U.S. patent application number 14/844914 was filed with the patent office on 2016-04-07 for b7-h1, a novel immunoregulatory molecule.
This patent application is currently assigned to MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH. The applicant listed for this patent is MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH. Invention is credited to Lieping Chen.
Application Number | 20160096889 14/844914 |
Document ID | / |
Family ID | 27036314 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160096889 |
Kind Code |
A1 |
Chen; Lieping |
April 7, 2016 |
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; (Hamden,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH |
Rochester |
MN |
US |
|
|
Assignee: |
MAYO FOUNDATION FOR MEDICAL
EDUCATION AND RESEARCH
Rochester
MN
|
Family ID: |
27036314 |
Appl. No.: |
14/844914 |
Filed: |
September 3, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14617500 |
Feb 9, 2015 |
|
|
|
14844914 |
|
|
|
|
12421310 |
Apr 9, 2009 |
8981063 |
|
|
14617500 |
|
|
|
|
12347492 |
Dec 31, 2008 |
9062112 |
|
|
12421310 |
|
|
|
|
09649108 |
Aug 28, 2000 |
|
|
|
12347492 |
|
|
|
|
09451291 |
Nov 30, 1999 |
6803192 |
|
|
09649108 |
|
|
|
|
Current U.S.
Class: |
435/69.6 |
Current CPC
Class: |
C07K 2319/74 20130101;
C07K 16/2827 20130101; C07K 14/70532 20130101; G01N 33/502
20130101; G01N 2333/70532 20130101; C07K 2317/526 20130101; G01N
33/505 20130101; C07K 2317/24 20130101; C07K 16/00 20130101; A61K
2035/124 20130101; C07K 14/47 20130101; C07K 2317/76 20130101; C07K
2317/524 20130101; A61K 38/00 20130101; A61K 39/39 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Goverment Interests
STATEMENT OF FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under grant
number CA079915 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-48. (canceled)
49. A method for recovering an antibody from hydridoma supernatant,
comprising culturing an antibody-producing hybridoma in vitro in
suitable medium to produce supernatant and recovering antibodies
produced by said antibody-producing hybridoma from said
supernatant, thereby obtaining said antibody, wherein said antibody
is an anti-B7-H1 antibody.
50. The method of claim 49, wherein said anti-B7-H1 antibody is an
anti-hB7-H1 antibody.
51. The method of claim 49, wherein said anti-B7-H1 antibody binds
specifically to a B7-H1 polypeptide comprising the amino acid
sequence set forth in SEQ ID NO:1.
52. The method of claim 49, wherein said anti-B7-H1 antibody is a
chimeric monoclonal antibody.
53. The method of claim 49, wherein said anti-B7-H1 antibody is a
humanized monoclonal antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/617,500, filed Feb. 9, 2015, which is a continuation of U.S.
application Ser. No. 12/421,310, filed on Apr. 9, 2009 (now U.S.
Pat. No. 8,981,063), which is continuation of U.S. application
Serial application Ser. No. 12/347,492, filed Dec. 31, 2008 (now
U.S. Pat. No. 9,062,112), which is a continuation of U.S. patent
application Ser. No. 09/649,108, filed Aug. 28, 2000 (now
abandoned), which is a continuation-in-part of U.S. application
Ser. No. 09/451,291, filed Nov. 30, 1999 (now U.S. Pat. No.
6,803,192), all of which are hereby expressly incorporated by
reference into the present application.
BACKGROUND OF THE INVENTION
[0003] The invention is generally in the field of immunoregulation,
and specifically T cell response regulation.
[0004] 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
[0005] 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 on 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 the
polypeptides, 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.
[0006] 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, by 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 sequence with SEQ ID
NO:2 or SEQ ID NO:4. The nucleic acid sequence can also encode
functional fragments of these B7-H1 polypeptides.
[0007] 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 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, or 32, 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.
[0008] 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.
[0009] 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-H 1
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. In addition, in
any of the methods, the T cell can be a helper T cell, e.g., a T
cell that helps an effector (e.g., a cytotoxic T lymphocyte (CTL)
or B cell antibody) response. An antibody response can be, for
example, an IgM, IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, or IgA
antibody response. Co-stimulation of a T cell by any of the B7-H1
agents can result in an increase in the level of CD40 ligand on the
surface of the T cell.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] "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.
[0014] 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."
[0015] 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.
[0016] An "isolated DNA" means DNA free of one or both of the genes
that flank the gene containing the DNA of interest in the genome of
the organism in which the gene containing the DNA 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. It will be apparent from the
foregoing that isolated DNA does not mean a DNA present among
hundreds to millions of other DNA molecules within, for example,
cDNA or genomic DNA libraries or genomic DNA restriction digests
in, for example, a restriction digest reaction mixture or an
electrophoretic gel slice.
[0017] 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 be an effector (e.g., CTL or
antibody-producing B cell) response, a helper response providing
help for one or more effector (e.g., CTL or antibody-producing B
cell) responses, or a suppressive response.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] As used herein, the term "antibody" refers not only to whole
antibody molecules, but also to antigen-binding fragments, e.g.,
Fab, F(ab')2, Fv, and single chain Fv fragments. Also included are
chimeric antibodies.
[0023] 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.
[0024] 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.
[0025] 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
[0026] 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.
[0027] FIG. 2a is a depiction of the amino acid sequence of hB7-H1
(SEQ ID NO:1). The signal peptide, Ig-V-like domain, Ig-C-like
domain, and transmembrane ("TM") domain are indicated. Potential
N-linked glycosylation sites are indicated by *.
[0028] FIG. 2b is a depiction of the amino acid sequences of the
extracellular domains of hB7-H1 (SEQ ID NO:10), human B7-1 (hB7-1;
SEQ ID NO:11), and human B7-2 (hB7-2; SEQ ID NO:12) aligned for
maximum homology. Identical amino acid residues are shaded in bold
and conserved residues are boxed. Conserved cysteine residues are
indicated by *.
[0029] FIG. 3 is a photograph of a Northern blot showing expression
of hB7-H1 mRNA in various human tissues.
[0030] FIG. 4a is a series of two-dimensional fluorescence flow
cytometry histograms showing cell surface expression of hB7-H1 on
resting CD3+ T cells, CD19+ B cells, and CD 14+ monocytes.
[0031] FIG. 4b is a series of two-dimensional fluorescence flow
cytometry histograms showing cell surface expression of hB7-H1 on
activated CD3+ T cells, CD19+ B cells, and CD 14+ monocytes.
[0032] 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 ("hB7-H1/293 cells"), or Raji cells.
[0033] FIG. 5b is a series of fluorescence flow cytometry
histograms showing the binding of hB7-H1Ig and antibody to CD28 to
Jurkat cells.
[0034] FIG. 6a is a line graph showing the ability of immobilized
hB7-H1Ig to co-stimulate the proliferative response of human T
cells to immobilized antibody specific for human CD3.
[0035] FIG. 6b is a line graph showing the ability of soluble
hB7-H1Ig to co-stimulate the proliferative response of human T
cells to irradiated allogeneic PBMC.
[0036] FIGS. 7a-7d are a series of line graphs showing the ability
of hB7-H1Ig, human B7-1Ig, or antibody specific for human CD28 to
co-stimulate the production of interleukin-(IL-)10 (FIG. 7a),
interferon-.gamma. (IFN-.gamma.) (FIG. 7b), IL-2 (FIG. 7c), or IL-4
(FIG. 7d) by human T cells responding to immobilized antibody
specific for CD3. FIG. 7e is a line graph showing the ability of
various concentrations of hB7-H1Ig to co-stimulate the production
of IL-2 by human T cells responding to immobilized antibody
specific for CD3.
[0037] FIG. 8a is a bar graph showing the ability of antibody
specific for human 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 human
B7-1.
[0038] FIG. 8b is a bar graph showing the ability of antibody
specific for human 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.
[0039] FIG. 9a is a series of two-dimensional fluorescence flow
cytometry profiles showing the relative proportion of T cells in
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.
[0040] 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.
[0041] FIG. 10 is a depiction of the nucleotide sequence of cDNA
encoding mB7-H1 (SEQ ID NO:4).
[0042] FIG. 11 is a depiction of the amino acid sequence of mB7-H1
(SEQ ID NO:3).
[0043] FIG. 12a1-2 are depictions of the amino acid sequence of
mB7-H1 (SEQ ID NO:3) aligned with the amino acid sequence of hB7-H1
(SEQ ID NO:1). The signal peptide, IgV-like domain, IgC-like
domain, transmembrane ("TM") domain, and cytoplasmic domain
("cytoplasmic") of mB7-H1 are indicated.
[0044] FIG. 12b1-3 are depictions of the amino acid sequences of
mB7-H1, mouse B7-1 (mB7-1; SEQ ID NO:13), mouse B7-2 (mB7-2: SEQ ID
NO:14), and mB7h/B7KF-1 (SEQ ID NO:15) aligned for maximum
homology. Identical amino acid residues are shaded and conserved
amino acid residues are boxed. Conserved cysteine residues are
indicated by *.
[0045] FIG. 13 is a series of fluorescence flow cytometry
histograms showing relative levels of cell surface expression of
mB7-H1 on resting and activated mouse CD3+ T cells, mouse B220+ B
cells, and Mac-1+ mouse macrophages.
[0046] FIG. 14a is a line graph showing the ability of various
concentrations of immobilized mB7-H1Ig or immobilized control Ig to
co-stimulate the in vitro proliferative response of mouse T cells
to a suboptimal dose of immobilized antibody specific for mouse
CD3.
[0047] FIG. 14b is a pair of line graphs showing the ability of
immobilized mB7-H1Ig, immobilized control Ig, or soluble antibody
specific for mouse CD28 to co-stimulate the in vitro proliferative
response of wild type ("wt") C57BL/6 mouse (left graph) or
CD28-deficient ("CD28.sup.-/-) C57BL/6 mouse (right graph) T cells
to two suboptimal doses of immobilized antibody specific for mouse
CD3.
[0048] FIG. 14c is a bar graph showing the ability of immobilized
mB7-H1Ig or immobilized control Ig to co-stimulate the in vitro
proliferative response of purified CD4+ or CD8+ mouse T cells to a
suboptimal dose of immobilized antibody specific for mouse CD3.
[0049] FIG. 15a is a series of line graphs showing the ability of
immobilized mB7-H1Ig, immobilized control Ig, or soluble antibody
specific for mouse CD28 ("Anti-CD28") to stimulate the in vitro
production (on days one, two, and three after initiation of the
cultures) of various cytokines by C57BL/6 mouse T cells in response
to immobilized antibody specific for mouse CD3.
[0050] FIG. 15b is a line graph showing the effect of immobilized
mB7-H1Ig on the in vitro production (18, 24, 36, and 48 hours after
initiation of the cultures) of IL-2 by C57BL/6 mouse T cells
responding to immobilized antibody specific for mouse CD3 and
co-stimulated by soluble antibody specific for mouse CD28
("Anti-CD28").
[0051] FIG. 16a and FIG. 16b are fluorescence flow cytometry
histograms showing lack of surface expression of murine B7-1 (FIG.
16a) and mB7-H1 (FIG. 16b) on P815 cells transfected with a control
expression vector ("mock.P815").
[0052] FIG. 17a and FIG. 17b are fluorescence flow cytometry
histograms showing surface expression of murine B7-1 (FIG. 17a) and
lack of surface expression of mB7-H1 (FIG. 17b) on P815 cells
transfected with an expression vector containing a nucleic acid
sequence encoding murine B7-1 ("mB7-1.sup.+ P815").
[0053] FIG. 18a and FIG. 18b are fluorescence flow cytometry
histograms showing lack of surface expression of mB7-1 (FIG. 18a)
and showing surface expression of mB7-H1 (FIG. 18b) on P815 cells
transfected with an expression vector containing a nucleic acid
sequence encoding mB7-H1 ("mB7-H1.sup.+ P815").
[0054] FIG. 19a and FIG. 19b 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. 19a) or an
expression vector containing a nucleic acid sequence encoding
mB7-H1 (FIG. 19b).
[0055] FIG. 20a is a pair of line graphs showing the ability of
control mock-transfected P815 cells ("mock.P815"), P815 cells
transfected with an expression vector containing a cDNA sequence
encoding mB7-H1 ("mB7-H1.sup.+ P815"), or P815 cells transfected
with an expression vector containing a cDNA sequence encoding mB7-1
("mB7-1.sup.+ P815") to activate allospecific C57BL/6 mouse CTL in
vitro. The effector cell populations were tested for cytotoxic
activity ("% Lysis") at various effector to target cell (E/T)
ratios against wild type P815 (left graph) and control EL4 (right
graph) target cells.
[0056] FIG. 20b is a pair of line graphs showing the ability of
control mock-transfected P815 cells ("mock. P815"), P815 cells
transfected with an expression vector containing a cDNA sequence
encoding mB7-H1 ("mB7-H1.sup.+ P815"), or P815 cells transfected
with an expression vector containing a cDNA sequence encoding mB7-1
("mB7-1.sup.+ P815") to activate tumor-specific DBA/2 mouse CTL in
vivo. The effector cell populations were tested for cytotoxic
activity ("% Lysis") at various E/T ratios against wild type P815
(left graph) and control L1210 (right graph) target cells.
[0057] FIG. 21 is a pair of line graphs showing the in vitro
proliferative responses to various concentrations of keyhole limpet
hemacyanin (KLH) of draining lymph node (left graph) or spleen
(right graph) T cells from C57BL/6 immunized intraperitoneally
(i.p.) with trinitrophenol (TNP) conjugated KLH (TNP-KLH) in
incomplete Freund's adjuvant and subsequently injected i.p. with
mB7-H1Ig or control Ig.
[0058] FIG. 22 is a series of bar graphs showing the relative
levels (as measured by ELISA) of IgG1, IgG2a, IgG2b, IgG3, or IgM
antibodies specific for TNP in the sera of C57BL/6 mice injected
i.p. with TNP-KLH in phosphate buffered saline and either control
Ig, mB7-H1Ig, or mB7-1Ig.
[0059] FIG. 23a is a series of fluorescence flow cytometry
histograms showing the relative levels of cell surface expression
of CD40 ligand (CD40L) (as measured by the binding of an antibody
specific for mouse CD40L ("anti-CD40L")) on purified mouse CD4+ T
cells (at 4 and 24 hours after initiation of the cultures)
activated by immobilized antibody specific for mouse CD3
("anti-CD3") coated onto the bottoms of tissue culture wells at a
concentration of 200 ng/ml and co-stimulated by immobilized control
Ig, immobilized mB7-H1Ig, or soluble antibody specific for mouse
CD28 ("anti-CD28").
[0060] FIG. 23b is a series of fluorescence flow cytometry
histograms showing the relative levels of cell surface expression
of CD40 ligand (CD40L) (as measured by the binding of an antibody
specific for mouse CD40L ("anti-CD40L")) on purified mouse CD4+ T
cells (at 4 and 24 hours after initiation of the cultures)
activated by immobilized antibody specific for mouse CD3
("anti-CD3") coated onto the bottoms of tissue culture wells at a
concentration of 1,000 ng/ml and co-stimulated by immobilized
control Ig, immobilized mB7-H1Ig, or soluble antibody specific for
mouse CD28 ("anti-CD28").
DETAILED DESCRIPTION
[0061] 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.
[0062] 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).
[0063] 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 CD14+ monocytes constitutively expressed the molecule
on their surface. Activation of T cells increased expression such
that about 30% expressed cell-surface hB7-H1.
[0064] Activation resulted in about 90% of monocytes expressing
hB7-1H, but only about 6% of B cells expressed it after
activation.
[0065] 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.
[0066] 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 was
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.
[0067] In addition, using a strategy similar to 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) and has the same domain structure as
hB7-H1. Moreover, mB7-H1 has a similar tissue distribution to
hB7-H1. mB7-H1 co-stimulated the response of mouse T cells with its
effect being more potent on CD4+ than on CD8+ T cells. In addition,
like hB7-H1, mB7-H1 co-stimulates the production of high levels of
IL-10 by T cells. mB7-H1 also enhanced the production of both
interferon-.gamma. (IFN-.gamma.) and granulocyte macrophage-colony
stimulating factor (GM-CSF) by T cells. While mB7-H1 showed no
significant ability to enhance CTL responses, it did greatly
increase antibody responses and, in particular, IgG2a antibody
responses. Finally, co-stimulation of T cells with mB7-H1 caused an
increase in the level of CD40 ligand (CD40L) on the surface of the
T cells.
[0068] B7-H1 can be useful as an augmenter of immune responses
(e.g., helper T cell and antibody 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) its 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)).
[0069] B7-H1 Nucleic Acid Molecules
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.,)(BLAST and NBLAST) are used (See
http://www.ncbi.nlm.nih.gov).
[0078] 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.
[0079] The invention also encompasses: (a) vectors that contain any
of the foregoing B7-H1-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.
[0080] 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.
[0081] 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 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.
[0082] 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.sup.r, G418.sup.r), dihydrofolate reductase
(DHFR), hygromycin-B-phosphotransferase (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 heavy chain.
[0083] 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.
[0084] Polypeptides and Polypeptide Fragments
[0085] 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. The
polypeptides can be any of those described above but with one or
more (e.g., one, two, three, four, five, six, seven, eight, nine,
10, 12, 14, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or
more) conservative substitutions.
[0086] 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].
[0087] Polypeptides and fragments of the invention also include
those described above, but modified for in vivo use by the
addition, at 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] Methods of Co-Stimulating a T Cell
[0092] The methods of the invention involve contacting a T cell
with a B7-H1 polypeptide of the invention, or a functional fragment
thereof, in order to co-stimulate the T cell. Such polypeptides or
functional fragments can have amino acid sequences identical to
wild-type sequences or they can contain one or more (e.g., one,
two, three, four, five, six, seven, eight, nine, 10, 12, 14, 17,
20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) conservative
substitutions. 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.
[0093] 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).
[0094] The B7-H1 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)).
[0095] 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.
[0096] In Vivo Approaches
[0097] 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-acceptable 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] Ex Vivo Approaches
[0106] 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.
[0107] 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
particularly 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.
[0108] 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.
[0109] 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.
[0110] Methods of Screening for Compounds that Inhibit or Enhance
Immune Responses.
[0111] 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).
Useful B7-H1 polypeptides include those with amino acid sequences
identical to wild-type sequences or they can contain one or more
(e.g., one, two, three, four, five, six, seven, eight, nine, 10,
12, 14, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more)
conservative substitutions. The B7-H1 polypeptide 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.
[0112] 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.
[0113] 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.
[0114] B7-H1 Antibodies
[0115] 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.
[0116] 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.
[0117] 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, 522-25; Veroeyan et al. (1988) Science 239,
1534; and Beidler et al. (1988) J. Immunol. 141, 4053-60.
[0118] 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.
[0119] The following examples are meant to illustrate, not limit,
the invention.
Example 1
Materials and Methods
[0120] Cloning of hB7-H1 cDNA and Construction of Ig Fusion
Proteins.
[0121] 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 analogous 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.
[0122] DNA Transfection.
[0123] 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 fluorescence flow cytometry (FFC)
with an antiserum specific for hB7-H1 or anti-B7-1 monoclonal
antibody (mAb) (PharMingen), respectively.
[0124] Mice and Cell Lines.
[0125] Female C57BL/6 (B6), DBA/2, and BALB/c mice were purchased
from the National Cancer Institute (Frederick, Md.). CD28.sup.-/-
mice with a B6 genetic background were kindly provided by Dr. Moses
Rodrigues (Department of Immunology, Mayo Clinic, Rochester,
Minn.). P815 mastocytoma, L1210 lymphoma, EL4 mouse T-cell lymphoma
and 293 human kidney epithelial cells were purchased from the
American Type Culture Collection (Manassas, Va.). Cell lines were
maintained in a complete medium containing RPMI-1640 (Life
Technologies, Rockville, Md.) supplemented with 10% fetal bovine
serum (FBS) (HyClone, Logan, Utah), 25 mM HEPES, penicillin G (100
U/ml) and streptomycin sulfate (100 .mu.g/ml).
[0126] T-Cell and Cytokine Assays.
[0127] For human T cell studies, 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).
[0128] For mixed lymphocyte reaction (MLR) assays, purified human T
cells (2.times.10.sup.5 cells/well) were co-cultured in triplicate
with allogeneic human 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 (10 .mu.g/ml) was
also included in the assays of cell proliferation and cytokine
secretion to completely neutralize any contaminating endotoxin.
[0129] For mouse T cell studies, T cells were purified by passing
lymph node or spleen cells through a nylon wool column. CD4+ or
CD8+ T cells were positively selected by magnetic sorting using
FITC-conjugated mAb against CD4 or CD8 and microbeads coated with
antibody specific for fluorescein isothiocyanate (FITC)
(MiltenyiBiotec, Auburn, Calif.) according to the manufacturer's
instructions. The purity of isolated CD4.sup.+ and CD8.sup.+ T
cells was >95% by FFC with mAb specific for mouse CD4 and CD8,
respectively. Purified T cells at 2.times.10.sup.6/ml from mouse
spleens were cultured in 96-well plates that were pre-coated with
mAb specific for mouse CD3 in the presence of mB7-H1Ig or control
mouse IgG2a ("control Ig") also coated onto the culture well
bottoms. mAb specific for mouse CD28 (2.5 .mu.g/ml) was used in
soluble form as a positive control co-stimulator. Proliferation of
T cells was determined by incorporation of [.sup.3H]-thymidine (1.0
.mu.Ci/well) added 15 h before harvesting of the 3-day cultures.
[.sup.3H]-thymidine incorporation was determined by a MicroBeta
TriLux liquid scintillation counter (Wallac, Turku, Finland). To
detect cytokines, supernatants were collected between 18-72 h of
culture and the concentrations of IFN-.gamma., IL-2, IL-10, IL-4,
and GM-CSF were measured by sandwich ELISA following the
manufacturer's (PharMingen) instructions.
[0130] Nucleic Acid Analysis.
[0131] Northern blot analysis of human RNA was carried out using
commercially available human multiple tissue Northern blot
membranes (Clontech, Palo Alto, Calif.). Membranes were incubated
in ExpressHyb hybridization solution (Clontech) for 30 min at
68.degree. C. The random-primed cDNA probe was full length hB7-H1
encoding cDNA (870 bp) labeled using [.sup.32P]-dCTP. A
.sup.32P-labeled 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 membranes were washed 3 times in 2.times.SSC containing 0.05%
SDS, and were then exposed at -80.degree. C. to x-ray film.
[0132] Tissue distribution of mB7-H1 mRNA was carried out using
commercially available multiple tissue mouse RNA dot blot membranes
(Clontech) according to the manufacturer's instructions. The
random-primed cDNA probe was full-length mB7-H1 encoding cDNA and
was labeled using [.sup.32P]-dCTP. The hybridization was performed
for 16 h at 65.degree. C. After washing four times with 2.times.SSC
containing 0.05% SDS, the membranes were exposed at -80.degree. C.
to x-ray films.
[0133] Antibodies and Fusion Proteins.
[0134] Rabbit antibodies against mB7-H1 protein were prepared by
Cocalico Biologicals (Reamstown, Pa.) by immunization of rabbits
with a keyhole limpet hemocyanin (KLH)-conjugated hydrophilic
peptide spanning amino acids 95-119 of mB7-H1 ("peptide 95-119")
(GNAALQITDVKLQDAGVYCCIISYG) (SEQ ID NO:16). Polyclonal antibody was
purified from rabbit serum using an affinity column containing
insoluble matrix material conjugated with the peptide 95-119. Both
ELISA and FFC analysis of COS cells transfected with an expression
vector containing cDNA encoding mB7-H1 demonstrated that the
polyclonal antibody bound specifically to mB7-H1. Purified mAb
specific for mouse CD3 and mouse CD28 and FITC-conjugated mAb
specific for mouse CD4, mouse CD8, and mouse CD40L, phycoerythrin
(PE)-conjugated mAb specific for mouse CD3, mouse B220 and mouse
Mac-1 were purchased from PharMingen (SanDiego, Calif.).
FITC-conjugated goat antibody specific for rabbit IgG was purchased
from Southern Biotechnology Associates (Birmingham, Ala.). Purified
rabbit IgG and hamster IgG were purchased from Rockland
(Gilbertsville, Pa.).
[0135] To prepare the mB7-H1 Ig fusion protein, cDNA encoding the
mB7-H1 extracellular domain was generated by RT-PCR using the sense
primer 5'-CAGGAATTCACCATGAGGATATTTGCTG-3' (SEQ ID NO:17) and the
anti-sense primer 5'-CATCAGATCTATGTGAGTCCTGTTCTGTG-3' (SEQ ID
NO:18) from mouse T cell mRNA. After digestion with EcoRI and
BglII, the PCR products were fused to the CH2-CH3 domain of mouse
IgG2a heavy chain in the expression plasmid pmIgV [Dong et al.
(1999) Nature Med. 5, 1365-1369]. The resulting plasmid, pmB7-H1Ig,
was transfected into CHO cells. Stably transfected cells were
cultured in serum-free CHO media (Life Technologies). The mB7-H1Ig
in the supernatants was purified using a protein G-Sepharose column
(Pierce, Rockford, Ill.) and dialyzed into LPS-free PBS. The
endotoxin concentration was less than 1 pg/mg of purified protein
according to the limulus amebocyte lysate assays (CAPE COD, Woods
Hole, Mass.). The mB7-1Ig fusion protein containing the extra
cellular domain of mB7-1 fused to the CH2-CH3 domain of mouse
IgG-2a heavy chain was prepared by an analogous method.
[0136] Fluorescence Flow Cytometry Analysis.
[0137] 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 a control.
[0138] To prepare activated human 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-.gamma.
(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')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.
[0139] For indirect immunofluorescence analysis of mouse cells, the
cells were incubated with the antibodies at 4.degree. C. for 30 min
in the presence of blocking mAb specific for CD16/32 (Fc receptor)
(Pharmingen). The cells were washed and further incubated with
FITC-conjugated anti-rabbit IgG. The cells were then stained with
PE-conjugated mAb specific for mouse CD3, mouse B220, or mouse
Mac-1. Fluorescence was analyzed with a FACS Calibur flow cytometer
and analyzed with Cell Quest software (Becton Dickinson, Mountain
View, Calif.). To prepare activated mouse T cells,
nylon-wool-purified mouse T cells (>75% CD3.sup.+ cells) at a
concentration of 2.times.10.sup.6/ml were cultured with mAb
specific for mouse CD28 (5 .mu.g/ml) and mouse CD3 (5 .mu.g/ml).
For preparation of activated mouse B cells, mouse splenocytes were
cultured with LPS (10 .mu.g/ml; Sigma, St. Louis, Mo.). Mouse
macrophages were obtained from the peritoneal cavities of mice
which had been injected with thioglycoilate 7 days before. For
activation, the mouse peritoneal exudate cells (PEC) were cultured
with IFN-.gamma. (10 U/ml) and LPS (100 ng/ml). All cultures were
harvested and the cells analyzed at 48 h. To detect CD40L
expression, CD4.sup.+ T cells were purified by magnetic sorting
(see above), cultured as indicated, and incubated with
FITC-conjugated mAb to CD40L.
[0140] Cytotoxic T-Lymphocyte (CTL) Generation.
[0141] To generate alloantigen-specific CTL activity in vitro,
nylon wool purified T cells (2.5.times.10.sup.6/ml) from B6
splenocytes were stimulated in 24-well tissue culture plates with
irradiated (10,000 rads) mock.P815, mB7-1.sup.+ P815, or
mB7-H1.sup.+ P815 cells (2.5.times.10.sup.5/ml) for 5 days. After
the 5-day stimulation, CTL activities against P815 (H-2d) and EL4
(H-2b) were measured in a standard .sup.51Cr release assay [Chen et
al. (1994) J. Exp. Med. 179, 523-532; Li et al. (1996) J. Exp. Med.
183, 639-644].
[0142] To generate tumor-specific CTL activity in vivo, DBA/2 mice
were inoculated subcutaneously (s.c.) with 1.times.10.sup.6
mock.P815, mB7-1.sup.+ P815, or mB7-H1.sup.+ P815 cells. The
draining lymph nodes were removed 7-10 d after tumor injection and
the suspended lymph node cells (3.times.10.sup.6/ml) were
re-stimulated in 24 well tissue culture plates with wild type
irradiated (10,000 rads) P815 cells (3.times.10.sup.5/ml) for 5
days. The cells were harvested and their CTL activity was measured
in a standard .sup.51Cr release assay against wild type P815 tumor
target cells.
[0143] In Vivo Induction and Assay of TNP-Specific Antibody.
[0144] Trinitrophenol (TNP) conjugated to KLH (TNP-KLH; 100
.mu.g/mouse) (Biosearch Technologies, Novato, Calif.) in phosphate
buffered saline (PBS) was injected i.p. into B6 mice on day 0. On
days 1 and 4, the mice were injected i.p. with 100 .mu.g of control
Ig, mB7-1Ig, or mB7-H1Ig. Sera were collected on days 7 and 14. To
measure TNP-specific antibodies in the sera, 0.3 mg/ml TNP-BSA
(Biosearch Technologies) was coated onto the well-bottoms of
96-well ELISA plates overnight at 4.degree. C. Non-specific binding
sites in the ELISA plates were blocked with 10% FBS in PBS for 90
min at room temperature. After extensive washing, samples (diluted
by 1/200- 1/2000 with PBS) were added and incubated for 2 h. The
plates were then washed and biotinylated rat antibodies specific
for mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (PharMingen) were added
to the wells. The plates were further incubated for 1 h at room
temperature. After washing the plates, horseradish peroxidase
(HRP)-conjugated streptavidin (Caltag Laboratories, Burlingame,
Calif.) was added to the wells and the plates were incubated for 1
h at room temperature. The plates were washed and the solutions in
all wells was measured. 3,3',5,5'-tetramethyl-benzidine substrate
(Sigma) was added to the wells. The OD.sub.450 for the solutions in
all wells was measured.
[0145] T Cell Proliferation in Response to KLH.
[0146] B6 mice were immunized with 100 .mu.g TNP-KLH in IFA s.c. or
in PBS i.p. on day 0 and were injected i.p. with 100 .mu.g of
either mB7-H1Ig or control Ig on days 1 and 4. To detect T cell
responses to KLH, draining lymph nodes and spleens were removed
from immunized mice on day 7 and 14, respectively. Suspended lymph
node or spleen cells were cultured with KLH at 1.56-100 .mu.g/ml as
indicated. T cell proliferation in response to KLH was determined
by addition of 1 .mu.Ci/well [.sup.3H]-thymidine 15 h before
harvesting of the 3-day cultures. [.sup.3H]-thymidine incorporation
was measured with a MicroBeta TriLux liquid scintillation counter
(Wallac).
Example 2
Molecular Cloning and Expression Pattern of the hB7-H1 Gene
[0147] 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).
[0148] 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.
[0149] 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-H1 Ig
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 CD 19+ 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.
[0150] 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
[0151] 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 mAb specific for human CD3. 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 mAb specific for human CD3, also
immobilized on the culture plates. In the absence of mAb specific
for human CD3, 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
[0152] 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 mAb specific
for human CD28 in the presence of mAb specific for human 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 mAb specific for human CD3 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 mAb specific for human 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.
[0153] 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 mAb specific for human IL-2 on
T cell proliferation and IL-10 production in B7H-mediated
co-stimulation were tested. Similar to T cell proliferation induced
by B7-1-COS cells and immobilized mAb specific for human CD3, T
cell proliferation induced by hB7-H1-COS cells and mAb specific for
human CD3 was blocked by inclusion of mAb specific for human IL-2
(FIG. 8a). Furthermore, IL-10 secretion from hB7-H1Ig-co-stimulated
T cells was also inhibited by mAb specific for human IL-2 (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
[0154] 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 mAb specific
for human CD3 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
[0155] 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
[0156] 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). A cDNA fragment encoding full-length mB7-H1
was cloned into the pcDNA3 vector (Invitrogen, Carlsbad, Calif.) to
give mB7-H1.pcDNA3.
[0157] mB7-H1, like hB7-H1, is a type I transmembrane protein of
290 amino acids that has 69% overall amino acid homology to hB7-H1
(FIG. 12a). Similar to other members of B7 family, mB7-H 1 consists
of an Ig V-like domain, an Ig C-like domain, a hydrophobic
transmembrane domain and a cytoplasmic tail. mB7-H1 shares 20%
homology to mouse B7-1, 14% to mouse B7-2, and 19% to mouse
B7h/B7RP-1, based on analysis using McVector 6.5 software (Clustal
W Program) (FIG. 12b).
[0158] RNA analysis revealed that mB7-H1 mRNA is abundant in mouse
heart, spleen, lung, skeletal muscle and liver, and less abundant
but present in mouse kidney, liver, thymus, and thyroid. Thus, the
expression pattern of mB7-H1 mRNA is similar to that of human B7-H1
mRNA. Negligible expression of the mB7-H1 mRNA was observed in
pancreas and testis.
[0159] FFC analysis using the anti-mB7-H1 antibody showed that
resting mouse CD3.sup.+ T cells do not express mB7-H1 (FIG. 13,
upper panel). However, a small fraction of B220.sup.+ mouse B cells
and Mac-1.sup.+ mouse macrophages expressed a low level of mB7-H1
(FIG. 13, upper panel). Stimulation of mouse T cells with
antibodies specific for mouse CD3 and mouse CD28 moderately
increased the mB7-H1 expression on T cells. Activation of mouse B
cells with LPS and macrophages with LPS plus IFN-.gamma.
significantly increased the expression of mB7-H1 on their surfaces
(FIG. 13, lower panel). Thus, mB7-H1, like hB7-H1, is an inducible
cell surface molecule.
Example 8
Co-Stimulation of Mouse T Cell Proliferation by mb7-H1
[0160] To investigate the costimulatory effect of mB7-H1,
nylon-wool purified mouse T cells were activated with a suboptimal
dose of mAb specific for mouse CD3 (coated onto culture well
bottoms at a concentration of 200 ng/ml) and co-stimulated with
various concentrations of mB7-H1Ig. mB7-H1Ig enhanced T cell
proliferation by up to 5-fold compared to control Ig (FIG. 14a).
The costimulatory effect of the mB7-H1Ig was dose-dependent and
dependent on the presence of mAb specific for mouse CD3 since in
the absence of mAb specific for mouse CD3, mB7-H1Ig (up to a
concentration of mB7-H1Ig 10 .mu.g/ml) failed to stimulate the
proliferation of T cells. When nylon-wool purified mouse T cells
were cultured with 293 cells transfected with either mB7-H1.pcDNA3
or control vector in the presence of suboptimal doses of mAb
specific for mouse CD3, mB7-H1-transfected 293 cells also enhanced
T cell proliferation substantially compared to the T cell
proliferation in the presence of the control vector-transfected 293
cells. Thus, similar to hB7-H1, mB7-H1 costimulates T cell
proliferation.
[0161] The role of CD28 in mB7-H1 costimulation was evaluated by
comparing the effects of mB7-H1Ig costimulation on T cells isolated
from CD28.sup.-/- mice and from normal mice. Nylon wool purified
mouse T cells were activated with two suboptimal doses of antibody
specific for mouse CD3 (coated onto the well bottoms of 96 well
tissue culture plates at a concentration of 0.125 .mu.g/ml or 0.25
.mu.g/ml) and either soluble antibody specific for mouse CD28 (2.5
.mu.g/ml) or mB7-H1Ig or control Ig (both coated onto the well
bottoms of 96 well tissue culture plates at a concentration of 10
.mu.g/ml). As shown in FIG. 14b, while there was no co-stimulatory
effect of anti-CD28 mAb on CD28.sup.-/- T cells, mB7-H1Ig, induced
the proliferation of both CD28.sup.-/- (FIG. 14b, right panel) and
CD28.sup.+/+ ("wt"; FIG. 14b, left panel) T cells to a similar
degree. Therefore, mB7-H1 can co-stimulate T cell growth in a
CD28-independent fashion.
[0162] In order to test whether mB7-H1 preferentially co-stimulates
CD4.sup.+ or CD8.sup.+ T cells, purified CD4.sup.+ and CD8.sup.+ T
cells were stimulated with mB7-H1Ig (same concentration as in the
experiment shown in FIG. 14b) and mAb specific for mouse CD3
(coated onto the well bottoms of 96 well tissue culture plates at a
concentration of 200 ng/ml). Proliferation of CD4.sup.+ T cells was
enhanced about 10 fold by mB7-H1Ig and the proliferation of
CD8.sup.+ T cells was only enhanced 2-3 fold mB7-H1Ig (FIG. 14c).
Thus, the co-stimulatory effect of mB7-H1 is more potent on CD4+ T
cells than on CD8+ cells.
Example 9
Co-Stimulation of Cytokine Production by mB7-H1
[0163] The levels of IL-10, IFN-.gamma., IL-2, IL-4 and GM-CSF
produced by T cells activated with mAb specific for mouse CD3 and
co-stimulated with either mB7-H1Ig or anti-CD28 mAb were measured.
FIG. 15a shows that mB7-H1Ig, similar to mAb specific for mouse
CD28, co-stimulates the production of high levels of IL-10 in the
day 3 cultures. IL-10 was not detectable at day 3 when T cells were
treated with either control Ig and mAb specific for mouse CD3 or
mAb specific for mouse CD3 alone. mB7-H1 and mAb specific for mouse
CD28 enhanced the production of IFN-.gamma. and GM-CSF. In contrast
to mAb specific for mouse CD28, which induced high levels of IL-2
and IL-4, mB7-H1 Ig induced no or negligible levels of IL-2 and
IL-4 at all time points (FIG. 15a). Thus, mB7-H1 and hB7-H1
co-stimulate the production of a similar spectrum of cytokines.
[0164] Since IL-2 was undetectable in culture supernatants from
mB7-H1Ig co-stimulated cultures, it seemed possible that mB7-H1
ligation inhibited IL-2 secretion. To test this possibility, the
effect of mB7-H1 on IL-2 secretion by T cells activated by mAb
specific for mouse CD3 and co-stimulated with mAb specific for
mouse CD28 was tested. FIG. 15b shows that inclusion of immobilized
mB7-H1Ig (at concentrations up to 10 .mu.g/ml) in the culture
resulted in a small decrease in IL-2 production during the 18-48 h
culture period that was statistically insignificant; in several
repeat experiments no statistically significant decrease was ever
seen. Similarly, mB7-H1Ig did not inhibit IL-2 production in
cultures in which T cells were activated by mAb specific for mouse
CD3 alone (FIG. 15b). The results thus indicate that mB7-H1
ligation does not inhibit the production of IL-2.
Example 10
Expression of mB7-H1 on Transfected P815 Tumor Cells and Decreased
Growth Rate of the Transfected P815 Cells in Mice
[0165] Mouse (DBA/2) P815 mastocytoma cells were stably transfected
with the expression plasmid (mB7-H1.pcDNA3) containing the coding
sequence for mB7-H1 using FUGENE.TM. (Roche, Mannheim, Germany)
according to the manufacturer's instructions. The transfected cells
were selected in complete medium containing G418 (1 mg/ml; Life
Technologies) and were subsequently cloned by limiting dilution.
mB7-H1 expressing P815 cells were identified by FFC using the
above-described anti-mB7-H1 polyclonal antibody preparation. A
representative clone (mB7-H1.sup.+ P815) was selected for further
studies. P815 clones transfected with the pc DNA vector (mock.P815)
or MB7-1 (mB7-1.sup.+ P815) were generated similarly [Chen et al.
(1994) J. Exp. Med. 179, 523-532]. Using a PE-conjugated rat
polyclonal antibody specific for mB7-H1 ("anti-mB7H/PE"), mB7-H1
expression was detected by FFC on the mB7H-H1.sup.+ P815 cells
(FIG. 18b) but not on either mock transfected P815 cells
("mock.P815") (FIG. 16b) or P815 cells transfected with a construct
encoding murine B7-1 ("mB7-1.sup.+ P815") (FIG. 17b). On the other
hand, the mB7-1.sup.+ P815 cells were stained with a
FITC-conjugated mAb specific for murine B7-1 ("anti-mB7-1-FITC")
(FIG. 17a). Furthermore, inclusion of the mB7-H1 peptide used to
make the polyclonal anti-mB7-H1 antibody in the staining reaction
mixture completely blocked binding of the polyclonal anti-mB7-H1
antibody to the mB7-H1.sup.+ P815 cells.
[0166] 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-H1.sup.+ P815 cells. The growth rate of the mock.P815 cells was
significantly greater in 4 out of 5 injected mice (FIG. 19a) than
in the 5 mice injected with mB7-H1.sup.+ P815 (FIG. 19b). These
findings indicate that the mB7-H1.sup.+ P81.5 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.
Example 11
mB7-H1 Costimulation Fails to Enhance Allogeneic and Syngeneic CTL
Responses
[0167] To examine the effect of mB7-H1 on the generation of
allogeneic CTL in vitro, nylon wool-purified T cells from B6 mice
(H-2b) were co-cultured with irradiated mock.P815 (H-2d),
mB7-1.sup.+ P815, or mB7-H1.sup.+ P815 cells for 5 days and the CTL
activity of cells harvested from the cultures was tested against
wild-type P815 target cells in standard .sup.51Cr release assays at
the indicated effector to target cell ratios ("E/T ratio"). As
depicted in FIG. 20a, mB7-H1.sup.+ P815 cells and mock.P815 cells
were poor stimulators of CTL activity. In contrast, mB7-1.sup.+
P815 cells elicited strong P815-specific CTL activity. The CTL
induced by mB7-1 were alloantigen-specific since they did not lyse
responder (B6) H-2 haplotype (H-2b) EL4 tumor target cells. Thus,
mB7-H1 expression does not facilitate the generation of allogeneic
CTL.
[0168] The ability of mB7-H1.sup.+ P815 cells to stimulate P815
tumor specific CTL in vivo was tested. DBA/2 mice were injected
s.c. with mock.P815, mB7-1.sup.+ P815, or mB7-H1.sup.+ P815 cells.
Tumor-draining lymph nodes were removed 7 days later and T cells
isolated from them were cultured with wild-type P815 cells for 5
days. Cells harvested from the cultures were tested for CTL
activity against wild-type P815 target cells in a standard
.sup.51Cr release assay at the indicated E/T ratios (FIG. 20b).
Effector cells from mice injected with mB7-H1.sup.+ P815 showed
slightly increased CTL activity against P815 cells compared to
effector cells from the mock.P815-injected mice; this difference in
CTL activity was however statistically insignificant. In contrast,
mB7-1.sup.+ P815 cells elicited strong CTL activity. CTL activity
was P815 tumor-specific since syngeneic L1210 tumor target cells
were not lysed. Thus, expression of mB7-H1 in P815 cells does not
enhance the induction of CTL activity against P815 tumor
antigens.
Example 12
B7-H1 Costimulation Amplifies Antigen-Specific T Helper Cell
Responses, T Cell-Dependent Humoral Responses, and Expression of
CD40L on T Cells
[0169] To investigate the effect of mB7-H1 costimulation on T
helper cell function, B6 mice were immunized with TNP-conjugated
KLH on day 0, and were injected with mB7-H1Ig at day 1 and day 4.
The in vitro proliferative responses of T cells obtained from both
lymph nodes and spleens of the immunized mice to various
concentrations of KLH were measured. As shown in FIG. 21, T cells
from both spleens and lymph nodes of TNP-KLH-immunized mice
proliferated in response to KLH in a dose dependent fashion.
Administration of mB7-H1Ig to TNP-KLH-immunized mice amplified the
subsequent in vitro proliferative responses of T cells by up to 2-3
fold. These results indicate that mB7-H1 co-stimulation enhances T
helper cell responses in vivo.
[0170] The effect of mB7-H1 co-stimulation on the generation of
antigen-specific antibodies to TNP was investigated in a system
well recognized as measuring helper T cell-dependent antibody
responses [Marrack and Kappler (1975) J. Immunol. 114, 1116-1125;
Romano et al. (1975) Proc. Natl. Acad. Sci. USA 72, 4555-4558].
Thus, the level of antibodies specific for TNP in the sera of mice
immunized with TNP-KLH was measured after treatment with control
Ig, mB7-1Ig, or mB7-H1Ig. In preliminary experiments, a significant
increase in the total anti-TNP IgG level was observed in sera of
mice immunized with TNP-KLH and treated with mB7-H1Ig compared to
sera of mice immunized with TNP-KLH and treated with control mIg.
The relative levels of IgM and individual IgG subclass (IgG1,
IgG2a, IgG2b and IgG3) anti-TNP antibodies elicited by TNP-KLH
immunization and various costimulations were each measured. As
shown in FIG. 22, the amount of TNP-specific IgG2a antibody was
increased significantly in the sera of mice immunized with TNP-KLH
and treated with mB7-H1Ig. The effect was different from that
elicited by mB7-1Ig in which the levels of antibodies specific for
TNP of other IgG subclasses (IgG1 and IgG2b) were also
significantly increased (FIG. 22). Thus, mB7-H1 costimulation
enhances T helper cell proliferation and T helper-dependent
antibody responses, particularly IgG2a responses.
[0171] The CD40-CD40 ligand (CD40L) interaction in T helper cell-B
cell interactions is critical for the generation of antibody
responses and for Ig class switching [Calderhead et al. (2000)
Curr. Top. Microbiol. Immunol. 245, 73-99]. The effect of
co-stimulation with mB7-H1Ig on the level of CD40L on T cells was
investigated. Purified CD4+ T cells from B6 mice were stimulated
with a suboptimal concentration of mAb specific for mouse CD3 in
the presence of mB7-H1Ig or mAb specific for mouse CD28. Expression
of CD40L on T cells was detected with a mAb specific for mouse
CD40L by FFC. mB7-H1Ig co-stimulation upregulated CD40L rapidly
(25.3% after 4 h incubation) compared to co-stimulation with
control IgG (6.6%) or antibody specific for mouse CD28 (10.5%)
(FIG. 23a). The level of CD40L was also higher after 24 h on T
cells co-stimulated with mB7-H1Ig than on T cells co-stimulated
with the either control Ig or mB7-1Ig (FIG. 23a). Similar results
were obtained with using an optimal dose of mAb specific for mouse
CD3 for activation (FIG. 23b). Thus, triggering of the B7-H1
counter-receptor on T cells rapidly upregulates the expression of
CD40L.
[0172] 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
181290PRTHomo sapiens 1Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr
Tyr Trp His Leu Leu1 5 10 15Asn Ala Phe Thr Val Thr Val Pro Lys Asp
Leu Tyr Val Val Glu Tyr 20 25 30Gly Ser Asn Met Thr Ile Glu Cys Lys
Phe Pro Val Glu Lys Gln Leu 35 40 45Asp Leu Ala Ala Leu Ile Val Tyr
Trp Glu Met Glu Asp Lys Asn Ile 50 55 60Ile Gln Phe Val His Gly Glu
Glu Asp Leu Lys Val Gln His Ser Ser65 70 75 80Tyr Arg Gln Arg Ala
Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95Ala Ala Leu Gln
Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110Arg Cys
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120
125Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
130 135 140Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu
Gly Tyr145 150 155 160Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp
His Gln Val Leu Ser 165 170 175Gly Lys Thr Thr Thr Thr Asn Ser Lys
Arg Glu Glu Lys Leu Phe Asn 180 185 190Val Thr Ser Thr Leu Arg Ile
Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205Cys Thr Phe Arg Arg
Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220Val Ile Pro
Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His225 230 235
240Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
245 250 255Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys
Lys Cys 260 265 270Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp
Thr His Leu Glu 275 280 285Glu Thr 2902870DNAHomo sapiens
2atgaggatat ttgctgtctt tatattcatg acctactggc atttgctgaa cgcatttact
60gtcacggttc ccaaggacct atatgtggta gagtatggta gcaatatgac aattgaatgc
120aaattcccag tagaaaaaca attagacctg gctgcactaa ttgtctattg
ggaaatggag 180gataagaaca ttattcaatt tgtgcatgga gaggaagacc
tgaaggttca gcatagtagc 240tacagacaga gggcccggct gttgaaggac
cagctctccc tgggaaatgc tgcacttcag 300atcacagatg tgaaattgca
ggatgcaggg gtgtaccgct gcatgatcag ctatggtggt 360gccgactaca
agcgaattac tgtgaaagtc aatgccccat acaacaaaat caaccaaaga
420attttggttg tggatccagt cacctctgaa catgaactga catgtcaggc
tgagggctac 480cccaaggccg aagtcatctg gacaagcagt gaccatcaag
tcctgagtgg taagaccacc 540accaccaatt ccaagagaga ggagaagctt
ttcaatgtga ccagcacact gagaatcaac 600acaacaacta atgagatttt
ctactgcact tttaggagat tagatcctga ggaaaaccat 660acagctgaat
tggtcatccc agaactacct ctggcacatc ctccaaatga aaggactcac
720ttggtaattc tgggagccat cttattatgc cttggtgtag cactgacatt
catcttccgt 780ttaagaaaag ggagaatgat ggatgtgaaa aaatgtggca
tccaagatac aaactcaaag 840aagcaaagtg atacacattt ggaggagacg
8703290PRTMus musculus 3Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala
Cys Cys His Leu Leu1 5 10 15Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp
Leu Tyr Val Val Glu Tyr 20 25 30Gly Ser Asn Val Thr Met Glu Cys Arg
Phe Pro Val Glu Arg Glu Leu 35 40 45Asp Leu Leu Ala Leu Val Val Tyr
Trp Glu Lys Glu Asp Glu Gln Val 50 55 60Ile Gln Phe Val Ala Gly Glu
Glu Asp Leu Lys Pro Gln His Ser Asn65 70 75 80Phe Arg Gly Arg Ala
Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn 85 90 95Ala Ala Leu Gln
Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110Cys Cys
Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu 115 120
125Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
130 135 140Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly
Tyr Pro145 150 155 160Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His
Gln Pro Val Ser Gly 165 170 175Lys Arg Ser Val Thr Thr Ser Arg Thr
Glu Gly Met Leu Leu Asn Val 180 185 190Thr Ser Ser Leu Arg Val Asn
Ala Thr Ala Asn Asp Val Phe Tyr Cys 195 200 205Thr Phe Trp Arg Ser
Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile 210 215 220Ile Pro Glu
Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp225 230 235
240Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val
245 250 255Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu
Lys Cys 260 265 270Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp
Thr Gln Phe Glu 275 280 285Glu Thr 2904873DNAMus
musculusCDS(1)...(870) 4atg agg ata ttt gct ggc att ata ttc aca gcc
tgc tgt cac ttg cta 48Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala
Cys Cys His Leu Leu1 5 10 15cgg gcg ttt act atc acg gct cca aag gac
ttg tac gtg gtg gag tat 96Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp
Leu Tyr Val Val Glu Tyr 20 25 30ggc agc aac gtc acg atg gag tgc aga
ttc cct gta gaa cgg gag ctg 144Gly Ser Asn Val Thr Met Glu Cys Arg
Phe Pro Val Glu Arg Glu Leu 35 40 45gac ctg ctt gcg tta gtg gtg tac
tgg gaa aag gaa gat gag caa gtg 192Asp Leu Leu Ala Leu Val Val Tyr
Trp Glu Lys Glu Asp Glu Gln Val 50 55 60att cag ttt gtg gca gga gag
gag gac ctt aag cct cag cac agc aac 240Ile Gln Phe Val Ala Gly Glu
Glu Asp Leu Lys Pro Gln His Ser Asn65 70 75 80ttc agg ggg aga gcc
tcg ctg cca aag gac cag ctt ttg aag gga aat 288Phe Arg Gly Arg Ala
Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn 85 90 95gct gcc ctt cag
atc aca gac gtc aag ctg cag gac gca ggc gtt tac 336Ala Ala Leu Gln
Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110tgc tgc
ata atc agc tac ggt ggt gcg gac tac aag cga atc acg ctg 384Cys Cys
Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu 115 120
125aaa gtc aat gcc cca tac cgc aaa atc aac cag aga att tcc gtg gat
432Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
130 135 140cca gcc act tct gag cat gaa cta ata tgt cag gcc gag ggt
tat cca 480Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly
Tyr Pro145 150 155 160gaa gct gag gta atc tgg aca aac agt gac cac
caa ccc gtg agt ggg 528Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His
Gln Pro Val Ser Gly 165 170 175aag aga agt gtc acc act tcc cgg aca
gag ggg atg ctt ctc aat gtg 576Lys Arg Ser Val Thr Thr Ser Arg Thr
Glu Gly Met Leu Leu Asn Val 180 185 190acc agc agt ctg agg gtc aac
gcc aca gcg aat gat gtt ttc tac tgt 624Thr Ser Ser Leu Arg Val Asn
Ala Thr Ala Asn Asp Val Phe Tyr Cys 195 200 205acg ttt tgg aga tca
cag cca ggg caa aac cac aca gcg gag ctg atc 672Thr Phe Trp Arg Ser
Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile 210 215 220atc cca gaa
ctg cct gca aca cat cct cca cag aac agg act cac tgg 720Ile Pro Glu
Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp225 230 235
240gtg ctt ctg gga tcc atc ctg ttg ttc ctc att gta gtg tcc acg gtc
768Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val
245 250 255ctc ctc ttc ttg aga aaa caa gtg aga atg cta gat gtg gag
aaa tgt 816Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu
Lys Cys 260 265 270ggc gtt gaa gat aca agc tca aaa aac cga aat gat
aca caa ttc gag 864Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp
Thr Gln Phe Glu 275 280 285gag acg taa 873Glu Thr 29053616DNAHomo
sapiensCDS(73)...(942) 5cccacgcgtc cgcagcttcc cgaggctccg caccagccgc
gcttctgtcc gcctgcaggg 60cattccagaa 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 10cat ttg ctg aac gca ttt act gtc acg gtt ccc aag
gac cta tat gtg 159His Leu Leu Asn Ala Phe Thr Val Thr Val Pro Lys
Asp Leu Tyr Val 15 20 25gta gag tat ggt agc aat atg aca att gaa tgc
aaa ttc cca gta gaa 207Val Glu Tyr Gly Ser Asn Met Thr Ile Glu Cys
Lys Phe Pro Val Glu30 35 40 45aaa caa tta gac ctg gct gca cta att
gtc tat tgg gaa atg gag gat 255Lys Gln Leu Asp Leu Ala Ala Leu Ile
Val Tyr Trp Glu Met Glu Asp 50 55 60aag aac att att caa ttt gtg cat
gga gag gaa gac ctg aag gtt cag 303Lys Asn Ile Ile Gln Phe Val His
Gly Glu Glu Asp Leu Lys Val Gln 65 70 75cat agt agc tac aga cag agg
gcc cgg ctg ttg aag gac cag ctc tcc 351His Ser Ser Tyr Arg Gln Arg
Ala Arg Leu Leu Lys Asp Gln Leu Ser 80 85 90ctg gga aat gct gca ctt
cag atc aca gat gtg aaa ttg cag gat gca 399Leu Gly Asn Ala Ala Leu
Gln Ile Thr Asp Val Lys Leu Gln Asp Ala 95 100 105ggg gtg tac cgc
tgc atg atc agc tat ggt ggt gcc gac tac aag cga 447Gly Val Tyr Arg
Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg110 115 120 125att
act gtg aaa gtc aat gcc cca tac aac aaa atc aac caa aga att 495Ile
Thr Val Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile 130 135
140ttg gtt gtg gat cca gtc acc tct gaa cat gaa ctg aca tgt cag gct
543Leu Val Val Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala
145 150 155gag ggc tac ccc aag gcc gaa gtc atc tgg aca agc agt gac
cat caa 591Glu Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp
His Gln 160 165 170gtc ctg agt ggt aag acc acc acc acc aat tcc aag
aga gag gag aag 639Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys
Arg Glu Glu Lys 175 180 185ctt ttc aat gtg acc agc aca ctg aga atc
aac aca aca act aat gag 687Leu Phe Asn Val Thr Ser Thr Leu Arg Ile
Asn Thr Thr Thr Asn Glu190 195 200 205att ttc tac tgc act ttt agg
aga tta gat cct gag gaa aac cat aca 735Ile Phe Tyr Cys Thr Phe Arg
Arg Leu Asp Pro Glu Glu Asn His Thr 210 215 220gct gaa ttg gtc atc
cca gaa cta cct ctg gca cat cct cca aat gaa 783Ala Glu Leu Val Ile
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu 225 230 235agg act cac
ttg gta att ctg gga gcc atc tta tta tgc ctt ggt gta 831Arg Thr His
Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val 240 245 250gca
ctg aca ttc atc ttc cgt tta aga aaa ggg aga atg atg gat gtg 879Ala
Leu Thr Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val 255 260
265aaa aaa tgt ggc atc caa gat aca aac tca aag aag caa agt gat aca
927Lys Lys Cys Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp
Thr270 275 280 285cat ttg gag gag acg taatccagca ttggaacttc
tgatcttcaa gcagggattc 982His Leu Glu Glu Thr 290tcaacctgtg
gtttaggggt tcatcggggc tgagcgtgac aagaggaagg aatggacccg
1042tgggatgcag gcaatgtggg acttaaaagg cccaagcact gaaaatggaa
cctggcgaaa 1102gcagaggagg agaatgaaga aagatggagt caaacaggga
gcctggaggg agaccttgat 1162actttcaaat gcctgagggg ctcatcgacg
cctgtgacag ggagaaagga tacttctgaa 1222caaggagcct ccaagcaaat
catccattgc tcatcctagg aagacgggtt gagaatccct 1282aatttgaggg
tcagttcctg cagaagtgcc ctttgcctcc actcaatgcc tcaatttctt
1342ttctgcatga ctgagagtct cagtgttgga acgggacagt atttatgtat
gagtttttcc 1402tatttatttt gagtctgtga ggtcttcttg tcatgtgagt
gtggttgtga atgatttctt 1462ttgaagatat attgtagtag atgttacaat
tttgtcgcca aactaaactt gctgcttaat 1522gatttgctca catctagtaa
aacatggagt atttgtaagg tgcttggtct cctctataac 1582tacaagtata
cattggaagc ataaagatca aaccgttggt tgcataggat gtcaccttta
1642tttaacccat taatactctg gttgacctaa tcttattctc agacctcaag
tgtctgtgca 1702gtatctgttc catttaaata tcagctttac aattatgtgg
tagcctacac acataatctc 1762atttcatcgc tgtaaccacc ctgttgtgat
aaccactatt attttaccca tcgtacagct 1822gaggaagcaa acagattaag
taacttgccc aaaccagtaa atagcagacc tcagactgcc 1882acccactgtc
cttttataat acaatttaca gctatatttt actttaagca attcttttat
1942tcaaaaacca tttattaagt gcccttgcaa tatcaatcgc tgtgccaggc
attgaatcta 2002cagatgtgag caagacaaag tacctgtcct caaggagctc
atagtataat gaggagatta 2062acaagaaaat gtattattac aatttagtcc
agtgtcatag cataaggatg atgcgagggg 2122aaaacccgag cagtgttgcc
aagaggagga aataggccaa tgtggtctgg gacggttgga 2182tatacttaaa
catcttaata atcagagtaa ttttcattta caaagagagg tcggtactta
2242aaataaccct gaaaaataac actggaattc cttttctagc attatattta
ttcctgattt 2302gcctttgcca tataatctaa tgcttgttta tatagtgtct
ggtattgttt aacagttctg 2362tcttttctat ttaaatgcca ctaaatttta
aattcatacc tttccatgat tcaaaattca 2422aaagatccca tgggagatgg
ttggaaaatc tccacttcat cctccaagcc attcaagttt 2482cctttccaga
agcaactgct actgcctttc attcatatgt tcttctaaag atagtctaca
2542tttggaaatg tatgttaaaa gcacgtattt ttaaaatttt tttcctaaat
agtaacacat 2602tgtatgtctg ctgtgtactt tgctattttt atttatttta
gtgtttctta tatagcagat 2662ggaatgaatt tgaagttccc agggctgagg
atccatgcct tctttgtttc taagttatct 2722ttcccatagc ttttcattat
ctttcatatg atccagtata tgttaaatat gtcctacata 2782tacatttaga
caaccaccat ttgttaagta tttgctctag gacagagttt ggatttgttt
2842atgtttgctc aaaaggagac ccatgggctc tccagggtgc actgagtcaa
tctagtccta 2902aaaagcaatc ttattattaa ctctgtatga cagaatcatg
tctggaactt ttgttttctg 2962ctttctgtca agtataaact tcactttgat
gctgtacttg caaaatcaca ttttctttct 3022ggaaattccg gcagtgtacc
ttgactgcta gctaccctgt gccagaaaag cctcattcgt 3082tgtgcttgaa
cccttgaatg ccaccagctg tcatcactac acagccctcc taagaggctt
3142cctggaggtt tcgagattca gatgccctgg gagatcccag agtttccttt
ccctcttggc 3202catattctgg tgtcaatgac aaggagtacc ttggctttgc
cacatgtcaa ggctgaagaa 3262acagtgtctc caacagagct ccttgtgtta
tctgtttgta catgtgcatt tgtacagtaa 3322ttggtgtgac agtgttcttt
gtgtgaatta caggcaagaa ttgtggctga gcaaggcaca 3382tagtctactc
agtctattcc taagtcctaa ctcctccttg tggtgttgga tttgtaaggc
3442actttatccc ttttgtctca tgtttcatcg taaatggcat aggcagagat
gatacctaat 3502tctgcatttg attgtcactt tttgtacctg cattaattta
ataaaatatt cttatttatt 3562ttgttacttg gtaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaa 3616625PRTHomo sapiens 6Met Ala Ile Ser
Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val1 5 10 15Leu Met Ser
Ala Gln Glu Ser Trp Ala 20 2575PRTUnknownknown family is Bovidae
7Lys Phe Glu Arg Gln1 5815PRTHomo sapiens 8Met Asp Asp Gln Arg Asp
Leu Ile Ser Asn Asn Glu Gln Leu Pro1 5 10 1594PRTRattus rattus 9Lys
Asp Glu Leu110210PRTHomo sapiens 10Val Glu Tyr Gly Ser Asn Met Thr
Ile Glu Cys Lys Phe Pro Val Glu1 5 10 15Lys Gln Leu Asp Leu Ala Ala
Leu Ile Val Tyr Trp Glu Met Glu Asp 20 25 30Lys Asn Ile Ile Gln Phe
Val His Gly Glu Glu Asp Leu Lys Val Gln 35 40 45His Ser Ser Tyr Arg
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser 50 55 60Leu Gly Asn Ala
Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala65 70 75 80Gly Val
Tyr Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg 85 90 95Ile
Thr Val Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile 100 105
110Leu Val Val Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala
115 120 125Glu Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp
His Gln 130 135 140Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys
Arg Glu Glu Lys145 150 155 160Leu Phe Asn Val Thr Ser Thr Leu Arg
Ile Asn Thr Thr Thr Asn Glu 165 170 175Ile Phe Tyr Cys Thr Phe Arg
Arg Leu Asp Pro Glu Glu Asn His Thr 180 185 190Ala Glu Leu Val Ile
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu 195 200 205Arg Thr
21011205PRTHomo sapiens 11Lys Glu Val Lys Glu Val Ala Thr Leu Ser
Cys Gly His Asn Val Ser1 5 10 15Val Glu Glu Leu Ala Gln Thr Arg Ile
Tyr Trp Gln Lys Glu Lys Lys 20 25 30Met Val Leu Thr Met Met Ser Gly
Asp Met Asn Ile Trp Pro Glu Tyr 35 40 45Lys Asn Arg Thr Ile Phe Asp
Ile Thr Asn Asn Leu Ser Ile Val
Ile 50 55 60Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val
Val Leu65 70 75 80Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu
Ala Glu Val Thr 85 90 95Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser
Ile Ser Asp Phe Glu 100 105 110Ile Pro Thr Ser Asn Ile Arg Arg Ile
Ile Cys Ser Thr Ser Gly Gly 115 120 125Phe Pro Glu Pro His Leu Ser
Trp Leu Glu Asn Gly Glu Glu Leu Asn 130 135 140Ala Ile Asn Thr Thr
Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala145 150 155 160Val Ser
Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met 165 170
175Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp
180 185 190Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn Leu Leu 195
200 20512218PRTHomo sapiens 12Ala Tyr Phe Asn Glu Thr Ala Asp Leu
Pro Cys Gln Phe Ala Asn Ser1 5 10 15Gln Asn Gln Ser Leu Ser Glu Leu
Val Val Phe Trp Gln Asp Gln Glu 20 25 30Asn Leu Val Leu Asn Glu Val
Tyr Leu Gly Lys Glu Lys Phe Asp Ser 35 40 45Val His Ser Lys Tyr Met
Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp 50 55 60Thr Leu Arg Leu His
Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln65 70 75 80Cys Ile Ile
His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln 85 90 95Met Asn
Ser Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile 100 105
110Val Pro Ile Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys
115 120 125Ser Ser Ile His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val
Leu Leu 130 135 140Arg Thr Lys Asn Ser Thr Ile Glu Tyr Asp Gly Ile
Met Gln Lys Ser145 150 155 160Gln Asp Asn Val Thr Glu Leu Tyr Asp
Val Ser Ile Ser Leu Ser Val 165 170 175Ser Phe Pro Asp Val Thr Ser
Asn Met Thr Ile Phe Cys Ile Leu Glu 180 185 190Thr Asp Lys Thr Arg
Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu 195 200 205Asp Pro Gln
Pro Pro Pro Asp His Ile Pro 210 21513306PRTMus musculus 13Met Ala
Cys Asn Cys Gln Leu Met Gln Asp Thr Pro Leu Leu Lys Phe1 5 10 15Pro
Cys Pro Arg Leu Ile Leu Leu Phe Val Leu Leu Ile Arg Leu Ser 20 25
30Gln Val Ser Ser Asp Val Asp Glu Gln Leu Ser Lys Ser Val Lys Asp
35 40 45Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His Glu Asp Glu
Ser 50 55 60Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val Val Leu
Ser Val65 70 75 80Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys
Asn Arg Thr Leu 85 90 95Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu
Gly Leu Val Leu Ser 100 105 110Asp Arg Gly Thr Tyr Ser Cys Val Val
Gln Lys Lys Glu Arg Gly Thr 115 120 125Tyr Glu Val Lys His Leu Ala
Leu Val Lys Leu Ser Ile Lys Ala Asp 130 135 140Phe Ser Thr Pro Asn
Ile Thr Glu Ser Gly Asn Pro Ser Ala Asp Thr145 150 155 160Lys Arg
Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro Lys Pro Arg Phe 165 170
175Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile Asn Thr Thr Ile
180 185 190Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser Ser Gln
Leu Asp 195 200 205Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu
Ile Lys Tyr Gly 210 215 220Asp Ala His Val Ser Glu Asp Phe Thr Trp
Glu Lys Pro Pro Glu Asp225 230 235 240Pro Pro Asp Ser Lys Asn Thr
Leu Val Leu Phe Gly Ala Gly Phe Gly 245 250 255Ala Val Ile Thr Val
Val Val Ile Val Val Ile Ile Lys Cys Phe Cys 260 265 270Lys His Arg
Ser Cys Phe Arg Arg Asn Glu Ala Ser Arg Glu Thr Asn 275 280 285Asn
Ser Leu Thr Phe Gly Pro Glu Glu Ala Leu Ala Glu Gln Thr Val 290 295
300Phe Leu30514309PRTMus musculus 14Met Asp Pro Arg Cys Thr Met Gly
Leu Ala Ile Leu Ile Phe Val Thr1 5 10 15Val Leu Leu Ile Ser Asp Ala
Val Ser Val Glu Thr Gln Ala Tyr Phe 20 25 30Asn Gly Thr Ala Tyr Leu
Pro Cys Pro Phe Thr Lys Ala Gln Asn Ile 35 40 45Ser Leu Ser Glu Leu
Val Val Phe Trp Gln Asp Gln Gln Lys Leu Val 50 55 60Leu Tyr Glu His
Tyr Leu Gly Thr Glu Lys Leu Asp Ser Val Asn Ala65 70 75 80Lys Tyr
Leu Gly Arg Thr Ser Phe Asp Arg Asn Asn Trp Thr Leu Arg 85 90 95Leu
His Asn Val Gln Ile Lys Asp Met Gly Ser Tyr Asp Cys Phe Ile 100 105
110Gln Lys Lys Pro Pro Thr Gly Ser Ile Ile Leu Gln Gln Thr Leu Thr
115 120 125Glu Leu Ser Val Ile Ala Asn Phe Ser Glu Pro Glu Ile Lys
Leu Ala 130 135 140Gln Asn Val Thr Gly Asn Ser Gly Ile Asn Leu Thr
Cys Thr Ser Lys145 150 155 160Gln Gly His Pro Lys Pro Lys Lys Met
Tyr Phe Leu Ile Thr Asn Ser 165 170 175Thr Asn Glu Tyr Gly Asp Asn
Met Gln Ile Ser Gln Asp Asn Val Thr 180 185 190Glu Leu Phe Ser Ile
Ser Asn Ser Leu Ser Leu Ser Phe Pro Asp Gly 195 200 205Val Trp His
Met Thr Val Val Cys Val Leu Glu Thr Glu Ser Met Lys 210 215 220Ile
Ser Ser Lys Pro Leu Asn Phe Thr Gln Glu Phe Pro Ser Pro Gln225 230
235 240Thr Tyr Trp Lys Glu Ile Thr Ala Ser Val Thr Val Ala Leu Leu
Leu 245 250 255Val Met Leu Leu Ile Ile Val Cys His Lys Lys Pro Asn
Gln Pro Ser 260 265 270Arg Pro Ser Asn Thr Ala Ser Lys Leu Glu Arg
Asp Ser Asn Ala Asp 275 280 285Arg Glu Thr Ile Asn Leu Lys Glu Leu
Glu Pro Gln Ile Ala Ser Ala 290 295 300Lys Pro Asn Ala
Glu30515322PRTMus musculus 15Met Gln Leu Lys Cys Pro Cys Phe Val
Ser Leu Gly Thr Arg Gln Pro1 5 10 15Val Trp Lys Lys Leu His Val Ser
Ser Gly Phe Phe Ser Gly Leu Gly 20 25 30Leu Phe Leu Leu Leu Leu Ser
Ser Leu Cys Ala Ala Ser Ala Glu Thr 35 40 45Glu Val Gly Ala Met Val
Gly Ser Asn Val Val Leu Ser Cys Ile Asp 50 55 60Pro His Arg Arg His
Phe Asn Leu Ser Gly Leu Tyr Val Tyr Trp Gln65 70 75 80Ile Glu Asn
Pro Glu Val Ser Val Thr Tyr Tyr Leu Pro Tyr Lys Ser 85 90 95Pro Gly
Ile Asn Val Asp Ser Ser Tyr Lys Asn Arg Gly His Leu Ser 100 105
110Leu Asp Ser Met Lys Gln Gly Asn Phe Ser Leu Tyr Leu Lys Asn Val
115 120 125Thr Pro Gln Asp Thr Gln Glu Phe Thr Cys Arg Val Phe Met
Asn Thr 130 135 140Ala Thr Glu Leu Val Lys Ile Leu Glu Glu Val Val
Arg Leu Arg Val145 150 155 160Ala Ala Asn Phe Ser Thr Pro Val Ile
Ser Thr Ser Asp Ser Ser Asn 165 170 175Pro Gly Gln Glu Arg Thr Tyr
Thr Cys Met Ser Lys Asn Gly Tyr Pro 180 185 190Glu Pro Asn Leu Tyr
Trp Ile Asn Thr Thr Asp Asn Ser Leu Ile Asp 195 200 205Thr Ala Leu
Gln Asn Asn Thr Val Tyr Leu Asn Lys Leu Gly Leu Tyr 210 215 220Asp
Val Ile Ser Thr Leu Arg Leu Pro Trp Thr Ser Arg Gly Asp Val225 230
235 240Leu Cys Cys Val Glu Asn Val Ala Leu His Gln Asn Ile Thr Ser
Ile 245 250 255Ser Gln Ala Glu Ser Phe Thr Gly Asn Asn Thr Lys Asn
Pro Gln Glu 260 265 270Thr His Asn Asn Glu Leu Lys Val Leu Val Pro
Val Leu Ala Val Leu 275 280 285Ala Ala Ala Ala Phe Val Ser Phe Ile
Ile Tyr Arg Arg Thr Arg Pro 290 295 300His Arg Ser Tyr Thr Gly Pro
Lys Thr Val Gln Leu Glu Leu Thr Asp305 310 315 320His Ala1625PRTMus
musculus 16Gly Asn Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp
Ala Gly1 5 10 15Val Tyr Cys Cys Ile Ile Ser Tyr Gly 20 251728DNAMus
musculus 17caggaattca ccatgaggat atttgctg 281829DNAMus musculus
18catcagatct atgtgagtcc tgttctgtg 29
* * * * *
References