U.S. patent application number 11/351617 was filed with the patent office on 2006-06-22 for mammalian cell surface antigens; related reagents.
This patent application is currently assigned to Schering Corporation. Invention is credited to Daniel M. Gorman, Troy D. Randall, Albert Zlotnik.
Application Number | 20060135756 11/351617 |
Document ID | / |
Family ID | 27362079 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060135756 |
Kind Code |
A1 |
Gorman; Daniel M. ; et
al. |
June 22, 2006 |
Mammalian cell surface antigens; related reagents
Abstract
Purified genes encoding a T cell surface antigen from a mammal,
reagents related thereto including purified proteins, specific
antibodies, and nucleic acids encoding this antigen are provided.
Methods of using said reagents and diagnostic kits are also
provided.
Inventors: |
Gorman; Daniel M.; (Palo
Alto, CA) ; Randall; Troy D.; (Saranac Lake, NY)
; Zlotnik; Albert; (San Diego, CA) |
Correspondence
Address: |
DNAX RESEARCH, INC.;LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
Schering Corporation
Kenilworth
NJ
|
Family ID: |
27362079 |
Appl. No.: |
11/351617 |
Filed: |
February 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09545998 |
Apr 10, 2000 |
7025962 |
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11351617 |
Feb 10, 2006 |
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08911423 |
Aug 14, 1997 |
6111090 |
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09545998 |
Apr 10, 2000 |
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60027901 |
Oct 7, 1996 |
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60023419 |
Aug 16, 1996 |
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Current U.S.
Class: |
536/23.5 ;
435/320.1; 435/325; 435/69.1; 530/350; 530/388.22 |
Current CPC
Class: |
A61P 37/02 20180101;
Y10S 435/81 20130101; A61K 2039/55516 20130101; C07K 14/47
20130101; A61K 39/39 20130101; C07K 16/28 20130101; C07K 2317/70
20130101; A61P 37/04 20180101; A61K 38/00 20130101; C07K 2319/00
20130101; C07K 16/2878 20130101; C07K 14/705 20130101; C07K
14/70578 20130101; Y10S 435/975 20130101 |
Class at
Publication: |
536/023.5 ;
530/350; 435/069.1; 435/320.1; 435/325; 530/388.22 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/715 20060101
C07K014/715; C07K 16/28 20060101 C07K016/28 |
Claims
1. A substantially pure or recombinant 312C2 protein or
conservatively modified variants thereof, said protein: a) is
expressed on activated T cells; or b) specifically binds to
antibodies generated against. SEQ ID NO: 2 or 4.
2. A protein or peptide of claim 1, selected from the group
consisting of: a) a natural protein from a warm blooded animal
selected from the group of birds and mammals, including a rodent or
primate; b) a protein or peptide: i) comprising at least one
polypeptide segment of at least 14 amino acids of SEQ ID NO: 2; ii)
comprising at least one polypeptide segment of at least 14 amino
acids of SEQ ID NO: 4; or iii) which is not glycosylated; iv) which
is in a buffered solution; v) which is attached to a solid
substrate; vi) which exhibits a plurality of epitopes from SEQ ID
NO: 2 or 4; vii) which is detectably labeled; viii) which is a
synthetic polypeptide; ix) which is conjugated to a chemical
moiety; x) which is a 5-fold or less substitution from a natural
sequence; or xi) which is a deletion or insertion variant from a
natural sequence.
3. A protein or peptide of claim 1, comprising a sequence from the
extracellular or the intracellular portion of a 312C2.
4. A fusion protein comprising a peptide of claim 1.
5. A sterile composition comprising the protein of claim 1, and a
pharmaceutically acceptable carrier.
6. An antibody which specifically binds a protein or peptide of
claim 1.
7. An antibody of claim 6, wherein: a) said 312C2 is a mammalian
protein, including a mouse or human; b) said antibody is raised
against a purified peptide sequence of SEQ ID NO: 2 or 4; c) said
antibody is a monoclonal antibody; d) said antibody is detectably
labeled; e) said antibody is attached to a solid substrate; f) said
antibody is in a sterile composition; or g) said antibody is in a
buffered composition.
8. A method of purifying a 312C2 protein or peptide from other
materials in a mixture comprising contacting said mixture to an
antibody of claim 6, and separating bound 312C2 from other
materials.
9. An isolated or recombinant nucleic acid capable of encoding a
protein or peptide of claim 1.
10. A nucleic acid of claim 9, wherein said nucleic acid: a)
encodes a sequence of SEQ ID NO: 2 or 4; b) comprises a sequence of
SEQ ID NO: 1 or 3; or c) encodes a sequence from an extracellular
domain of a natural 312C2; d) encodes a sequence from an
intracellular domain of a natural 312C2; e) attached to a solid
substrate; f) is detectable labeled; or g) is in a sterile
composition.
11. An expression or replicating vector of claim 9.
12. A kit comprising: a) a substantially pure 312C2 or fragment of
claim 1; b) an antibody or receptor which specifically binds a
312C2; or c) a nucleic acid encoding a 312C2 or peptide.
13. A method for detecting in a sample the presence of a 312C2
nucleic acid, protein, or antibody, comprising testing said sample
with a kit of claim 12.
14. A method of modulating the physiology of a cell comprising
contacting said cell with: a) a substantially pure 312C2 or
fragment of claim 1; b) an antibody or binding partner which
specifically binds a 312C2; or c) a nucleic acid encoding a 312C2
or peptide.
15. The method of claim 14, wherein said cell is a T cell and said
modulating of physiology is: a) apoptosis of said T cell; or b)
activation of said T cell
16. A method of claim 14, wherein said cell is in a tissue and/or
in an organism.
17. A method of expressing a 312C2 comprising expressing a nucleic
acid of claim 9.
18. A cell, tissue, organ, or organism comprising a nucleic acid of
claim 9.
19. A recombinant nucleic acid comprising sequence at least about
70% identity over a stretch of at least about 30 nucleotides to a
312C2 nucleic acid sequence of SEQ ID NO: 1 or 3.
20. A nucleic acid of claim 19, further encoding a polypeptide
comprising at least about 60% identity over a stretch of at least
about 20 amino acids to a 312C2 sequence of SEQ ID NO: 2 or 4.
21. A method of treating a host mammal having an abnormal immune
response by administering to said mammal an effective dose of: a)
an antibody or binding partner which binds specifically to a 312C2;
b) a substantially pure 312C2 protein or peptide thereof; or c) a
nucleic acid encoding a 312C2 peptide.
22. The method of claim 21, wherein said abnormal immune response
is characterized by: a) a T-cell immune deficiency; b) chronic
inflammation; or c) tissue rejection.
Description
[0001] The present filing is a conversion from U.S. Provisional
Patent Applications 60/023,419, filed. Aug. 16, 1996 and
60/027,901, filed Oct. 7, 1996, to a U.S. Utility Patent
Application.
FIELD OF THE INVENTION
[0002] The present invention generally pertains to molecules that
control activation and expansion of mammalian cells, especially
mammalian immune system cells. The invention provides purified
genes, proteins, antibodies, and related reagents useful, for
example, to regulate activation, development, differentiation, and
function of various cell types, including hematopoietic cells. In
particular, the invention provides mammalian 312C2 genes, gene
products, compositions, and methods for using these.
BACKGROUND OF THE INVENTION
[0003] The activation of resting T cells is critical to most immune
responses and allows these cells to exert their regulatory or
effector capabilities. See Paul (ed; 1993) Fundamental Immunology
3d ed., Raven Press, N.Y. Increased adhesion between T cells and
antigen presenting cells (APC) or other forms of primary stimuli,
e.g., immobilized monoclonal antibodies (mAb), can potentiate the
T-cell receptor signals. T-cell activation and T cell expansion
depends upon engagement of the T-cell receptor (TCR) and
co-stimulatory signals provided by accessory cells. See, e.g.,
Jenkins and Johnson (1993) Curr. Opin. Immunol. 5:361-367; Bierer
and Hahn (1993) Semin. Immunol. 5:249-261; June, et al. (1990)
Immunol. Today 11:211-216; and Jenkins (1994) Immunity 1:443-446. A
major, and well-studied, co-stimulatory interaction for T cells
involves either CD28 or CTLA-4 on T cells with either B7 or B70
(Jenkins (1994) Immunity 1:443-446). Recent studies on CD28
deficient mice (Shahinian, et al. (1993) Science 261:609-612;
Green, et al. (1994) Immunity 1:501-508) and CTLA-4 immunoglobulin
expressing transgenic mice (Ronchese, et al. (1994) J. Exp. Med.
179:809-817) have revealed deficiencies in some T-cell responses
though these mice have normal primary immune responses and normal
CTL responses to lymphocytic choriomeningitis virus and vesicular
stomatitis virus. As a result, both these studies conclude that
other co-stimulatory molecules must be supporting T-cell function.
However, identification of these molecules which mediate distinct
costimulatory signals has been difficult.
[0004] The inability to modulate activation signals prevents
control of inappropriate developmental or physiological responses
in the immune system. The present invention provides at least one
alternative costimulatory molecule, agonists and antagonists of
which will be useful in modulating a plethora of immune
responses.
SUMMARY OF THE INVENTION
[0005] The present invention is based, in part, upon the discovery
of a family of proteins which appear to act as a costimulator of T
cell activation. In particular, the invention provides mammalian,
e.g., rodent and primate, genes designated m312C2 and h312C2,
respectively, which are expressed in the thymus, and are induced on
T cells and spleen cells following activation. Engagement of 312C2
appears stimulate proliferation of T cell clones, antigen-specific
proliferation and cytokine production by T cells, and appears to
potentiate T cell expansion or apoptosis. The mouse and human
embodiments are described in greater detail, but the invention
encompasses related mammalian genes, proteins, antibodies, and uses
thereof. Functional equivalents exhibiting significant sequence
homology are available from other mammalian and non-mammalian
species. Moreover, the ligand of 312C2 can function as its binding
partner to stimulate other cells expressing the antigen.
[0006] The present invention provides a substantially pure or
recombinant 312C2 protein or peptide fragment thereof. The protein
or polypeptide is expressed, e.g., activated T cells or
specifically binds to antibodies generated against SEQ ID NO: 2 or
4. Some embodiments involve a protein or peptide selected from a
protein or peptide from a warm blooded animal selected from the
group of birds and mammals, including a rodent or primate. The
groups further consist of a protein or peptide which comprises at
least one polypeptide segment of SEQ ID NO: 2 or 4; is not
glycosylated; is in a buffered solution; is attached to a solid
substrate; exhibits a plurality of epitopes from SEQ ID NO: 2 or 4;
is synthetically labeled; is conjugated to a chemical moiety; is a
5-fold or less substitution from a natural sequence; or is a
deletion or insertion variant from a natural sequence. The protein
or peptide can comprise a sequence from the extracellular or the
intracellular portion of a 312C2; or be a fusion protein.
[0007] The invention also provides a recombinant nucleic acid
comprising sequence at least about 70% identity over a stretch of
at least about 30nucleotides to a 312C2 nucleic acid sequence of
SEQ ID NO: 1, 3 or 5, useful, e.g., as a probe or PCR primer for a
related gene. Another embodiment further encodes a polypeptide
sharing a plurality of specific epitopes and comprising at least
about 60% identity over a stretch of at least about 20 amino acids
to a 312C2 sequence of SEQ ID NO: 2 or 4.
[0008] Another embodiment is a sterile composition comprising a
312C2 protein and a pharmaceutically acceptable carrier. Other
compositions may combine said entities with an agonist or
antagonist of other T cell signaling molecules, e.g., signaling
entities through the T cell receptor, CD40, CD40 ligand, CTLA-8,
CD28, B7, B70, BAS-1, SLAM, etc.
[0009] The invention also embraces an antibody which specifically
-binds a 312C2 protein or peptide, e.g., wherein the 312C2 is a
mammalian protein, including a mouse; the antibody is raised
against a purified 312C2 peptide sequence of SEQ ID NO: 2 or 4; the
antibody is a monoclonal antibody; the antibody is detectably
labeled; the antibody is attached to a solid substrate; or the
antibody is in a sterile or buffered composition. The antibodies
also make available a method of purifying a 312C2 protein or
peptide from other materials in a mixture comprising contacting the
mixture to an anti-312C2 antibody, and separating bound 312C2 from
other materials.
[0010] Another aspect of the invention is an isolated or
recombinant nucleic acid capable of encoding a 312C2 protein or
peptide, including a nucleic acid which encodes a mature
polypeptide sequence of SEQ ID NO: 2 or 4; which includes a
sequence of SEQ ID NO: 1, 3 or 5; which encodes a sequence from an
extracellular domain of a natural 312C2; which encodes a sequence
from an intracellular domain of a natural 312C2; which is
detectably labeled; which is attached to a solid substrate; or is
in a sterile composition. Such nucleic acid embodiments also
include an expression or replicating vector.
[0011] Also provided is a method of expressing a 312C2 peptide by
expressing a nucleic acid encoding a 312C2 polypeptide. The
invention also provides a cell, tissue, organ, or organism
comprising a nucleic acid encoding a 312C2 peptide.
[0012] The invention also provides a kit containing a substantially
pure 312C2 or fragment; an antibody or receptor which specifically
binds a 312C2; or a nucleic acid, or its complement, encoding a
312C2 or peptide. This kit also provides methods for detecting in a
sample the presence of a nucleic acid, protein, or antibody,
comprising testing said sample with such a kit.
[0013] The invention also supplies methods of modulating the
physiology of a cell comprising contacting said cell with a
substantially pure 312C2 or a fragment thereof; or with an antibody
or ligand which specifically binds a 312C2; or with a nucleic acid
encoding a 312C2 or a peptide fragment thereof. Certain preferred
embodiments include a method where the cell is a T cell and the
modulating of physiology is activation of the T cell or apoptosis
of the T cell; or where the cell is in a tissue and/or in an
organism.
[0014] The invention further provides a method of treating a
patient having an abnormal immune response by administering an
effective dose of an antibody or binding partner specific for
312C2; a 312C2 protein or polypeptide; or a nucleic acid encoding a
312C2 peptide. The abnormal immune response is characterized by a T
cell immune deficiency; chronic inflammation; or tissue
rejection.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0015] The terms "nucleic acid", "probe", or "primer" include
reference to a deoxyribonucleotide, ribonucleotide, or mixed
polymer in either single- or double-stranded form, and unless
otherwise limited, encompasses known analogs of natural
polynucleotides that hybridize to nucleic acids in manner similar
to naturally occurring polynucleotides. Unless otherwise indicated,
a particular nucleic acid sequence includes the perfect
complementary sequence thereof. Eukaryotic nucleic acids are
nucleic acids from eukaryotic cells, preferably cells of
multicellular eukaryotes.
[0016] Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively.
Numeric ranges are inclusive of the numbers defining the range. The
terms defined below are more fully defined by reference to the
Specification as a whole.
[0017] The term "recombinant" when used with reference to a cell,
or nucleic acid, or vector, includes reference to a cell, or
nucleic acid, or vector, that has been modified by the introduction
of a heterologous nucleic acid or the alteration of a native
nucleic acid to a form not native to that cell, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed, or not expressed
at all.
[0018] The term "subsequence" in the context of a referenced
nucleic acid sequence includes reference to a contiguous sequence
from the nucleic acid having fewer nucleotides in length than the
referenced nucleic acid. In the context of a referenced protein,
polypeptide, or peptide sequence (collectively, "protein"),
"subsequence" refers to a contiguous sequence from the referenced
protein having fewer amino acids than the referenced protein. The
terminus of such subsequences include all combinations consistent
with a defined length.
[0019] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0020] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations", which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes equivalents which
encompass every possible silent variation of the nucleic acid. One
of skill will recognize that each codon in a nucleic acid (except
AUG, which is ordinarily the only codon for methionine) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid which encodes a polypeptide
is implicit in each described sequence. Substitutions with
functionally equivalent unusual nucleotides or analogs are
intended, e.g., inositol, etc.
[0021] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. The following six groups each contain amino acids that are
conservative substitutions for one another: [0022] 1) Alanine (A),
Serine (S), Threonine (T); [0023] 2) Aspartic acid (D), Glutamic
acid (E); [0024] 3) Asparagine (N), Glutamine (Q); [0025] 4)
Arginine (R), Lysine (K); [0026] 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); and [0027] 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins
W. H. Freeman and Company.
[0028] By "contiguous amino acids from" in the context of a
specified number of amino acid residues from a specified sequence,
is meant a sequence of amino acids of the specified number from
within the specified reference sequence which has the identical
order of amino acids each of which is directly adjacent to the same
amino acids as in the reference sequence.
[0029] The term "polypeptide" as used herein includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least about
12 amino acids, typically at least about 16 amino acids, preferably
at least about 20 amino acids, and, in particularly preferred
embodiments, at least about 30 or more amino acids, e.g., 40, 50,
60, 70, 80, 100, etc. The ends may be are virtually all
combinations consistent with length.
[0030] The term "plurality of non-overlapping fragments"
encompasses a series of polypeptide fragments or segments. A
plurality includes 2, 3, 4, 5, etc., polypeptide fragments.
[0031] The terms "biologically pure" or "isolated" refer to
material which is substantially or essentially free from components
which normally accompany or interact with it as found in its
naturally occurring environment. The isolated material optionally
comprises material not found with the material in its natural
environment.
[0032] The phrase "encodes a protein" in the context of nucleic
acids includes those nucleic acids encoding naturally occurring
proteins or derivatives of natural proteins, but which are
deliberately modified or engineered to no longer hybridize to a
natural gene encoding the protein of natural origin under the
stated conditions.
[0033] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison may be
conducted by the local homology algorithm of Smith and Waterman
(1981) Adv. Appl. Math. 2:482; by the homology alignment algorithm
of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-445; by the
search for similarity method of Pearson and Lipman (1988) Proc.
Natl. Acad. Sci. USA 85:2444; by computerized implementations of
these algorithms (including, but not limited to CLUSTAL in the
PC/Gene program by Intelligenetics, Mountain View, Calif., GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group (GCG), 575 Science Dr.,
Madison, Wis., USA); the CLUSTAL program is well described by
Higgins and Sharp (1988) Gene 73:237-244 and Higgins and Sharp
(1989) CABIOS 5:151-153; Corpet, et al. (1988) Nucleic Acids
Research 16:10881-90; Huang, et al. (1992) Computer applications in
the Biosciences 8:155-65, and Pearson, et al. (1994.) Methods in
Molecular Biology 24:307-31. Alignment is also often performed by
inspection and manual alignment.
[0034] The terms "identical" or "sequence identity" in the context
of two nucleic acid or polypeptide sequences includes reference to
the residues in the two sequences which are the same when aligned
for maximum correspondence over a specified comparison window. When
percentage of sequence identity is used in reference to proteins it
is recognized that residue positions which are not identical often
differ by conservative amino acid substitutions, where amino acid
residues are substituted for other amino acid residues with similar
chemical properties (e.g. charge or hydrophobicity) and therefore
do not change the functional properties of the molecule. Where
sequences differ in conservative substitutions, the percent
sequence identity may be adjusted upwards to correct for the
conservative nature of the substitution. Means for making this
adjustment are well-known to those of skill in the art. Typically
this involves scoring a conservative substitution as a partial
rather than a full mismatch, thereby increasing the percentage
sequence identity. Thus, for example, where an identical amino acid
is given a score of 1 and a non-conservative substitution is given
a score of zero, a conservative substitution is given a score
between zero and 1. The scoring of conservative substitutions is
calculated, e.g., according to the algorithm of Meyers and Miller
(1988) Computer Applic. Biol. Sci. 4:11-17, e.g., as implemented in
the program PC/GENE (Intelligenetics, Mountain View, Calif.,
USA).
[0035] The terms "substantial identity" or "similarity" of
polynucleotide sequences means that a polynucleotide comprises a
sequence that has at least 60% sequence identity, preferably at
least 80%, more preferably at least 90%, and most preferably at
least 95%, compared to a reference sequence using, e.g., the
programs described above (preferably BLAST) using standard
parameters. One indication that two nucleic acid sequences are
substantially identical is that the polypeptide which the first
nucleic acid encodes is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid.
[0036] Another indication that two nucleic acid sequences have
substantial identity is that the two molecules hybridize to each
other under "moderate stringency hybridization conditions" (or
"moderate conditions") or better. Exemplary "moderate stringency
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 40.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar or higher stringency. Nucleic
acids which do not hybridize to each other under moderate
stringency hybridization conditions are still substantially
identical if the polypeptides which they encode are substantially
identical. This occurs, e.g., when a copy of a nucleic acid is
created, e.g., using the maximum codon degeneracy permitted by the
genetic code.
[0037] The terms "substantial identity" or "similarity" in the
context of a peptide indicates that a peptide comprises a sequence
with at least 60% sequence identity to a reference sequence,
usually at least 70%, preferably 80%, more preferably 85%, most
preferably at least 90% or 95% sequence identity to the reference
sequence over a specified comparison window. Preferably, optimal
alignment is conducted using the homology alignment algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48:443. An indication
that two peptide sequences are substantially identical is that one
peptide is immunologically reactive with antibodies raised against
the second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. Generally, similarity is determined
using a comparison window having a length of any number from 20
contiguous positions at various positions in the respective
molecules to the number of residues in the full-length core region
sequence, where the comparison window is within the core
sequence.
[0038] The terms "oligonucleotide" or "polynucleotide" probes
include reference to both double stranded and single stranded DNA
or RNA. The terms also refer to synthetically or recombinantly
derived sequences essentially free of non-nucleic acid
contamination.
[0039] As used herein, "contact" or "contacting" means to place in
direct physical association, e.g., mixing of solutions.
[0040] "Biological sample" as used herein is a sample of biological
tissue or fluid that contains, or is being tested for presence of,
312C2 protein, or another of described composition, e.g., nucleic
acid or protein. Such samples include, but are not limited to,
sputum, amniotic fluid, blood, blood cells, e.g., white cells, or
tissue, e.g., spleen, thymus, bone marrow, or lymph node.
Biological samples may also include sections of tissues such as
frozen sections taken for histological purposes. Examples of
biological samples include a cell sample from nervous, muscular,
glandular or epithelial tissue or from the immune system (e.g., T
cells). A biological sample is typically obtained from a eukaryotic
organism, preferably a multicellular eukaryotes such as insect,
protozoa, birds, fish, reptiles, and preferably a mammal such as
rat, mice, cow, dog, guinea pig, pig, goat, or rabbit, and most
preferably a primate such as macaques, chimpanzees, or humans.
[0041] An "expression vector" is a nucleic acid construct,
typically generated recombinantly or synthetically, with a series
of specified nucleic acid elements which permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed,
and a promoter.
[0042] The phrase "functional effects" in the-context of assays for
testing compounds affecting the 312C2 includes the determination of
any parameter that is indirectly or directly under the influence of
the 312C2. It includes changes such as increases or decreases of
transcription or second messenger or lymphokine release.
[0043] By "selectively hybridizing" or "selective hybridization" or
"selectively hybridizes" is meant hybridization, under stringent
hybridization conditions, of a nucleic acid sequence to a specified
nucleic acid target sequence to a detectably greater degree than
its hybridization to non-target nucleic acid sequences and/or to
the substantial exclusion of non-target nucleic acids. Selectively
hybridizing sequences typically have at least 80% sequence
identity, usually 90% sequence identity, preferably 95% identity,
more preferably 98% identity, and most preferably 100% sequence
identity (i.e., complementary) with each other over lengths which
typically start from about 10 nucleotides, e.g., 13, 17, 20, 23,
26, 29, 32, etc. "Percentage of sequence identity" is determined by
comparing two optimally aligned sequences over a comparison window,
wherein the portion of the polynucleotide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
[0044] The terms "stringent conditions" or "stringent hybridization
conditions" refer to conditions under which a probe will hybridize
to its target sequence, to a detectably greater degree than other
sequences. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point(.TM.) for the specific sequence at a defined
ionic strength and pH. The T.sub.m is the temperature (under
defined ionic strength and pH) at which 50% of a complementary
target sequence hybridizes to a perfectly matched probe. Typically,
stringent conditions will be those in which the salt concentration
is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. Exemplary low stringency conditions include
hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1%
SDS at 37.degree. C., and a wash in 2.times.SSC at 50.degree. C.
Exemplary high stringency conditions include hybridization in 50%
for mamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.1.times.SSC at 60.degree. C.
[0045] "Stringent hybridization conditions" or "stringent
conditions" in the context of nucleic acid hybridization assay
formats are sequence dependent, and are different under different
environmental parameters. An extensive guide to the hybridization
of nucleic acids is found e.g., in Tijssen (1993) Laboratory
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Acid Probes Part I, Chapter 2 "Overview of principles
of hybridization and the strategy of nucleic acid probe assays",
Elsevier, N.Y. Stringent conditions are sequence-dependent and will
be different in different circumstances. Longer sequences hybridize
specifically at higher temperatures.
[0046] By "hybridization complex" is meant a duplex nucleic acid
sequence formed by selective hybridization of two single-stranded
nucleic acid sequences with each other.
[0047] By "host cell" is meant a cell which is manipulated to
contain and, in certain instances, express a molecule, usually a
nucleic acid. Host cells may be prokaryotic cells such as E. coli ,
or eukaryotic cells such as yeast, insect, amphibian, or mammalian
cells.
[0048] The term "antibody" also includes antigen binding forms of
antibodies (e.g., Fab, F(ab).sub.2). The term "antibody" refers to
a polypeptide substantially encoded by an immunoglobulin gene or
immunoglobulin genes, or fragments thereof which specifically bind
and recognize an analyte (antigen). Antibodies exist, e.g., as
intact immunoglobulins or as a number of well characterized
fragments e.g., produced by digestion with various peptidases.
Thus, for example, pepsin digests an antibody below the disulfide
linkages in the hinge region to produce F(ab)'.sub.2, a dimer of
Fab which itself is a light chain joined to V.sub.H-C.sub.H1 by a
disulfide bond. The F(ab)'.sub.2 may be reduced under mild
conditions to break the disulfide linkage in the hinge region,
thereby converting the F(ab)'.sub.2 dimer into an Fab' monomer. The
Fab' monomer is essentially an Fab with part of the hinge region,
see, e.g., Paul (ed.) (1993) Fundamental Immunology, 3rd ed., Raven
Press, N.Y. While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that such fragments may be synthesized de novo either
chemically or by utilizing recombinant DNA methodology. Thus, the
term antibody, as used herein, is often functionally equivalent to
antibody fragments such as single chain Fv, chimeric antibodies
(i.e., comprising constant and variable regions from different
species), humanized antibodies (i.e., comprising a complementarity
determining region (CDR) from a non-human source) and
heteroconjugate antibodies (e.g., bispecific antibodies).
[0049] By "immunologically reactive conditions" is meant conditions
which allow an antibody, generated to a particular epitope, to bind
to that epitope to a detectably greater degree than the antibody
binds to substantially all other epitopes. Immunologically reactive
conditions are dependent upon the format of the antibody binding
reaction and typically are those utilized in immunoassay protocols.
See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold
Spring Harbor Publications, New York, for a description of
immunoassay formats and conditions.
[0050] By "antibody reactive to a protein" is meant the protein is
"specifically immunoreactive with an antibody."
[0051] The phrase "specifically immunoreactive with an antibody",
or "specifically binds to an antibody" when referring to a protein
or peptide, refers to a binding reaction between an antibody and a
protein having an epitope recognized by the antigen binding site of
the antibody. This binding reaction is determinative of the
presence of a protein having the recognized epitope amongst the
presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a protein having the recognized
epitope and bind, if at all, to a detectably lesser degree to other
proteins lacking the epitope which are present in the sample.
[0052] Specific binding to an antibody under such conditions may
require an antibody that is selected for its specificity for a
particular protein. For example, antibodies raised to the 312C2 of
SEQ ID NO: 2 or 4 can be selected from to obtain antibodies
specifically immunoreactive with that particular protein and not
with other proteins. The proteins used as immunogens can be in
native conformation or denatured, e.g., so as to provide a linear
epitope. Preferably, antibody preparations which specifically
recognize multiple epitopes will be used.
[0053] A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with a
protein. See Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
[0054] By "antigen" is meant a substance to which an antibody can
be generated and/or to which the antibody is specifically
immunoreactive with. An antibody immunologically reactive with a
particular antigen can be generated in vivo or by recombinant
methods such as selection of libraries of recombinant antibodies in
phage or similar vectors. See, e.g., Huse et al. (1989) Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546; and
Vaughan et al. (1996) Nature Biotechnology, 14:309-314.
[0055] By "transfected" is meant the introduction of a nucleic acid
into a eukaryotic cell where the nucleic acid may be incorporated
into the genome of the cell (i.e., chromosome, plasmid, or
mitochondrial DNA), converted into an autonomous replicon, or
transiently expressed (e.g., transfected mRNA). The transfection
can be in vivo or ex vivo. "Ex vivo" means outside the body of the
organism from which a cell or cells is obtained or from which a
cell line is isolated. Ex vivo transfection is preferably followed
by re-infusion of the cells back into the organism. In contrast, by
"in vivo" is meant within the body of the organism from which the
cell was obtained or from which a cell line is isolated.
[0056] The term "binding composition" refers to molecules that bind
with specificity to 312C2, e.g., in a cell adhesion pairing type
fashion, or an antibody-antigen interaction. It also includes
compounds, e.g., proteins, which specifically associate with 312C2,
including in a natural physiologically relevant protein-protein
interaction, either covalent or non-covalent. The molecule may be a
polymer, or chemical reagent. A functional analog may be an antigen
with structural modifications, or it may be a molecule which has a
molecular shape which interacts with the appropriate binding
determinants. The compounds may serve as agonists or antagonists of
the binding interaction, see, e.g., Goodman, et al. (eds.) (1990)
Goodman & Gilman's: The Pharmacological Bases of Therapeutics
(8th ed.), Pergamon Press.
[0057] Substantially pure typically means that the protein is free
from other contaminating proteins, nucleic acids, or other
biologicals with which it is associated in the original source
organism. Purity may be assayed by standard methods, typically by
weight, and will ordinarily be at least about 40% pure, generally
at least about 50% pure, often at least about 60% pure, typically
at least about 80% pure, preferably at least about 90% pure, and in
most preferred embodiments, at least about 95% pure. Carriers or
excipients will often be added, or aqueous buffers or organic
solvents used in certain situations.
II. General
[0058] The present invention provides amino acid sequences and DNA
sequences encoding various mammalian proteins which are antigens
found in the early stages of T cell activation, e.g., which can
activate a T cell. Among these proteins are antigens which
modulate, e.g., induce or prevent proliferation or differentiation
of T cells, among other physiological effects. The full length
antigens, and fragments, or antagonists will be useful in
physiological modulation of cells expressing the antigen. The
proteins will also be useful as antigens, e.g., immunogens, for
raising antibodies to various epitopes on the protein, both linear
and conformational epitopes. The molecule may be useful in defining
or isolating functional T cell or NK cell subsets.
[0059] A cDNA encoding mouse 312C2 was isolated from an activated
pro T-cell cDNA library, see Kelner, et al. (1994) Science
266:13995-1399. The mouse 312C2 cDNA contains a stretch of about
1073 bp in length and contained one large open reading frame
encoding a type I transmembrane protein. Structural features
include an N-terminal leader sequence of about 19 amino acids, an
extracellular region of about 153 amino acids, a hydrophobic
presumptive membrane spanning portion of about 25 amino acids, and
a presumptive cytoplasmic domain of about 50 amino acids. See SEQ
ID NO: 2. A human cDNA was isolated using the mouse clone to probe
a human anergic T cell library designated HY06. See SEQ ID NO: 3
and 4. A transmembrane region may begin at about amino acid 155 and
end at about amino acid 185 based on hydrophobicity analysis. The
rodent and primate sequences can be aligned.
[0060] 312C2 exhibits structural motifs characteristic of a member
of the TNF receptor family, with numerous cysteine repeats.
Compare, e.g., with the CD40, OX40, TNF receptor, NGF receptor, and
FASL receptor. The intracellular portion 312C2 does not contain a
death domain as described, e.g., in Pan, et al. (1997) Science
277:815-818; and Sheridan, et al. (1997) Science 277:818-821. Lack
of the death domain motif may indicate that 312C2 is likely to work
in the control of proliferation rather than apoptosis.
[0061] As used herein, the term "mouse 312C2" shall encompass, when
used in a protein context, a protein having amino acid sequence
shown in SEQ ID NO: 2, or a significant fragment of such a protein,
or another highly homologous protein derived from mouse. The term
"human 312C2" shall encompass, when used in a protein context, a
protein having amino acid sequence shown in SEQ ID NO: 4, or a
significant fragment of such a protein, or another highly
homologous protein derived from human.
[0062] The natural antigens are capable of mediating various
biochemical responses which lead to biological or physiological
responses in target cells. The embodiments characterized herein are
from mouse and human, but other species and tissue specific
variants exist. Additional sequences for proteins in other
mammalian species, e.g., primates and rodents, should also be
available. See below. The descriptions below are directed, for
exemplary purposes, to a mouse or human 312C2, but are likewise
applicable to related embodiments from other species.
III. Purified 312C2
[0063] The mouse 312C2 nucleic acid sequence is shown in SEQ ID NO:
1, and the amino acid sequence is shown in SEQ ID NO: 2, the human
312C2 nucleic acid sequence is shown in SEQ ID NO: 3, and the
corresponding amino acid sequence is shown in SEQ ID NO: 4. A
reverse translation of the human 312C2 sequence is shown in SEQ ID
NO: 5. These amino acid sequences, provided amino to carboxy, are
important in providing sequence information about the antigen
allowing for distinguishing the protein from other proteins and
exemplifying numerous variants. Moreover, the peptide sequences
allow preparation of peptides to generate antibodies to recognize
such segments, and nucleotide sequences allow preparation of
oligonucleotide probes, both of which are strategies for detection
or isolation, e.g., cloning, of genes encoding such sequences.
[0064] The mouse 312C2 nucleotide and predicted amino-acid
sequence, particularly the predicted leader sequence runs from
about Met1 through Gly19, though natural boundaries may be
different, also depending upon cell type. A polyadenylation signal
occurs at nucleotide position 1010. The poly A tail begins at
position 1034. See SEQ ID NO: 1 and 2. The transmembrane domain is
predicted to encompass amino acids beginning at about 154through
about 179.
[0065] In human the putative leader sequence runs from about Met1
through about Leu18. The transmembrane domain is predicted to begin
at out amino acid 153 through about 182. Again the natural
boundaries may vary.
[0066] Antibodies to these proteins typically bind to a 312C2 with
high affinity, e.g., at least about 100 nM, usually better than
about 30 nM, preferably better than about 10 nM, and more
preferably at better than about 3 nM. Homologous proteins would be
found in mammalian species other than mouse, e.g., primates or
rodents. Non-mammalian species should also possess structurally or
functionally related genes and proteins, e.g., birds or
amphibians.
[0067] Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect polypeptide
solubility, including temperature electrolyte environment, size and
molecular characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the polypeptide is
used ranges from about 4.degree. C. to about 65.degree. C. Usually
the temperature at use is greater than about 18.degree. C. For
diagnostic purposes, the temperature will usually be about room
temperature or warmer, but less than the denaturation temperature
of components in the assay. For therapeutic purposes, the
temperature will usually be body temperature, typically about
37.degree. C. for humans and mice, though under certain situations
the temperature may be raised or lowered in situ or in vitro.
[0068] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents in a manner which approximates natural
lipid bilayer interactions. In certain contexts, e.g., Western
blots, the protein will be denatured, and/or attached to a solid
substrate, e.g., in an affinity column.
[0069] The solvent and electrolytes will usually be a biologically
compatible buffer, of a type used for preservation of biological
activities, and will usually approximate a physiological aqueous
solvent. Usually the solvent will have a neutral pH, typically
between about 5 and 10, and preferably about 7.5. On some
occasions, one or more detergents will be added, typically a mild
non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS
(3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), or a
low enough concentration as to avoid significant disruption of
structural or physiological properties of the protein.
[0070] A. Physical Variants
[0071] This invention also encompasses proteins or peptides having
substantial amino acid sequence identity with the amino acid
sequence of the 312C2. The variants include species, polymorphic,
or allelic variants.
[0072] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. See also Needleham, et al. (1970) J.
Mol. Biol. 48:443-453; Sankoff, et al. (1983) Chapter One in Time
Warps, String Edits, and Macromolecules: The Theory and Practice of
Sequence Comparison, Addison-Wesley, Reading, Mass.; and software
packages from IntelliGenetics, Mountain View, Calif.; and the
University of Wisconsin Genetics Computer Group, Madison, Wis.
Sequence identity changes when considering conservative
substitutions as matches. Conservative substitutions typically
include substitutions within the following groups: glycine,
alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. Homologous amino acid sequences are
typically intended to include natural polymorphic or allelic and
interspecies variations in each respective protein sequence.
Typical homologous proteins or peptides will have from 25-100%
identity (if gaps can be introduced), to 50-100% identity (if
conservative substitutions are included) with the amino acid
sequence of the 312C2. Identity measures will be at least about
35%, generally at least about 40%, often at least about 50%,
typically at least about 60%, usually at least about 70%,
preferably at least about 80%, and more preferably at least about
90%.
[0073] The isolated 312C2 DNA can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and
inversions of nucleotide stretches. These modifications result in
novel DNA sequences which encode these antigens, their derivatives,
or proteins having similar physiological, immunogenic, antigenic,
or other functional activity. These modified sequences can be used
to produce mutant antigens or to enhance expression. Enhanced
expression may involve gene amplification, increased transcription,
increased translation, and other mechanisms. "Mutant 312C2"
encompasses a polypeptide otherwise falling within the sequence
identity definition of the 312C2 as set forth above, but having an
amino acid sequence which differs from that of 312C2 as normally
found in nature, whether by way of deletion, substitution, or
insertion. This generally includes proteins having significant
identity with a protein having sequence of SEQ ID NO: 2, and as
sharing various biological activities, e.g., antigenic or
immunogenic, with those sequences, and in preferred embodiments
contain most of the full length disclosed sequences. Preferred
variants will share a plurality of immune epitopes with the recited
sequences, or equivalent proteins. Full length sequences will
typically be preferred, though truncated versions will also be
useful, likewise, genes or proteins found from natural sources are
typically most desired. Similar concepts apply to different 312C2
proteins, particularly those found in various warm blooded animals,
e.g., mammals and birds. These descriptions are generally meant to
encompass all 312C2 proteins, not limited to the particular mouse
or human embodiments specifically discussed.
[0074] 312C2 mutagenesis can also be conducted by making amino acid
insertions or deletions. Substitutions, deletions, insertions, or
any combinations may be generated to arrive at a final construct.
Insertions include amino- or carboxy-terminal fusions. Random
mutagenesis can be conducted at a target codon and the expressed
mutants can then be screened for the desired activity. Methods for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known in the art, e.g., by M13 primer
mutagenesis or polymerase chain reaction (PCR) techniques. See,
e.g., Sambrook, et al. (1989); Ausubel, et al. (1987 and
Supplements); and Kunkel, et al. (1987) Methods in Enzymol.
154:367-382.
[0075] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
A similar concept applies to heterologous nucleic acid
sequences.
[0076] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
target-binding or other segments may be "swapped" between different
new fusion polypeptides or fragments. See, e.g., Cunningham, et al.
(1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol.
Chem. 263:15985-15992.
[0077] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence, e.g., PCR techniques.
[0078] B. Functional Variants
[0079] The blocking of physiological response to 312C2s may result
from the inhibition of binding of the antigen to its binding
partner, e.g., another of itself, likely through competitive
inhibition. Thus, in vitro assays of the present invention will
often use isolated protein, membranes from cells expressing a
membrane associated recombinant 312C2, soluble fragments comprising
antigen binding segments of these proteins, or fragments attached
to solid phase substrates. These assays will also allow for the
diagnostic determination of the effects of either binding segment
mutations and modifications, or antigen mutations and
modifications, e.g., 312C2 analogs. Structure-activity analysis
will be performed with mutational variants.
[0080] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to antigen or
binding fragments compete with a test compound for binding to the
protein, e.g., of natural protein sequence.
[0081] "Derivatives" of 312C2 antigens include amino acid sequence
mutants from naturally occurring forms, glycosylation variants, and
covalent or aggregate conjugates with other chemical moieties.
Covalent derivatives can be prepared by linkage of functionalities
to groups which are found in 312C2 amino acid side chains or at the
N- or C-termini, e.g., by standard means. See, e.g., Lundblad and
Noyes (1988) Chemical Reagents for Protein Modification, vols. 1-2,
CRC Press, Inc., Boca Raton, Fla.; Hugli (ed.) (1989) Techniques in
Protein Chemistry, Academic Press, San Diego, Calif.; and Wong
(1991) Chemistry of Protein Conjugation and Cross Linking, CRC
Press, Boca Raton, Fla.
[0082] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. See, e.g., Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also
embraced are versions of the peptides with the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0083] Fusion polypeptides between 312C2s and other homologous or
heterologous proteins are also provided. Many cytokine receptors or
other surface proteins are multimeric,. e.g., homodimeric entities,
and a repeat construct may have various advantages, including
lessened susceptibility to proteolytic cleavage. Typical examples
are fusions of a reporter polypeptide, e.g., luciferase, with a
segment or domain of a protein, e.g., a receptor-binding segment,
so that the presence or location of the fused ligand may be easily
determined. See, e.g., Dull, et al., U.S. Pat. No. 4,859,609. Other
gene fusion partners include bacterial .beta.-galactosidase, trpE,
Protein A, .beta.-lactamase, alpha amylase, alcohol dehydrogenase,
yeast alpha mating factor, and detection or purification tags such
as a FLAG sequence of His6 sequence. See, e.g., Godowski, et al.
(1988) Science 241:812-816.
[0084] Fusion peptides will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, e.g., in Sambrook, et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed.), vols. 1-3, Cold Spring
Harbor Laboratory; and Ausubel, et al. (eds.) (1993) Current
Protocols in Molecular Biology, Greene and Wiley, N.Y. Techniques
for synthesis of polypeptides are described, e.g., in Merrifield
(1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science
232:341-347; Atherton, et al. (1989) Solid Phase Peptide Synthesis:
A Practical Approach, IRL Press, Oxford; and Grant (1992) Synthetic
Peptides: A User's Guide, W. H. Freeman, NY.
[0085] This invention also contemplates the use of derivatives of
312C2s other than variations in amino acid sequence or
glycosylation. Such derivatives may involve covalent or aggregative
association with chemical moieties. Covalent or aggregative
derivatives will be useful as immunogens, as reagents in
immunoassays, or in purification methods such as for affinity
purification of binding partners, e.g., other antigens. A 312C2 can
be immobilized by covalent bonding to a solid support such as
cyanogen bromide-activated SEPHAROSE, by methods which are well
known in the art, or adsorbed onto polyolefin surfaces, with or
without glutaraldehyde cross-linking, for use in the assay or
purification of anti-312C2 antibodies or an alternative binding
composition. Western blot techniques are also common. The 312C2s
can also be labeled with a detectable group, e.g., for use in
diagnostic assays. Purification of 312C2 may be effected by an
immobilized antibody or complementary binding partner.
[0086] A solubilized 312C2 or fragment of this invention can be
used as an immunogen for the production of antisera or antibodies
specific for binding to the antigen or fragments thereof. Purified
antigen can be used to screen or purify monoclonal antibodies or
antigen-binding fragments, encompassing antigen binding fragments
of natural antibodies, e.g., Fab, Fab', F(ab).sub.2, etc. Purified
312C2s can also be used as a reagent to detect antibodies generated
in response to the presence of elevated levels of the antigen or
cell fragments containing the antigen, both of which may be
diagnostic of an abnormal or specific physiological or disease
condition. This invention contemplates antibodies raised against
amino acid sequences encoded by nucleotide sequence shown in SEQ ID
NO: 1, or fragments of proteins containing it. In particular, this
invention contemplates antibodies having binding affinity to or
being raised against specific fragments which are predicted to lie
outside of the lipid bilayer, both extracellular or
intracellular.
[0087] The present invention contemplates the isolation of
additional closely related species variants. Southern and Northern
blot analysis should establish that similar genetic entities exist
in other mammals. It is likely that 312C2s are widespread in
species variants, e.g., rodents, lagomorphs, carnivores,
artiodactyla, perissodactyla, and primates.
[0088] The invention also provides means to isolate a group of
related antigens displaying both distinctness and similarities in
structure, expression, and function. Elucidation of many of the
physiological effects of the molecules will be greatly accelerated
by the isolation and characterization of additional distinct
species variants of them. In particular, the present invention
provides useful probes for identifying additional homologous
genetic entities in different species.
[0089] The isolated genes will allow transformation of cells
lacking expression of a corresponding 312C2, e.g., either species
types or cells which lack corresponding antigens and exhibit
negative background activity. This should allow analysis of the
function of 312C2 in comparison to untransformed control cells.
[0090] Dissection of critical structural elements which effect the
various activation or differentiation functions mediated through
these antigens is possible using standard techniques of modern
molecular biology, particularly in comparing members of the related
class. See, e.g., the homolog-scanning mutagenesis technique
described in Cunningham, et al. (1989) Science 243:1339-1336; and
approaches used in O'Dowd, et al. (1988) J. Biol. Chem.
263:15985-15992; and Lechleiter, et al. (1990) EMBO J. 9:4381-4390.
Structure activity relationship can be analyzed using variants.
[0091] Intracellular functions would probably involve segments of
the antigen which are normally accessible to the cytosol. However,
protein internalization may occur under certain circumstances, and
interaction between intracellular components and "extracellular"
segments may occur. The specific segments of interaction of 312C2
with other intracellular components may be identified by
mutagenesis or direct biochemical means, e.g., cross-linking or
affinity methods. Structural analysis by crystallographic or other
physical methods will also be applicable. Further investigation of
the mechanism of signal transduction will include study of
associated components which may be isolatable by affinity methods
or by genetic means, e.g., complementation analysis of mutants.
[0092] Further study of the expression and control of 312C2 will be
pursued. The controlling elements associated with the antigens
should exhibit differential physiological, developmental, tissue
specific, or other expression patterns. Upstream or downstream
genetic regions, e.g., control elements, are of interest. In
particular, physiological or developmental variants, e.g., multiple
alternatively processed forms of the mouse antigen have been found.
See, e.g., SEQ ID NO: 1. Thus, differential splicing of message may
lead to an assortment of membrane bound forms, soluble forms, and
modified versions of antigen.
[0093] With human 312C2, 6 alternatively processed forms have been
isolated. Clone A8, a truncated form of 312C2, is missing 7 amino
acids immediately after the transmembrane domain. See SEQ ID NO: 6.
Clone A5 is identical to 312C2 for the first 105 amino acids. It is
believed that the divergence may be due to an unspliced intron. See
SEQ ID NO: 7. Clone G10 is identical to 312C2 for the first 202
amino acids, but then varies in the 11 amino acids after the
transmembrane domain and is 76 amino acids longer in the
intracellular domain. The intracellular domain of G10, like that of
312C2, does not contain a death domain. See SEQ ID NO: 8.
[0094] Structural studies of the antigens will lead to design of
new antigens, particularly analogs exhibiting agonist or antagonist
properties on the molecule. This can be combined with previously
described screening methods to isolate antigens exhibiting desired
spectra of activities.
IV. Antibodies
[0095] Antibodies can be raised to various 312C2s, including
species, polymorphic, or allelic variants, and fragments thereof,
both in their naturally occurring forms and in their recombinant
forms. Additionally, antibodies can be raised to 312C2s in either
their active forms or in their inactive forms, including native or
denatured versions. Anti-idiotypic antibodies are also
contemplated.
[0096] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the antigens can be
raised by immunization of animals with conjugates of the fragments
with immunogenic proteins. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be
screened for binding to normal or defective 312C2s, or screened for
agonistic or antagonistic activity, e.g., mediated through the
antigen or its binding partner. Antibodies may be agonistic or
antagonistic, e.g., by sterically blocking ligand binding. These
monoclonal antibodies will usually bind with at least a K.sub.D of
about 1 mM, more usually at least about 300 .mu.M, typically at
least about 100 .mu.M, more typically at least about 30 .mu.M,
preferably at least about 10 .mu.M, and more preferably at least
about 3 .mu.M or better.
[0097] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they can be screened for ability to bind to the antigens without
inhibiting binding by a partner. As neutralizing antibodies, they
can be useful in competitive binding assays. They will also be
useful in detecting or quantifying 312C2 protein or its binding
partners. See, e.g., Chan (ed.) (1987) Immunology: A Practical
Guide, Academic Press, Orlando, Fla.; Price and Newman (eds.)
(1991) Principles and Practice of Immunoassay, Stockton Press,
N.Y.; and Ngo (ed.) (1988) Nonisotopic Immunoassay, Plenum Press,
N.Y. Cross absorptions or other tests will identify antibodies
which exhibit various spectra of specificities, e.g., unique or
shared species specificities.
[0098] Further, the antibodies, including antigen binding
fragments, of this invention can be potent antagonists that bind to
the antigen and inhibit functional binding or inhibit the ability
of a binding partner to elicit a biological response. They also can
be useful as non-neutralizing antibodies and can be coupled to
toxins or radionuclides so that when the antibody binds to antigen,
a cell expressing it, e.g., on its surface, is killed. Further,
these antibodies can be conjugated to drugs or other therapeutic
agents, either directly or indirectly by means of a linker, and may
effect drug targeting.
[0099] Antigen fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. An antigen and its fragments
may be fused or covalently linked to a variety of immunogens, such
as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid,
etc. See Microbiology, Hoeber Medical Division, Harper and Row,
1969; Landsteiner (1962) Specificity of Serological Reactions,
Dover Publications, New York; Williams, et al. (1967) Methods in
Immunology and Immunochemistry, vol. 1, Academic Press, New York;
and Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH
Press, NY, for descriptions of methods of preparing polyclonal
antisera.
[0100] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.), Academic Press, New York; and particularly in Kohler and
Milstein (1975) in Nature 256:495-497, which discusses one method
of generating monoclonal antibodies.
[0101] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. The
polypeptides and antibodies of the present invention may be used
with or without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents, teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; Moore, et al., U.S. Pat. No. 4,642,334; and Queen, et
al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.
[0102] The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can be
prepared where the antibodies are linked to a solid support. See,
e.g., Wilchek et al. (1984) Meth. Enzymol. 104:3-55.
[0103] Antibodies raised against each 312C2 will also be useful to
raise anti-idiotypic antibodies. These will be useful in detecting
or diagnosing various immunological conditions related to
expression of the respective antigens.
V. Nucleic Acids
[0104] The described peptide sequences and the related reagents are
useful in detecting, isolating, or identifying a DNA clone encoding
312C2, e.g., from a natural source. Typically, it will be useful in
isolating a gene from mammal, and similar procedures will be
applied to isolate genes from other species, e.g., warm blooded
animals, such as birds and mammals. Cross hybridization will allow
isolation of 312C2 from other species. A number of different
approaches should be available to successfully isolate a suitable
nucleic acid clone.
[0105] The purified protein or defined peptides are useful for
generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold
Spring Harbor Press. Alternatively, the 312C2 can be used as a
specific binding reagent, and advantage can be taken of its
specificity of binding, much like an antibody would be used.
[0106] For example, the specific binding composition could be used
for screening of an expression library made from a cell line which
expresses a 312C2. The screening can be standard staining of
surface expressed antigen, or by panning. Screening of
intracellular expression can also be performed by various staining
or immunofluorescence procedures. The binding compositions could be
used to affinity purify or sort out cells expressing the
protein.
[0107] The peptide segments can also be used to predict appropriate
oligonucleotides to screen a library. The genetic code can be used
to select appropriate oligonucleotides useful as probes for
screening. See, e.g., SEQ ID NO: 1 or 3. In combination with
polymerase chain reaction (PCR) techniques, synthetic
oligonucleotides will be useful in selecting correct clones from a
library. Complementary sequences will also be used as probes,
primers, or antisense strands. Based upon identification of the
likely extracellular domain, various fragments should be
particularly useful, e.g., coupled with anchored vector or poly-A
complementary PCR techniques or with complementary DNA of other
peptides.
[0108] This invention contemplates use of isolated DNA or fragments
to encode a biologically active corresponding 312C2 polypeptides.
See SEQ ID NO: 5. In addition, this invention covers isolated or
recombinant DNA which encodes a biologically active, e.g.,
antigenic, protein or polypeptide which is capable of hybridizing
under appropriate conditions with the DNA sequences described
herein. Said biologically active protein or polypeptide can be an
intact antigen, or fragment, and have an amino acid sequence
disclosed in, e.g., SEQ ID NO: 2 or 4. Further, this invention
covers the use of isolated or recombinant DNA, or fragments
thereof, which encode proteins which are homologous to a 312C2 or
which was isolated using cDNA encoding a 312C2 as a probe. The
isolated DNA can have the respective regulatory sequences in the 5'
and 3' flanks, e.g., promoters, enhancers, poly-A addition signals,
and others.
[0109] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other components which naturally accompany a native sequence, e.g.,
ribosomes, polymerases, and/or flanking genomic sequences from the
originating species. The term embraces a nucleic acid sequence
which has been removed from its naturally occurring environment,
and includes recombinant or cloned DNA isolates and chemically
synthesized analogs or analogs biologically synthesized by
heterologous systems. A substantially pure molecule includes
isolated forms of the molecule. Generally, the nucleic acid will be
in a vector or fragment less than about 50 kb, usually less than
about 30 kb, typically less than about 10 kb, and preferably less
than about 6 kb.
[0110] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
minor heterogeneity. This heterogeneity is typically found at the
polymer ends or portions not critical to a desired biological
function or activity.
[0111] A "recombinant" nucleic acid is defined either by its method
of production or its structure. In reference to its method of
production, e.g., a product made by a process, the process is use
of recombinant nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence, typically selection or
production. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants. Thus; e.g.,
products made by transforming cells with any unnaturally occurring
vector is encompassed, as are nucleic acids comprising sequence
derived using any synthetic oligonucleotide process. Such is often
done to replace a codon with a redundant codon encoding the same or
a conservative amino acid, while typically introducing or removing
a sequence recognition site.
[0112] Alternatively, it is performed to join together nucleic acid
segments of desired functions to generate a single genetic entity
comprising a desired combination of functions not found in the
commonly available natural forms. Restriction enzyme recognition
sites are often the target of such artificial manipulations, but
other site specific targets, e.g., promoters, DNA replication
sites, regulation sequences, control sequences, or other useful
features may be incorporated by design. A similar concept is
intended for a recombinant, e.g., fusion, polypeptide. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode polypeptides similar to fragments of these
antigens, and fusions of sequences from various different species
variants. Alternatively, a heterologous promoter may be inserted
upstream from a natural gene.
[0113] A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally at
least about 22 nucleotides, ordinarily at least about 29
nucleotides, more often at least about 35 nucleotides, typically at
least about 41 nucleotides, usually at least about 47 nucleotides,
preferably at least about 55 nucleotides, and in particularly
preferred embodiments will be at least about 60 or more
nucleotides.
[0114] A DNA which codes for a 312C2 protein will be particularly
useful to identify genes, mRNA, and cDNA species which code for
related or homologous proteins, as well as DNAs which code for
homologs proteins from different species. There are likely
homologues in other species, including primates, rodents, and
birds. Various 312C2 proteins should be homologous and are
encompassed herein. However, even proteins that have a more distant
evolutionary relationship to the antigen can readily be isolated
under appropriate conditions using these sequences if they are
sufficiently homologous. Primate 312C2 proteins are of particular
interest.
[0115] Recombinant clones derived from the genomic sequences, e.g.,
containing introns, will be useful for transgenic studies,
including, e.g., transgenic cells and organisms, and for gene
therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt
(ed.) Encyclopedia of Immunology, Academic Press, San Diego, pp.
1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991)
Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson
(1987)(ed.) Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, IRL Press, Oxford; and Rosenberg (1992) J. Clinical
Oncology 10:180-199.
[0116] Substantial homology in the nucleic acid sequence comparison
context means either that the segments, or their complementary
strands, when compared, are identical when optimally aligned, with
appropriate nucleotide insertions or deletions, in at least about
50% of the nucleotides, generally at least about 58%, ordinarily at
least about 65%, often at least about 71%, typically at least about
77%, usually at least about 85%, preferably at least about 95 to
98% or more, and in particular embodiments, as high as about 99% or
more of the nucleotides. Alternatively, substantial homology exists
when the segments will hybridize under selective hybridization
conditions, to a strand, or its complement, typically using a
sequence of 312C2, e.g., in SEQ ID NO: 1 or 3. Typically, selective
hybridization will occur when there is at least about 55% homology
over a stretch of at least about 30 nucleotides, preferably at
least about 75% over a stretch of about 25 nucleotides, and most
preferably at least about 90% over about 20 nucleotides. See,
Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length of homology
comparison, as described, may be over longer stretches, and in
certain embodiments will be over a stretch of at least about 17
nucleotides, usually at least about 28 nucleotides, typically at
least about 40 nucleotides, and preferably at least about 75 to 100
or more nucleotides.
[0117] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., usually in excess of about 37.degree. C.,
typically in excess of about 55.degree. C., preferably in excess of
about 70.degree. C. Stringent salt conditions will ordinarily be
less than about 1000 mM, usually less than about 400 mM, typically
less than about 250 mM, preferably less than about 150 mM. However,
the combination of parameters is much more important than the
measure of any single parameter. See, e.g., Wetmur and Davidson
(1968) J. Mol. Biol. 31:349-370.
[0118] 312C2 from other mammalian species can be cloned and
isolated by cross-species hybridization of closely related species.
Homology may be relatively low between distantly related species,
and thus hybridization of relatively closely related species is
advisable. Alternatively, preparation of an antibody preparation
which exhibits less species specificity may be useful in expression
cloning approaches.
VI. Making 312C2; Mimetics
[0119] DNA which encodes the 312C2 or fragments thereof can be
obtained by chemical synthesis, screening cDNA libraries, or
screening genomic libraries prepared from a wide variety of cell
lines or tissue samples. See, e.g., Okayama and Berg (1982) Mol.
Cell. Biol. 2:161-170; Gubler. and Hoffman (1983) Gene 25:263-269;
and Glover (ed.) (1984) DNA Clonina: A Practical Approach, IRL
Press, Oxford. Alternatively, the sequences provided herein provide
useful PCR primers or allow synthetic or other preparation of
suitable genes encoding a 312C2; including, naturally occurring
embodiments.
[0120] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length 312C2 or fragments which can in
turn, e.g., be used to generate polyclonal or monoclonal
antibodies; for binding studies; for construction and expression of
modified molecules; and for structure/function studies.
[0121] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. See, e.g., Pouwels, et al. (1985 and Supplements) Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y.; and Rodriguez, et al.
(1988)(eds.) Vectors: A Survey of Molecular Clonina Vectors and
Their Uses, Buttersworth, Boston, Mass.
[0122] For purposes of this invention, DNA sequences are operably
linked when they are functionally related to each other. For
example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression. See e.g., Rodriguez, et al., Chapter 10,
pp. 205-236; Balbas and Bolivar (1990) Methods in Enzymology
185:14-37; and Ausubel, et al. (1993) Current Protocols in
Molecular Biology, Greene and Wiley, N.Y.
[0123] Representative examples of suitable expression vectors
include pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
See, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-199.
[0124] It will often be desired to express a 312C2 polypeptide in a
system which provides a specific or defined glycosylation pattern.
See, e.g., Luckow and Summers (1988) Bio/Technology 6:47-55; and
Kaufman (1990) Meth. Enzymol. 185:487-511.
[0125] The 312C2, or a fragment thereof, may be engineered to be
phosphatidyl inositol (PI) linked to a cell membrane, but can be
removed from membranes by treatment with a phosphatidyl inositol
cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. This
releases the antigen in a biologically active form, and allows
purification by standard procedures of protein chemistry. See,
e.g., Low (1989) Biochim. Biophys. Acta 988:427-454; Tse, et al.
(1985) Science 230:1003-1008; and Brunner, et al. (1991) J. Cell
Biol. 114:1275-12.83.
[0126] Now that the 312C2 has been characterized, fragments or
derivatives thereof can be prepared by conventional processes for
synthesizing peptides. These include processes such as are
described in Stewart and Young (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York; and Villafranca (ed.)
(1991) Techniques in Protein Chemistry II, Academic Press, San
Diego, Calif.
VII. Uses
[0127] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
the general description for T cell mediated conditions, or below in
the description of kits for diagnosis. The antigen is useful as a
marker, e.g., to identify T or NK cell subsets, or as a positive
selection marker to fractionate immune subsets.
[0128] This invention also provides reagents with significant
therapeutic value. The 312C2 (naturally occurring or recombinant),
fragments thereof, and antibodies thereto, along with compounds
identified as having binding affinity to 312C2, should be useful in
the treatment of conditions associated with abnormal physiology or
development, including abnormal proliferation, e.g., cancerous
conditions, or degenerative conditions. In particular, modulation
of development of lymphoid cells will be achieved by appropriate
therapeutic treatment using the compositions provided herein. For
example, a disease or disorder associated with abnormal expression
or abnormal signaling by a 312C2 should be a likely target for an
agonist or antagonist of the antigen. The antigen plays a role in
regulation or development of hematopoietic cells, e.g., lymphoid
cells, which affect immunological responses, e.g., autoimmune
disorders.
[0129] In particular, the antigen will likely provide a
costimulatory signal to T cell activation. Thus, the 312C2 will
likely mediate T cell interactions with other cell types. These
interactions lead, in particular contexts, to cell proliferation,
enhanced cytokine synthesis by the cells, and consequential
amplification of T cell proliferation.
[0130] Moreover, the 312C2 or antagonists could redirect T cell
responses, e.g., towards a Th0/Th1 pathway, or towards a Th2 type
response. Among these agonists should be various antibodies which
recognize the appropriate epitopes, e.g., which mimic binding of
312C2 to its ligand. Alternatively, antibody antagonists may bind
to epitopes which sterically can block partner binding.
[0131] Conversely, antagonists of 312C2, such as the naturally
occurring secreted form of 312C2 or blocking antibodies, may
provide a selective and powerful way to block immune responses in
abnormal situations, e.g., autoimmune disorders, including
rheumatoid arthritis, systemic lupus erythrematosis (SLE),
Hashimoto's autoimmune thyroiditis, as well as acute and chronic
inflammatory responses in which T cell activation, expansion,
and/or immunological T cell memory play an important role. See also
Samter, et al. (eds.) Immunological Diseases vols. 1 and 2, Little,
Brown and Co. Suppression of T cell activation! expansion, and/or
cytokine release by the naturally occurring secreted form of 312C2,
which can be produced in large quantities by recombinant methods,
or by blocking antibodies, should be effective in many disorders in
which abnormal or undesired T cell responses are of importance,
e.g., in a transplantation resection situation.
[0132] In addition, certain combination compositions with other
modulators of T cell signaling would be useful. Such other
signaling molecules include TcR reagents, CD40, CD40L, CTLA-8,
CD28, SLAM, FAS, and their respective antagonists.
[0133] Various abnormal conditions are known in each of the cell
types shown to possess 312C2 mRNA by Northern blot analysis. See
Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck &
Co., Rahway, N.J.; Thorn, et al. Harrison's Principles of Internal
Medicine, McGraw-Hill, N.Y.; and Weatherall, et al. (eds.) Oxford
Textbook of Medicine, Oxford University Press, Oxford. Many other
medical conditions and diseases involve T cells or are T cell
mediated, and many of these will be responsive to treatment by an
agonist or antagonist provided herein. See, e.g., Stites and Terr
(eds; 1991) Basic and Clinical Immunology, Appleton and Lange,
Norwalk, Conn.; and Samter, et al. (eds) Immunological Diseases
Little, Brown and Co. These problems should be susceptible to
prevention or treatment using compositions provided herein.
[0134] 312C2 antibodies can be purified and then administered to a
patient, veterinary or human. These reagents can be combined for
therapeutic use with additional active or inert ingredients, e.g.,
in conventional pharmaceutically acceptable carriers or diluents,
e.g., immunogenic adjuvants, along with physiologically innocuous
stabilizers, excipients, buffers, or preservatives. These
combinations can be sterile filtered and placed into dosage forms
as by lyophilization in dosage vials or storage in stabilized
aqueous preparations. This invention also contemplates use of
antibodies or binding fragments thereof, including forms which are
not complement binding. Sterile compositions of nucleic acids and
proteins are also contemplated.
[0135] Drug screening using 312C2 or fragments thereof can be
performed to identify compounds having binding affinity to or other
relevant biological effects on 312C2 functions, including isolation
of associated components. Subsequent biological assays can then be
utilized to determine if the compound has intrinsic stimulating
activity and is therefore a blocker or antagonist in that it blocks
the activity of the antigen. Likewise, a compound having intrinsic
stimulating activity can activate the signal pathway and is thus an
agonist in that it simulates the activity of 312C2. This invention
further contemplates the therapeutic use of blocking antibodies to
312C2 as antagonists and of stimulatory antibodies, e.g., A12, as
agonists. This approach should be particularly useful with other
312C2 species variants.
[0136] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medicants administered. Thus, treatment dosages should be
titrated to optimize safety and efficacy. Typically, dosages used
in vitro may provide useful guidance in the amounts useful for in
situ administration of these reagents. Animal testing of effective
doses for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds.) (1990) Goodman and
Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.
(1990),. Mack Publishing Co., Easton, Pa. Methods for
administration are discussed therein and below, e.g., for oral,
intravenous, intraperitoneal, or intramuscular administration,
transdermal diffusion, and others. Pharmaceutically acceptable
carriers will include water, saline, buffers, and other compounds
described, e.g., in the Merck Index, Merck & Co., Rahway, N.J.
Dosage ranges would ordinarily be expected to be in amounts lower
than 1 mM concentrations, typically less than about 10 .mu.M
concentrations, usually less than about 100 nM, preferably less
than about 10 .mu.M (picomolar), and most preferably less than
about 1 fM (femtomolar), with an appropriate carrier. Slow release
formulations, or a slow release apparatus will often be utilized
for continuous or long term administration. See, e.g., Langer
(1990) Science 249:1527-1533.
[0137] 312C2, fragments thereof, and antibodies to it or its
fragments, antagonists, and agonists, may be administered directly
to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in
many conventional dosage formulations. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Each carrier should
be both pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and not
injurious to the patient. Formulations include those suitable for
oral, rectal, nasal, topical, or parenteral (including
subcutaneous, intramuscular, intravenous and intradermal)
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods well known in
the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)
Goodman and Gilman's: The Pharmacological Bases of Therapeutics,
8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences,
17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al.
(eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,
Dekker, N.Y.; Lieberman, et al. (eds.). (1990) Pharmaceutical
Dosage Forms: Tablets, Dekker, N.Y.; and Lieberman, et al. (eds.)
(1990) Pharmaceutical Dosage Forms: Disperse Systems, Dekker, N.Y.
The therapy of this invention may be combined with or used in
association with other agents, e.g., other modulators of T cell
activation, e.g., CD40, CD40 ligand, CD28, CTLA-4, B7, B70, SLAM, T
cell receptor signaling entities, or their respective
antagonists.
[0138] Both the naturally occurring and the recombinant form of the
312C2s of this invention are particularly useful in kits and assay
methods which are capable of screening compounds for binding
activity to the proteins. Several methods of automating assays have
been developed in recent years so as to permit screening of tens of
thousands of compounds in a short period. See, e.g., Fodor, et al.
(1991) Science 251:767-773, which describes means for testing of
binding affinity by a plurality of defined polymers synthesized on
a solid substrate. The development of suitable assays can be
greatly facilitated by the availability of large amounts of
purified, soluble 312C2 as provided by this invention.
[0139] Other methods can be used to determine the critical residues
in the 312C2-312C2 ligand interactions. Mutational analysis can be
performed, e.g., see Somoza, et al. (1993) J. Exptl. Med.
178:549-558, to determine specific residues critical in the
interaction and/or signaling. Both extracellular domains, involved
in the homophilic interaction, or intracellular domain, which
provides interactions important in intracellular signaling.
[0140] For example, antagonists can normally be found once the
antigen has been structurally defined, e.g., by tertiary structure
data. Testing of potential interacting analogs is now possible upon
the development of highly automated assay methods using a purified
312C2. In particular, new agonists and antagonists will be
discovered by using screening techniques described herein. Of
particular importance are compounds found to have a combined
binding affinity for a spectrum of 312C2 molecules, e.g., compounds
which can serve as antagonists for species variants of 312C2.
[0141] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing a 312C2. Cells may be isolated
which express a 312C2 in isolation from other molecules. Such
cells, either in viable or fixed form, can be used for standard
binding partner binding assays. See also, Parce, et al. (1989)
Science 246:243-247; and Owicki, et al. (1990) Proc. Nat'l Acad.
Sci. USA 87:4007-4011, which describe sensitive methods to detect
cellular responses.
[0142] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to a 312C2 and is described in detail in
Geysen, European Patent Application 84/03564, published on Sep. 13,
1984. First, large numbers of different small peptide test
compounds are synthesized on a solid substrate, e.g., plastic pins
or some other appropriate surface, see Fodor, et al. (1991). Then
all the pins are reacted with solubilized, unpurified or
solubilized, purified 312C2, and washed. The next step involves
detecting bound 312C2.
[0143] Rational drug design may also be based upon structural
studies of the molecular shapes of the 312C2 and other effectors or
analogs. Effectors may be other proteins which mediate other
functions in response to binding, or other proteins which normally
interact with 312C2. One means for determining which sites interact
with specific other proteins is a physical structure determination,
e.g., x-ray crystallography or 2 dimensional NMR techniques. These
will provide guidance as to which amino acid residues form
molecular contact regions. For a detailed description of protein
structural determination, see, e.g., Blundell and Johnson (1976)
Protein Crystallography, Academic Press, New York. Structure from
related TcR family genes will also provide further insight.
[0144] VIII. Kits
[0145] This invention also contemplates use of 312C2 proteins,
fragments thereof, peptides, and their fusion products in a variety
of diagnostic kits and methods for detecting the presence of
another 312C2 or binding partner. Typically the kit will have a
compartment containing either a defined 312C2 peptide or gene
segment or a reagent which recognizes one or the other, e.g., 312C2
fragments or antibodies.
[0146] A kit for determining the binding affinity of a test
compound to a 312C2 would typically comprise a test compound; a
labeled compound, for example a binding partner or antibody having
known binding affinity for 312C2; a source of 312C2 (naturally
occurring or recombinant); and a means for separating bound from
free labeled compound, such as a solid phase for immobilizing the
molecule. Once compounds are screened, those having suitable
binding affinity to the antigen can be evaluated in suitable
biological assays, as are well known in the art, to determine
whether they act as agonists or antagonists to the signaling
pathway. The availability of recombinant 312C2 polypeptides also
provide well defined standards for calibrating such assays.
Histological analysis is also possible.
[0147] A preferred kit for determining the concentration of, e.g.,
a 312C2 in a sample would typically comprise a labeled compound,
e.g., binding partner or antibody, having known binding affinity
for the antigen; a source of antigen (naturally occurring or
recombinant) and a means for separating the bound from free labeled
compound, e.g., a solid phase for immobilizing the 312C2.
Compartments containing reagents, and instructions, will normally
be provided.
[0148] Antibodies, including antigen binding fragments, specific
for the 312C2 or fragments are useful in diagnostic applications to
detect the presence of elevated levels of 312C2 and/or its
fragments. Such diagnostic assays can employ lysates, live cells,
fixed cells, immunofluorescence, cell cultures, body fluids, and
further can involve the detection of antigens related to the
antigen in serum, or the like. Diagnostic assays may be homogeneous
(without a separation step between free reagent and antigen-binding
partner complex) or heterogeneous (with a separation step). Various
commercial assays exist, such as radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay
(EIA), enzyme-multiplied immunoassay technique (EMIT),
substrate-labeled fluorescent immunoassay (SLFIA), and the like.
See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525;
Harlow and Lane (1980) Antibodies: A Laboratory Manual, CSH Press,
NY; and Coligan, et al. (eds.) (1993) Current Protocols in
Immunology, Greene and Wiley, N.Y.
[0149] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against a 312C2, as such may be diagnostic
of various abnormal states. For example, overproduction of 312C2
may result in production of various immunological reactions which
may be diagnostic of abnormal physiological states, particularly in
proliferative cell conditions such as cancer or abnormal activation
or differentiation.
[0150] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the labels either labeled or unlabeled antibody or
binding partner, or labeled 312C2 is provided. This is usually in
conjunction with other additives, such as buffers, stabilizers,
materials necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also contain
instructions for proper use and disposal of the contents after use.
Typically the kit has compartments for each useful reagent.
Desirably, the reagents are provided as a dry lyophilized powder,
where the reagents may be reconstituted in an aqueous medium
providing appropriate concentrations of reagents for performing the
assay. Kits may be for solution determination, or histology in
tissue samples.
[0151] Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification or may be modified in a variety of ways. For example,
labeling may be achieved by covalently or non-covalently joining a
moiety which directly or indirectly provides a detectable signal.
In any of these assays, the binding partner, test compound, 312C2,
or antibodies thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups radiolabels
such as .sup.125I, enzymes (U.S. Pat. No. 3,645,090) such as
peroxidase and alkaline phosphatase, and fluorescent labels (U.S.
Pat. No. 3,940,475) capable of monitoring the change in
fluorescence intensity, wavelength shift, or fluorescence
polarization. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0152] There are also numerous methods of separating the bound from
the free 312C2, or alternatively the bound from the free test
compound. The 312C2 can be immobilized on various matrixes followed
by washing. Suitable matrixes include plastic such as an ELISA
plate, filters, and beads. See, e.g., Coligan, et al. (eds.) (1993)
Current Protocols in Immunology, Vol. 1, Chapter 2, Greene and
Wiley, NY. Other suitable separation techniques include, without
limitation, the fluorescein antibody magnetizable particle method
described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and
the double antibody magnetic particle separation as described in
U.S. Pat. No. 4,659,678.
[0153] Methods for linking proteins or their fragments to the
various labels have been extensively reported in the literature and
do not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the use
of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin such
as maleimide, for linkage, or the like. Fusion proteins will also
find use in these applications.
[0154] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of a 312C2. These sequences can be used as probes for detecting
levels of the 312C2 message in samples from patients suspected of
having an abnormal condition, e.g., cancer or developmental
problem. Since the antigen is a marker for activation, it may be
useful to determine the numbers of activated T cells to determine,
e.g., when additional suppression may be called for. The
preparation of both RNA and DNA nucleotide sequences, the labeling
of the sequences, and the preferred size of the sequences has
received ample description and discussion in the literature. See,
e.g., Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci.
79:4381-4385; Caskey (1987) Science 236:962-967; and Wilchek et al.
(1988) Anal. Biochem. 171:1-32. Histological analysis may also be
performed.
[0155] Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1:89-97. Other kits may be used to evaluate T cell
subsets, e.g., analysis or isolation, using conservative or
destructive means.
IX. Methods for Isolating 312C2 Specific Binding Partners
[0156] The 312C2 protein should interact with a ligand based, e.g.,
upon its similarity in structure and function to other cell surface
antigens exhibiting similar structure and cell type specificity of
expression. Methods to isolate a ligand are made available by the
ability to make purified 312C2 for screening programs. Soluble or
other constructs using the 312C2 sequences provided herein will
allow for screening or isolation of 312C2 specific ligands. Many
methods exist for expression cloning, panning, affinity isolation,
cross-linking, genetic selection, or other means to identify a
receptor ligand.
[0157] A variety of different assays for detecting compounds
capable of binding to 312C2 are used in the present invention. For
instance, the binding of a test compound to 312C2 or a peptide
fragment thereof can be measured directly, in the presence or
absence of 312C2 polypeptide. This latter type of assay is called a
direct binding assay. In addition, compounds which inhibit the
binding of 312C2 to specific, preferably monoclonal, antibodies can
be identified in competitive binding assays. Both direct binding
assays and competitive binding assays can be used in a variety of
different formats, similar to the formats used in immunoassays and
receptor binding assays. For a description of different formats for
binding assays, including competitive binding assays and direct
binding assays, see Basic and Clinical Immunology 7th Edition (D.
Stites and A. Terr ed.) 1991; Enzyme Immunoassay, E. T. Maggio,
ed., CRC Press, Boca Raton, Fla. (1980); and "Practice and Theory
of Enzyme Immunoassays," P. Tijssen, Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publishers B.
V. Amsterdam (1985), each of which is incorporated herein by
reference.
[0158] In competitive binding assays, for example, the sample
compound can compete with a labeled analyte for specific binding
sites on a binding agent bound to a solid surface. In this type of
format, the labeled analyte can be labeled 312C2 and the binding
agent can be an antibody bound to a solid phase. Alternatively, the
labeled analyte can be labeled antibody and the binding agent can
be a solid phase wild type 312C2 or a fragment thereof. The
concentration of labeled analyte bound to the capture agent is
inversely proportional to the ability of a test compound to compete
in the binding assay. The amount of inhibition of labeled analyte
by the test compound depends on the binding assay conditions and on
the concentrations of binding agent, labeled analyte, and test
compound that are used. Under specified assay conditions, a
compound is said to be capable of inhibiting the binding of 312C2
to a specific antibody in a competitive binding assay, if the
amount of binding of the labeled analyte to the binding agent is
decreased by 50% or preferably 90% or more. When a direct binding
assay format is used, a test compound is said to bind an 312C2 when
the signal measured is twice the background level or higher.
[0159] In a competitive binding assay, the sample compound competes
with labeled protein for binding to a specific binding agent. As
described above, the binding agent may be bound to a solid surface
to effect separation of bound labeled protein from the unbound
labeled protein. Alternately, the competitive binding assay may be
conducted in liquid phase, and any of a variety of techniques known
in the art may be used to separate the bound labeled protein from
the unbound labeled protein. Following separation, the amount of
bound labeled protein is determined. The amount of protein present
in the sample is inversely proportional to the amount of labeled
protein binding.
[0160] Alternatively, a homogeneous binding assay may be performed
in which a separation step is not needed. In these type of binding
assays, the label on the protein is altered by the binding of the
protein to its specific binding agent. This alteration in the
labeled protein results in a decrease or increase in the signal
emitted by label, so that measurement of the label at the end of
the binding assay allows for detection or quantitation of the
protein.
[0161] The binding assay formats described herein employ labeled
assay components. The label can be in a variety of forms. The label
may be coupled directly or indirectly to the desired component of
the assay according to methods well known in the art. A wide
variety of labels may be used. The component may be labeled by any
one of several methods. Traditionally, a radioactive label
incorporating .sup.3H .sup.125I, .sup.35S, .sup.14C, or .sup.32P is
used. Non-radioactive labels include ligands which bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled ligand. The choice of label depends on sensitivity
required, ease of conjugation with the compound, stability
requirements, and available instrumentation. For a review of
various labeling or signal producing systems which may be used, see
U.S. Pat. No. 4,391,904, which is incorporated herein by
reference.
[0162] Alternatively, an expression library can be screened for
specific binding to 312C2, e.g., by cell sorting, or other
screening to detect subpopulations which express such a binding
component. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA
90:11267-11271. Alternatively, a panning method may be used. See,
e.g., Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA
84:3365-3369. A two-hybrid selection system may also be applied
making appropriate constructs with the available 312C2 sequences.
See, e.g., Fields and Song (1989) Nature 340:245-246.
[0163] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the invention to specific embodiments.
EXAMPLES
General Methods
[0164] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology, Greene and
Wiley, New York; Innis, et al. (eds.)(1990) PCR Protocols: A Guide
to Methods and Applications, Academic Press, N.Y. Methods for
protein purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification" in Methods in Enzymology vol. 182, and other
volumes in this series; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia, Piscataway, N.J.,
or Bio-Rad, Richmond, Calif. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;
and Crowe, et al. (1992) QIAexpress: The High Level Expression
& Protein Purification System QIAGEN, Inc., Chatsworth, Calif.
Cell culture techniques are described in Doyle, et al. (eds.)
(1994) Cell and Tissue Culture: Laboratory Procedures, John Wiley
and Sons, NY.
[0165] Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunology vols 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Methods in Enzymology
volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and
163.
[0166] Assays for vascular biological activities are well known in
the art. They will cover angiogenic and angiostatic activities in
tumor, or other tissues, e.g., arterial smooth muscle proliferation
(see, e.g., Koyoma, et. al. (1996) Cell 87:1069-1078), monocyte
adhesion to vascular epithelium (see McEvoy, et al. (1997) J. Exp.
Med. 185:2069-2077), etc. See also Ross (1993) Nature 362:801-809;
Rekhter and Gordon (1995) Am. J. Pathol. 147:668-677; Thyberg, et
al. (1990) Athersclerosis 10:966-990; and Gumbiner (1996) Cell
84:345-357.
[0167] Assays for neural cell biological activities are described,
e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules 10,
Elsevier; Methods in Neurosciences Academic Press; and Neuromethods
Humana Press, Totowa, N.J. Methodology of developmental systems is
described, e.g., in Meisami (ed.) Handbook of Human Growth and
Developmental Biology CRC Press; and Chrispeels (ed.) Molecular
Techniques and Approaches in Developmental Biology
Interscience.
[0168] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y. Fluorescent labeling of appropriate reagents was
performed by standard methods.
Example 1
Cloning of Mouse 312C2
Antibodies and Flow-Cytometric Sorting
[0169] .alpha..beta.TcR+CD4-CD8- (DN) thymocytes were sorted using
CD4/CD8a-PE and TcR.alpha..beta.-FITC mAbs (PharMingen, San Diego,
Calif.). See Zlotnik, et al. (1992) J. Immunol. 4:1211-1215. The
sorted cells (approximately 5.times.10.sup.5) were stimulated on
solid-phase anti-CD3 for 24 h and were then expanded and cultured
in IL-2 (500 U/ml) and IL-7 (100 U/ml) for one week (to
approximately 1.times.10.sup.8 cells). Cells were either harvested
after one week in culture or stimulated again for 6 h on anti-CD3
and then harvested. See Kelner, et al. (1994) Science
266:1395-1399.
Construction of Directional cDNA Libraries
[0170] Poly (A)+ RNA from anti-CD3 stimulated .alpha..beta.DN
thymocytes or unstimulated .alpha..beta.DN thymocytes was used to
synthesize first strand cDNA by using NotI/Oligo-dT primer
(Gibco-BRL, Gaithersburg, Md.), Double-stranded cDNA was
synthesized, ligated with BstXI adaptors, digested with NotI, size
fractionated for >0.5 kilobase pairs (kb) and ligated into the
NotI/BstXI sites of PJFE-14, a derivative of the pCDSR.alpha.
vector. See Takebe, et al. Mol. Cell Biol. 8:466-472.
Electro-competent E. coli DH10.alpha. cells (Gibco-BRL) were used
for transformation. Total number of independent clones of the cDNA
libraries were 1.2.times.10.sup.6 for stimulated .alpha..beta.DN
and 8.times.10.sup.5 for unstimulated .alpha..beta.DN thymocytes,
respectively.
Library Subtraction
[0171] The PCR-based subtraction system developed by Wang and Brown
(1991) Proc. Natl. Acad. Sci. USA 88:11505-11509, was modified to
apply to plasmid cDNA libraries. A cDNA library specific for
activated .alpha..beta.DN thymocytes was generated using 100 .mu.g
of the unstimulated .alpha..beta.DN cDNA library DNA digested with
XbaI, NotI, and ScaI as driver DNA and 5 .mu.g of the stimulated
.alpha..beta.DN cDNA library DNA as tracer DNA. Following
restriction digestion, the driver DNA was treated with DNA
polymerase Klenow fragment to fill-in the restriction sites. After
ethanol precipitation, the DNA was dissolved in 100 .mu.l of water,
heat-denatured and mixed with 100 .mu.l (100 .mu.g) of Photoprobe
biotin (Vector Laboratories, Burlingame, Calif.). The driver DNA
was then irradiated with a 270-W sunlamp on ice for 20 min. 50
.mu.l more Photoprobe biotin was added and the biotinylation
reaction was repeated. After butanol extraction, the
photobiotinylated DNA (driver-U) was ethanol-precipitated and
dissolved in 30 .mu.l of 10 mM Tris-HCl and 1 mM EDTA, pH 8 (TE).
As tracer DNA, 5 .mu.g of stimulated .alpha..beta.DN cDNA was
digested with XbaI and NotI; ethanol precipitated; and dissolved in
4 .mu.l of TE (tracer-S). Tracer-S was mixed-with 15 .mu.l of
driver-U, 1 .mu.l (10 .mu.g) of E. coli tRNA (Sigma, St. Louis,
Mo.), and 20 .mu.l of 2.times.hybridization buffer (1.5 M NaCl, 10
mM EDTA, 50 mM HEPES, pH 7.5, 0.2% SDS), overlaid with mineral oil,
and heat-denatured. The sample tube was immediately transferred
into a 68.degree. C. water bath and incubated for 20 h. The
reaction mixture was then subjected to streptavidin treatment
followed by phenol/chloroform extraction. Subtracted DNA was
precipitated, dissolved in 12 .mu.l of TE, mixed with 8 .mu.l of
driver-U and 20 .mu.l of 2.times. hybridization buffer, and then
incubated at 68.degree. C. for 2 h. After streptavidin treatment,
the remaining DNA was ligated with 250 ng of a purified XbaI / NotI
fragment of pJFE-14 and then transformed into electro-competent E.
coli cells to generate the activation specific .alpha..beta.DN
subtracted library (S1). 100 independent clones were randomly
picked and screened by hybridization using a cocktail of known
cytokine cDNAs. Plasmid DNA's were prepared from clones that did
not hybridize to the cytokine probes. These clones were grouped by
insert size and further characterized by DNA sequencing. Clones
corresponding to the 312C2 were isolated.
Example 2
Cellular Expression of Mouse 312C2
[0172] A probe specific for cDNA encoding mouse 312C2 was used to
determine tissue distribution of the antigen. All probes were
labeled by random priming.
[0173] The results showed that 312C2 was expressed most abundantly
in T cells, in particular, certain subsets of activated T cells.
Thymus, spleen, and lymph node appeared to have more expression
than other tissues. Expression levels are: thymus +; Th1 subset
++++; Th2 subset ++++, NK1.1+T cells ++; .alpha..beta. T cells ++;
pro T cells +; CD4+ cells ++; CD8+ cells ++; and activated spleen
cells +. A message was also detected in certain pro-, pre-, and
mature B cell lines. The signal in the following cell types
suggested that expression is very low to virtually absent in lung,
heart, kidney, macrophage, stroma, brain, liver, muscle, and
testes.
Example 3
Purification of 312C2 Protein
[0174] Multiple transfected cell lines are screened for one which
expresses the antigen at a high level compared with other cells.
Various cell lines are screened and selected for their favorable
properties in handling. Natural 312C2 can be isolated from natural
sources, or by expression from a transformed cell using an
appropriate expression vector. Purification of the expressed
protein is achieved by standard procedures, or may be combined with
engineered means for effective purification at high efficiency from
cell lysates or supernatants. FLAG or His.sub.6 segments can be
used for such purification features.
[0175] By Northern analysis, it is clear that 312C2 is expressed in
various Th1, Th2, CD4+, CD8+, NK1.1+, pro-, pre-, and
.alpha..beta.CD4-CD8-T cells. 312C2 is also expressed in thymus and
activated spleen cells. Cells expressing 312C2 typically contain a
transcript of about 1.3 kb, corresponding to the size of the cloned
312C2 cDNA. Transcripts for 312C2 have not been detected in heart,
kidney, macrophage, stroma, brain, liver, muscle, testes
tissue.
[0176] The structural homology of 312C2 to the TNF receptor family,
suggests a broad function of this molecule. 312C2, as an activation
molecule, likely mediates enhanced Ag-specific proliferative
responses on T cells, or induction of apoptosis of these cells.
312C2 agonists, or antagonists, may also act as a co-stimulatory
molecule for T-cell activation, and may in fact, cause a shift of T
helper cell types, e.g., from Th1 to Th2, or Th2 to Th1 . Thus,
312C2 may be useful in the treatment of abnormal immune disorders,
e.g., T cell immune deficiencies, chronic inflammation, or tissue
rejection.
[0177] The mouse 312C12 protein exhibits structural features
characteristic of a cell surface antigen. The protein is easily
detected on particular cell types, others express lesser amounts.
The 312C2 antigen should be present in the identified tissue types
and the interaction of the antigen with its binding partner should
be important for mediating various aspects of cellular physiology
or development.
Example 4
Isolation of Homologous 312C2 Genes
[0178] The 312C2 cDNA can be used as a hybridization probe to
screen a library from a desired source, e.g., a primate cell cDNA
library. Many different species can be screened both for stringency
necessary for easy hybridization, and for presence using a probe.
Appropriate hybridization conditions will be used to select for
clones exhibiting specificity of cross hybridization. Specifically,
the mouse 312C2 cDNA clone was used to probe the HY06 human anergic
T cell library. A clone of about 1006 bp encoding a predicted
polypeptide of 241 amino acids was isolated.
[0179] Screening by hybridization using degenerate probes based
upon the peptide sequences will also allow isolation of appropriate
clones. Alternatively, use of appropriate primers for PCR screening
will yield enrichment of appropriate nucleic acid clones. See SEQ
ID NO: 5.
[0180] Similar methods are applicable to isolate either species,
polymorphic, or allelic variants. Species variants are isolated
using cross-species hybridization techniques based upon isolation
of a full length isolate or fragment from one species as a
probe.
[0181] Alternatively, antibodies raised against mouse 312C2 will be
used to screen for cells which express cross-reactive proteins from
an appropriate, e.g., cDNA library. The purified protein or defined
peptides are useful for generating antibodies by standard methods,
as described above. Synthetic peptides or purified protein are
presented to an immune system to generate monoclonal or polyclonal
antibodies. See, e.g., Coligan (1991) Current Protocols in
Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press. The resulting
antibodies are used for screening, panning, or sorting.
Example 5
Expression and Tissue Distribution of Human 312C2
[0182] Southern and PCR analysis of various hematopoietic cells and
tissues was performed as described above. Expression was detected
in several cell lines and tissues, most notably, stimulated
dendritic cell library, some activated T cell clones, activated
PBMCs, NK clones, Th1 , Th2 cells, pre-T cells, pro-T cells. Spleen
and lung tissue had detectable levels of 312C2.
Example 6
Preparation of Antibodies Specific for 312C2
[0183] Synthetic peptides or purified protein are presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (199.1) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold
Spring Harbor Press. Polyclonal serum, or hybridomas may be
prepared. In appropriate situations, the binding reagent is either
labeled as described above, e.g., fluorescence or otherwise, or
immobilized to a substrate for panning methods.
Example 7
Chromosomal mapping of 312C2
[0184] Chromosome spreads are prepared. In situ hybridization is
performed on chromosome preparations obtained from
phytohemagglutinin-stimulated lymphocytes cultured for 72 h.
5-bromodeoxyuridine is added for the final seven hours of culture
(60 .mu.g/ml of medium), to ensure a posthybridization chromosomal
banding of good quality.
[0185] An appropriate fragment, e.g., a PCR fragment, amplified
with the help of primers on total B cell cDNA template, is cloned
into an appropriate vector. The vector is labeled by
nick-translation with .sup.3H. The radiolabeled probe is hybridized
to metaphase spreads as described in Mattei, et al. (1985) Hum.
Genet. 69:327-331.
[0186] After coating with nuclear track emulsion (KODAK NTB2),
slides are exposed, e.g., for 18 days at 4.degree. C. To avoid any
slipping of silver grains during the banding procedure, chromosome
spreads are first stained with buffered Giemsa solution and
metaphase photographed. R-banding is then performed by the
fluorochrome-photolysis-Giemsa (FPG) method and metaphases
rephotographed before analysis.
Example 8
Isolation of Variants from Individuals
[0187] Mutational 312C2 variants from individuals having abnormal
immune responses are isolated by standard methods. For example,
affected cells, e.g., lymphocytes, are isolated as described, e.g.,
in Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY.
cDNA libraries are constructed as described above and probed with
the mouse or human 312C2 clone. Isolated clones are then sequenced
and compared to the human or mouse clone.
[0188] Alternatively, PCR techniques are also be employed to
isolate variants. See, e.g., Innis, et al. (eds.)(1990) PCR
Protocols: A Guide to Methods and Applications, Academic Press,
N.Y.
Example 9
Immunohistochemical Localization
[0189] The antibody described in Example 6 is used to identify
expression of 312C2 in various tissues. Methods for
immunohistochemical staining are described, e.g., in Sheehan, et
al. (eds.) (1987) Theory and Practice of Histotechnology, Battelle
Press, Columbus, Ohio.
Example 10
Soluble Molecules
[0190] Soluble constructs of 312C2 are made, e.g., as described in
Ausubel, et al. (1987 and Supplements) Current Protocols in
Molecular Biology, Greene and Wiley, NY; and Coligan, et al (eds.)
(1995 and periodic supplements) Current Protocols in Protein
Science, John Wiley and Sons, NY. Briefly, the transmembrane
portion of 312C2 is truncated. The nucleic acid encoding the
remaining portions of the molecule is subcloned into an appropriate
vector and the protein expressed through a suitable host cell.
[0191] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described above are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled. All references cited herein are incorporated
herein by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
Sequence CWU 1
1
* * * * *