the T cell. Contacting
of the T cell with the B7-H1 polypeptide will preferably be at
substantially the same time as activation. Activation can be, for
example, by exposing the T cell to an antibody that binds to the
TCR or one of the polypeptides of the CD3 complex that is
physically associated with the TCR. Alternatively, the T cell can
be exposed to either an alloantigen (e.g., a MHC alloantigen) on,
for example, an antigen presenting cell (APC) (e.g., a dendritic
cell, a macrophage, a monocyte, or a B cell) or an antigenic
peptide produced by processing of a protein antigen by any of the
above APC and presented to the T cell by MHC molecules on the
surface of the APC. The T cell can be a CD4+ T cell or a CD8+ T
cell. The B7-H1 molecule can be added to the solution containing
the cells, or it can be expressed on the surface of an APC, e.g.,
an APC presenting an alloantigen or an antigen peptide bound to an
MHC molecule. Alternatively, if the activation is in vitro, the
B7-H1 molecule can be bound to the floor of a the relevant culture
vessel, e.g. a well of a plastic microtiter plate.
[0075] The methods can be performed in vitro, in vivo, or ex vivo.
In vitro application of B7-H1 can be useful, for example, in basic
scientific studies of immune mechanisms or for production of
activated T cells for use in either studies on T cell function or,
for example, passive immunotherapy. Furthermore, B7-H1 could be
added to in vitro assays (e.g., in T cell proliferation assays)
designed to test for immunity to an antigen of interest in a
subject from which the T cells were obtained. Addition of B7-H1 to
such assays would be expected to result in a more potent, and
therefore more readily detectable, in vitro response. However, the
methods of the invention will preferably be in vivo or ex vivo (see
below).
[0076] The B7-H 1 proteins and variants thereof are generally
useful as immune response-stimulating therapeutics. For example,
the polypeptides of the invention can be used for treatment of
disease conditions characterized by immunosuppression: e.g.,
cancer, AIDS or AIDS-related complex, other virally or
environmentally-induced conditions, and certain congenital immune
deficiencies. The compounds may also be employed to increase immune
function that has been impaired by the use of radiotherapy of
immunosuppressive drugs such as certain chemotherapeutic agents,
and therefore are particularly useful when given in conjunction
with such drugs or radiotherapy. In addition, in view of the
ability of B7-H1 to co-stimulate the production of especially high
levels of IL-10, B7-H1 molecules can be used to treat conditions
involving cellular immune responses, e.g., inflammatory conditions,
e.g., those induced by infectious agents such Mycobacterium
tuberculosis or M leprae), or other pathologic cell-mediated
responses such as those involved in autoimmune diseases (e.g.,
rheumatoid arthritis (RA), multiple sclerosis (MS), or
insulin-dependent diabetes mellitus (IDDM)).
[0077] These methods of the invention can be applied to a wide
range of species, e.g., humans, non-human primates, horses, cattle,
pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters,
rats, and mice.
[0078] In Vivo Approaches
[0079] In one in vivo approach, the B7-H1 polypeptide (or a
functional fragment thereof) itself is administered to the subject.
Generally, the compounds of the invention will be suspended in a
pharmaceutically-accept- able carrier (e.g., physiological saline)
and administered orally or by intravenous infusion, or injected
subcutaneously, intramuscularly, intraperitoneally, intrarectally,
intravaginally, intranasally, intragastrically, intratracheally, or
intrapulmonarily. They are preferably delivered directly to an
appropriate lymphoid tissue (e.g. spleen, lymph node, or
mucosal-associated lymphoid tissue (MALT)). The dosage required
depends on the choice of the route of administration, the nature of
the formulation, the nature of the patient's illness, the subject's
size, weight, surface area, age, and sex, other drugs being
administered, and the judgment of the attending physician. Suitable
dosages are in the range of 0.01-100.0 .mu.g/kg. Wide variations in
the needed dosage are to be expected in view of the variety of
polypeptides and fragments available and the differing efficiencies
of various routes of administration. For example, oral
administration would be expected to require higher dosages than
administration by i.v. injection. Variations in these dosage levels
can be adjusted using standard empirical routines for optimization
as is well understood in the art. Administrations can be single or
multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or
more fold). Encapsulation of the polypeptide in a suitable delivery
vehicle (e.g., polymeric microparticles or implantable devices) may
increase the efficiency of delivery, particularly for oral
delivery.
[0080] Alternatively, a polynucleotide containing a nucleic acid
sequence encoding the B7-H1 polypeptide or functional fragment can
be delivered to an appropriate cell of the animal. Expression of
the coding sequence will preferably be directed to lymphoid tissue
of the subject by, for example, delivery of the polynucleotide to
the lymphoid tissue. This can be achieved by, for example, the use
of a polymeric, biodegradable microparticle or microcapsule
delivery vehicle, sized to optimize phagocytosis by phagocytic
cells such as macrophages. For example, PLGA
(poly-lacto-co-glycolide) microparticles approximately 1-10 .mu.m
in diameter can be used. The polynucleotide is encapsulated in
these microparticles, which are taken up by macrophages and
gradually biodegraded within the cell, thereby releasing the
polynucleotide. Once released, the DNA is expressed within the
cell. A second type of microparticle is intended not to be taken up
directly by cells, but rather to serve primarily as a slow-release
reservoir of nucleic acid that is taken up by cells only upon
release from the micro-particle through biodegradation. These
polymeric particles should therefore be large enough to preclude
phagocytosis (i.e., larger than 5 .mu.m and preferably larger than
20 .mu.m.
[0081] Another way to achieve uptake of the nucleic acid is using
liposomes, prepared by standard methods. The vectors can be
incorporated alone into these delivery vehicles or co-incorporated
with tissue-specific antibodies. Alternatively, one can prepare a
molecular conjugate composed of a plasmid or other vector attached
to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine
binds to a ligand that can bind to a receptor on target cells
[Cristiano et al. (1995), J. Mol. Med. 73, 479]. Alternatively,
lymphoid tissue specific targeting can be achieved by the use of
lymphoid tissue-specific transcriptional regulatory elements (TRE)
such as a B lymphocyte, T lymphocyte, or dendritic cell specific
TRE. Lymphoid tissue specific TRE are known [Thompson et al.
(1992), Mol. Cell. Biol. 12, 1043-1053; Todd et al. (1993), J. Exp.
Med. 177, 1663-1674; Penix et al. (1993), J. Exp. Med. 178,
1483-1496]. Delivery of "naked DNA" (i.e., without a delivery
vehicle) to an intramuscular, intradermal, or subcutaneous site, is
another means to achieve in vivo expression.
[0082] In the relevant polynucleotides (e.g., expression vectors)
the nucleic acid sequence encoding the B7-H1 polypeptide or
functional fragment of interest with an initiator methionine and
optionally a targeting sequence is operatively linked to a promoter
or enhancer-promoter combination.
[0083] Short amino acid sequences can act as signals to direct
proteins to specific intracellular compartments. For example,
hydrophobic signal peptides (e.g., MAISGVPVLGFFIIAVLMSAQESWA (SEQ
ID NO:6)) are found at the amino terminus of proteins destined for
the ER. While the sequence KFERQ (SEQ ID NO:7) (and other closely
related sequences) is known to target intracellular polypeptides to
lysosomes, other sequences (e.g., MDDQRDLISNNEQLP (SEQ ID NO:8)
direct polypeptides to endosomes. In addition, the peptide sequence
KDEL (SEQ ID NO:9) has been shown to act as a retention signal for
the ER. Each of these signal peptides, or a combination thereof,
can be used to traffic the B7-H1 polypeptides or functional
fragments of the invention as desired. DNAs encoding the B7-H1
polypeptides or functional fragments containing targeting signals
will be generated by PCR or other standard genetic engineering or
synthetic techniques.
[0084] A promoter is a TRE composed of a region of a DNA molecule,
typically within 100 basepairs upstream of the point at which
transcription starts. Enhancers provide expression specificity in
terms of time, location, and level. Unlike a promoter, an enhancer
can function when located at variable distances from the
transcription site, provided a promoter is present. An enhancer can
also be located downstream of the transcription initiation site. To
bring a coding sequence under the control of a promoter, it is
necessary to position the translation initiation site of the
translational reading frame of the peptide or polypeptide between
one and about fifty nucleotides downstream (3') of the promoter.
The coding sequence of the expression vector is operatively linked
to a transcription terminating region.
[0085] Suitable expression vectors include plasmids and viral
vectors such as herpes viruses, retroviruses, vaccinia viruses,
attenuated vaccinia viruses, canary pox viruses, adenoviruses and
adeno-associated viruses, among others.
[0086] Polynucleotides can be administered in a pharmaceutically
acceptable carrier. Pharmaceutically acceptable carriers are
biologically compatible vehicles which are suitable for
administration to a human, e.g., physiological saline. A
therapeutically effective amount is an amount of the polynucleotide
which is capable of producing a medically desirable result (e.g.,
an enhanced T cell response) in a treated animal. As is well known
in the medical arts, the dosage for any one patient depends upon
many factors, including the patient's size, body surface area, age,
the particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. Dosages will vary, but a preferred dosage for
administration of polynucleotide is from approximately 10.sup.6 to
10.sup.12 copies of the polynucleotide molecule. This dose can be
repeatedly administered, as needed. Routes of administration can be
any of those listed above.
[0087] Ex Vivo Approaches
[0088] Peripheral blood mononuclear cells (PBMC) can be withdrawn
from the patient or a suitable donor and exposed ex vivo to an
activating stimulus (see above) and a B7-H1 polypeptide or
polypeptide fragment (whether in soluble form or attached to a sold
support by standard methodologies). The PBMC containing highly
activated T cells are then introduced into the same or a different
patient.
[0089] An alternative ex vivo strategy can involve transfecting or
transducing cells obtained from the subject with a polynucleotide
encoding an B7-H1 polypeptide or functional fragment-encoding
nucleic acid sequences described above. The transfected or
transduced cells are then returned to the subject. While such cells
would preferably be hemopoietic cells (e.g., bone marrow cells,
macrophages, monocytes, dendritic cells, or B cells) they could
also be any of a wide range of types including, without limitation,
fibroblasts, epithelial cells, endothelial cells, keratinocytes, or
muscle cells in which they act as a source of the B7-H1 polypeptide
or functional fragment for as long as they survive in the subject.
The use of hemopoietic cells, that include the above APC, would be
particular advantageous in that such cells would be expected to
home to, among others, lymphoid tissue (e.g., lymph nodes or
spleen) and thus the B7-H1 polypeptide or functional fragment would
be produced in high concentration at the site where they exert
their effect, i.e., enhancement of an immune response. In addition,
if APC are used, the APC expressing the exogenous B7-H1 molecule
can be the same APC that presents an alloantigen or antigenic
peptide to the relevant T cell. The B7-H1 can be secreted by the
APC or expressed on its surface. Prior to returning the recombinant
APC to the patient, they can optionally be exposed to sources of
antigens or antigenic peptides of interest, e.g., those of tumors,
infectious microorganisms, or autoantigens. The same genetic
constructs and trafficking sequences described for the in vivo
approach can be used for this ex vivo strategy. Furthermore, tumor
cells, preferably obtained from a patient, can be transfected or
transformed by a vector encoding a B7-H1 polypeptide or functional
fragment thereof. The tumor cells, preferably treated with an agent
(e.g., ionizing irradiation) that ablates their proliferative
capacity, are then returned to the patient where, due to their
expression of the exogenous B7-H1 (on their cell surface or by
secretion), they can stimulate enhanced tumoricidal T cell immune
responses. It is understood that the tumor cells which, after
transfection or transformation, are injected into the patient, can
also have been originally obtained from an individual other than
the patient.
[0090] The ex vivo methods include the steps of harvesting cells
from a subject, culturing the cells, transducing them with an
expression vector, and maintaining the cells under conditions
suitable for expression of the B7-H1 polypeptide or functional
fragment. These methods are known in the art of molecular biology.
The transduction step is accomplished by any standard means used
for ex vivo gene therapy, including calcium phosphate, lipofection,
electroporation, viral infection, and biolistic gene transfer.
Alternatively, liposomes or polymeric microparticles can be used.
Cells that have been successfully transduced are then selected, for
example, for expression of the coding sequence or of a drug
resistance gene. The cells may then be lethally irradiated (if
desired) and injected or implanted into the patient.
[0091] Methods of Screening for Compounds that Inhibit or Enhance
Immune Responses.
[0092] The invention provides methods for testing compounds (small
molecules or macromolecules) that inhibit or enhance an immune
response. Such a method can involve, e.g., culturing a B7-H1
polypeptide of the invention (or a functional fragment thereof)
with T cells in the presence of a T cell stimulus (see above). The
B7-H1 molecule can be in solution or membrane bound (e.g.,
expressed on the surface of the T cells) and it can be natural or
recombinant. Compounds that inhibit the T cell response will likely
be compounds that inhibit an immune response while those that
enhance the T cell response will likely be compounds that enhance
an immune response.
[0093] The invention also relates to using B7-H1 or functional
fragments thereof to screen for immunomodulatory compounds that can
interact with B7-H1. One of skill in the art would know how to use
standard molecular modeling or other techniques to identify small
molecules that would bind to T cell interactive sites of B7-H1 .
One such example is provided in Broughton (1997) Curr. Opin. Chem.
Biol. 1, 392-398.
[0094] A candidate compound whose presence requires at least
1.5-fold (e.g., 2-fold, 4-fold, 6-fold, 10-fold, 150-fold,
1000-fold, 10,000-fold, or 100,000-fold) more B7-H1 in order to
achieve a defined arbitrary level of T cell activation than in the
absence of the compound can be useful for inhibiting an immune
response. On the other hand, a candidate compound whose presence
requires at least 1.5 fold (e.g., 2-fold, 4-fold, 6-fold, 10-fold,
100-fold, 1000-fold, 10,000 fold, or 100,000-fold) less B7-H1 to
achieve a defined arbitrary level of T cell activation than in the
absence of the compound can be useful for enhancing an immune
response. Compounds capable of interfering with or modulating B7-H1
function are good candidates for immunosuppressive immunoregulatory
agents, e.g., to modulate an autoimmune response or suppress
allogeneic or xenogeneic graft rejection.
[0095] B7-H1 Antibodies
[0096] The invention features antibodies that bind to either or
both of the B7-H1 polypeptides or fragments of such polypeptides.
Such antibodies can be polyclonal antibodies present in the serum
or plasma of animals (e.g., mice, rabbits, rats, guinea pigs,
sheep, horses, goats, cows, or pigs) which have been immunized with
the relevant B7-H1 polypeptide or peptide fragment using methods,
and optionally adjuvants, known in the art. Such polyclonal
antibodies can be isolated from serum or plasma by methods known in
the art. Monoclonal antibodies that bind to the above polypeptides
or fragments are also embodied by the invention. Methods of making
and screening monoclonal antibodies are well known in the art.
[0097] Once the desired antibody-producing hybridoma has been
selected and cloned, the resultant antibody can be produced in a
number of methods known in the art. For example, the hybridoma can
be cultured in vitro in a suitable medium for a suitable length of
time, followed by the recovery of the desired antibody from the
supernatant. The length of time and medium are known or can be
readily determined.
[0098] Additionally, recombinant antibodies specific for B7-H1,
such as chimeric and humanized monoclonal antibodies comprising
both human and non-human portions, are within the scope of the
invention. Such chimeric and humanized monoclonal antibodies can be
produced by recombinant DNA techniques known in the art, for
example, using methods described in Robinson et al., International
Patent Publication PCT/US86/02269; Akira et al., European Patent
Application 184,187; Taniguchi, European Patent Application
171,496; Morrison et al., European Patent Application 173,494;
Neuberger et al., PCT Application WO 86/01533; Cabilly et al., U.S.
Pat. No. 4,816,567; Cabilly et al., European Patent Application
125,023; Better et al. (1988) Science 240, 1041-43; Liu et al.
(1987) J. Immunol. 139, 3521-26; Sun et al. (1987) PNAS 84, 214-18;
Nishimura et al. (1987) Canc. Res. 47, 999-1005; Wood et al. (1985)
Nature 314, 446-49; Shaw et al. (1988) J. Natl. Cancer Inst. 80,
1553-59; Morrison, (1985) Science 229, 1202-07; Oi et al. (1986)
BioTechniques 4, 214; Winter, U.S. Pat. No. 5,225,539; Jones et al.
(1986) Nature 321, 552-25; Veroeyan et al. (1988) Science 239,
1534; and Beidler et al. (1988) J. Immunol. 141, 4053-60.
[0099] Also included within the scope of the invention are antibody
fragments and derivatives which contain at least the functional
portion of the antigen binding domain of an antibody that binds
specifically to B7-H1. Antibody fragments that contain the binding
domain of the molecule can be generated by known techniques. For
example, such fragments include, but are not limited to:
F(ab').sub.2 fragments which can be produced by pepsin digestion of
antibody molecules; Fab fragments which can be generated by
reducing the disulfide bridges of F(ab').sub.2 fragments; and Fab
fragments which can be generated by treating antibody molecules
with papain and a reducing agent. See, e.g., National Institutes of
Health, 1 Current Protocols In Immunology, Coligan et al., ed. 2.8,
2.10 (Wiley Interscience, 1991). Antibody fragments also include Fv
(e.g., single chain Fv (scFv)) fragments, i.e., antibody products
in which there are no constant region amino acid residues. Such
fragments can be produced, for example, as described in U.S. Pat.
No. 4,642,334 which is incorporated herein by reference in its
entirety.
[0100] The following examples are meant to illustrate, not limit,
the invention.
EXAMPLE 1
Materials and Methods
[0101] Cloning of hB7-H1 cDNA and construction of Ig fusion
proteins. The 5' and 3' ends of hB7-H1 cDNA were amplified by PCR
from a human placenta cDNA library synthesized by SMART PCR cDNA
synthesis kit (Clontech, Palo Alto, Calif.). The primer pairs used
for the PCR were derived from the placenta library plasmid and from
the expressed sequence tag (EST) clone AA292201. A cDNA clone that
included an orf encoding hB7-H1 of hB7-H1 cDNA was amplified by PCR
from the same cDNA library by specific primers and cloned into the
pcDNA3 vector (Invitrogen, Carlsbad, Calif.) and sequenced. The
amino acid sequences of hB7-H1, B7-1 and B7-2 were analyzed using
the ClustalW algorithm with BLOSUM 30 matrix (MacVector, Oxford
Molecular Group). The hB7-H1Ig fusion protein was prepared by
fusing the extracellular domain of hB7H-1 to the CH2-CH3 domain of
mouse IgG2a in the expression plasmid pmIgV and the resulting
construct was transfected into CHO cells. An identical method was
also used for preparation of B7-1Ig, CTLA4Ig and ICOSIg fusion
proteins. The fusion proteins were purified from culture
supernatants by passage over a Protein G-Sepharose affinity columns
(Pharmacia, Uppsala, Sweden) and the purified fusion proteins were
dialyzed into endotoxin-free PBS.
[0102] DNA transfection. Plasmids containing nucleic acid sequences
encoding full length hB7-H1 (pcDNA3-hB7-H1), B7-1 (pCDM8-B7.1) or
control parental vectors without coding sequences were transfected
into 293 cells or COS cells by calcium phosphate or DEAE-Dextran
transfection (Promega, Madison, Wis.). After 48 hours of
incubation, the expression levels of hB7-H1 or B7-1 on
transfectants were determined by FACS analysis with an antiserum
specific for hB7-H1 or anti-B7-1 monoclonal antibody (mAb)
(PharMingen), respectively.
[0103] T-cell and cytokine assays. PBMC were isolated from the
blood of healthy human volunteer donors by Ficoll-Hypaque gradient
centrifugation. The PBMC were passed through a nylon wool column to
obtain purified T cells (.about.85% of CD3.sup.+ cells), or were
subjected to further purification (>95% of CD3.sup.+ cells)
using an anti-CD4/8 MACS magnetic bead system (Miltenyl Biotec,
Germany). For co-stimulation assays, purified T cells at a
concentration of 1.times.10.sup.5 cells/well were cultured in
triplicate in 96-well flat-bottomed microtiter tissue culture
plates that were pre-coated overnight with antibody specific for
human CD3 (HIT3a, PharMingen, Palo Alto, Calif.) and either
hB7-H1Ig (5 .mu.g/ml) or control Ig (purified mouse IgG2a or murine
4-1BBIg fusion protein). In some experiments, the microtiter wells
were coated with only antibody specific for CD3 and B7-1- or
hB7-H1-transfected COS cells were used (10.sup.4 cells/well) as a
source of the co-stimulatory molecules. To measure cytokine
production, supernatants were collected at 24, 48 and 72 hours
after initiation of the cultures and the concentrations of IL-2,
IL-4, IFN-.gamma. and IL-10 were determined by sandwich ELISA
(PharMingen) according to the manufacturer's instructions. Wells
containing B7-1Ig or antibody specific for human CD28 (CD28.2,
PharMingen) were included for comparison or as a positive control,
respectively. T cell proliferation was determined by the addition
of 1.0 .mu.Ci [.sup.3H]-thymidine per well on day 2 followed by at
least 18 hours of additional culture. Incorporated
[.sup.3H]-thymidine was determined using a MicroBeta TriLux liquid
scintillation counter (Wallac, Finland).
[0104] For mixed lymphocyte reaction (MLR) assays, purified T cells
(2.times.10.sup.5 cells/well) were co-cultured in triplicate with
allogeneic PBMC (4000 Rad-irradiated) at 2.times.10.sup.5
cells/well in the presence of soluble hB7-H1Ig or control Ig. Four
days later, T cell proliferation was determined by
[.sup.3H]-thymidine incorporation. Neutralizing mAb specific for
human IL-2 (Clone MQ1-17H12, PharMingen) was added at 8 .mu.g/ml in
the beginning of T cell cultures. Polymyxin B at 10 .mu.g/ml was
also included in the assays of cell proliferation and cytokine
secretion to completely neutralize any contaminating endotoxin.
[0105] Nucleic acid analysis. Northern blot analysis was carried
out using commercially available human multiple tissue Northern
blots (Clontech, Palo Alto, Calif.). The membrane was incubated in
ExpressHyb hybridization solution (Clontech) for 30 min at
68.degree. C. The random-primed cDNA probe was full length hB7-H1
coding cDNA (870 bp), and was labeled using .sup.32P-dCTP. The
human .beta.-actin cDNA probe (2.0 kb) was used as a control.
Hybridization was carried out for 1 hr at 68.degree. C., the
membrane was washed 3 times in 2.times.SSC containing 0.05% SDS,
and was then exposed at -70.degree. C. to x-ray film.
[0106] Flow cytometry analysis. To prepare an antiserum specific
for hB7-H1, mice were immunized with purified hB7-H1Ig emulsified
in complete Freund's adjuvant (Sigma) and boosted three times with
hB7-H1Ig in incomplete Freund's adjuvant. Serum was collected and
the specificity was determined by ELISA and by FACS staining
(1:1000 dilution) of hB7-H1 cDNA-transfected 293 cells or COS
cells. Pre-injection mouse serum was used as control.
[0107] To prepare activated T and B cells, freshly isolated human
PBMC (10.times.10.sup.6 cells/ml) were activated with 5 .mu.g/ml of
PHA (Sigma) or 10 .mu.g/ml of LPS (Sigma), respectively. For
preparation of activated monocytes, adherent PBMCs were cultured in
1,500 IU/ml of recombinant human IFN-.alpha. (Biosource, Camarillo,
Calif.) and 100 ng/ml of LPS. All cultures were harvested and
analyzed at 48 hours. For direct immunofluorescence staining, T
cells were incubated at 4.degree. C. with 1 .mu.g of either
fluorescein-(FITC) or phycoerythrin-(PE) conjugated mAb for 30 min
and analyzed by FACScan flow cytometry (Becton Dickinson, Mountain
View, Calif.) with Cell Quest software (Becton Dickinson) as
described previously. The mAb specific for CD3 (UCHT1), CD4
(RPA-T4), CD8 (RPA-T8), CD14 (M5E2), CD19 (B43), CD28 (CD28.2),
CD80 (BB1) were purchased from PharMingen. For indirect
immunofluorescence staining, cells were first incubated with
anti-hB7-H1 antibody (1:1000), 5 .mu.g of ICOSIg or CTLA4Ig at
4.degree. C. After 30 min, the cells were washed and further
incubated with FITC-(Biosource, Camarillo, Calif.) or PE-conjugated
(Southern Biotechnology Associates, Inc., Birmingham, Ala.) goat
anti-human or anti-mouse IgG F(ab').sub.2 for 30 min at 4.degree.
C. The human or mouse IgG1 protein (Sigma) or mouse 4-1BBIg (mouse
4-1BB extracellular domain fused with the Fc of human IgG1 or mouse
IgG2a) was used as control Ig. In some experiments, Fc receptors
were blocked by human or mouse Ig before incubation with FITC- or
PE-conjugated mAbs.
EXAMPLE 2
Molecular Cloning and Expression Pattern of the hB7-H1 Gene
[0108] A homology search of the human cDNA EST database using
published human B7-1 and B7-2 amino acid sequences revealed an EST
sequence (GeneBank #AA292201) encoding a homologue to human B7-1
and B7-2 molecules. The 5'- and 3'-sequences were obtained by
several independent reverse transcriptase-coupled polymerase chain
reactions (RT-PCR) from a human placenta cDNA library utilizing
vector and EST sequences as primers. A 3,616 bp fragment that
included the hB7-H1 encoding orf was cloned and sequenced (SEQ ID
NO:5) (FIG. 1). The coding sequence for hB7-H1 (SEQ ID NO:2) spans
nucleotides 72-951 of SEQ ID NO:5. The amino acid sequence of
full-length hB7-H1 (SEQ ID NO:1) is shown in FIG. 2a. The
extracellular domain of hB7-H1 has greater homology to B7-1 (20%
amino acid identity) than to B7-2 (15%) (FIG. 2b) whereas its
cytoplasmic domain is highly divergent from that of B7-1 and B7-2
based on analysis using the McVector 6.5 software. The open reading
frame of the gene encodes a type I transmembrane protein of 290
amino acids consisting of a 22 amino acid signal peptide, Ig V-like
domain, and Ig C-like domains, a hydrophobic transmembrane domain
and a cytoplasmic tail of 30 amino acids (FIG. 2a). Four structural
cysteines (labeled by stars in FIG. 2b), which are apparently
involved in forming the disulfide bonds of the Ig V and Ig C
domains are well conserved in all B7 members (FIG. 2b) [Fargeas, C.
A. et al. (1995) J. Exp. Med. 182, 667-675; Bajorath, J. et al.
(1994) Protein Sci. 3, 2148-50; Linsley, P. S. et al. (1994)
Immunity 1, 793-801; Inaba, K. et al. (1994) J. Exp. Med. 180,
1849-60; Freeman, G. J. et al. (1995) Immunity 2, 523-532]. In
addition, the tyrosine residue in B7-1 (at position 87) and in B7-2
(at position 82) of the Ig V-like domain is conserved in hB7-H1 (at
position 81) (FIG. 2b).
[0109] Northern blot analysis revealed that expression of the
hB7-H1 mRNA was abundant in heart, skeletal muscle, placenta and
lung but was weak in thymus, spleen, kidney and liver (FIG. 3). The
hB7-H1 mRNA was not detectable in brain, colon, small intestine and
peripheral blood mononuclear cells (PBMC). In most of the tissues
in which hB7-H1 mRNA was detectable, two transcripts of
approximately 4.1 and 7.2 kb were found.
[0110] An expression plasmid containing the extracellular domain of
hB7-H1 fused in frame with the Fc portion (CH2 and CH3-domains) of
the mouse IgG2a was constructed. The resulting product, hB7-H1Ig
fusion protein, was purified from the supernatants of CHO cells
transfected with the plasmid and was used for immunization of the
mice to prepare a hB7-H1-specific antiserum. Fluorescence flow
cytometry analysis using the hB7-H1-specific antiserum showed that
freshly isolated CD3+ T and CD19+ B cells express negligible levels
of hB7-H1 while a fraction (.about.16%) of CD14+ monocytes
constitutively express hB7-H1. HB7-H1 can, however, be up-regulated
by cell activation. Approximately 30% of PHA-treated CD3+ T cells
and 90% of CD14+ monocytes (treated with IFN-.gamma. and LPS)
express hB7-H1. Only 6% of CD19+ B cells after LPS activation
express hB7-H1 (FIG. 4). Confirmatory results were obtained by
RT-PCR analysis.
[0111] Transfection of the plasmid pcDNA3-hB7-H1 into 293 cells
(B7-H1/293 cells) led to the expression of hB7-H1 as detected by
hB7-H1-specific antiserum (FIG. 5a). The binding of antibody was
eliminated by the inclusion of soluble hB7-H1Ig in the staining
mixture (FIG. 5a, arrow), thereby demonstrating specificity of the
antiserum. Neither CTLA4Ig nor ICOSIg bound to hB7-H1/293 cells.
Although both CTLA4Ig and ICOSIg bound to Raji cells, the binding
was not blocked by the inclusion of hB7-H1Ig (FIG. 5a, arrows).
Taken together with the observation that hB7-H1Ig did not bind to
Jurkat cells (FIG. 5b, right panel), despite their constitutive
expression of CD28 (FIG. 5b, left panel), the above results
indicate that hB7-H1 is not a ligand for CD28, CTLA-4, or ICOS.
EXAMPLE 3
Co-Stimulation of T Cell Proliferation by hB7-H1 Ligation
[0112] To assess whether hB7-H1 co-stimulates T-cell growth, T
cells purified (>95% purity) from PBMC of healthy human donors
were stimulated with hB7-H1Ig in the presence of suboptimal doses
of anti-CD3 mAb. T cell proliferation in 3-day cultures was
determined by incorporation of [.sup.3H]-thymidine. hB7-H1Ig,
immobilized on culture plates, enhanced T cell proliferation up to
10-fold compared to the control Ig in the presence of 5-20 ng/ml of
anti-CD3 mAb, also immobilized on the culture plates. In the
absence of anti-CD3 antibody, hB7-H1Ig at a concentration up to 5
.mu.g/ml induced no T cell proliferation (FIG. 6a). If hB7-H1Ig was
included in the cultures without immobilization, its co-stimulatory
effect was significantly decreased. Consistent with this
observation, the inclusion of soluble hB7-H1Ig at levels of 0.6-5
.mu.g/ml in allogeneic MLR moderately (.about.2-fold) increased the
proliferation of T cells (FIG. 6b). Thus, hB7-H1 can promote and
co-stimulate proliferative responses of T cells to polyclonal T
cell stimuli and to allogeneic antigens.
EXAMPLE 4
hB7-H1 Co-Stimulation Preferentially Induces the Production of
IL-10 and the Co-Stimulatory Effect Requires IL-2
[0113] The levels of IL-2, IL-4, IFN-.gamma., and IL-10 produced by
T cells after co-stimulation with hB7-H1Ig, B7-1Ig, or anti-CD28
mAb in the presence of anti-CD3 (FIG. 7a-7d) were measured. Similar
to B7-1Ig and anti-CD28, immobilized hB7-H1Ig antibody dramatically
increased the production of IL-10 by T cells in response to
immobilized anti-CD3 antibody after stimulation for 48 and 72 hours
(FIG. 7a). IL-10 was not detected if T cells were co-stimulated
with immobilized control Ig. The level of IFN-.gamma. was also
significantly elevated by co-stimulation with immobilized hB7-H1Ig
(FIG. 7b). In contrast to B7-1Ig and anti-CD28, hB7-H1Ig
co-stimulated low or negligible levels of IL-2 (FIG. 7c) and IL-4
(FIG. 7d), respectively. These observations were reproducible in
six independent experiments. These results show that co-stimulation
by hB7-H1 preferentially stimulates the production of IL-10.
[0114] The production of IL-2, although low, peaked at 24 hours
upon hB7-H1 co-stimulation (FIG. 7c), while IL-10 secretion started
to increase only after 48 and 72 hours (FIG. 7a). Increasing
concentrations of hB7-H1Ig led to a small increase (<1 ng/ml) of
IL-2 secretion (FIG. 7e). To determine the roles of the
early-produced IL-2, the effects of anti-IL-2 mAb on T cell
proliferation and IL-10 production in B7H-mediated co-stimulation
were tested. Similar to proliferation induced by B7-1-COS cells and
immobilized anti-CD3 antibody T cell proliferation induced by
hB7-H1-COS cells and anti-CD3 antibody was blocked by inclusion of
anti-IL-2 mAb (FIG. 8a). Furthermore, IL-10 secretion from
hB7-H1Ig-co-stimulated T cells was also inhibited by anti-IL-2 mAb
(FIG. 8b). Therefore, the hB7-H1 co-stimulation of both T cell
growth and IL-10 secretion is an IL-2-dependent process.
EXAMPLE 5
hB7-H1 Co-Stimulation Increases Apoptosis of Activated T Cells
[0115] To determine the effect of hB7-H1 ligation on the viability
of activated T cells, the proportion of live T cells remaining
after activation with an optimally activating dose of anti-CD3
antibody in the presence of immobilized hB7-H1Ig was determined by
trypan blue staining. A consistent decrease of alive T cells was
observed. At the end of culture, T cells were stained with annexin
V and propidium iodide (PI) to distinguish the early phase and late
phase of apoptosis, respectively. The apoptotic cells in early
phase (annexin V-positive, PI-negative) were significantly
increased to 24.8% in the presence of hB7-H1Ig compared to 14.2% in
the absence of hB7-H1Ig in 5 experiments (P=0.001). A
representative experiment is shown in FIG. 9a (upper panel).
Similar results were obtained using hB7-H1Ig-treated Jurkat cells
(control Ig: 38.3% vs. hB7-H1Ig: 54.6%) (FIG. 9a, lower panel). The
increased apoptosis was associated with upregulation of Fas and
FasL expression on hB7-H1 co-stimulated T cells (FIG. 9b). These
results indicated that hB7-H1 co-stimulation increased
activation-induced T cell apoptosis moderately, and the increased
apoptosis was associated with elevated expression of Fas and
FasL.
EXAMPLE 6
Production of Monoclonal Antibodies Specific for hB7-H1
[0116] Using standard protocols, BALB/c mice were immunized with
purified hB7-H1Ig and splenocytes from the immunized mice were
fused with X63-AG8.653 mouse myeloma cells. Five hybridoma lines
were found to secrete antibodies specific for hB7-H1 in that, as
detected by fluorescence flow cytometry, culture supernatants from
these hybridoma lines positively stained hB7-H1/293 cells but did
not stain control vector/293 cells. Furthermore, some of the
antibodies inhibited the co-stimulatory activity of hB7-H1.
EXAMPLE 7
Molecular Cloning and Expression Pattern of a Mouse B7-H1 (mB7-H1)
Gene
[0117] Starting with two overlapping mouse EST clones (AA823166 and
AA896104), and using a strategy similar to that for the hB7-H1
gene, a cDNA fragment that included an orf encoding mB7-H1 was
cloned. The coding sequence for mB7-H1 (SEQ ID NO:4) (FIG. 10) was
obtained and the amino acid sequence of mB7-H1 (SEQ ID NO:3) (FIG.
11) was derived from it. The length of mB7-H1 is identical to that
of hB7-H1 and it has the same conserved cysteine residues found in
hB7-H1 (see Example 2).
[0118] As for hB7-H1, mB7-H1 was found to be expressed on very few
B cells but on a high proportion of macrophages. The proportion of
expressing macrophages was similarly increased by activation. Also
like hB7-H1, mB7-H1 co-stimulated mouse T cell proliferation, as
well as the production of IL-10, IFN-.gamma., and low levels of
IL-2.
EXAMPLE 8
Expression of mB7-H1 on Transfected P815 Tumor Cells and Decreased
Growth Rate of the Transfected P815 Cells in Mice
[0119] Mouse (DBA/2) P815 mastocytoma cells were stably transfected
with a construct containing the coding sequence for mB7-H1
(mB7H.P815 cells). Using a PE-conjugated rat polyclonal antibody
specific for mB7-H1 (anti-mB7H/PE), mB7-H1 expression was detected
fluorescence flow cytometry on the mB7H.P815 cells (FIG. 14b) but
not on either mock transfected P815 cells (mock.P815) (FIG. 12b) or
P815 cells transfected with a construct encoding murine B7-1
(mB7-1.P815) (FIG. 13b). On the other hand, the mB7-1.P815 cells
were stained with a FITC-conjugated mAb specific for murine B7-1
(anti-mB7-1-FITC) (FIG. 13a).
[0120] Groups (5 mice per group) of DBA/2 mice were injected
subcutaneously (s.c.) with either 2.times.10.sup.5 mock.P815 or
mB7-1.P815 cells. The growth rate of the mock.P815 cells was
significantly greater in 4 out of 5 injected mice (FIG. 15a) than
in the 5 mice injected with mB7H.P815 (FIG. 15b). These findings
indicate that the mB7H-P815 cells were significantly more
immunogenic than mock.P815 cells and, therefore, that expression of
mB7-H1 expression by P815 cells enhances their ability to elicit
protective immunity.
[0121] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
12 1 290 PRT Homo sapiens 1 Met Arg Ile Phe Ala Val Phe Ile Phe Met
Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro
Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile
Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala
Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80
Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85
90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val
Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg
Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln
Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu
Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile
Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr
Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205
Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210
215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr
His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly
Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met
Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys
Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 2 870 DNA
Homo sapiens 2 atgaggatat ttgctgtctt tatattcatg acctactggc
atttgctgaa cgcatttact 60 gtcacggttc ccaaggacct atatgtggta
gagtatggta gcaatatgac aattgaatgc 120 aaattcccag tagaaaaaca
attagacctg gctgcactaa ttgtctattg ggaaatggag 180 gataagaaca
ttattcaatt tgtgcatgga gaggaagacc tgaaggttca gcatagtagc 240
tacagacaga gggcccggct gttgaaggac cagctctccc tgggaaatgc tgcacttcag
300 atcacagatg tgaaattgca ggatgcaggg gtgtaccgct gcatgatcag
ctatggtggt 360 gccgactaca agcgaattac tgtgaaagtc aatgccccat
acaacaaaat caaccaaaga 420 attttggttg tggatccagt cacctctgaa
catgaactga catgtcaggc tgagggctac 480 cccaaggccg aagtcatctg
gacaagcagt gaccatcaag tcctgagtgg taagaccacc 540 accaccaatt
ccaagagaga ggagaagctt ttcaatgtga ccagcacact gagaatcaac 600
acaacaacta atgagatttt ctactgcact tttaggagat tagatcctga ggaaaaccat
660 acagctgaat tggtcatccc agaactacct ctggcacatc ctccaaatga
aaggactcac 720 ttggtaattc tgggagccat cttattatgc cttggtgtag
cactgacatt catcttccgt 780 ttaagaaaag ggagaatgat ggatgtgaaa
aaatgtggca tccaagatac aaactcaaag 840 aagcaaagtg atacacattt
ggaggagacg 870 3 290 PRT Mus musculus 3 Met Arg Ile Phe Ala Gly Ile
Ile Phe Thr Ala Cys Cys His Leu Leu 1 5 10 15 Arg Ala Phe Thr Ile
Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn
Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu 35 40 45 Asp
Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val 50 55
60 Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn
65 70 75 80 Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys
Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp
Ala Gly Val Tyr 100 105 110 Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp
Tyr Lys Arg Ile Thr Leu 115 120 125 Lys Val Asn Ala Pro Tyr Arg Lys
Ile Asn Gln Arg Ile Ser Val Asp 130 135 140 Pro Ala Thr Ser Glu His
Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro 145 150 155 160 Glu Ala Glu
Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly 165 170 175 Lys
Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val 180 185
190 Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys
195 200 205 Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu
Leu Ile 210 215 220 Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn
Arg Thr His Trp 225 230 235 240 Val Leu Leu Gly Ser Ile Leu Leu Phe
Leu Ile Val Val Ser Thr Val 245 250 255 Leu Leu Phe Leu Arg Lys Gln
Val Arg Met Leu Asp Val Glu Lys Cys 260 265 270 Gly Val Glu Asp Thr
Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu 275 280 285 Glu Thr 290
4 873 DNA Mus musculus CDS (1)...(870) 4 atg agg ata ttt gct ggc
att ata ttc aca gcc tgc tgt cac ttg cta 48 Met Arg Ile Phe Ala Gly
Ile Ile Phe Thr Ala Cys Cys His Leu Leu 1 5 10 15 cgg gcg ttt act
atc acg gct cca aag gac ttg tac gtg gtg gag tat 96 Arg Ala Phe Thr
Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 ggc agc
aac gtc acg atg gag tgc aga ttc cct gta gaa cgg gag ctg 144 Gly Ser
Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu 35 40 45
gac ctg ctt gcg tta gtg gtg tac tgg gaa aag gaa gat gag caa gtg 192
Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val 50
55 60 att cag ttt gtg gca gga gag gag gac ctt aag cct cag cac agc
aac 240 Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser
Asn 65 70 75 80 ttc agg ggg aga gcc tcg ctg cca aag gac cag ctt ttg
aag gga aat 288 Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu
Lys Gly Asn 85 90 95 gct gcc ctt cag atc aca gac gtc aag ctg cag
gac gca ggc gtt tac 336 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln
Asp Ala Gly Val Tyr 100 105 110 tgc tgc ata atc agc tac ggt ggt gcg
gac tac aag cga atc acg ctg 384 Cys Cys Ile Ile Ser Tyr Gly Gly Ala
Asp Tyr Lys Arg Ile Thr Leu 115 120 125 aaa gtc aat gcc cca tac cgc
aaa atc aac cag aga att tcc gtg gat 432 Lys Val Asn Ala Pro Tyr Arg
Lys Ile Asn Gln Arg Ile Ser Val Asp 130 135 140 cca gcc act tct gag
cat gaa cta ata tgt cag gcc gag ggt tat cca 480 Pro Ala Thr Ser Glu
His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro 145 150 155 160 gaa gct
gag gta atc tgg aca aac agt gac cac caa ccc gtg agt ggg 528 Glu Ala
Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly 165 170 175
aag aga agt gtc acc act tcc cgg aca gag ggg atg ctt ctc aat gtg 576
Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val 180
185 190 acc agc agt ctg agg gtc aac gcc aca gcg aat gat gtt ttc tac
tgt 624 Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr
Cys 195 200 205 acg ttt tgg aga tca cag cca ggg caa aac cac aca gcg
gag ctg atc 672 Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala
Glu Leu Ile 210 215 220 atc cca gaa ctg cct gca aca cat cct cca cag
aac agg act cac tgg 720 Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln
Asn Arg Thr His Trp 225 230 235 240 gtg ctt ctg gga tcc atc ctg ttg
ttc ctc att gta gtg tcc acg gtc 768 Val Leu Leu Gly Ser Ile Leu Leu
Phe Leu Ile Val Val Ser Thr Val 245 250 255 ctc ctc ttc ttg aga aaa
caa gtg aga atg cta gat gtg gag aaa tgt 816 Leu Leu Phe Leu Arg Lys
Gln Val Arg Met Leu Asp Val Glu Lys Cys 260 265 270 ggc gtt gaa gat
aca agc tca aaa aac cga aat gat aca caa ttc gag 864 Gly Val Glu Asp
Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu 275 280 285 gag acg
taa 873 Glu Thr 290 5 3616 DNA Homo sapiens CDS (73)...(942) 5
cccacgcgtc cgcagcttcc cgaggctccg caccagccgc gcttctgtcc gcctgcaggg
60 cattccagaa ag atg agg ata ttt gct gtc ttt ata ttc atg acc tac
tgg 111 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp 1 5 10
cat ttg ctg aac gca ttt act gtc acg gtt ccc aag gac cta tat gtg 159
His Leu Leu Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val 15
20 25 gta gag tat ggt agc aat atg aca att gaa tgc aaa ttc cca gta
gaa 207 Val Glu Tyr Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val
Glu 30 35 40 45 aaa caa tta gac ctg gct gca cta att gtc tat tgg gaa
atg gag gat 255 Lys Gln Leu Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu
Met Glu Asp 50 55 60 aag aac att att caa ttt gtg cat gga gag gaa
gac ctg aag gtt cag 303 Lys Asn Ile Ile Gln Phe Val His Gly Glu Glu
Asp Leu Lys Val Gln 65 70 75 cat agt agc tac aga cag agg gcc cgg
ctg ttg aag gac cag ctc tcc 351 His Ser Ser Tyr Arg Gln Arg Ala Arg
Leu Leu Lys Asp Gln Leu Ser 80 85 90 ctg gga aat gct gca ctt cag
atc aca gat gtg aaa ttg cag gat gca 399 Leu Gly Asn Ala Ala Leu Gln
Ile Thr Asp Val Lys Leu Gln Asp Ala 95 100 105 ggg gtg tac cgc tgc
atg atc agc tat ggt ggt gcc gac tac aag cga 447 Gly Val Tyr Arg Cys
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg 110 115 120 125 att act
gtg aaa gtc aat gcc cca tac aac aaa atc aac caa aga att 495 Ile Thr
Val Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile 130 135 140
ttg gtt gtg gat cca gtc acc tct gaa cat gaa ctg aca tgt cag gct 543
Leu Val Val Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala 145
150 155 gag ggc tac ccc aag gcc gaa gtc atc tgg aca agc agt gac cat
caa 591 Glu Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His
Gln 160 165 170 gtc ctg agt ggt aag acc acc acc acc aat tcc aag aga
gag gag aag 639 Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg
Glu Glu Lys 175 180 185 ctt ttc aat gtg acc agc aca ctg aga atc aac
aca aca act aat gag 687 Leu Phe Asn Val Thr Ser Thr Leu Arg Ile Asn
Thr Thr Thr Asn Glu 190 195 200 205 att ttc tac tgc act ttt agg aga
tta gat cct gag gaa aac cat aca 735 Ile Phe Tyr Cys Thr Phe Arg Arg
Leu Asp Pro Glu Glu Asn His Thr 210 215 220 gct gaa ttg gtc atc cca
gaa cta cct ctg gca cat cct cca aat gaa 783 Ala Glu Leu Val Ile Pro
Glu Leu Pro Leu Ala His Pro Pro Asn Glu 225 230 235 agg act cac ttg
gta att ctg gga gcc atc tta tta tgc ctt ggt gta 831 Arg Thr His Leu
Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val 240 245 250 gca ctg
aca ttc atc ttc cgt tta aga aaa ggg aga atg atg gat gtg 879 Ala Leu
Thr Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val 255 260 265
aaa aaa tgt ggc atc caa gat aca aac tca aag aag caa agt gat aca 927
Lys Lys Cys Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr 270
275 280 285 cat ttg gag gag acg taatccagca ttggaacttc tgatcttcaa
gcagggattc 982 His Leu Glu Glu Thr 290 tcaacctgtg gtttaggggt
tcatcggggc tgagcgtgac aagaggaagg aatggacccg 1042 tgggatgcag
gcaatgtggg acttaaaagg cccaagcact gaaaatggaa cctggcgaaa 1102
gcagaggagg agaatgaaga aagatggagt caaacaggga gcctggaggg agaccttgat
1162 actttcaaat gcctgagggg ctcatcgacg cctgtgacag ggagaaagga
tacttctgaa 1222 caaggagcct ccaagcaaat catccattgc tcatcctagg
aagacgggtt gagaatccct 1282 aatttgaggg tcagttcctg cagaagtgcc
ctttgcctcc actcaatgcc tcaatttctt 1342 ttctgcatga ctgagagtct
cagtgttgga acgggacagt atttatgtat gagtttttcc 1402 tatttatttt
gagtctgtga ggtcttcttg tcatgtgagt gtggttgtga atgatttctt 1462
ttgaagatat attgtagtag atgttacaat tttgtcgcca aactaaactt gctgcttaat
1522 gatttgctca catctagtaa aacatggagt atttgtaagg tgcttggtct
cctctataac 1582 tacaagtata cattggaagc ataaagatca aaccgttggt
tgcataggat gtcaccttta 1642 tttaacccat taatactctg gttgacctaa
tcttattctc agacctcaag tgtctgtgca 1702 gtatctgttc catttaaata
tcagctttac aattatgtgg tagcctacac acataatctc 1762 atttcatcgc
tgtaaccacc ctgttgtgat aaccactatt attttaccca tcgtacagct 1822
gaggaagcaa acagattaag taacttgccc aaaccagtaa atagcagacc tcagactgcc
1882 acccactgtc cttttataat acaatttaca gctatatttt actttaagca
attcttttat 1942 tcaaaaacca tttattaagt gcccttgcaa tatcaatcgc
tgtgccaggc attgaatcta 2002 cagatgtgag caagacaaag tacctgtcct
caaggagctc atagtataat gaggagatta 2062 acaagaaaat gtattattac
aatttagtcc agtgtcatag cataaggatg atgcgagggg 2122 aaaacccgag
cagtgttgcc aagaggagga aataggccaa tgtggtctgg gacggttgga 2182
tatacttaaa catcttaata atcagagtaa ttttcattta caaagagagg tcggtactta
2242 aaataaccct gaaaaataac actggaattc cttttctagc attatattta
ttcctgattt 2302 gcctttgcca tataatctaa tgcttgttta tatagtgtct
ggtattgttt aacagttctg 2362 tcttttctat ttaaatgcca ctaaatttta
aattcatacc tttccatgat tcaaaattca 2422 aaagatccca tgggagatgg
ttggaaaatc tccacttcat cctccaagcc attcaagttt 2482 cctttccaga
agcaactgct actgcctttc attcatatgt tcttctaaag atagtctaca 2542
tttggaaatg tatgttaaaa gcacgtattt ttaaaatttt tttcctaaat agtaacacat
2602 tgtatgtctg ctgtgtactt tgctattttt atttatttta gtgtttctta
tatagcagat 2662 ggaatgaatt tgaagttccc agggctgagg atccatgcct
tctttgtttc taagttatct 2722 ttcccatagc ttttcattat ctttcatatg
atccagtata tgttaaatat gtcctacata 2782 tacatttaga caaccaccat
ttgttaagta tttgctctag gacagagttt ggatttgttt 2842 atgtttgctc
aaaaggagac ccatgggctc tccagggtgc actgagtcaa tctagtccta 2902
aaaagcaatc ttattattaa ctctgtatga cagaatcatg tctggaactt ttgttttctg
2962 ctttctgtca agtataaact tcactttgat gctgtacttg caaaatcaca
ttttctttct 3022 ggaaattccg gcagtgtacc ttgactgcta gctaccctgt
gccagaaaag cctcattcgt 3082 tgtgcttgaa cccttgaatg ccaccagctg
tcatcactac acagccctcc taagaggctt 3142 cctggaggtt tcgagattca
gatgccctgg gagatcccag agtttccttt ccctcttggc 3202 catattctgg
tgtcaatgac aaggagtacc ttggctttgc cacatgtcaa ggctgaagaa 3262
acagtgtctc caacagagct ccttgtgtta tctgtttgta catgtgcatt tgtacagtaa
3322 ttggtgtgac agtgttcttt gtgtgaatta caggcaagaa ttgtggctga
gcaaggcaca 3382 tagtctactc agtctattcc taagtcctaa ctcctccttg
tggtgttgga tttgtaaggc 3442 actttatccc ttttgtctca tgtttcatcg
taaatggcat aggcagagat gatacctaat 3502 tctgcatttg attgtcactt
tttgtacctg cattaattta ataaaatatt cttatttatt 3562 ttgttacttg
gtaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 3616 6 25 PRT Homo
sapiens 6 Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile
Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala 20 25 7 5 PRT
Bovidae 7 Lys Phe Glu Arg Gln 1 5 8 15 PRT Homo sapiens 8 Met Asp
Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro 1 5 10 15 9 4
PRT Rattus rattus 9 Lys Asp Glu Leu 1 10 210 PRT Homo sapiens 10
Val Glu Tyr Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu 1 5
10 15 Lys Gln Leu Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu
Asp 20 25 30 Lys Asn Ile Ile Gln Phe Val His Gly Glu Glu Asp Leu
Lys Val Gln 35 40 45 His Ser Ser Tyr Arg Gln Arg Ala Arg Leu Leu
Lys Asp Gln Leu Ser 50 55 60 Leu Gly Asn Ala Ala Leu Gln Ile Thr
Asp Val Lys Leu Gln Asp Ala 65 70 75 80 Gly Val Tyr Arg Cys Met Ile
Ser Tyr Gly Gly Ala Asp Tyr Lys Arg 85 90 95 Ile Thr Val Lys Val
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile 100 105 110 Leu Val Val
Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala 115 120 125 Glu
Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln 130 135
140 Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys
145 150 155 160 Leu Phe Asn Val Thr Ser Thr Leu Arg Ile Asn Thr Thr
Thr Asn Glu 165 170 175 Ile Phe Tyr Cys Thr Phe Arg Arg Leu Asp Pro
Glu Glu Asn His Thr 180 185 190 Ala Glu Leu Val Ile Pro Glu Leu Pro
Leu Ala His Pro Pro Asn Glu 195 200 205 Arg Thr 210 11 205 PRT Homo
sapiens 11 Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn
Val Ser 1 5 10 15 Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp
Gln
Lys Glu Lys Lys 20 25 30 Met Val Leu Thr Met Met Ser Gly Asp Met
Asn Ile Trp Pro Glu Tyr 35 40 45 Lys Asn Arg Thr Ile Phe Asp Ile
Thr Asn Asn Leu Ser Ile Val Ile 50 55 60 Leu Ala Leu Arg Pro Ser
Asp Glu Gly Thr Tyr Glu Cys Val Val Leu 65 70 75 80 Lys Tyr Glu Lys
Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr 85 90 95 Leu Ser
Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu 100 105 110
Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly 115
120 125 Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu
Asn 130 135 140 Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu Thr Glu
Leu Tyr Ala 145 150 155 160 Val Ser Ser Lys Leu Asp Phe Asn Met Thr
Thr Asn His Ser Phe Met 165 170 175 Cys Leu Ile Lys Tyr Gly His Leu
Arg Val Asn Gln Thr Phe Asn Trp 180 185 190 Asn Thr Thr Lys Gln Glu
His Phe Pro Asp Asn Leu Leu 195 200 205 12 218 PRT Homo sapiens 12
Ala Tyr Phe Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser 1 5
10 15 Gln Asn Gln Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln
Glu 20 25 30 Asn Leu Val Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys
Phe Asp Ser 35 40 45 Val His Ser Lys Tyr Met Gly Arg Thr Ser Phe
Asp Ser Asp Ser Trp 50 55 60 Thr Leu Arg Leu His Asn Leu Gln Ile
Lys Asp Lys Gly Leu Tyr Gln 65 70 75 80 Cys Ile Ile His His Lys Lys
Pro Thr Gly Met Ile Arg Ile His Gln 85 90 95 Met Asn Ser Glu Leu
Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile 100 105 110 Val Pro Ile
Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys 115 120 125 Ser
Ser Ile His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu 130 135
140 Arg Thr Lys Asn Ser Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser
145 150 155 160 Gln Asp Asn Val Thr Glu Leu Tyr Asp Val Ser Ile Ser
Leu Ser Val 165 170 175 Ser Phe Pro Asp Val Thr Ser Asn Met Thr Ile
Phe Cys Ile Leu Glu 180 185 190 Thr Asp Lys Thr Arg Leu Leu Ser Ser
Pro Phe Ser Ile Glu Leu Glu 195 200 205 Asp Pro Gln Pro Pro Pro Asp
His Ile Pro 210 215
* * * * *
References