WO2000006732A2 - Polynucleotide encoding an autoantigen associated with endometriosis - Google Patents

Polynucleotide encoding an autoantigen associated with endometriosis Download PDF

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Publication number
WO2000006732A2
WO2000006732A2 PCT/US1999/017284 US9917284W WO0006732A2 WO 2000006732 A2 WO2000006732 A2 WO 2000006732A2 US 9917284 W US9917284 W US 9917284W WO 0006732 A2 WO0006732 A2 WO 0006732A2
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Prior art keywords
seq
nucleic acid
acid sequence
group
ncav
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PCT/US1999/017284
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French (fr)
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WO2000006732A9 (en
WO2000006732A3 (en
Inventor
Said A. El Shami
Surendra Nath Menon
Cynthia K. French
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Diagnostic Products Corporation
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Application filed by Diagnostic Products Corporation filed Critical Diagnostic Products Corporation
Priority to AU52444/99A priority Critical patent/AU5244499A/en
Publication of WO2000006732A2 publication Critical patent/WO2000006732A2/en
Priority to AU37460/00A priority patent/AU3746000A/en
Priority to PCT/US2000/006742 priority patent/WO2001007616A1/en
Publication of WO2000006732A3 publication Critical patent/WO2000006732A3/en
Publication of WO2000006732A9 publication Critical patent/WO2000006732A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity

Definitions

  • the present invention relates to T cell receptor beta chain variable region nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins and inhibitors of such proteins or nucleic acid molecules.
  • the present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and/or inhibitors, as well as their use to regulate an immune response in an animal. BACKGROUND OF THE INVENTION
  • the immune system of an animal is characterized by its ability to respond to a diverse set of antigenic determinants, or epitopes. This response is reflected through T and B lymphocytes, also referred to as T cells and B cells, respectively.
  • T and B lymphocytes also referred to as T cells and B cells, respectively.
  • the immune system comprising these specialized cells, recognizes and processes foreign pathogens and macromolecules. Lymphocytes individually exhibit high specificity in recognition of particular molecular structures of antigens. The structural properties are recognized by T cell receptors, which act as antigen receptors.
  • T cell receptors are members of the immunoglobulin superfamily.
  • a TCR molecule comprises two polypeptide chains, generally an alpha chain and a beta chain.
  • Each chain comprises an amino terminal variable region domain (V) and a carboxyl terminal constant region domain (C), and can be designated with or ⁇ when indicating the particular chain of origin.
  • V and C regions are encoded by V region or C region genes, respectively.
  • Each domain can be stabilized by a disulfide bond between two conserved cysteine residue pairs on each chain.
  • Each chain is anchored to the cell membrane by a hydrophobic transmembrane domain, which typically spans the entire lipid bilayer of the membrane. A short carboxyl domain extends into the cytoplasm.
  • the and ⁇ chains of the TCR are encoded by gene segments analogous to the variable region (V), the diversity region (D), the joining region (J) and the constant region (C) of immunoglobulin genes.
  • Diversity in the TCR repertoire arises, in part, from the rearrangement of V, D and J regions and from the insertion of or deletion of nucleotides at the junction between the V, D and J regions.
  • TCR beta chain sequences e.g., Malisson et al., Cell, vol. 37, pp. 1101-1110, 1984; Patten et al, Nature, vol. 312, pp. 40-46, 1984; Davis et al, Nature, vol. 334, pp. 395-402, 1988; Hood et al., U.S. Patent No. 4,886,743, issued Dec. 12, 1989; and Makrides et al, U.S. Patent No. 5,552,300, issued Sep. 3, 1996.
  • T cells play a pivotal role in the differentiation and regulation of immune cells.
  • Previous investigators have studied diseases in which there appears to be improper immune regulation, such as autoimmunity and some forms of immunodeficiency, and have implicated T cells in the pathogenesis of such diseases.
  • situations exist in which clonal or oligoclonal expansion of a particular T cell population, identified by the presence of a particular TCR can be representative of a disease state.
  • a T cell leukemia or lymphoma e.g., Hood et al., ibid.
  • a TCR acts as a unique tumor marker since the TCR is stably rearranged and presented on the surface of the cell.
  • the present invention relates to T cell receptor nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins and inhibitors of such proteins or nucleic acid molecules.
  • the present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and/or inhibitors, as well as their use to regulate an immune response in an animal.
  • the present invention also includes methods to detect T cell expansion using reagents including or derived from such nucleic acid molecules, proteins and/or antibodies, as well as the use of such methods to diagnose abnormal states or disease in an animal.
  • BRIEF DESCRIPTION OF THE FIGURES Fig. 1 illustrates PCR amplified TCR V ⁇ DNA using DNA from tissue of normal dogs and dogs having lymphoma.
  • Figs. 2A, 2B and 2C illustrate fingerprints of TCR V ⁇ DNA from a normal dog.
  • Figs. 3 A, 3B and 3C illustrate fingerprints of TCR V ⁇ DNA from a dog having lymphoma.
  • Fig. 4 illustrates a comparison between fingerprints of TCR V ⁇ DNA from a normal dog and a dog having lymphoma.
  • TCR V ⁇ T cell receptor beta chain variable region
  • isolated TCR V ⁇ nucleic acid molecules antibodies directed against TCR V ⁇ proteins, and compounds derived therefrom that regulate the immune response of an animal.
  • a TCR V ⁇ protein can refer to a TCR V ⁇ protein or a homolog thereof.
  • TCR V ⁇ refers to a molecule that can include the variable (V) region, alone or in combination with the diversity (D) and/or joining (J) regions of a TCR beta chain.
  • V, D and J regions of a TCR beta chain can vary as a result of any given recombination event between genes encoding such V, D and J regions.
  • Typical consensus sequences used to identify the junction between the V, D and J regions are also known to one of skill in the art, thereby enabling the identification of the size and sequence of the V, D or J regions of a TCR beta chain from a novel nucleic acid or amino acid sequence.
  • Compounds derived from TCR V ⁇ proteins or nucleic acid molecules of the present invention include compounds including at least a portion of, or designed using, such proteins or nucleic acid molecules.
  • the phrase "regulate an immune response” refers to modulating the activity of cells involved in an immune response.
  • the term “regulate” can refer to increasing or decreasing an immune response Regulation of an immune response can be determined using methods known in the art as well as methods disclosed herein.
  • the terms isolated TCR V ⁇ proteins and isolated TCR V ⁇ nucleic acid molecules refers to TCR V ⁇ proteins and TCR V ⁇ nucleic acid molecules derived from mammals, preferably canids, more preferably dogs, and, as such, can be obtained from their natural source, or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of these proteins, nucleic acid molecules, antibodies, and compounds derived therefrom as therapeutic compositions to regulate the immune response of an animal as well as in other applications, such as those disclosed below.
  • One embodiment of the present invention is an isolated protein that includes a TCR V ⁇ protein.
  • a or “an” entity refers to one or more of that entity; for example, a protein refers to one or more proteins or at least one protein.
  • the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.
  • the terms “comprising”, “including”, and “having” can be used interchangeably.
  • an isolated, or biologically pure, protein is a protein that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the protein has been purified.
  • An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.
  • an isolated TCR V ⁇ protein of the present invention can be a full-length protein or any homolog of such a protein.
  • Full- length proteins can refer to proteins having the V, D, J and C regions of a beta chain or one or more of such regions.
  • a homolog refers to one or more or at least one homolog.
  • An isolated protein of the present invention, including a homolog can be identified in a straight-forward manner by the protein's ability to elicit an immune response against a TCR V ⁇ protein or to bind to a major histocompatability (MHC) molecule or superantigen.
  • MHC major histocompatability
  • TCR V ⁇ homologs include TCR V ⁇ proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog includes at least one epitope capable of eliciting an immune response against a TCR V ⁇ protein, and/or of binding to an antibody directed against a TCR V ⁇ protein.
  • amino acids e.g., a truncated version of the protein, such as a peptide
  • derivatized e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of
  • the animal when the homolog is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a natural TCR V ⁇ protein.
  • the ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art.
  • epitope refers to the smallest portion of a protein or other antigen capable of selectively binding to the antigen binding site of an antibody. It is well accepted by those skilled in the art that the minimal size of a protein epitope is about six to seven amino acids.
  • Other examples of TCR V ⁇ protein homologs include those homologs that are capable of binding to MHC, in the presence or absence of peptide, or superantigen. The ability of a protein to bind to MHC or superantigen can be measured using various methods well known to those of skill in the art.
  • TCR V ⁇ protein homologs can be the result of natural allelic variation or natural mutation.
  • TCR V ⁇ protein homologs of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • TCR V ⁇ proteins of the present invention are encoded by TCR V ⁇ nucleic acid molecules.
  • a TCR V ⁇ nucleic acid molecule includes nucleic acid sequences related to a natural TCR V ⁇ gene. It is to be noted that the term "a nucleic acid molecule”, “a gene” or “a nucleic acid sequence” refers to one or more or at least one nucleic acid molecule, gene or nucleic acid sequence, respectively.
  • a TCR V ⁇ gene includes all regions of the gene such as regulatory regions that control production of the TCR V ⁇ protein encoded by the gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions.
  • a gene that "includes” or “comprises” a sequence may include that sequence in one contiguous array, or may include the sequence as fragmented exons.
  • the term "coding region” refers to a continuous linear array of nucleotides that translates into a protein.
  • a full-length coding region is that coding region that is translated into a full- length, i.e., a complete, protein as would be initially translated in its natural milieu, prior to any post-translational modifications.
  • a TCR V ⁇ gene of the present invention includes the nucleic acid sequence SEQ ID NO:l, as well as the complement of SEQ ID NO:l.
  • Nucleic acid sequence SEQ ID NO:l represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaV ⁇ 3 3gl , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nCaV ⁇ 3 381 comprises the coding region for the V, D and J regions of TCRV ⁇ 3 (also referred to herein as hcV ⁇ 3).
  • the complement of SEQ ID NO:l (represented herein by SEQ ID NO:3) refers to the nucleic acid sequence of the strand complementary to the strand having SEQ ID NO: 1, which can easily be determined by those skilled in the art.
  • a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a double helix with) the strand for which the sequence is cited.
  • SEQ ID NO:l (as well as other nucleic acid and protein sequences presented herein) represents an apparent nucleic acid sequence of the nucleic acid molecule encoding a TCR V ⁇ protein of the present invention.
  • a TCR V ⁇ gene of the present invention includes the nucleic acid sequence SEQ ID NO:4, as well as the complement of SEQ ID NO:4 represented by SEQ ID NO:6.
  • Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaV ⁇ 4 408 , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nCaV ⁇ 4 408 comprises the coding region for the V, D and J regions of TCRV ⁇ 4 (also referred to herein as hcV ⁇ 4).
  • a TCR V ⁇ gene of the present invention includes the nucleic acid sequence SEQ LD NO:9, as well as the complement of SEQ LD NO:9 represented by SEQ ID NO: 11.
  • Nucleic acid sequence SEQ ID NO: 9 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaV ⁇ l2 408 , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nCaV ⁇ l2 40g comprises the coding region for the V, D and J regions of TCRV ⁇ l2 (also referred to herein as hcV ⁇ l2).
  • a TCR V ⁇ gene of the present invention includes the nucleic acid sequence SEQ LD NO:98, as well as the complement of SEQ ED NO:98 represented by SEQ LD NO: 100.
  • Nucleic acid sequence SEQ ID NO:98 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaV ⁇ 72 438 , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nCaV ⁇ 72 438 comprises the coding region for the V, D and J regions of TCRV ⁇ 72 (also referred to herein as hcV ⁇ 72).
  • a TCR V ⁇ gene of the present invention includes the nucleic acid sequence SEQ ID NO: 19, as well as the complement of SEQ ID NO: 19 represented by SEQ ED NO: 19.
  • Nucleic acid sequence SEQ ID NO: 19 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaV ⁇ 21 462 , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nCaV ⁇ 21 462 comprises the coding region for the V, D and J regions of TCRV ⁇ 21 (also referred to herein as hcV ⁇ 21).
  • a TCR V ⁇ gene of the present invention includes the nucleic acid sequence SEQ ID NO:22, as well as the complement of SEQ ID NO:22 represented by SEQ LD NO:22.
  • Nucleic acid sequence SEQ ED NO:22 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaV ⁇ 54 417 , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nCaV ⁇ 54 417 comprises the coding region for the V, D and J regions of TCRV ⁇ 54 (also referred to herein as dtb54).
  • a TCR V ⁇ gene of the present invention includes the nucleic acid sequence SEQ LD NO:25, as well as the complement of SEQ LD NO:25 represented by SEQ ID NO:25.
  • Nucleic acid sequence SEQ ID NO:25 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaV ⁇ l82 423 , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nCaV ⁇ l82 423 comprises the coding region for the V, D and J regions of TCRV ⁇ l82 (also referred to herein as dtbl82).
  • a TCR V ⁇ gene or nucleic acid molecule can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 1 , SEQ LD NO:3, SEQ LD NO:4, SEQ ED NO:6, SEQ ID NO:9, SEQ ED NO: 11, SEQ LD NO:98, SEQ ID NO: 100, or any other TCR V ⁇ nucleic acid sequence cited herein.
  • An allelic variant of a TCR V ⁇ nucleic acid molecule including SEQ ID NO: 1, SEQ LD NO:3, SEQ LD NO:4, SEQ LD NO:6, SEQ ID NO:9, SEQ ID NO: 11, SEQ LD NO:98 and SEQ ID NO: 100 is a nucleic acid molecule that occurs at essentially the same locus (or loci) in the genome as the nucleic acid molecule including SEQ ED NO: 1, SEQ LD NO:3, SEQ LD NO:4, SEQ ID NO:6, SEQ ED NO:9, SEQ ID NO: 11, SEQ ED NO:98 and SEQ LD NO: 100, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence.
  • allelic variants usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared.
  • Allelic variants of genes or nucleic acid molecules can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions), or can involve alternative splicing of a nascent transcript, thereby bringing alternative exons into juxtaposition.
  • Allelic variants are well known to those skilled in the art and would be expected to be found within a given genome, since the respective genomes are diploid, and sexual reproduction will result in the reassortment of alleles.
  • an isolated TCR V ⁇ protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a gene encoding a TCR V ⁇ protein (i.e., to a TCR V ⁇ gene).
  • the minimal size of a TCR V ⁇ protein of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridize under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein.
  • the size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the TCR V ⁇ nucleic acid molecule and the complementary nucleic acid sequence. It can easily be understood that the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered (i.e., localized) in distinct regions on a given nucleic acid molecule. Stringent hybridization conditions are determined based on defined physical properties of the gene to which the nucleic acid molecule is being hybridized, and can be defined mathematically.
  • Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al, 1984, Anal Biochem. 138, 267-284.
  • the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (expressed as molarity (M), in moles/liter), the hybridization temperature (°C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybridized.
  • these variables are inserted into a standard mathematical formula to calculate the melting temperature, or T m , of a given nucleic acid molecule.
  • T m is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands:
  • T m 81.5°C + 16.6 log M + 0.41(% G + C) - 500/n - 0.61(% formamide).
  • T d dissociation temperature
  • T d 4(G + C) + 2(A + T).
  • a temperature of 5°C below T d is used to detect hybridization between perfectly matched molecules.
  • T m decreases about 1°C for each 1% of mismatched base-pairs for hybrids greater than about 150 bp
  • T d decreases about 5°C for each mismatched base-pair for hybrids below about 50 bp.
  • Conditions for hybrids between about 50 and about 150 base-pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art. These simple procedures allow one skilled in the art to set the hybridization conditions (by altering, for example, the salt concentration, the formamide concentration or the temperature) so that only nucleic acid hybrids with greater than a specified % base-pair mismatch will hybridize. Stringent hybridization conditions are commonly understood by those skilled in the art to be those experimental conditions that will allow about 30% base-pair mismatch (i.e., about 70% identity).
  • nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropriate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybridize with a given nucleic acid molecule under stringent hybridization conditions and similarly whether the nucleic acid molecule will hybridize under conditions designed to allow a desired amount of base pair mismatch.
  • Hybridization reactions are often carried out by attaching the nucleic acid molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled probe suspended in a hybridization solution.
  • a solid support such as a membrane
  • Examples of common hybridization reaction techniques include the well-known Southern and northern blotting procedures.
  • the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.
  • nucleic acid molecule that hybridizes under stringent hybridization conditions with a specific or known canine nucleic acid molecule of about 150 bp in length
  • the average G + C content of canine DNA includes about 35%, about 36%, about 37%, about 38%, about 39%, about 41%, about 42%, about 43%, about 44%, about 45%, with about 40% being preferred.
  • the unknown nucleic acid molecules would be attached to a support membrane, and the specified 150 bp nucleic acid molecule would be labeled, e.g. with a radioactive tag.
  • the hybridization reaction could be carried out in a solution comprising 2X SSC and 0% formamide, at a temperature of about 37°C (low stringency conditions).
  • Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20X SSC (175.3 gram NaCI and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC.
  • the skilled artisan would calculate the washing conditions required to allow up to 30%) base-pair mismatch. For example, in a wash solution comprising IX SSC and 0% formamide, the T m of perfect hybrids would be about 80.8°C:
  • hybridization washes would be carried out at a temperature of about 50.8°C. It is within the skill of one in the art to calculate the hybridization temperature based on the formulae and G/C content disclosed herein.
  • a preferred TCR V ⁇ nucleic acid molecule includes a nucleic acid molecule which has greater than about 50 base pairs and which hybridizes under conditions which preferably allow about 20% base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10% base pair mismatch and even more preferably under conditions which allow about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ED NO:3, SEQ LD NO:6, SEQ ID NO:8, SEQ ID NO:l 1, SEQ LD NO:13, SEQ LD NO:100, SEQ ED NO: 18, SEQ ID NO:30, SEQ LD NO:33, SEQ ED NO:36, SEQ ED NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ED NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO
  • Another preferred TCR V ⁇ nucleic acid molecule of the present invention includes a nucleic acid molecule which has greater than about 150 base pairs and which hybridizes under conditions which preferably allow about 30% base pair mismatch, more preferably under conditions which allow about 25% base pair mismatch, more preferably under conditions which allow about 20%) base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10% base pair mismatch and even more preferably under conditions which allow about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ LD NO:6, SEQ ID NO:8, SEQ ID NO:l l, SEQ LD NO:13, SEQ LD NO:100, SEQ ID NO:18, SEQ LD NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ LD NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:60, S
  • Another preferred TCR V ⁇ nucleic acid molecule of the present invention includes a nucleic acid molecule which has greater than about 200 base pairs and which hybridizes under conditions which preferably allow about 30%) base pair mismatch, more preferably under conditions which allow about 25% base pair mismatch, more preferably under conditions which allow about 20% base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10%> base pair mismatch and even more preferably under conditions which allow about 5%> base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ LD NO:8, SEQ ID NO: 11, SEQ LD NO: 13, SEQ ID NO: 100, SEQ ID NO:18, SEQ LD NO:21, SEQ ID NO:30, SEQ ID NO:33, SEQ LD NO:36, SEQ LD NO:39, SEQ ID NO:42, and/or the complement of a nucleic acid molecule that encodes a protein having an
  • nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 49°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ED NO:3, SEQ LD NO:30, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61 or SEQ LD NO:62.
  • nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 56°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ED NO:5, SEQ LD NO:8, SEQ ID NO:33, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ED NO: 63, SEQ ID NO: 64 or SEQ ED NO:65.
  • nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 53°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 11 , SEQ LD NO: 13, SEQ ED NO:36, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:66, SEQ ED NO:67 or SEQ ID NO: 68.
  • nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0%o formamide, at a temperature of about 41°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 100, SEQ LD NO: 18, SEQ ID NO:39, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:69, SEQ ED NO: 70 or SEQ LD NO:71.
  • nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 29°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:21, SEQ ED NO:23, SEQ LD NO:42, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:72, SEQ ED NO:73 or SEQ ED NO:74.
  • a preferred TCR V ⁇ protein includes a protein encoded by a nucleic acid molecule which has greater than about 50 base pairs and which hybridizes under conditions which preferably allow about 20%> base pair mismatch, more preferably under conditions which allow about 15%) base pair mismatch, more preferably under conditions which allow about 10%) base pair mismatch and even more preferably under conditions which allow about 5%o base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ LD NO:2, SEQ LD NO:5, SEQ ID NO:10, SEQ LD NO:15, SEQ LD NO:20, SEQ LD NO:23, SEQ ID NO:26, SEQ ED NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ LD NO:38, SEQ LD NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ LD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:
  • Another preferred TCR V ⁇ protein of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 150 base pairs and which hybridizes under conditions which preferably allow about 30%> base pair mismatch, more preferably under conditions which allow about 25% base pair mismatch, more preferably under conditions which allow about 20%> base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10% base pair mismatch and even more preferably under conditions which allow about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ LD NO:2, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ LD NO:20, SEQ ED NO:23, SEQ ID NO:26, SEQ LD NO:29, SEQ ID NO:32, SEQ ED NO:35, SEQ ID NO:38, SEQ LD NO:41, SEQ ED NO:44, SEQ ID NO:47, SEQ LD NO:60, SEQ
  • Another preferred TCR V ⁇ protein of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 50 base pairs which is preferably about 80%> identical, more preferably about 85% identical, more preferably about 90%) identical, and even more preferably about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ LD NO:3, SEQ LD NO:6, SEQ LD NO:8, SEQ LD NO:l l, SEQ LD NO:13, SEQ ED NO:100, SEQ ID NO:18, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ LD NO:66, SEQ LD NO:67, SEQ
  • Yet another preferred TCR V ⁇ of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 150 base pairs which is preferably about 70% identical, more preferably about 75% identical, more preferably about 80%) identical, more preferably about 85%o identical, more preferably about 90%> identical and even more preferably about 95%> identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ED NO:13, SEQ LD NO:100, SEQ ED NO:18, SEQ ID NO:30, SEQ LD NO:33, SEQ ID NO:36, SEQ ID NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61, SEQ ED NO:62, SEQ ED NO:63, SEQ LD NO:64, SEQ ID NO:65, SEQ
  • Yet another preferred TCR V ⁇ of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 200 base pairs which is preferably about 10% identical, more preferably about 75% identical, more preferably about 80%) identical, more preferably about 85%> identical, more preferably about 90%> identical and even more preferably about 95%o identical to a nucleic acid molecule having the nucleic acid sequence SEQ ED NO:3, SEQ ID NO:6, SEQ LD NO:8, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ED NO:100, SEQ ID NO:18, SEQ ID NO:21, SEQ ED NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ LD NO:
  • the minimal size of such a nucleic acid molecule is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if the nucleic acid molecule is AT-rich.
  • the minimal size of a nucleic acid molecule used to encode a TCR V ⁇ protein homolog of the present invention is from about 12 to about 18 nucleotides in length.
  • the minimal size of a TCR V ⁇ protein homolog of the present invention is from about 4 to about 6 amino acids in length.
  • nucleic acid molecule of the present invention can include a portion of a gene, an entire gene, or multiple genes.
  • the preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired.
  • fragment thereof and portions thereof are intended to be used interchangeably, and have a minimal size as disclosed herein.
  • the minimal size of a protein or nucleic acid molecule of the present invention also can include a portion of a protein or nucleic acid molecule that is less than 100%) identical to another protein or nucleic acid molecule, when determined using hybridization or computer alignment methods disclosed herein.
  • a fragment of a hcV ⁇ 3 protein of the present invention is at least about 15 residues, preferably 20 residues and more preferably 25 residues in length; a fragment of a hcV ⁇ 4 protein of the present invention is at least about 10 residues, preferably 15 residues and more preferably 20 residues in length; a fragment of a hcV ⁇ l2 protein of the present invention is at least about 11 residues, preferably 15 residues and more preferably 20 residues in length; a fragment of a hcV ⁇ 72 protein of the present invention is at least about 18 residues, preferably 25 residues and more preferably 30 residues in length; or a fragment of a hcV ⁇ 21 protein of the present invention is at least about 13 residues, preferably 20 residues and more preferably 25 residues in length.
  • a nucleic acid molecule fragment of a hcV ⁇ 3 nucleic acid molecule of the present invention is at least about 33 nucleotides, preferably 35 nucleotides and more preferably 40 nucleotides in length;
  • a fragment of a hcV ⁇ 4 nucleic acid molecule of the present invention is at least about 21 nucleotides, preferably 25 nucleotides and more preferably 30 nucleotides in length;
  • a fragment of a hcV ⁇ l2 nucleic acid molecule of the present invention is at least about 19 nucleotides, preferably 25 nucleotides and more preferably 30 nucleotides in length;
  • Suitable protein fragments of the present invention include functional portions of a TCR V ⁇ protein of the present invention including, but not limited to, epitopes, MHC and/or peptide recognition sequences, antigen recognition sequences, superantigen recognition sequences, framework V regions and hypervariable V regions.
  • Preferred functional portions of a TCR V ⁇ protein include the V, D or J regions.
  • More preferred functional portions of a TCR V ⁇ protein include: the putative signal peptide encoded by about nucleotide 1 to nucleotide 51, the V region encoded by about nucleotide 52 to about nucleotide 333, and the D/J region encoded by about nucleotide 334 to about nucleotide 381 of SEQ LD NO: 1 ; the putative signal peptide encoded by nucleotide 25 to nucleotide 69, the V region encoded by nucleotide 70 to about nucleotide 351, and the D/J region encoded by about nucleotide 352 to about nucleotide 408 of SEQ LD NO:4; the putative signal peptide encoded by nucleotide 7 to nucleotide 63, the V region encoded by nucleotide 64 to about nucleotide 339, and the D/J region encoded by about nucleotide 340 to about nucleotide 408 of S
  • the junction between the V and D region can vary but that typically the carboxyl end of the V region contains one or more of the amino acid residues alanine or serine following a cysteine.
  • a conserved carboxyl V region sequence comprises the amino acids CASS.
  • a V region of the present invention can include the amino acid sequence SEQ ED NO:60, SEQ LD NO:61, SEQ ED NO:62, SEQ ED NO:63, SEQ ID NO:64, SEQ LD NO:65, SEQ LD NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ LD NO:71, SEQ ID NO: 72, SEQ LD NO: 73, SEQ ED NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ LD NO:77, SEQ LD NO:78, SEQ LD NO: 79 or SEQ LD NO: 80.
  • the present invention includes nucleic acid molecules having nucleic acid sequences encoding such proteins, which can be identified using the nucleic acid sequences disclosed herein and standard codon usage known to those of skill in the art; such as disclosed, for example, in Lehninger, Biochemistry, Worth Publishers, Inc., 1975, which is incorporated herein by this reference in its entirety.
  • codons encoding serine include ACA, ACC, AGT, AGC or ACG
  • codons that encode alanine include GCA, GCC, GCT or GCG.
  • One embodiment of a TCR V ⁇ protein of the present invention is a fusion protein that includes a TCR V ⁇ protein-containing domain attached to one or more fusion segments.
  • a fusion protein refers to one or more or at least one fusion protein.
  • Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; deliver a TCR V ⁇ protein or portion thereof to a desired target; act as an immunopotentiator to enhance an immune response against a TCR V ⁇ protein; and/or assist in purification of a TCR V ⁇ protein (e.g., by affinity chromatography).
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts increased immunogenicity to a protein, and/or simplifies purification of a protein).
  • Fusion segments can be joined to amino and/or carboxyl termini of the TCR V ⁇ -containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a TCR V ⁇ protein.
  • Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a TCR V ⁇ -containing domain.
  • Preferred fusion segments include a metal binding domain (e.g., a poly- histidine segment); an immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); and/or a "tag" domain (e.g., at least a portion of -galactosidase, a strep tag peptide, a T7 tag peptide, a FlagTM peptide, or other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies).
  • a metal binding domain e.g., a poly- histidine segment
  • an immunoglobulin binding domain e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains
  • a sugar binding domain e.g., a maltose binding domain
  • More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, FL; and an SIO peptide.
  • a preferred fusion protein of the present invention includes a TCR V ⁇ protein of the present invention linked to a TCR alpha chain in such a manner that the beta chain and alpha chain fold correctly to form a functional dimer.
  • Another preferred fusion protein includes a TCR V ⁇ protein of the present invention linked to at least a portion of the constant region of an immunoglobulin in such a manner that crystallization of the V beta protein is enhanced by the presence of the immunoglobulin sequence.
  • a TCR V ⁇ protein of the present invention is isolated (including isolation of the natural protein or production of the protein by recombinant or synthetic techniques) from canids.
  • a preferred isolated protein of the present invention is an isolated protein that is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence including SEQ ED NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 100, SEQ ID NO:18, SEQ LD NO:21, SEQ ID NO:24, SEQ ED NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ LD NO:39, SEQ ED NO:42, SEQ ED NO:45, SEQ ED NO:48, a complement of SEQ ID NO:50, SEQ ID NO:51, SEQ LD NO:52, SEQ ID NO:53, SEQ LD NO:54, SEQ ID NO:55, SEQ ED NO:56, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ
  • a preferred isolated protein of the present invention is a protein encoded by at least one of the following nucleic acid molecules:nCaV ⁇ 3 3g] , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390 , nCaV ⁇ 21 396 , nCaV ⁇ 21 348 , nCaV ⁇ 54 417 , nC
  • Another preferred isolated protein is encoded by a nucleic acid molecule having nucleic acid sequence SEQ ID NO:l, SEQ ED NO:4, SEQ ID NO:7, SEQ LD NO:9, SEQ ID NO: 12, SEQ ID NO:98, SEQ ID NO: 17, SEQ LD NO: 19, SEQ LD NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ D NO:31, SEQ LD NO:34, SEQ ED NO:37, SEQ LD NO:40, SEQ ED NO:43, SEQ LD NO:46, SEQ LD NO:50, SEQ LD NO:51, SEQ LD NO:52, SEQ ED NO:53, SEQ LD NO:54, SEQ ID NO:55, SEQ ED NO:56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO: 60, SEQ ID NO:61, SEQ YD NO:62, SEQ ID NO:63,
  • TCR V ⁇ protein of about 127 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaV ⁇ 3 127 , the amino acid sequence of which is presented in SEQ ID NO:2, assuming an open reading frame having a first codon spanning from nucleotide 1 through nucleotide 3 of SEQ LD NO:l and a last codon spanning from nucleotide 379 through nucleotide 381 of SEQ LD NO : 1.
  • the partial putative signal sequence extends from nucleotide 1 to nucleotide 51 of SEQ ID NO:l.
  • PCaV ⁇ 3 110 contains about 110 amino acids, extending from residue 18 through residue 127 of SEQ LD NO:2.
  • the nucleic acid molecule encoding PCaV ⁇ 3 no is denoted herein as nCaV ⁇ 3 330 , extending from nucleotide 52 through nucleotide 381 of SEQ ID NO: 1.
  • the coding region encoding PCaV ⁇ 4 ]2g is presented herein as nCaV ⁇ 4 384 , which has the nucleotide sequence SEQ LD NO: 7 (the coding strand) and SEQ ID NO: 8 (the complementary strand).
  • the putative signal sequence extends from nucleotide 25 to nucleotide 69 of SEQ ED NO:4.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 4 U3 contains about 113 amino acids, extending from residue 60 through residue 128 of SEQ ID NO:5.
  • the nucleic acid molecule encoding PCaV ⁇ 4 113 is denoted herein as nCaV ⁇ 4 339 , extending from nucleotide 70 through nucleotide 408 of SEQ ID NO:4.
  • Translation of SEQ LD NO: 9, the coding strand of nCaV ⁇ l2 408 yields a protein of about 134 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaV ⁇ l2 134 , the amino acid sequence of which is presented in SEQ ID NO: 10, assuming an open reading frame having an initiation codon spanning from nucleotide 7 through nucleotide 9 of SEQ ID NO:9 and a last codon spanning from nucleotide 406 through nucleotide 408 of SEQ LD NO:9.
  • the coding region encoding PCaV ⁇ 12 134 is presented herein as nCaV ⁇ l2 402 , which has the nucleotide sequence SEQ ID NO: 12 (the coding strand) and SEQ ID NO: 13 (the complementary strand).
  • the putative signal sequence extends from nucleotide 7 to nucleotide 63 of SEQ ID NO:9.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ l2 115 contains about 115 amino acids, extending from residue 20 through residue 134 of SEQ ID NO: 10.
  • the nucleic acid molecule encoding PCaV ⁇ l2 ] 15 is denoted herein as nCaV ⁇ l2 345 , extending from nucleotide 64 through nucleotide 408 of SEQ ID NO:9.
  • the coding region encoding PCaV ⁇ 72 133 is presented herein as nCaV ⁇ 72 399 , which has the nucleotide sequence SEQ ED NO: 17 (the coding strand) and SEQ LD NO: 18 (the complementary strand).
  • the putative signal sequence extends from nucleotide 85 to nucleotide 141 of SEQ ID NO:98.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 72 114 contains about 114 amino acids, extending from residue 20 through residue 133 of SEQ ID NO:98.
  • the nucleic acid molecule encoding PCaV ⁇ 72 1 ]4 is denoted herein as nCaV ⁇ 72 342 , extending from nucleotide 142 through nucleotide 438 of SEQ ID NO: 19.
  • the coding region encoding PCaV ⁇ 21 !30 is presented herein as nCaV ⁇ 21 390 , which extends from nucleotide 73 to nucleotide 462 of SEQ ID NO: 19.
  • the putative signal sequence extends from nucleotide 73 to nucleotide 114 of SEQ LD NO: 19.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 21 116 contains about 116 amino acids, extending from residue 15 through residue 130 of SEQ ID NO: 19.
  • the nucleic acid molecule encoding PCaV ⁇ 21 116 is denoted herein as nCaV ⁇ 21 348 , extending from nucleotide 115 through nucleotide 462 of SEQ ID NO: 19.
  • Translation of SEQ ID NO:22, the coding strand of nCaV ⁇ 54 417 yields a protein of about 135 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaV ⁇ 54 135 , the amino acid sequence of which is presented in SEQ ID NO:23, assuming an open reading frame having an initiation codon spanning from nucleotide 13 through nucleotide 15 of SEQ ID NO:22 and a last codon spanning from nucleotide 415 through nucleotide 417 of SEQ ID NO:22.
  • the coding region encoding PCaV ⁇ 54 !35 is presented herein as nCaV ⁇ 54 405 , which extends from nucleotide 13 to nucleotide 417 of SEQ ID NO:22.
  • the putative signal sequence extends from nucleotide 13 to nucleotide 69 of SEQ ID NO:22.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 54 116 contains about 116 amino acids, extending from residue 20 through residue 135 of SEQ LD NO:22.
  • the nucleic acid molecule encoding PCaV ⁇ 54 116 is denoted herein as nCaV ⁇ 54 348 , extending from nucleotide 70 through nucleotide 417 of SEQ ID NO:22.
  • SEQ LD NO:25 the coding strand of nCaV ⁇ l82 423 , yields a protein of about 128 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaV ⁇ 182 ]28 , the amino acid sequence of which is presented in SEQ LD NO:26, assuming an open reading frame having an initiation codon spanning from nucleotide 40 through nucleotide 43 of SEQ ID NO:25 and a last codon spanning from nucleotide 421 through nucleotide 423 of SEQ ED NO:25.
  • the coding region encoding PCaV ⁇ 182 128 is presented herein as nCaV ⁇ l82 384 , which extends from nucleotide 40 to nucleotide 423 of SEQ ED NO:25.
  • the putative signal sequence extends from nucleotide 40 to nucleotide 96 of SEQ ED NO:25.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ l82 109 contains about 109 amino acids, extending from residue 20 through residue 128 of SEQ LD NO:25.
  • nucleic acid molecule encoding PCaV ⁇ 182 ]09 is denoted herein as nCaV ⁇ l82 327 , extending from nucleotide 97 through nucleotide 423 of SEQ ID NO:25.
  • SEQ ID NO:28 the coding strand of nCaV ⁇ 3 333 , yields a protein of about 111 amino acids containing the beta chain V region, denoted herein as PCaV ⁇ 3 ⁇ n , the amino acid sequence of which is presented in SEQ DD NO:29, assuming an open reading frame having a first codon spanning from nucleotide 1 through nucleotide 3 of SEQ ID NO:28 and a last codon spanning from nucleotide 331 through nucleotide 333 of SEQ ED NO:28.
  • the complement of SEQ DD NO:28 is SEQ DD NO:30.
  • SEQ ED NO:31 the coding strand of nCaV ⁇ 4 351 , yields a protein of about 109 amino acids containing the beta chain V region, denoted herein as PCaV ⁇ 4 109 , the amino acid sequence of which is presented in SEQ ED NO:32, assuming an open reading frame having a first codon spanning from nucleotide 25 through nucleotide 27 of SEQ ID NO:31 and a last codon spanning from nucleotide 349 through nucleotide 351 of SEQ TD NO:31.
  • the complement of SEQ TD NO:31 is SEQ TD NO:33.
  • SEQ ID NO: 34 the coding strand of nCaV ⁇ 12 339 , yields a protein of about 111 amino acids containing the beta chain V region, denoted herein as PCaV ⁇ l2 lu , the amino acid sequence of which is presented in SEQ LD NO:35, assuming an open reading frame having a first codon spanning from nucleotide 7 through nucleotide 9 of SEQ LD NO:34 and a last codon spanning from nucleotide 337 through nucleotide 339 of SEQ ED NO:34.
  • the complement of SEQ ID NO:34 is SEQ LD NO:35.
  • SEQ ED NO:37 the coding strand of nCaV ⁇ 72 423 , yields a protein of about 113 amino acids containing the beta chain V region, denoted herein as PCaV ⁇ 72 113 , the amino acid sequence of which is presented in SEQ ID NO:38, assuming an open reading frame having a first codon spanning from nucleotide 85 through nucleotide 87 of SEQ ED NO:37 and a last codon spanning from nucleotide 421 through nucleotide 423 of SEQ ID NO:37.
  • the complement of SEQ LD NO:37 is SEQ TD NO:39.
  • SEQ ID NO:43 the coding strand of nCaV ⁇ 54 354 , yields a protein of about 114 amino acids containing the beta chain V region, denoted herein as PCaV ⁇ 54 ] ]4 , the amino acid sequence of which is presented in SEQ LD NO:44, assuming an open reading frame having a first codon spanning from nucleotide 13 through nucleotide 15 of SEQ ID NO:43 and a last codon spanning from nucleotide 352 through nucleotide 354 of SEQ ID NO:43.
  • the complement of SEQ LD NO:43 is SEQ LD NO:45.
  • SEQ LD NO:46 the coding strand of nCaV ⁇ l82 369 , yields a protein of about 110 amino acids containing the beta chain V region, denoted herein as PCaV ⁇ 182, , 0 , the amino acid sequence of which is presented in SEQ LD NO:47, assuming an open reading frame having a first codon spanning from nucleotide 40 through nucleotide 42 of SEQ ED NO:46 and a last codon spanning from nucleotide 367 through nucleotide 369 of SEQ ID NO:46.
  • the complement of SEQ LD NO:46 is SEQ LD NO:48.
  • Preferred TCR V ⁇ proteins of the present invention include proteins that are at least about 65%), preferably at least about 70% > , even more preferably at least about 75%, and even more preferably at least about 80% identical to PCaV ⁇ 3 127 ; are at least about 69%), preferably at least about 75%, even more preferably at least about 80%, and even more preferably at least about 85% identical to PCaV ⁇ 4 128 ; are at least about 57%, preferably at least about 60%, even more preferably at least about 65%, and even more preferably at least about 70% identical to PCaV ⁇ l2 134 ; are at least about 65%, preferably at least about 70%o, even more preferably at least about 75%, and even more preferably at least about 80% identical to PCaV ⁇ 72 133 ; or are at least about 60%, preferably at least about 65%), even more preferably at least about 70%, and even more preferably at least about 75%) identical to PCaV ⁇ 21 130 .
  • TCR V ⁇ proteins comprising PCaV ⁇ 3 127 , PCaV ⁇ 3 ⁇ n , PCaV ⁇ 3 U0 , PCaV ⁇ 4 128 , PCaV ⁇ 4 113 , PCaV ⁇ 4 109 , PCaV ⁇ l2 134 , PCaV ⁇ l2 , PCaV ⁇ l2 115 , PCaV ⁇ 72 133 , PCaV ⁇ 72 113 , PCaV ⁇ 72 U4 , PCaV ⁇ 21 130 , PCaV ⁇ 21 108 , PCaV ⁇ 21 116 , PCaV ⁇ 54 135 , PCaV ⁇ 54 114 , PCaV ⁇ 54 116 , PCaV ⁇ 182 128 , PCaV ⁇ 182, ]0 , PCaV ⁇ 182 109 and fragments thereof; and proteins encoded by allelic variants of nucleic acid molecules encoding proteins PCaV ⁇ 3 127 , PCaV ⁇ 3 l u , PCaV ⁇ 3 ] 10
  • PCaV ⁇ 72 133 PCaV ⁇ 72 113 , PCaV ⁇ 72 H4 , PCaV ⁇ 21 130 , PCaV ⁇ 21 108 , PCaV ⁇ 21 116 , PCaV ⁇ 54 135 , PCaV ⁇ 54 ] 14 , PCaV ⁇ 54 116 , PCaV ⁇ 182 128 , PCaV ⁇ 182, 10 and/or PCaV ⁇ 182, 09 ,and fragments thereof.
  • TCR V ⁇ proteins of the present invention include proteins having amino acid sequences that are at least about 65%), preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to SEQ ED NO:2; at least about 69%, preferably at least about 75%, even more preferably at least about 80%>, and even more preferably at least about 85 > identical to SEQ ED NO:5; are at least about 51%, preferably at least about 60%, even more preferably at least about 65%, and even more preferably at least about 70% identical to SEQ LD NO: 10; are at least about 65%, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to SEQ ID NO: 15; or are at least about 60%, preferably at least about 65%, even more preferably at least about 70%, and even more preferably at least about 75% identical to SEQ LD NO:20.
  • TCR V ⁇ proteins comprising amino acid sequences SEQ LD NO:2, SEQ ED NO:5, SEQ LD NO:10, SEQ TD NO:15, SEQ ED NO:20, SEQ DD NO:23, SEQ DD NO:26, SEQ ED NO:29, SEQ DD NO:32, SEQ DD NO:35, SEQ ID NO:38, SEQ LD NO:41, SEQ LD NO:44, SEQ ID NO:47, SEQ ID NO:60, SEQ LD NO:61, SEQ ID NO:62, SEQ ED NO:63, SEQ ED NO:64, SEQ ID NO:65, SEQ DD NO:66, SEQ DD NO:67, SEQ DD NO:68, SEQ DD NO:69, SEQ DD NO:70, SEQ DD NO:71, SEQ DD NO:72, SEQ DD NO:73, SEQ DD NO:74, SEQ DD NO:75, SEQ DD NO:70, SEQ
  • SEQ DD NO:77 SEQ DD NO:78, SEQ ED NO:79, SEQ DD NO:80 and/or proteins encoded by the complement of a nucleic acid sequence including SEQ ID NO:50, SEQ ID NO:51, SEQ ED NO:52, SEQ ID NO:53, SEQ DD NO:54, SEQ TD NO:55, SEQ TD NO:56; and TCR V ⁇ proteins encoded by allelic variants of nucleic acid molecules encoding TCR V ⁇ proteins having amino acid sequences SEQ TD NO:2, SEQ TD NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ LD NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ LD NO:32, SEQ ED NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ LD NO:44, SEQ ED NO:47, SEQ LD NO:60, SEQ LD NO:
  • a preferred isolated protein of the present invention comprises a protein selected from the group consisting of: (a) an isolated protein having an amino acid sequence that is at least about 55 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:29, SEQ ID NO:60, SEQ LD NO:61, and SEQ ID NO: 62, or a fragment thereof that is at least about 25 amino acids in length; (b) an isolated protein having an amino acid sequence that is at least about 75 percent identical to an amino acid sequence selected from the group consisting of SEQ ED NO:5, SEQ LD NO:32, SEQ DD NO:63, SEQ ED NO:64, and SEQ TD NO:65, or a fragment thereof that is at least about 15 amino acids in length; (c) an isolated protein having an amino acid sequence that is at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ TD NO: 10, SEQ LD NO:35, SEQ ED NO:66, SEQ LD NO:67, and S
  • a preferred isolated protein of the present invention comprises a protein selected from the group consisting of:(a) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ DD NO:28, SEQ ID NO:50, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ DD NO: 61, and SEQ ID NO: 62, or a fragment thereof that is at least about 25 nucleotides in length; (b) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:4, SEQ DD NO:7, SEQ ID NO:31, SEQ DD NO:51, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:63, SEQ TD NO:
  • a preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Compare function by maximum matching within the program DNAsis Version 2.1.
  • Additional preferred TCR V ⁇ proteins of the present invention include proteins encoded by nucleic acid molecules comprising at least a portion of nCaV ⁇ 3 381 , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCa
  • TCR V ⁇ proteins encoded by nucleic acid molecules having nucleic acid sequences comprising at least a portion of SEQ ID NO: 1, SEQ ED NO:4, SEQ ID NO:7, SEQ LD NO:9, SEQ ED NO: 12, SEQ LD NO:98, SEQ DD NO: 17, SEQ DD NO: 19, SEQ ID NO:22, SEQ ED NO:25, SEQ ED NO:28, SEQ DD NO:31, SEQ DD NO:34, SEQ DD NO:37, SEQ DD NO:40, SEQ DD NO:43, SEQ DD NO:46, SEQ DD NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ DD NO:54, SEQ ID NO:55, SEQ DD NO: 56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ DD NO:60, SEQ ED NO:61, SEQ ID NO:
  • the present invention also includes mimetopes of TCR V ⁇ proteins of the present invention.
  • a mimetope of a TCR V ⁇ protein of the present invention refers to any compound that is able to mimic the activity of a TCR V ⁇ protein of the present invention, often because the mimetope has a structure that mimics the particular TCR V ⁇ protein. It is to be noted that the term "a mimetope" refers to one or more or at least one mimetope.
  • Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation such as all-D retro peptides; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non- proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids.
  • Such mimetopes can be designed using computer-generated structures of proteins of the present invention.
  • Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides, RNA or other organic molecules, non- organic molecules and screening such samples by affinity chromatography techniques using the corresponding binding partner.
  • nucleic acid molecule homolog refers to one or more or at least one nucleic acid molecule homologs.
  • an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified.
  • An isolated TCR V ⁇ nucleic acid molecule of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis.
  • Isolated TCR V ⁇ nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a TCR V ⁇ protein of the present invention.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • the minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybrid with the complementary sequence of another nucleic acid molecule.
  • the minimal size of a TCR V ⁇ nucleic acid molecule of the present invention is from about 12 to about 18 nucleotides in length.
  • a TCR V ⁇ nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, ibid..
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof.
  • Nucleic acid molecule homologs can be selected by hybridization with a TCR V ⁇ nucleic acid molecule or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least one epitope of a TCR V ⁇ protein).
  • An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one TCR V ⁇ protein of the present invention, examples of such proteins being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a TCR V ⁇ protein.
  • a preferred nucleic acid molecule of the present invention when administered to an animal, is capable of regulating an immune response in an animal.
  • a nucleic acid molecule can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound.
  • a nucleic acid molecule of the present invention can encode an immunoregulatory protein (e.g., a cell-bound or soluble TCR V ⁇ protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection (i.e, as a genetic vaccine) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine.
  • an immunoregulatory protein e.g., a cell-bound or soluble TCR V ⁇ protein of the present invention
  • One embodiment of the present invention is a TCR V ⁇ nucleic acid molecule comprising all or part of nucleic acid molecules nCaV ⁇ 3 3gl , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390 , nCaV ⁇ 21 396 , nCaV ⁇ 21 348 , nCaV ⁇ 54 417
  • Another preferred nucleic acid molecule comprises SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ED NO:7, SEQ ED NO:8, SEQ LD NO:9, SEQ ED NO: 11, SEQ LD NO: 12, SEQ TD NO: 13, SEQ ED NO:98, SEQ DD NO:100, SEQ DD NO:17, SEQ DD NO:18, SEQ ID NO:19, SEQ TD NO:21, SEQ TD NO:22, SEQ DD NO:24, SEQ ID NO:25, SEQ DD NO:27,SEQ DD NO:28, SEQ DD NO:30, SEQ DD NO:31, SEQ DD NO:33, SEQ DD NO:34, SEQ DD NO:36, SEQ ID NO:37, SEQ DD NO:39, SEQ ED NO:40, SEQ TD NO:42, SEQ DD NO:43, SEQ TD NO:45, SEQ
  • nucleic acid molecules can include nucleotides in addition to those included in the SEQ DD NOs, such as, but not limited to, a full-length gene, a full-length coding region, a nucleic acid molecule encoding a fusion protein, or a nucleic acid molecule encoding a multivalent therapeutic compound.
  • a TCR V ⁇ nucleic acid molecule of the present invention encodes a protein that is at least about 65%, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to PCaV ⁇ 3 127 ; are at least about 69%, preferably at least about 75%, even more preferably at least about 80%, and even more preferably at least about 85%> identical to PCaV ⁇ 4 128 ; are at least about 57%, preferably at least about 60%, even more preferably at least about 65%), and even more preferably at least about 70%> identical to PCaV ⁇ l2 134 ; are at least about 65%o, preferably at least about 70%, even more preferably at least about 15%, and even more preferably at least about 80% identical to PCaV ⁇ 72 133 ; or are at least about 60%), preferably at least about 65%>, even more preferably at least about 70%, and even more preferably at least about 75% identical to PCaV ⁇ 21 130 .
  • nucleic acid molecule encoding PCaV ⁇ 3 ]27 , PCaV ⁇ 3 ln , PCaV ⁇ 3 1]0 , PCaV ⁇ 4 ]2g , PCaV ⁇ 4 113 , PCaV ⁇ 4 109 , PCaV ⁇ l2 134 , PCaV ⁇ l2 m , PCaV ⁇ l2 115 , PCaV ⁇ 72 133 , PCaV ⁇ 72 113 , PCaV ⁇ 72 U4 , PCaV ⁇ 21 130 , PCaV ⁇ 21 10g , PCaV ⁇ 21 U6 , PCaV ⁇ 54 135 , PCaV ⁇ 54 114 , PCaV ⁇ 54 116 , PCaV ⁇ 182 128 , PCaV ⁇ 182 ⁇ o and/or PCaV ⁇ 182 109 , fragments thereof, complements thereof, and/or an allelic variant of such a nucleic acid molecule.
  • a TCR V ⁇ nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least about 65%>, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80%) identical to SEQ ID NO:2; are at least about 69%>, preferably at least about 75%>, even more preferably at least about 80%>, and even more preferably at least about 85% identical to SEQ ID NO:5; are at least about 57%>, preferably at least about 60%, even more preferably at least about 65%>, and even more preferably at least about 70% identical to SEQ LD NO: 10; are at least about 65%>, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to SEQ ED NO: 15; or at least about 60%, preferably at least about 65%o, even more preferably at least about 70%>, and even more preferably at least about 75% identical to SEQ LD NO:2;
  • the present invention also includes a TCR V ⁇ nucleic acid molecule encoding a protein having at least a portion of SEQ LD NO:2, SEQ TD NO:5, SEQ ID NO: 10, SEQ ED NO: 15, SEQ LD NO:20, SEQ LD NO:23, SEQ LD NO:26, SEQ ED NO:29, SEQ LD NO:32, SEQ ID NO:35, SEQ ED NO:38, SEQ DD NO:41, SEQ DD NO:44, SEQ DD NO:47, SEQ DD NO:60, SEQ DD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ DD NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ DD NO:69, SEQ DD NO:70, SEQ DD NO:71, SEQ DD NO:72, SEQ ID NO:73, SEQ DD NO:74, SEQ
  • a TCR V ⁇ nucleic acid molecule of the present invention is at least about 69%, preferably at least about 75%, even more preferably at least about 80%), and even more preferably at least about 85%> identical to nCaV ⁇ 3 381 ; is at least about 15%, preferably at least about 80%>, even more preferably at least about 85%, and even more preferably at least about 90%> identical to nCaV ⁇ 4 408 ; is at least about 72%>, preferably at least about 80%>, even more preferably at least about 85%, and even more preferably at least about 90%> identical to nCaV ⁇ l2 408 ; is at least about 60%>, preferably at least about 65%>, even more preferably at least about 70%>, and even more preferably at least about 75% identical to nCaV ⁇ 72 43g ; or is at least about 3 %>, preferably at least about 45%o, even more preferably at least about 50%>, and even more preferably at least about 55
  • nucleic acid molecule comprising nCaV ⁇ 3 381 , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 40g , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390 , nCaV ⁇ 21 396 , nCaV ⁇ 21 348 , nCaV ⁇ 54 417 , nCaV ⁇ 54 405 ,nCaV ⁇ 54
  • a TCR V ⁇ nucleic acid molecule of the present invention comprises a nucleic acid sequence that is at least about 69%, preferably at least about 75%), even more preferably at least about 80%>, and even more preferably at least about 85%o identical to SEQ ED NO:l; is at least about 75%, preferably at least about 80%), even more preferably at least about 85%, and even more preferably at least about 90% identical to SEQ ED NO:4; is at least about 72%, preferably at least about 80%, even more preferably at least about 85%, and even more preferably at least about 90% identical to SEQ ED NO:9; is at least about 60%o, preferably at least about 65%, even more preferably at least about 70%, and even more preferably at least about 75% identical to SEQ ID NO:98; or is at least about 38%, preferably at least about 45%, even more preferably at least about 50%, and even more preferably at least about 55% identical to SEQ ID NO: 19.
  • the present invention also includes a TCR V ⁇ nucleic acid molecule comprising at least a portion of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ HD NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ DD NO:9, SEQ ID NO: 11, SEQ DD NO:12, SEQ DD NO:13, SEQ DD NO:98, SEQ DD NO:100, SEQ ID NO:17, SEQ TD NO:18, SEQ HD NO:19, SEQ ID NO:21, SEQ TD NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ DD NO:27,SEQ DD NO:28, SEQ DD NO:30, SEQ ID NO:31, SEQ LD NO:33, SEQ HD NO:34, SEQ HD NO:36, SEQ TD NO:37, SEQ TD NO:39, SEQ TD NO.40, SEQ H) NO:42, SEQ TD NO:43
  • a preferred isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ DD NO:3, SEQ DD NO:6, SEQ DD NO:8, SEQ DD NO:l l, SEQ DD NO: 13, SEQ ID NO: 100, SEQ TD NO: 18, SEQ LD NO:21, SEQ ID NO:24, SEQ ED NO:27, SEQ LD NO:30, SEQ ID NO:33, SEQ LD NO:36, SEQ LD NO:39, SEQ LD NO:42, SEQ HD NO:45, SEQ LD NO:48, a complement of SEQ ID NO:50, SEQ ID NO:51, SEQ HD NO:52, SEQ TD NO:53, SEQ TD NO:54, SEQ ED NO:55, SEQ LD NO:56, and/or the complement of a nucleic acid
  • a preferred isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence that is any of the following: (a) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:l, SEQ DD NO:3, SEQ DD NO:28, SEQ DD NO:30 and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:60, SEQ ED NO:61, SEQ DD NO:62, and the complement of a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:60, SEQ TD NO:61 , and SEQ TD NO:62, or a fragment thereof that is at least about 25 nucleotides in length; (b) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ HD NO:6, SEQ
  • a preferred isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding a protein selected from the group consisting of: (a) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ DD NO:28, SEQ DD NO:50, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:60, SEQ LD NO:61, and SEQ DD NO:62, or a fragment thereof that is at least about 25 nucleotides in length; (b) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ HD NO:7, SEQ ID NO:31, SEQ DD NO: 51, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of
  • Another embodiment of the present invention is an isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ DD NO: 1 , SEQ ID NO:3, SEQ ID NO:28, and SEQ LD NO:30, or a fragment thereof, wherein said fragment has an at least a 20 contiguous nucleotide region identical in sequence to a 20 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ DD NO:3, SEQ ID NO:28, and SEQ ID NO:30; (b) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ DD NO:4, SEQ DD NO:6, SEQ DD NO:7, SEQ TD NO:8, SEQ ID NO:31, and SEQ DD NO:33, or a fragment thereof, wherein said fragment has an at least
  • a homolog having an at least "x" contiguous nucleotide region identical in sequence to an "x" contiguous nucleotide region of a nucleic acid molecule selected from the group consisting of SEQ DD NO:"y refers to an "x"-nucleotide in length nucleic acid molecule that is identical in sequence to an "x"-nucleotide portion of SEQ ID NO:"y", as well as to nucleic acid molecules that are longer in length than "x".
  • the additional length may be in the form of nucleotides that extend from either the 5 ' or the 3' end(s) of the contiguous identical "x"-nucleotide portion.
  • the 5' and/or 3' extensions can include one or more extensions that have no identity to an immunoregulatory molecule of the present invention, as well as extensions that show similarity or identity to cited nucleic acids sequences or portions thereof.
  • nucleic acid sequences of certain TCR V ⁇ nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain other TCR V ⁇ nucleic acid molecules.
  • nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention.
  • Preferred libraries to screen or from which to amplify nucleic acid molecules include mammalian cDNA libraries as well as genomic DNA libraries.
  • preferred DNA sources from which to amplify nucleic acid molecules include mammalian cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.
  • the present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising TCR V ⁇ nucleic acid molecules.
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
  • the minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention.
  • a preferred oligonucleotide of the present invention has a maximum size of about 100 nucleotides.
  • a preferred oligonucleotide of the present invention has a minimum size of about 12 nucleotides.
  • an oligonucleotide of the present invention has a size from about 12 nucleotides to about 30 nucleotides and more preferably from about 15 nucleotides to about 25 nucleotides.
  • a preferred isolated oligonucleotide of the present invention comprises a unique nucleic acid sequence within a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ HD NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ED NO:7, SEQ ID NO: 8, SEQ ED NO:9, SEQ DD NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, SEQ LD NO:98, SEQ LD NO: 100, SEQ LD NO: 17, SEQ ED NO: 18, SEQ LD NO: 19, SEQ LD NO:21, SEQ LD NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ DD NO:27,SEQ DD NO:28, SEQ ID NO:30, SEQ TD NO:31, SEQ DD NO:33, SEQ DD NO:34, SEQ ID NO:36, SEQ TD NO:37, SEQ ⁇ D NO:
  • the present invention includes oligonucleotides that can be used, for example, as probes to identify nucleic acid molecules, primers to produce nucleic acid molecules, or therapeutic reagents to inhibit TCR V ⁇ protein production or activity (e.g., as antisense-, triplex formation-, ribozyme- and/or RNA drug-based reagents).
  • the present invention also includes the use of such oligonucleotides to protect animals from disease using one or more of such technologies.
  • Appropriate oligonucleotide-containing therapeutic compositions can be administered to an animal using techniques known to those skilled in the art.
  • Preferred oligonucleotides of the present invention include oligonucleotides comprising a unique nucleic acid sequence, as defined herein, within a nucleic acid molecule of the present invention or homologs thereof. Preferred homologs of an oligonucleotide are capable of priming a nucleic acid sequence.
  • One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
  • Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of TCR V ⁇ nucleic acid molecules of the present invention.
  • a recombinant molecule comprises a nucleic acid molecule of the present invention operatively linked to an expression vector.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and plant cells.
  • Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells, and more preferably in the cell types disclosed herein
  • expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences.
  • Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription.
  • Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.
  • Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those which function in bacterial, yeast, helminth or other endoparasite, or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p L and lambda p R and fusions that include such promoters), bacteriophage T7, ⁇ llac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as immediate early promoter), simian virus 40, retrovirus, act
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with mammals, such as canine or feline transcription control sequences.
  • nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein.
  • Preferred nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules include nCaV ⁇ 3 381 , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 3g4 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390 , nCaV
  • Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed TCR V ⁇ protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins.
  • suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention.
  • Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments.
  • t-PA tissue plasminogen activator
  • Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein.
  • a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment.
  • Eukaryotic recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
  • Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Preferred nucleic acid molecules with which to transform a cell include TCR V ⁇ nucleic acid molecules disclosed herein.
  • nucleic acid molecules with which to transform a cell include nCaV ⁇ 3 381 , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 40g , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390 , nCaV ⁇ 21 396 , nCaV ⁇ 21 348 , nCaV ⁇ 54 417 , nCaV ⁇ 54 405 , nCaV ⁇
  • Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines).
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing TCR V ⁇ proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention.
  • Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), other insect, other animal and plant cells.
  • Preferred host cells include bacterial, mycobacterial, yeast, helminth, insect and mammalian cells.
  • More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Pichia, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CN-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, Chinese hamster ovary (CHO) cells, Ltk cells and Nero cells.
  • Particularly preferred host cells are Escherichia coli, including E.
  • coli K-12 derivatives Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 0 3987 and SR-11 0 4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CN-1 cells; COS cells; Nero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse ⁇ LH/3T3 cells, LMTK 31 cells and/or HeLa cells.
  • the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences, examples of which are disclosed herein.
  • a recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein.
  • a recombinant molecule refers to one or more or at least one recombinant molecule, host cell or recombinant cell, respectively.
  • Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including TCR V ⁇ nucleic acid molecules encoding one or more proteins of the present invention and one or more other nucleic acid molecules encoding other therapeutic compounds, as disclosed herein (e.g., to produce multivalent vaccines).
  • Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation.
  • transcription control signals e.g., promoters, operators, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
  • Isolated TCR V ⁇ proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins.
  • an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein.
  • a preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective, medium refers to any medium in which a cell is cultured to produce a TCR V ⁇ protein of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
  • Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
  • the present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to a TCR V ⁇ protein of the present invention or a mimetope thereof (e.g., anti-TCR V ⁇ antibodies).
  • a TCR V ⁇ protein of the present invention or a mimetope thereof (e.g., anti-TCR V ⁇ antibodies).
  • the term "selectively binds to" a TCR V ⁇ protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention.
  • Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid., and Harlow, et al, 1988, Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid.
  • An anti- TCR V ⁇ antibody of the present invention preferably selectively binds to a TCR V ⁇ protein in such a way as to inhibit the function of that protein.
  • Isolated antibodies of the present invention can include antibodies in serum, or antibodies that have been purified to varying degrees.
  • Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to one or more epitopes.
  • a preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies.
  • antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce TCR V ⁇ proteins of the present invention.
  • Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition.
  • Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention.
  • antibodies of the present invention can be used (a) as reagents in assays to detect TCR V ⁇ protein, (b) as reagents in assays to modulate cellular activity through a TCR V ⁇ protein (e.g., mimicking ligand binding to TCR V ⁇ protein), and/or (c) as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants.
  • antibodies of the present invention can be used to target compounds (e.g., nucleic acid molecules, drugs or proteins) to antigen presenting cells. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the compounds using techniques known to those skilled in the art. Suitable compounds are known to those skilled in the art.
  • compositions of the present invention include at least one of the following therapeutic compounds: an isolated TCR V ⁇ protein of the present invention or a mimetope thereof, an isolated TCR V ⁇ nucleic acid molecule of the present invention, an isolated antibody that selectively binds to a TCR V ⁇ protein of the present invention, an inhibitor of TCR V ⁇ function identified by its ability to bind to a TCR V ⁇ protein of the present invention and inhibit binding of a TCR V ⁇ protein to MHC, and a mixture thereof (i.e., combination of at least two of the compounds).
  • a therapeutic compound refers to a compound that, when administered to an animal in an effective manner, is able to treat, ameliorate, and/or prevent a disease.
  • proteins, nucleic acid molecules, antibodies and inhibitors of the present invention are disclosed herein.
  • the present invention also includes a therapeutic composition comprising at least one TCR V ⁇ -based compound of the present invention in combination with at least one additional therapeutic compound. Examples of such compounds are disclosed herein.
  • Therapeutic compositions of the present invention can be administered to any animal susceptible to such therapy, preferably to mammals, and more preferably to dogs.
  • a therapeutic composition of the present invention is administered to an animal in an effective manner such that the composition is capable of regulating an immune response in that animal.
  • Therapeutic compositions of the present invention can be administered to animals prior to onset of a disease (i.e., as a preventative vaccine) and/or can be administered to animals after onset of a disease in order to treat the disease (i.e., as a therapeutic vaccine).
  • Preferred diseases to prevent or treat include autoimmune diseases, allergic reactions, infectious diseases and cancer.
  • compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
  • excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • a therapeutic composition can include an adjuvant.
  • Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen.
  • Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., Flt-3 ligand, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (EL-3), interleukin 4 (LL-4), interleukin 5 (E -5), interleukin 6 (LL-6), interleukin 7 (LL-7), interleukin 8 (LL-8), interleukin 10 (LL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGLF), transforming growth factor beta, RANTES (regulated upon activation, normal T cell expressed and presumably secreted), macrophage
  • a therapeutic composition can contain one or more adjuvants.
  • a therapeutic composition can include a carrier.
  • Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
  • a therapeutic composition can contain one or more carriers.
  • a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal.
  • a controlled release formulation comprises a composition of the present invention in a controlled release vehicle.
  • Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
  • Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ.
  • Preferred controlled release formulations are biodegradable (i.e., bioerodible).
  • a preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition to regulate an immune response in an animal.
  • the therapeutic composition is preferably released over a period of time ranging from about 1 to about 12 months.
  • a controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
  • Therapeutic compositions of the present invention can be administered to animals prior to and/or after onset of disease.
  • Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art.
  • a suitable single dose is a dose that is capable of regulating the immune response in an animal when administered one or more times over a suitable time period.
  • a preferred single dose of a protein, mimetope or antibody therapeutic composition is from about 1 microgram (g) to about 10 milligrams (mg) of the therapeutic composition per kilogram body weight of the animal.
  • Booster vaccinations can be administered from about 2 weeks to several years after the original administration.
  • Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease.
  • a preferred administration schedule is one in which from about 10 g to about 1 mg of the therapeutic composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months.
  • Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, intraoccular, oral, transdermal and intramuscular routes.
  • a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a therapeutic protein or therapeutic RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNA drug) in the animal.
  • a therapeutic protein or therapeutic RNA e.g., antisense RNA, ribozyme, triple helix forms or RNA drug
  • Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a) administering a naked (i.e., not packaged in a viral coat or cellular membrane) nucleic acid as a genetic vaccine (e.g., as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or (b) administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle).
  • a naked nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle.
  • One or more nucleic acid molecules can be delivered to an animal.
  • a genetic (i.e., naked nucleic acid) vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent.
  • a genetic vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule.
  • Preferred genetic vaccines include at least a portion of a viral genome (i.e., a viral vector).
  • Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses, with those based on alphaviruses (such as Sindbis or Semliki forest virus), species- specific herpesviruses and poxviruses being particularly preferred.
  • Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production.
  • Particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron-A), Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a "strong" polyadenylation signal is also preferred.
  • Genetic vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, intranasal and oral routes of administration being preferred.
  • a preferred single dose of a genetic vaccine ranges from about 1 nanogram (ng) to about 600 g, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically.
  • Genetic vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or in a carrier (e.g., lipid-based vehicles).
  • a recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration.
  • the recombinant molecule is packaging- or replication-deficient and/or encodes an attenuated virus.
  • a number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses.
  • Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses.
  • a recombinant virus vaccine of the present invention When administered to an animal, infects cells within the immunized animal and directs the production of a therapeutic protein or RNA nucleic acid molecule that is capable of protecting the animal from disease caused by a parasitic helminth as disclosed herein.
  • a recombinant virus vaccine comprising a TCR V ⁇ nucleic acid molecule of the present invention is administered according to a protocol that results in the regulation of an immune response in an animal.
  • a preferred single dose of a recombinant virus vaccine of the present invention is from about 1 x 10 4 to about 1 x 10 8 virus plaque forming units (pfu) per kilogram body weight of the animal.
  • a recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention.
  • Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae andPichia pastoris), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells.
  • Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 10 8 to about 10 12 cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
  • the efficacy of a therapeutic composition of the present invention to regulate the immune response in an animal can be tested in a variety of ways including, but not limited to, detection of cellular immunity within the treated animal, determination of T cell activity (helper or cytotoxic T cell activity), identification of T cell repertoire, detection of immunoregulatory cytokines, e.g., E -2, IL-4, EL- 10, LL-12, levels, detection of antibody levels, determine tumor development or challenge of the treated animal with an infectious agent to determine whether the treated animal is resistant to disease.
  • therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art.
  • a therapeutic composition is used to treat a disease requiring immunological regulation, such as cancer, infectious diseases, autoimmune diseases or allergy.
  • Suitable cancers to treat include lymphomas or leukemias.
  • Suitable infectious diseases to treat include diseases caused by viral, bacterial, yeast, fungal or parasitic infection.
  • Suitable autoimmune diseases to treat include: autoimmune skin diseases, e.g. pemphigus foliaceous, pemphigus vulgaris, pemphigus vegetans, pemphigus erythematosus, bollous pemphigoid, discoid lupus, dermatomyositis or subcorneal pustular dermatosis; blood disorders, e.g.
  • autoimmune hemolytic anemia immune-mediated thrombocytopenia, aplstic anemia, pure red cell aplasia or immune mediated neutropenia
  • endocrine dysfuntion e.g. lymphocytic thyroditis, diabetes, hypoadrenocorticism or hypoparathyroidism
  • multi-system dysfunction e.g. systemic lupus erythematosus or Sjogren's syndrome
  • neurologic dysfunction e.g. myasthenia gravis, distemper and rabies post vaccinal encephalopathy or acute polyradiculoneuritis
  • musculoskeletal disease e.g.
  • rheumatoid arthritis idiopathic polyarthritis, plasmacytic-lymphocytic arthritis or polymyosutus.
  • Suitable allergies to treat include allergic dermatitis, atopic dermatitis, allergic rhinitis or allergic bronchitis.
  • One therapeutic composition of the present invention includes a TCR V ⁇ protein of the present invention, or a portion of such TCR V ⁇ protein that elicits a cytotoxic T cell response against a T cell bearing the TCR V ⁇ protein.
  • a preferred TCR V ⁇ protein comprises a soluble form of a TCR V ⁇ protein of the present invention, with a peptide of a TCR V ⁇ protein being more preferred.
  • a preferred TCR V ⁇ peptide is from about 5 to about 50 residues, more preferably from about 10 to about 40 residues and even more preferably from about 12 to about 30 residue in length.
  • One or more TCR V ⁇ proteins of the present invention, or portions of such TCR V ⁇ proteins can be used in a therapeutic composition.
  • a therapeutic composition comprising a TCR
  • V ⁇ peptide e.g. a portion of a polypeptide
  • a TCR V ⁇ peptide of the present invention can be linked to another molecule to assist in the delivery of the peptide to an animal.
  • a TCR V ⁇ peptide of the present invention is administered systemically to an animal.
  • One or more peptides can be used in a therapeutic composition.
  • Another therapeutic composition of the present invention includes an inhibitory compound that inhibits a TCR V ⁇ protein from binding to MHC.
  • An inhibitory compound is capable of substantially interfering with the function of a TCR V ⁇ protein susceptible to inhibition.
  • an inhibitory compound is administered in an amount and manner that inhibits an immune response that is sufficient to treat an animal for a disease that requires downregulation of an immune response.
  • One or more inhibitors can be used in a therapeutic composition.
  • Suitable inhibitory compounds include compounds that prevent the activation of an immunoregulatory cell through TCR V ⁇ by, for example, interfering with the binding of TCR V ⁇ protein to MHC by binding to either the TCR V ⁇ protein or MHC.
  • An example of an inhibitory compound is an antibody of the present invention, administered to an animal in an effective manner; i.e., an antibody of the present invention, is administered in an amount so as to be present in the animal at a titer that is sufficient, upon interaction of that antibody with a native TCR V ⁇ protein, to decrease TCR V ⁇ activity in an animal, at least temporarily.
  • Oligonucleotide nucleic acid molecules of the present invention can also be administered in an effective manner, thereby reducing expression of TCR V ⁇ proteins in order to interfere with TCR V ⁇ activity targeted in accordance with the present invention.
  • Peptides of TCR V ⁇ proteins of the present invention can also be administered in an effective manner, thereby reducing binding of TCR V ⁇ proteins to MHC in order to interfere with TCR V ⁇ activity targeted in accordance with the present invention.
  • an inhibitory compound is derived from a TCR V ⁇ protein of the present invention.
  • An inhibitory compound of TCR V ⁇ function can be identified using TCR V ⁇ proteins of the present invention.
  • One embodiment of the present invention is a method to identify a compound capable of inhibiting TCR V ⁇ function. Such a method includes the steps of: (a) contacting (e.g., combining, mixing) an isolated TCR V ⁇ protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein binds to a protein including MHC, and (b) determining if the putative inhibitory compound inhibits the binding of TCR V ⁇ to MHC.
  • Putative inhibitory compounds to screen include small organic molecules, antibodies (including mimetopes thereof), and ligand analogs. Such compounds can also be screened to identify those compounds that are substantially not toxic to a recipient animal.
  • One embodiment of the present invention is a method to detect expansion of T cells in an animal.
  • the present method utilizes the discovery by the inventors of unique sequences in a TCR V ⁇ and more particularly in the V region of a TCR V ⁇ that function, or can be used, as markers for T cells.
  • unique sequences refers to nucleic acid or amino acid sequences that are present in one TCR V ⁇ nucleic acid molecule or TCR V ⁇ protein, but not in another TCR V ⁇ nucleic acid molecule or TCR V ⁇ protein, respectively.
  • a unique sequence differentiates one TCR V ⁇ nucleic acid molecule or TCR V ⁇ protein of the present invention from another TCR V ⁇ nucleic acid molecule or TCR V ⁇ protein, respectively.
  • a unique sequence within hcV ⁇ 3, is not found within hcV ⁇ 4, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82, and a unique sequence within hcV ⁇ 4, is not found within hcV ⁇ 3, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82, and so on.
  • a unique sequence of the present invention can be used to detect expansion of T cells by, for example, determining the presence or absence of any one or more unique sequences, determining increased or decreased levels of molecules carrying one or more unique sequences compared with other unique sequences in the same animal and/or a different animal, or comparing levels of different TCR V ⁇ proteins in an animal. It is within the scope of the present invention that any unique sequence present in a TCR V ⁇ sequence of the present invention can be used in the practice of the present method, including those identified herein and those which will be identified based on the sequences of nucleic acid molecules or proteins disclosed herein.
  • unique sequences of the present invention include those sequences located within about the first 200 nucleotides or about the first 70 residues of the 5' or amino terminus of a nucleic acid molecule or a protein.
  • the suitable length of a unique sequence depends upon the reagent used to detect the presence of the unique sequence. For example, a suitable length of a unique sequence is from about 15 to about 30 nucleotides if the detection reagent is a DNA primer. Alternatively, a suitable length of a unique sequence is from about 50 to about 300 nucleotides if the detection reagent is a DNA hybridization probe. Alternatively, a suitable length of a unique sequence is at least about 5 amino acids if the detection reagent is an antibody.
  • one or more unique sequences can be identified, and one or more reagents can be used to identify such sequences, using a method of the present invention.
  • Any method of the present invention can be used to determine the presence, absence, amount or ratio of TCR V ⁇ 's of the present invention in an animal to determine T cell expansion and/or diagnose an abnormal state or a specific disease.
  • numbers of T cells in an animal are regulated. Expansion of total T cell numbers, or of a particular T cell clone, can represent an abnormal state or disease.
  • a particular T cell clone is expanded in a T cell lymphoma or leukemia.
  • information derived using the present methods is particularly useful because the inventors have discovered the seven different TCR V ⁇ proteins disclosed herein which can comprise at least about 95%> of the TCR V ⁇ repertoire in canids.
  • any diagnostic method of the present invention is useful for determining if an animal is susceptible to, has or is in remission from a disease.
  • T cell expansion is determined by detecting increased levels of one or more specific TCR V ⁇ molecules in a tissue sample isolated from an animal.
  • Increased levels of TCR V ⁇ molecules can be determined by comparing levels of two or more different TCR V ⁇ molecules in a tissue sample isolated from one animal, or levels of one or more TCR V ⁇ molecules in tissue samples from two or more different animals. Total amounts or ratios of particular TCR V ⁇ molecules can be determined by one of skill in the art depending upon the method used to detect the presence of a TCR V ⁇ molecule in a sample.
  • a ratio illustrative of increased levels of a particular TCR V ⁇ molecule is from about 2-fold to about 5-fold, more preferably from about 5-fold to about 25, and more preferably 10-fold more of one particular TCR V ⁇ molecule compared with another sample of the same TCR V ⁇ molecule or a different TCR V ⁇ molecule.
  • Methods to determine ratios are known to those in the art and include, for example, densitometry, spectrophotometry or determining counts per minute of isotopes.
  • One embodiment of the present invention is a method to detect expansion of T cells in an animal comprising detecting the presence of one or more T cell receptors having unique nucleic acid sequences within hcV ⁇ 3, hcV ⁇ 4, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82 nucleic acid molecules or hcV ⁇ 3, hcV ⁇ 4, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82 proteins by forming detectable products, wherein the increased level of a detectable product compared with another detectable product indicates expansion of the T cells.
  • a suitable sample containing T cells is isolated from an animal.
  • Samples containing T cell receptors can be isolated from the same or different animals. Control samples can be obtained from the same or different animals. The sample is contacted under appropriate conditions with one or more reagents capable of identifying one or more unique nucleic acid or amino acid sequences, respectively.
  • a reagent distinguishes one member of the group comprising hcV ⁇ 3, hcV ⁇ 4, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, hcV ⁇ 54 or hcV ⁇ l82 nucleic acid molecules or hcV ⁇ 3, hcV ⁇ 4, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, hcV ⁇ 54 or hcV ⁇ l82 proteins, preferably nCaV ⁇ 3 3gl , nCaV ⁇ 4 408 , nCaV ⁇ l2 408 , nCaV ⁇ 72 438 , nCaV ⁇ 21 462 , nCaV ⁇ 54 417 , nCaV ⁇ l82 423 , PCaV ⁇ 3 127 , PCaV ⁇ 4 128 , PCaV ⁇ l2 134 , PCaV ⁇ 72 133 , PCaV ⁇ 21 130 , PCaV ⁇ 54 135
  • a detectable product can comprise a nucleic acid molecule, a peptide, a protein or an antibody.
  • Preferred detectable products and methods to form detectable products are disclosed herein. It is within the skill of one in the art that methods to identify detectable products based on the product being detected.
  • the invention also provides novel reagents useful in the methods which have been described herein above.
  • the invention includes reagents capable of binding to unique nucleic acid or unique amino acid sequences contained within a TCR V ⁇ .
  • Preferred reagents include those which can differentiate one TCR V beta protein, or nucleic acid molecule, from another. Preferred reagents can distinguish TCR V beta proteins hcV ⁇ 2, hcV ⁇ 3, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82, or the nucleic acid molecules that encode them, from each other.
  • More preferred reagents can distinguish proteins comprising PCaV ⁇ 3 127 , PCaV ⁇ 3 uford PCaV ⁇ 3 U0 , PCaV ⁇ 4 128 , PCaV ⁇ 4 113 , PCaV ⁇ 4 109 , PCaV ⁇ l2 134 , PCaV ⁇ l2 ] U , PCaV ⁇ l2 115 , PCaV ⁇ 72 133 , PCaV ⁇ 72 113 , PCaV ⁇ 72 114 , PCaV ⁇ 21 130 , PCaV ⁇ 21 108 , PCaV ⁇ 21 116 , PCaV ⁇ 54 I35 , PCaV ⁇ 54 114 , PCaV ⁇ 54 116 , PCaV ⁇ l82 128 , PCaV ⁇ l82 110 an ⁇ VorPCaV ⁇ l82 109 from each other, or nucleic acid molecules comprising nCaV ⁇ 3 381 , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV
  • TCR V beta proteins comprising amino acid sequence SEQ TD NO:29, SEQ ED NO:32, SEQ ED NO:35, SEQ LD NO:38, SEQ ID NO:41, SEQ LD NO:44, SEQ TD NO:47 from each other.
  • a method of the present invention comprises: (a) contacting a sample, i.e., one or more samples, containing DNA from T cells with a reagent, i.e., one or more reagents, having specificity for a unique nucleic acid sequence; and (b) determining the presence of DNA carrying the unique nucleic acid sequences. Methods to determine the presence of the DNA are disclosed herein.
  • identifying the presence of a T cell receptor having unique sequence can be achieved by polymerase chain reaction (PCR) amplification techniques known to those of skill in the art.
  • the PCR amplification forms a detectable product comprising DNA.
  • An example of a suitable reagent to use in PCR amplification techniques is a DNA primer complementary to all or a portion of a unique nucleic acid sequence, referred to herein as a unique sequence primer.
  • a unique sequence primer is used in conjunction with the unique sequence primer in order o effect amplification.
  • This second primer can be complementary to a unique sequence or to a common sequence (i.e., a sequence shared by different TCR beta chain sequences).
  • the second primer is complementary to a common sequence and is chosen based on its distance from the unique sequence primer, i.e. based on ease of detection of PCR amplification product.
  • a preferred product of a PCR reaction is from about 100 to about 500 nucleotides, preferably from about 150 to about 450 nucleotides and more preferably from about 200 to about 400 nucleotides.
  • a suitable second primer includes a DNA primer complementary to a sequence in the constant region of a beta chain. Preferred second primers are described herein in the Examples section.
  • PCR Methods to resolve PCR products are well known to those of skill in the art. In addition, methods to quantitate the amount of PCR product produced in a PCR reaction are well known to those of skill in the art.
  • preferred unique nucleic acid sequences to be identified by PCR include unique sequences contained within SEQ LD NO:l, SEQ ED NO:3, SEQ ID NO:4, SEQ TD NO:6, SEQ HD NO:7, SEQ TD NO:8, SEQ HD NO:9, SEQ TD NO:l 1, SEQ ID NO: 12, SEQ TD NO: 13, SEQ ID NO:98, SEQ DD NO: 100, SEQ ID NO: 17, SEQ DD NO: 18, SEQ DD NO: 19, SEQ DD NO:21, SEQ DD NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ DD NO:27 and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ DD NO:60, SEQ DD NO:
  • More preferred unique nucleic acid sequences to be identified by PCR include SEQ TD NO:50, SEQ HD NO:51, SEQ TD NO:52, SEQ HD NO:53, SEQ TD NO:54, SEQ ID NO:55 and/or SEQ ID NO:56, or complements thereof.
  • Preferred second primers include primers located in the constant region sequence of a beta chain. More preferred second primers include SEQ ID NO:58 and SEQ ID NO:59, or complements thereof.
  • identifying the presence of a T cell receptor having a unique nucleic acid sequence can be achieved by nucleic acid, e.g., DNA, RNA, modified DNA or modified RNA, hybridization techniques using a nucleic acid probe.
  • the hybridization forms a detectable product comprising a hybrid between the nucleic acid and the reagent.
  • a suitable reagent for use with hybridization techniques include nucleic acid probes which are complementary to nucleic acid sequences that include all or a portion of a unique sequence.
  • the hybridization forms a detectable product comprising a hybrid between the nucleic acid and the reagent. It is within the skill of one in the art to design and produce suitable probes based on sequences of the present invention. The presence of a unique sequence in a sample from an animal is determined by detecting the hybridization of a "unique sequence" probe to that unique sequence in the TCR V ⁇ nucleic acid molecule.
  • Preferred unique nucleic acid sequences to be identified by nucleic acid hybridization include unique sequences contained within SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ DD NO:9, SEQ DD NO: 11, SEQ ED NO: 12, SEQ LD NO: 13, SEQ ID NO:98, SEQ ED NO:100, SEQ ID NO:17, SEQ LD NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ LD NO:22, SEQ LD NO:24, SEQ ED NO:25, SEQ DD NO:27 and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:
  • More preferred unique nucleic acid sequences to be identified by nucleic acid hybridization include unique sequences SEQ ID NO:50, SEQ ED NO:51, SEQ ID NO:52, SEQ LD NO:53, SEQ LD NO:54, SEQ ED NO:55 and/or SEQ LD NO:56.
  • a unique amino acid sequence of the present invention can be used to produce antibodies that bind specifically to the portion of a TCR V ⁇ protein that contains the unique amino acid sequence.
  • Such antibodies can be monoclonal or polyclonal, and produced using methods described herein.
  • the antibodies can be used to detect T cells having T cell receptors containing such unique sequences by contacting T cells isolated from an animal with the antibody under appropriate conditions known in the art that enable formation of a complex between an antibody and a T cell receptor in a specific manner, i.e., such that the antibody only binds specifically a particular TCR V ⁇ .
  • the complex between an antibody and a T cell receptor is a detectable product.
  • Preferred unique amino acid sequences to be identified using antibodies include antibodies that bind specifically to a unique amino acid sequence contained within SEQ ED NO:2, SEQ ID NO:5, SEQ HD NO: 10, SEQ ID NO: 15, SEQ ED NO:20, SEQ ID NO:23, SEQ TD NO:25, SEQ ID NO:29, SEQ TD NO:32, SEQ HD NO:35, SEQ ID NO:38, SEQ TD NO:41, SEQ ID NO:44, SEQ DD NO:47, SEQ DD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ DD NO:65, SEQ DD NO:66, SEQ TD NO:67, SEQ TD NO:68, SEQ TD NO:
  • Particularly preferred unique amino acid sequences to be identified using antibodies include antibodies that bind specifically to unique amino acid sequences encoded by a nucleic acid sequence SEQ ID NO: 50, SEQ ED NO:51, SEQ LD NO:52, SEQ ED NO:53, SEQ LD NO:54, SEQ LD NO:55, SEQ ED NO:56.
  • identifying the presence of a T cell receptor having a unique nucleic acid sequence can be achieved by PCR amplification and nucleic acid sequencing techniques.
  • Suitable reagents for use with such techniques include DNA primers which are complementary to common sequences (i.e., sequences shared by two or more TCR V ⁇ molecules) that flank all or a portion of a unique sequence. It is within the skill of one in the art to design and produce suitable primers based on sequences of the present invention.
  • the presence of a unique sequence in a sample from an animal is determined by sequencing the PCR product produced using the common sequence primers and identifying whether one or more nucleic acid sequences are present in the PCR product. An example of such method is described in Example 4 herein.
  • T cell expansion is determined by identifying the heterogeneity or homogeneity of nucleic acid sequence displayed in a DNA fingerprint profile of a given sequence in a given sample of PCR products.
  • Preferred reagents include, but are not limited to, DNA primers or probes complementary to unique nucleic acid sequences contained in SEQ LD NO: 1, SEQ ED NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ DD NO:7, SEQ DD NO:8, SEQ DD NO:9, SEQ DD NO:l l, SEQ DD NO:12, SEQ ID NO:13, SEQ TD NO:98, SEQ ⁇ D NO:100, SEQ TD NO:17, SEQ TD NO:18, SEQ TD NO:19, SEQ TD NO:21, SEQ ID NO:22, SEQ TD NO:24, SEQ ED NO:25, SEQ ED NO:27 and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ DD NO: 60, SEQ DD NO:61, SEQ LD NO:62, SEQ LD NO:63, SEQ ED NO:64, SEQ ED NO:
  • One embodiment of the present invention is a method of detecting T cell expansion in an animal comprising detecting the expansion of a T cell receptor having a unique amino acid sequence within a protein selected from the group consisting of hcV ⁇ 3, hcV ⁇ 4, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82 proteins by forming detectable products, wherein the increased level of a detectable product compared with another detectable product indicates expansion of the T cells.
  • V ⁇ proteins containing unique amino acid sequence include PCaV ⁇ 3 127 , PCaV ⁇ 3 l u , PCaV ⁇ 3 U0 , PCaV ⁇ 4 128 , PCaV ⁇ 4 113 , PCaV ⁇ 4 109 , PCaV ⁇ l2 134 , PCaV ⁇ l2 H 1 , PCaV ⁇ l2 115 , PCaV ⁇ 72 133 , PCaV ⁇ 72 113 , PCaV ⁇ 72 114 , PCaV ⁇ 21 130 , PCaV ⁇ 21 108 , PCaV ⁇ 21 116 , PCaV ⁇ 54 I35 , PCaV ⁇ 54 114 , PCaV ⁇ 54 U6 , PCaV ⁇ l82 128 , PCaV ⁇ 182, ]0 and/or PCaV ⁇ 182 109 .
  • samples useful a method of the present invention include samples from an animal that contains T cells.
  • a sample to be tested using a method of the present invention comprises blood, synovial fluid, lung lavage, saliva, spleen, thymus, tumors, granulomas, abscesses, edematous fluid, central nervous system fluid.
  • Preferred animals from which to isolate a sample of the present invention includes a mammal, more preferably a canid and more preferably a dog.
  • Any method of the present invention can be used to determine the disease state of an animal. Such methods can be used to determine the presence or absence of disease in an animal, including an animal susceptible to disease, an animal suspected of having disease, an animal having disease or an animal being or having been treated for a disease.
  • diseases that can be diagnosed using a method of the present invention include: various forms of cancer, e.g., lymphoma and leukemia; various autoimmune diseases, e.g., rheumatoid arthritis or diabetes; various infectious diseases, such as those caused by viruses, by a yeast, e.g.
  • T cells e.g. allergic dermatitis, atopic dermatitis, allergic bronchitis or allergic rhinitis.
  • a prefened embodiment of the present invention is a method to diagnose T cell cancer comprising: (a) contacting a sample containing DNA from an animal with a DNA primer that is complementary to a unique nucleic acid sequence within a nucleic acid molecule including nCaV ⁇ 3 38] , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390 ,
  • Another preferred embodiment of the present invention is a method to diagnose T cell cancer comprising: (a) contacting a sample containing DNA from an animal with a DNA primer that is complementary to a unique nucleic acid sequence within nucleic acid molecule selected from the group consisting of nCaV ⁇ 3 381 , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390
  • the present invention also includes a kit comprising one or more reagents of the present invention.
  • Preferred reagents include those which can differentiate TCR V beta proteins or nucleic acid molecules that encode such proteins.
  • Preferred TCR V beta proteins include hcV ⁇ 2, hcV ⁇ 3, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82, with PCaV ⁇ 3 127 , PCaV ⁇ 3 , PCaV ⁇ 3 110 , PCaV ⁇ 4 128 , PCaV ⁇ 4 113 , PCaV ⁇ 4 109 , PCaV ⁇ l2, 34 , PCaV ⁇ l2 , PCaV ⁇ l2 115 , PCaV ⁇ 72 133 , PCaV ⁇ 72 113 , PCaV ⁇ 72 114 , PCaV ⁇ 21 130 , PCaV ⁇ 21 108 , PCaV ⁇ 21 116 , PCaV ⁇ 54 135 , PCaV ⁇ 54
  • SEQ TD NO:77 SEQ TD NO:78, SEQ HD NO:79, SEQ TD NO: 80 and/or proteins encoded by the complement of a nucleic acid sequence including SEQ DD NO:50, SEQ ED NO:51, SEQ LD NO:52, SEQ HD NO:53, SEQ TD NO:54, SEQ TD NO:55, SEQ TD NO:56 being even more preferred.
  • Prefened TCR V beta nucleic acid molecules include hcV ⁇ 2, hcV ⁇ 3, hcV ⁇ l2, hcV ⁇ 72, hcV ⁇ 21, dtb54 or dtbl82, with nCaV ⁇ 3 381 , nCaV ⁇ 3 333 , nCaV ⁇ 3 330 , nCaV ⁇ 4 408 , nCaV ⁇ 4 384 , nCaV ⁇ 4 351 , nCaV ⁇ 4 339 , nCaV ⁇ l2 408 , nCaV ⁇ l2 402 , nCaV ⁇ l2 339 , nCaV ⁇ l2 345 , nCaV ⁇ 72 438 , nCaV ⁇ 72 399 , nCaV ⁇ 72 423 , nCaV ⁇ 72 342 , nCaV ⁇ 21 462 , nCaV ⁇ 21 390 , nC
  • SEQ DD NO:3 SEQ DD NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ DD NO:9, SEQ DD NO:l l, SEQ DD NO:12, SEQ DD NO:13, SEQ DD NO:98, SEQ DD NO:100, SEQ DD NO: 17, SEQ ID NO: 18, SEQ DD NO: 19, SEQ DD NO:21, SEQ ID NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ TD NO:27,SEQ ⁇ D NO:28, SEQ TD NO:30, SEQ TD NO:31, SEQ DD NO:33, SEQ DD NO:34, SEQ ED NO:36, SEQ ID NO:37, SEQ TD NO:39, SEQ ⁇ D NO:40, SEQ DD NO:42, SEQ ⁇ D NO:43, SEQ TD NO:45, SEQ TD NO:46, SEQ DD NO
  • TCR V ⁇ nucleic acid molecules Canine TCR V ⁇ nucleic acid molecules were produced as follows. RNA was purified from mitogen activated canine peripheral blood lymphocytes and resuspended in water at about 2.5 X 10 5 cell equivalent/microliter ( ⁇ l). About 5 ⁇ l of RNA and random hexanucleotide primers were used to synthesize cDNA using the First Strand cDNA SynthesisTM kit (available from Pharmacia, Uppsala, Sweden).
  • TCR V ⁇ genes were selectively PCR amplified from the cDNA using degenerate oligonucleotides designed using the conserved sequence motifs WYRQ and Y(Y/F)CA of T and B cell antigen receptors (Rast and Litman, Proc. Natl. Acad. Sci. USA, vol. 91, p. 9248, 1994).
  • the degenerate primer FR2 having the nucleic acid sequence 5' CCG AAT TCT GGT A(TC) C(GA) NCA 3' (SEQ LD NO:81) was used in combination with either the FR3A primer, having the nucleic acid sequence 5' CGG ATC CGC (GA)CA (GA)TA (GA)T A 3' (SEQ ID NO:82) or the primer FR3B, having the nucleic acid sequence 5' CGG ATC CGC (GA)CA (GA)A A(GA)T A 3' (SEQ ID NO:83).
  • First round PCR reactions were performed in about 50 ⁇ l of 50 mM KCI, 10 mM Tris-HCl, 0.01% gelatin (pH 8.3), 3.5 mM MgCl 2 , 0.2 mM of each of the four deoxyribonucleotide triphosphates (dNTP), about l ⁇ M of each primer and about 2.0 units Taq polymerase (available from Perkin- Elmer).
  • This reaction mixture was created after attempts using published methods failed.
  • 20 additional cycles of PCR using about 5 ⁇ l of the first round PCR sample as template under identical conditions were needed to obtain useful second round PCR product. No PCR products were identified after 30 cycles.
  • Second round PCR products were resolved by loading about 15 ⁇ l of the second round PCR product onto a 1.0% agarose gel in TBE buffer and staining the gel with ethidium bromide.
  • Plasmid DNA was purified using BioRad's Quantum PrepTM mini-prep kit (available from BioRad, Hercules, CA). Inserted sequence in 11 different plasmid samples were subjected to DNA sequence analysis using standard sequencing methods. Three different nucleic acid sequences were obtained. The 3 different nucleic acid molecules are referred to herein as hcvb3, hcvb4 and hcvbl2. The sequences obtained above indicated that incomplete nucleic acid molecules were obtained using the foregoing PCR procedure.
  • primers were designed using the nucleic acid sequences obtained above, including primer Phcvb3, having the nucleic acid sequence 5' CCA GAC CTG GGT CTT GTC G 3' (SEQ TD NO:84), primer Phcvb4, having the nucleic acid sequence 5' CTC TGT CCT GGG AGC TGA C 3' (SEQ ID NO:85), and primer Phcvbl2, having the nucleic acid sequence 5' TTG TTT GAT CTA GAG ACT GTG 3' (SEQ ID NO:86).
  • Each primer was then used in combination with the 5' vector primer T3 (available from available from Stratagene Cloning Systems, La Jolla, CA) to amplify PCR products representing V ⁇ genes from a canine PBL cDNA library generated in the ⁇ Zap II vector (available from Stratagene Cloning Systems, La Jolla, CA).
  • the C. familiaris mitogen activated PBMC cDNA library was constructed in the Uni-ZAP® XR vector (available from Stratagene Cloning Systems), using Stratagene' s ZAP-cDNA® Synthesis Kit and the manufacturer's protocol.
  • the mRNA was isolated from C. familiaris peripheral blood mononuclear cells 4 hours after they were activated by a polyclonal activating agent in culture.
  • PCR reaction were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min, about 94°C for about 30 sec, about 58 °C for about 30 sec, and about 72 °C for about 1 min. for about 35 cycles, then clonal extension was performed at about 72°C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 25 mM MgCl 2 , about 1 unit Amplitaq GoldTM (available from Perkin-Elmer Cetus), about 200 ⁇ M dNTP's and about 1 ⁇ M primer.
  • the resulting PCR products were gel purified, cloned and sequenced using standard methods.
  • the resulting nucleic acid sequences were aligned with the nucleic acid sequences obtained from the first set of PCR products to obtain a more complete sequence of hcvb3, hcvb4 and hcvbl2. Since the PCR primers used to generate the first set of PCR products were degenerate primers, the nucleic acid sequences of hcvb3, hcvb4 and hcvbl2 at the degenerate primer sites were ambiguous.
  • PCR primers corresponding to the newly derived extreme 5' ends of the hcvb3, hcvb4 and hcvbl2 nucleic acid molecules were designed.
  • primer 5'hcvb4 having the nucleic acid sequence 5' ACG GTG AAG GGC TAG CAC CT 3' SEQ ID NO:88
  • primer 5'hcvbl2 having the nucleic acid sequence 5' GCT GAA ATG GCC ACC GGC GT 3' (SEQ ID NO:89)
  • each were used in combination with a primer specific for a sequence in the constant region of a TCR beta chain (SED ID NO: 57) in PCR reactions using the cDNA library described above.
  • the resulting PCR products were purified, cloned into PCR2.1 vector (available from
  • SEQ ID NO: 1 includes the V, D and J regions of the sequenced PCR product.
  • the complement of SEQ TD NO: 1 is represented herein by SEQ ID NO:3.
  • nucleic acid molecule nCaV ⁇ 3 381 encodes a TCR V ⁇ protein of about 127 amino acids, denoted herein as PCaV ⁇ 3 127 , the amino acid sequence of which is presented in SEQ ID NO:2, assuming an open reading frame having a first codon spanning from nucleotide 1 through nucleotide 3 of SEQ TD NO:l and a last codon spanning from nucleotide 379 through nucleotide 381 of SEQ LD NO: 1.
  • the putative signal sequence extends from nucleotide 1 to nucleotide 51 of SEQ LD NO:l.
  • the proposed mature protein (i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved), denoted herein as PCaV ⁇ 3 110 , contains about 110 amino acids, extending from residue 18 through residue 127 of SEQ ID NO:2.
  • the nucleic acid molecule encoding PCaV ⁇ 3 U0 is denoted herein as nCaV ⁇ 3 330 , extending from nucleotide 52 through nucleotide 381 of SEQ ID NO:l.
  • Comparison of nucleic acid sequence SEQ DD NO:l with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 1 showed the most homology, i.e., about 69% identity, between SEQ ID NO: 1 and a human TCR ⁇ chain gene (Genbank Accession No. Z223040).
  • Comparison of amino acid sequence SEQ ID NO:2 with amino acid sequences reported in GenBank indicates that SEQ ID NO:2 showed the most homology, i.e., about 65% identity, between SEQ ID NO:2 and an Ovis aries TCR V ⁇ chain protein (Genbank Accession No. gi 2665554).
  • SEQ ID NO:4 A second clone (hcV ⁇ 4) was isolated, refened to herein as nCaV ⁇ 4 408 , the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO:4.
  • SEQ TD NO:4 includes the V, D and J regions of the sequenced PCR product.
  • the complement of SEQ ID NO:4 is represented herein by SEQ ID NO:6.
  • nucleic acid molecule nCaV ⁇ 4 408 encodes a TCR V ⁇ protein of about 128 amino acids, denoted herein as PCaV ⁇ 4 128 , the amino acid sequence of which is presented in SEQ ID NO:5, assuming an open reading frame having an initiation codon spanning from nucleotide 24 through nucleotide 26 of SEQ ID NO:4 and a last codon spanning from nucleotide 405 through nucleotide 407 of SEQ ID NO:4.
  • the coding region encoding PCaV ⁇ 4 128 is presented herein as nCaV ⁇ 4 384 , which has the nucleotide sequence SEQ ID NO:6 (the coding strand) and SEQ DD NO:7 (the complementary strand).
  • the putative signal sequence extends from nucleotide 25 to nucleotide 69 of SEQ TD NO:4.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 4 113 contains about 113 amino acids, extending from residue 60 through residue 128 of SEQ ID NO:5.
  • nucleic acid molecule encoding PCaV ⁇ 4 1 ]3 is denoted herein as nCaV ⁇ 4 339 , extending from nucleotide 70 through nucleotide 408 of SEQ TD NO:4.
  • nCaV ⁇ 4 339 The nucleic acid molecule encoding PCaV ⁇ 4 1 ]3 is denoted herein as nCaV ⁇ 4 339 , extending from nucleotide 70 through nucleotide 408 of SEQ TD NO:4.
  • SEQ ID NO:4 Comparison of nucleic acid sequence SEQ ID NO:4 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:4 showed the most homology, i.e., about 75% identity, between SEQ ID NO:4 and a human TCR ⁇ chain gene (Genbank Accession No. M97713).
  • SEQ ID NO:5 Comparison of amino acid sequence SEQ ID NO:5 with amino acid sequences reported in GenBank indicates that SEQ ID NO: 5 showed the most homology, i.e., about 69% identity, between SEQ ID NO:5 and an Ovis aries TCR V ⁇ chain protein (Genbank Accession No. gi 2665558).
  • SEQ ID NO:9 includes the V, D and J regions of the sequenced PCR product.
  • the complement of SEQ ID NO:9 is represented herein by SEQ ID NO: 11.
  • nucleic acid molecule nCaV ⁇ l2 408 encodes a TCR V ⁇ protein of about 134 amino acids, denoted herein as PCaV ⁇ l2 134 , the amino acid sequence of which is presented in SEQ ID NO: 10, assuming an open reading frame having an initiation codon spanning from nucleotide 7 through nucleotide 9 of SEQ ID NO:9 and a last codon spanning from nucleotide 406 through nucleotide 408 of SEQ ED NO:9.
  • the coding region encoding PCaV ⁇ l2 134 is presented herein as nCaV ⁇ l2 402 , which has the nucleotide sequence SEQ ID NO: 12 (the coding strand) and SEQ ID NO: 13 (the complementary strand).
  • the putative signal sequence extends from nucleotide 7 to nucleotide 63 of SEQ ID NO:9.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ l2 H5 contains about 115 amino acids, extending from residue 20 through residue 134 of SEQ ID NO: 10.
  • the nucleic acid molecule encoding PCaV ⁇ l2 115 is denoted herein as nCaV ⁇ l2 345 , extending from nucleotide 64 through nucleotide 408 of SEQ ID NO:9.
  • This example describes the isolation and sequencing of two additional canine TCR V ⁇ nucleic acid molecules.
  • Two canine TCR V ⁇ nucleic acid molecules were PCR amplified from the canine PBL cDNA library described above in Example 1.
  • a pair of primers was used to amplify DNA from the cDNA library.
  • the 5' vector primer T3, described in Example 1 was used in combination with primer Phcvb21, having the nucleic acid sequence 5' CTG TTG CCC ACG TTA GAG G 3' (SEQ DD NO:90) or primer Phcvb72, having the nucleic acid sequence 5' TTA CTG AAC TGC TGC ACT G 3' (SEQ ID NO:91).
  • PCR reaction were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min., about 94°C for about 30 sec, about 60°C for about 30 sec, about 72 °C for about 1 min. for about 35 cycles; then clonal extension was performed at about 72 °C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl 2 , about 1 unit Amplitaq Gold, about 200 ⁇ M dNTP's and about 1 ⁇ M primer.
  • the resultant PCR products obtained using standard PCR conditions were gel purified, cloned and sequenced.
  • the PCR products are refened to herein as hcvb21 or hcvb72, respectively.
  • sequences obtained were compared with sequences disclosed in Ito et al., Immuno genetics, vol. 38, p. 60, 1993 or Takano et al., Immunogenetics, vol. 40, p. 246, 1994.
  • the PCR products hcvb21 or hcvb72 were found to contain more 5' nucleic acid sequence than that disclosed in the above-referenced publications.
  • PCR amplification was performed using primers designed from the 5' sequence obtained from hcvb21 or hcvb72 nucleic acid molecules.
  • Primer 5'Phcvb21 having the nucleic acid sequence 5' GCT GCA GGA TTC GGC ACG AG 3' (SEQ ID NO:92) or primer 5'Phcvb72, having the nucleic acid sequence 5' TAC GAC TGT CAG CTT GGT CC 3' (SEQ ID NO:93), each were used in conjunction with a TCR beta constant region primer (SEQ ID NO:57) to amplify these sequences from mRNA prepared from canine concavalin A (ConA) activated PBMC.
  • the PCR products were cloned and sequenced using standard methods.
  • SEQ ID NO: 19 includes the V, D and J regions of the sequenced PCR product.
  • the complement of SEQ DD NO: 19 is represented herein by SEQ ID NO:21.
  • nucleic acid molecule nCaV ⁇ 21 462 encodes a TCR V ⁇ protein of about 130 amino acids, denoted herein as PCaV ⁇ 21 130 , the amino acid sequence of which is presented in SEQ ID NO:20, assuming an open reading frame having an initiation codon spanning from nucleotide 73 through nucleotide 75 of SEQ ID NO: 19 and a last codon spanning from nucleotide 460 through nucleotide 462 of SEQ ID NO: 19.
  • the coding region encoding PCaV ⁇ 21 130 is presented herein as nCaV ⁇ 21 390 , which extends from nucleotide 73 to nucleotide 462 of SEQ ID NO: 19.
  • the putative signal sequence extends from nucleotide 73 to nucleotide 114 of SEQ ID NO: 19.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 21 ] 16 contains about 116 amino acids, extending from residue 15 through residue 130 of SEQ ID NO: 19.
  • the nucleic acid molecule encoding PCaV ⁇ 21 1 ]6 is denoted herein as nCaV ⁇ 21 34g , extending from nucleotide 115 through nucleotide 462 of SEQ ID NO: 19.
  • SEQ ID NO: 19 is 221 nucleotides longer than at the 5'end of DTCRB21.
  • the sequences of SEQ ID NO: 19 and DTCR21 overlap by 176 nucleotides with 100% identity, corresponding to nucleotides 222 through 398 of SEQ LD NO:19.
  • An alignment of SEQ LD NO:19 with DTCR21 is shown in Table 1. TABLE 1. Nucleotide Sequence Comparison of hcvb21 and DTCRB21
  • SEQ ID NO: 19 CTCACCGTGA CA
  • the amino acid sequence SEQ HD NO:20 is 50 amino acids longer at the 5'end than the amino acid sequence encoded by DTCR21.
  • An alignment of SEQ ED NO:20 with the amino acid sequence encoded by DTCR21 is shown in Table 2. TABLE 2. Amino Acid Sequence Comparison of hcvb21 and DTCR21.
  • SEQ ID NO: 20 MLMLLLLLGP SSGLGALVFQ APSTMICKSG ATVQIQCQTV DLQATTVFWY DTCRB21
  • the amino acid sequence SEQ ID NO:20 is 50 amino acids longer at the 5'end than the amino acid sequence encoded by DTCR21.
  • An alignment of SEQ LD NO:20 with the amino acid sequence encoded by DTCR21 is shown in Table 2.
  • Comparison of nucleic acid sequence SEQ ID NO: 19 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 19 showed the most homology, i.e., about 38% identity, between SEQ HD NO: 19 and a dog TCR ⁇ chain gene (Genbank Accession No. M97510).
  • Comparison of amino acid sequence SEQ ID NO:20 with amino acid sequences reported in GenBank indicates that SEQ ID NO:20 showed the most homology, i.e., about 60% identity, between SEQ ID NO:20 and DTCR21 protein.
  • SEQ ID NO:98 includes the V, D and J regions of the sequenced PCR product.
  • the complement of SEQ ID NO:98 is represented herein by SEQ ID NO: 100.
  • nucleic acid molecule nCaV ⁇ 72 438 encodes a TCR V ⁇ protein of about 133 amino acids, denoted herein as PCaV ⁇ 72 133 , the amino acid sequence of which is presented in SEQ ID NO: 15, assuming an open reading frame having an initiation codon spanning from nucleotide 40 through nucleotide 42 of SEQ ID NO:98 and a last codon spanning from nucleotide 436 through nucleotide 438 of SEQ ID NO:98.
  • the coding region encoding PCaV ⁇ 72 133 is presented herein as nCaV ⁇ 72 399 , which has the nucleotide sequence SEQ ID NO: 17 (the coding strand) and SEQ LD NO: 18 (the complementary strand).
  • the putative signal sequence extends from nucleotide 40 to nucleotide 96 of SEQ TD NO:98.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 72 ]]4 contains about 114 amino acids, extending from residue 20 through residue 133 of SEQ HD NO: 15.
  • PCaV ⁇ 72 u4 is denoted herein as nCaV ⁇ 72 342 , extending from nucleotide 142 through nucleotide 438 of SEQ ID NO: 19.
  • sequence SEQ LD NO: 15 and the amino acid sequence encoded by DTB72 indicates that the identity between the two sequences is only 57%, when determined using the Compare function by maximum matching within the program DNAsisTM Version 2.1. An alignment of the two sequences is shown in Table 4. TABLE 4. Amino Acid Sequence Comparison of hcvb72 and DTB72
  • SEQ ID NO: 19 Comparison of nucleic acid sequence SEQ ID NO: 19 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 19 showed the most homology, i.e., about 60% identity, between SEQ ID NO: 19 and a feline leukemia virus transduced TCR ⁇ chain gene (Genbank Accession No. X05155). Comparison of amino acid sequence SEQ HD NO:20 with amino acid sequences reported in GenBank indicates that SEQ ID NO:20 showed the most homology, i.e., about 65% identity, between SEQ TD NO:20 and a a feline leukemia virus transduced TCR ⁇ chain protein (Genbank Accession No. RWMVTV).
  • Example 3 This example describes the production of two canine TCR V ⁇ nucleic acid molecules.
  • Two canine TCR V ⁇ nucleic acid molecules were PCR amplified from the canine PBL cDNA library described above in Example 1. A pair of primers was used to amplify DNA from the cDNA library.
  • sequences obtained were compared with sequences disclosed in Takano et al., ibid.
  • the PCR products dtb54 or dtbl 82 were found to contain more 5' nucleic acid sequence than that disclosed in the above-referenced publication.
  • PCR amplification was performed using primers designed from the 5' sequence obtained from dtb54 or dtbl 82 nucleic acid molecules.
  • Primer 5'Pdtb54 having the nucleic acid sequence 5' CAC GAG CCT GCC ATG TGC CC 3' (SEQ ID NO:96) or primer 5'Pdtbl82, having the nucleic acid sequence 5' GGC ACG AGC ACT GAG GAC CA 3' (SEQ TD NO:97), each were used in conjunction with a TCR beta constant region primer (SEQ DD NO:57) to amplify these sequences from mRNA prepared from canine concavalin A (ConA) activated PBMC.
  • the PCR products were cloned and sequenced using standard methods. A.
  • SEQ ID NO:22 The clone dtb54 was isolated, referred to herein as nCaV ⁇ 54 417 , the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO:22.
  • SEQ LD NO:22 includes the V, D and J regions of the sequenced PCR product.
  • the complement of SEQ ID NO:22 is represented herein by SEQ ID NO:24.
  • nucleic acid molecule nCaV ⁇ 54 417 encodes a TCR V ⁇ protein of about 135 amino acids, denoted herein as PCaV ⁇ 54 135 , the amino acid sequence of which is presented in SEQ ID NO:23, assuming an open reading frame having an initiation codon spanning from nucleotide 13 through nucleotide 15 of SEQ DD NO:22 and a last codon spanning from nucleotide 415 through nucleotide 417 of SEQ ID NO:22.
  • the coding region encoding PCaV ⁇ 54 ]35 is presented herein as nCaV ⁇ 54 405 , which extends from nucleotide 13 to nucleotide 417 of SEQ ID NO:22.
  • the putative signal sequence extends from nucleotide 13 to nucleotide 69 of SEQ ID NO:22.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 54 ]16 contains about 116 amino acids, extending from residue 20 through residue 135 of SEQ ID NO:22.
  • nCaV ⁇ 54 348 The nucleic acid molecule encoding PCaV ⁇ 54 1]6 is denoted herein as nCaV ⁇ 54 348 , extending from nucleotide 70 through nucleotide 417 of SEQ LD NO:22.
  • SEQ ID NO:25 includes the V, D and J regions of the sequenced PCR product.
  • SEQ ID NO:27 The complement of SEQ TD NO:25 is represented herein by SEQ ID NO:27.
  • nucleic acid molecule nCaV ⁇ l82 423 encodes a TCR V ⁇ protein of about 128 amino acids, denoted herein as PCaV ⁇ l 82 128 , the amino acid sequence of which is presented in SEQ ED NO: 26, assuming an open reading frame having an initiation codon spanning from nucleotide 40 through nucleotide 43 of SEQ ID NO:25 and a last codon spanning from nucleotide 421 through nucleotide 423 of SEQ ID NO:25.
  • the coding region encoding PCaV ⁇ l 82 128 is presented herein as nCaV ⁇ l82 384 , which extends from nucleotide 40 to nucleotide 423 of SEQ ID NO:25.
  • the putative signal sequence extends from nucleotide 40 to nucleotide 96 of SEQ ID NO:25.
  • the proposed mature protein i.e., canine TCR V ⁇ protein from which the signal sequence has been cleaved
  • PCaV ⁇ 182 contains about 109 amino acids, extending from residue 20 through residue 128 of SEQ ID NO:25.
  • nCaV ⁇ 182 327 The nucleic acid molecule encoding PCaV ⁇ 182 109 is denoted herein as nCaV ⁇ 182 327 , extending from nucleotide 97 through nucleotide 423 of SEQ ED NO:25.
  • This example describes the identification of unique TCR V ⁇ sequences by designing PCR primers that distinguish between different (TCR) V ⁇ nucleic acid molecules.
  • the primers were designed based on the nucleic acid sequences SEQ ID NO:l, SEQ LD NO:4, SEQ ID NO:9, SEQ LD NO:98, SEQ TD NO: 19, SEQ ID NO:22 or SEQ ID NO:25.
  • the inventors discovered unique sequences for each of the foregoing nucleic acid molecules that are not shared between each of the different molecules.
  • the inventors discovered which primers designed based on those unique sequences, and under what specific conditions, did not cross-prime between the different nucleic acid molecules.
  • V ⁇ genes several different V ⁇ primers to unique sequences had to be tested in order to find one that was specific to only one V ⁇ gene.
  • Some of the primers that were designed include: primer hcV ⁇ 3 unique, having the nucleic acid sequence 5' CGA CAA GAC CCA GGT CTG G 3' (SEQ ID NO:50), complementary to nucleotides 157 to 175 of SEQ ID NO: 1 ; primer hcV ⁇ 4 unique, having the nucleic acid sequence 5' GTC AGC TCC CAG GAC AGA G
  • each primer to specifically prime a specific TCR V ⁇ gene was tested by performing PCR reactions using each of the above primers in combination with a V ⁇ constant region primer including: primer C ⁇ l, having the nucleic acid sequence 5' GTG ACC TTC TGC AGA TCC TC 3' (SEQ ED NO:57); primer C ⁇ 2, having the nucleic acid sequence 5' AGC TCA GCT CCA CGT GGT C 3' (SEQ ID NO:58); or primer C ⁇ 3, having the nucleic acid sequence 5' TGC TGA ACC CAC TCG TGA C 3' (SEQ DD NO:59).
  • primers The specificity of these primers was first tested using 7 different DNA plasmids that contained nucleic acid molecules comprising either SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:9, SEQ LD NO:98, SEQ LD NO: 19, SEQ LD NO:22 or SEQ LD NO:25 as templates for different PCR reaction.
  • Each unique V ⁇ primer was used to amplify DNA from each template.
  • PCR reactions were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min., about 94°C for about 30 sec, about 60 °C for about 30 sec, about 72 °C for about 1 min.
  • PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl 2 , about 1 unit Amplitaq Gold, about 0.1 ⁇ M dNTP's and about 0.4 ⁇ M primer. The results indicated that PCR products were only present when the conect template and primer combination was used.
  • primer hcV ⁇ 3 unique was used with a plasmid containing SEQ ID NO: 1
  • primer hcV ⁇ 4 unique was used with a plasmid containing SEQ ID NO:4
  • primer hcV ⁇ l2 unique was used with a plasmid containing SEQ ID NO:9
  • primer hcV ⁇ 72 unique was used with a plasmid containing SEQ ID NO:98
  • primer hcV ⁇ 21 unique was used with a plasmid containing SEQ ID NO: 19
  • primer dtb54 unique was used with a plasmid containing SEQ ID NO:22 or primer dtbl82 unique was used with a plasmid containing SEQ ID NO:25.
  • lymph node cells or PBMC were isolated from 2 dogs known to have lymphoma (Haynes and Stoll) and a control dog.
  • cDNA samples from the cells were used as templates in separate PCR reactions using primer hcV ⁇ 3 unique, primer hcV ⁇ 4 unique, primer hcV ⁇ l2 unique, primer hcV ⁇ 72 unique, primer hcV ⁇ 21 unique, primer dtb54 unique or primer dtbl 82 unique, in combination with C ⁇ 3 primer (SEQ DD
  • PCR reactions were performed using the following conditions: Taq activation was performed at about 95°C for about 10 min., about 94°C for about 30 sec, about 60°C for about 30 sec, about 72°C for about 1 min. for about 35 cycles; then clonal extension was performed at about 72 °C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl 2 , about 1 unit Amplitaq Gold, about 0.1 ⁇ M dNTP's and about 0.4 ⁇ M primer. PCR products were resolved by electrophoresis on an about 1.2% LE agarose gel in TBE buffer and stained with ethidium bromide.
  • Fig. IA The results shown in Fig. IA indicated that PCR products using cells isolated from the control dog were generated using all 7 V ⁇ specific primers. In addition, the levels of PCR products were substantially the same using the 7 V ⁇ specific primers.
  • Fig. IB and IC indicated that the V ⁇ profiles of PCR products using cells isolated from the lymphoma dog Haynes, or the lymphoma dog Stoll, were different from the V ⁇ profile of the control dog.
  • T cells expressing hcV ⁇ 21 genes have been expanded in both patients, while T cells expressing the other 6 V ⁇ genes have decreased in proportion.
  • primers complementary to unique V ⁇ sequences of the present invention can be used to: (1) detect the presence of specific V ⁇ genes in a population of cells; (2) identify clonal expansion of cells expressing a particular V ⁇ gene; and (3) associate clonal T cell expansion with an abnormal state or disease; or (4) distinguish a general lymphoproliferative state involving polyclonal T cell activation from clonal T cell expansion.
  • Example 6 This example describes a method for DNA fingerprinting the junctional regions of rearranged V ⁇ genes to identify clonal expansion of T cells.
  • DNA sequence analysis of the junction between the V, D and J regions of a V ⁇ gene can determine whether a mRNA population that translates into a V ⁇ protein is homogenous or heterogeneous by looking at the fluorescent DNA fingerprint.
  • Fluorescent DNA fingerprints of V ⁇ mRNA populations were determined as follows. cDNA was prepared using standard methods from mitogen stimulated canine PBL cells isolated from a control dog and from lymph node cells isolated from the lymphoma dog Haynes or. The cDNA samples from either Haynes or the control dog were used as templates for PCR reactions using variable region primers in combination with constant region primers. Use of such primers were designed so that the resulting PCR products span the D/J junction of a V ⁇ cDNA.
  • PCR reactions were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min., about 94°C for about 30 sec, about 60°C for about 30 sec, about 72°C for about 1 min.
  • PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl 2 , about 1 unit Amplitaq Gold, about 0.1 ⁇ M dNTP's and about 0.4 ⁇ M primer.
  • Fig. 3 illustrates 4 histograms generated using cDNA from the lymphoma dog Haynes and V ⁇ primers hcV ⁇ l2 unique, hcV ⁇ 72 unique, hcV ⁇ 21 unique, dtb54 unique or dtbl 82 unique.
  • the histograms show that 3 of the 4 V ⁇ genes amplified have fingerprints similar to those seen with a heterogeneous population of T cells, such as shown in Fig. 2.
  • the histogram generated using the hcvb21 unique primer has a fingerprint which allows the unambiguous determination of the DNA sequence throughout the entire junctional region between the V, D, J and C regions. This result indicates that the sequence recognized by the hcvb21 unique primer was dominant among the canine PBL cell population from the lymphoma dog.
  • FIG. 4 A comparison of the histogram generated using the hcvb21 unique primer with cDNA from the normal dog with the histogram generated using the hcvb21 unique primer with cDNA from the lymphoma dog is shown in Fig. 4. The comparison illustrates that the difference between the two histograms can be used to determine clonal expansion of a single T cell and association of such expansion to an abnormal state or disease.
  • Example 7
  • This example describes the generation of T cell clones reactive to flea saliva allergens, and the characterization of the TCR V ⁇ genes used by the T cell receptors of the T cell clones.
  • T cell clones reactive to flea saliva antigens were generated from peripheral blood mononuclear cells (PBMC) isolated from the experimentally-induced flea allergic dog CPO2 (described in U.S. Patent No. 5,646,115, issued on July 8, 1997) as follows. Blood samples were isolated from experimentally-induced flea allergic dog CPO2. Peripheral blood lymphocytes (PBL) were harvested from each sample by centrifugation using a ficoll gradient (available from Pharmacia).
  • PBMC peripheral blood mononuclear cells
  • the PBL cells were cultured in about 5 ml cultures in culture medium containing LMDM and 10% fetal calf serum (available from Gibco, Gaithersburg, MD) at about 5 X 10 6 cells/ml in the presence of about 5 ⁇ g/ml flea saliva protein (prepared according to the methods described in U.S. Patent No. 5,646,115) for about 14 days, at about 37 °C, in about 5% CO 2 .
  • the incubated cells were harvested and the number of viable cells determined.
  • the viable cells were plated in 96 well round bottom plates at about 10 2 or about 10 3 cells per well per 200 ⁇ l of culture medium in the presence of about 10 5 autologous irradiated PBL (prepared according to the methods generally described in Coligan, ibid.) as antigen presenting cells (APC), about 5 ⁇ g/ml of flea saliva protein, and about 10 units/ml of recombinant hLL-2 for another 14 days.
  • Wells that contain growing cells were restimulated in situ by replacing about 150 ⁇ l of spent culture medium (i.e., medium in which cells had been grown) with about 150 ⁇ l of fresh culture medium, flea saliva protein and APC as described above.
  • T cell clones Seven different T cell clones were derived from cells isolated from CPO2. mRNA was prepared from about 10 6 cells from each of the 7 clones using standard methods. cDNA was prepared from the mRNA using methods described above in Example 1.
  • the cDNA samples from the 7 clones were then used as templates in PCR reactions to determine the presence of particular TCR V ⁇ molecules using the methods described above in Example 4. Analysis of the resulting PCR products indicated that 6 of the 7 clones expressed TCR using the dtbl 82 V beta chain, thereby indicating a bias in TCR V ⁇ usage in the T cell reactivity of CPO2 to flea saliva protein.

Abstract

This invention provides a polynucleotide encoding Repro-EN-1.0, a polypeptide associated with endometriosis. Auto-antibodies against Repro-EN-1.0 have been found in subjects diagnosed with endometriosis. This invention also provides methods of using this polynucleotide and polypeptide.

Description

POLYNUCLEOΗDE ENCODING AN AUTOANTIGEN ASSOCIATED WITH ENDOMETRIOSIS
FIELD OF THE INVENTION The present invention relates to T cell receptor beta chain variable region nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins and inhibitors of such proteins or nucleic acid molecules. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and/or inhibitors, as well as their use to regulate an immune response in an animal. BACKGROUND OF THE INVENTION
The immune system of an animal is characterized by its ability to respond to a diverse set of antigenic determinants, or epitopes. This response is reflected through T and B lymphocytes, also referred to as T cells and B cells, respectively. The immune system, comprising these specialized cells, recognizes and processes foreign pathogens and macromolecules. Lymphocytes individually exhibit high specificity in recognition of particular molecular structures of antigens. The structural properties are recognized by T cell receptors, which act as antigen receptors.
T cell receptors (TCR) are members of the immunoglobulin superfamily. A TCR molecule comprises two polypeptide chains, generally an alpha chain and a beta chain. Each chain comprises an amino terminal variable region domain (V) and a carboxyl terminal constant region domain (C), and can be designated with or β when indicating the particular chain of origin. V and C regions are encoded by V region or C region genes, respectively. Each domain can be stabilized by a disulfide bond between two conserved cysteine residue pairs on each chain. Each chain is anchored to the cell membrane by a hydrophobic transmembrane domain, which typically spans the entire lipid bilayer of the membrane. A short carboxyl domain extends into the cytoplasm.
The and β chains of the TCR are encoded by gene segments analogous to the variable region (V), the diversity region (D), the joining region (J) and the constant region (C) of immunoglobulin genes. Diversity in the TCR repertoire arises, in part, from the rearrangement of V, D and J regions and from the insertion of or deletion of nucleotides at the junction between the V, D and J regions.
Previous investigators have described TCR beta chain sequences, e.g., Malisson et al., Cell, vol. 37, pp. 1101-1110, 1984; Patten et al, Nature, vol. 312, pp. 40-46, 1984; Davis et al, Nature, vol. 334, pp. 395-402, 1988; Hood et al., U.S. Patent No. 4,886,743, issued Dec. 12, 1989; and Makrides et al, U.S. Patent No. 5,552,300, issued Sep. 3, 1996.
T cells play a pivotal role in the differentiation and regulation of immune cells. Previous investigators have studied diseases in which there appears to be improper immune regulation, such as autoimmunity and some forms of immunodeficiency, and have implicated T cells in the pathogenesis of such diseases. In addition, situations exist in which clonal or oligoclonal expansion of a particular T cell population, identified by the presence of a particular TCR, can be representative of a disease state. One example is the presence of malignancy which has resulted in a T cell leukemia or lymphoma (e.g., Hood et al., ibid.). In situations of T cell leukemias or lymphomas, a TCR acts as a unique tumor marker since the TCR is stably rearranged and presented on the surface of the cell.
In summary, there remains a need to develop methods and compounds useful for the detection and treatment of undesired immune responses involving T cells. SUMMARY OF THE INVENTION
The present invention relates to T cell receptor nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins and inhibitors of such proteins or nucleic acid molecules. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and/or inhibitors, as well as their use to regulate an immune response in an animal. The present invention also includes methods to detect T cell expansion using reagents including or derived from such nucleic acid molecules, proteins and/or antibodies, as well as the use of such methods to diagnose abnormal states or disease in an animal. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 illustrates PCR amplified TCR Vβ DNA using DNA from tissue of normal dogs and dogs having lymphoma.
Figs. 2A, 2B and 2C illustrate fingerprints of TCR Vβ DNA from a normal dog. Figs. 3 A, 3B and 3C illustrate fingerprints of TCR Vβ DNA from a dog having lymphoma.
Fig. 4 illustrates a comparison between fingerprints of TCR Vβ DNA from a normal dog and a dog having lymphoma.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides for isolated T cell receptor beta chain variable region (TCR Vβ) proteins, isolated TCR Vβ nucleic acid molecules, antibodies directed against TCR Vβ proteins, and compounds derived therefrom that regulate the immune response of an animal. A TCR Vβ protein can refer to a TCR Vβ protein or a homolog thereof. As used herein, the term "TCR Vβ" refers to a molecule that can include the variable (V) region, alone or in combination with the diversity (D) and/or joining (J) regions of a TCR beta chain. It is known to one of skill in the art that the size and sequence of V, D and J regions of a TCR beta chain can vary as a result of any given recombination event between genes encoding such V, D and J regions. Typical consensus sequences used to identify the junction between the V, D and J regions are also known to one of skill in the art, thereby enabling the identification of the size and sequence of the V, D or J regions of a TCR beta chain from a novel nucleic acid or amino acid sequence. Compounds derived from TCR Vβ proteins or nucleic acid molecules of the present invention include compounds including at least a portion of, or designed using, such proteins or nucleic acid molecules. As used herein, the phrase "regulate an immune response" refers to modulating the activity of cells involved in an immune response. The term "regulate" can refer to increasing or decreasing an immune response Regulation of an immune response can be determined using methods known in the art as well as methods disclosed herein. As used herein, the terms isolated TCR Vβ proteins and isolated TCR Vβ nucleic acid molecules refers to TCR Vβ proteins and TCR Vβ nucleic acid molecules derived from mammals, preferably canids, more preferably dogs, and, as such, can be obtained from their natural source, or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of these proteins, nucleic acid molecules, antibodies, and compounds derived therefrom as therapeutic compositions to regulate the immune response of an animal as well as in other applications, such as those disclosed below.
One embodiment of the present invention is an isolated protein that includes a TCR Vβ protein. It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a protein refers to one or more proteins or at least one protein. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.
As used herein, an isolated TCR Vβ protein of the present invention (i.e., a TCR Vβ protein) can be a full-length protein or any homolog of such a protein. Full- length proteins can refer to proteins having the V, D, J and C regions of a beta chain or one or more of such regions. It is to be noted that the term "a homolog" refers to one or more or at least one homolog. An isolated protein of the present invention, including a homolog, can be identified in a straight-forward manner by the protein's ability to elicit an immune response against a TCR Vβ protein or to bind to a major histocompatability (MHC) molecule or superantigen. Examples of TCR Vβ homologs include TCR Vβ proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog includes at least one epitope capable of eliciting an immune response against a TCR Vβ protein, and/or of binding to an antibody directed against a TCR Vβ protein. That is, when the homolog is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a natural TCR Vβ protein. The ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art. As used herein, the term "epitope" refers to the smallest portion of a protein or other antigen capable of selectively binding to the antigen binding site of an antibody. It is well accepted by those skilled in the art that the minimal size of a protein epitope is about six to seven amino acids. Other examples of TCR Vβ protein homologs include those homologs that are capable of binding to MHC, in the presence or absence of peptide, or superantigen. The ability of a protein to bind to MHC or superantigen can be measured using various methods well known to those of skill in the art.
TCR Vβ protein homologs can be the result of natural allelic variation or natural mutation. TCR Vβ protein homologs of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
TCR Vβ proteins of the present invention are encoded by TCR Vβ nucleic acid molecules. As used herein, a TCR Vβ nucleic acid molecule includes nucleic acid sequences related to a natural TCR Vβ gene. It is to be noted that the term "a nucleic acid molecule", "a gene" or "a nucleic acid sequence" refers to one or more or at least one nucleic acid molecule, gene or nucleic acid sequence, respectively. As used herein, a TCR Vβ gene includes all regions of the gene such as regulatory regions that control production of the TCR Vβ protein encoded by the gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions. As used herein, a gene that "includes" or "comprises" a sequence may include that sequence in one contiguous array, or may include the sequence as fragmented exons. As used herein, the term "coding region" refers to a continuous linear array of nucleotides that translates into a protein. A full-length coding region is that coding region that is translated into a full- length, i.e., a complete, protein as would be initially translated in its natural milieu, prior to any post-translational modifications. In one embodiment, a TCR Vβ gene of the present invention includes the nucleic acid sequence SEQ ID NO:l, as well as the complement of SEQ ID NO:l. Nucleic acid sequence SEQ ID NO:l represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaVβ33gl, the production of which is disclosed in the Examples. Nucleic acid molecule nCaVβ3381 comprises the coding region for the V, D and J regions of TCRVβ3 (also referred to herein as hcVβ3). The complement of SEQ ID NO:l (represented herein by SEQ ID NO:3) refers to the nucleic acid sequence of the strand complementary to the strand having SEQ ID NO: 1, which can easily be determined by those skilled in the art. Likewise, a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a double helix with) the strand for which the sequence is cited. It should be noted that since nucleic acid sequencing technology is not entirely error-free, SEQ ID NO:l (as well as other nucleic acid and protein sequences presented herein) represents an apparent nucleic acid sequence of the nucleic acid molecule encoding a TCR Vβ protein of the present invention.
In another embodiment, a TCR Vβ gene of the present invention includes the nucleic acid sequence SEQ ID NO:4, as well as the complement of SEQ ID NO:4 represented by SEQ ID NO:6. Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaVβ4408, the production of which is disclosed in the Examples. Nucleic acid molecule nCaVβ4408 comprises the coding region for the V, D and J regions of TCRVβ4 (also referred to herein as hcVβ4).
In another embodiment, a TCR Vβ gene of the present invention includes the nucleic acid sequence SEQ LD NO:9, as well as the complement of SEQ LD NO:9 represented by SEQ ID NO: 11. Nucleic acid sequence SEQ ID NO: 9 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaVβl2408, the production of which is disclosed in the Examples. Nucleic acid molecule nCaVβl240g comprises the coding region for the V, D and J regions of TCRVβl2 (also referred to herein as hcVβl2). In another embodiment, a TCR Vβ gene of the present invention includes the nucleic acid sequence SEQ LD NO:98, as well as the complement of SEQ ED NO:98 represented by SEQ LD NO: 100. Nucleic acid sequence SEQ ID NO:98 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaVβ72438, the production of which is disclosed in the Examples. Nucleic acid molecule nCaVβ72438 comprises the coding region for the V, D and J regions of TCRVβ72 (also referred to herein as hcVβ72).
In another embodiment, a TCR Vβ gene of the present invention includes the nucleic acid sequence SEQ ID NO: 19, as well as the complement of SEQ ID NO: 19 represented by SEQ ED NO: 19. Nucleic acid sequence SEQ ID NO: 19 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaVβ21462, the production of which is disclosed in the Examples. Nucleic acid molecule nCaVβ21462 comprises the coding region for the V, D and J regions of TCRVβ21 (also referred to herein as hcVβ21). In another embodiment, a TCR Vβ gene of the present invention includes the nucleic acid sequence SEQ ID NO:22, as well as the complement of SEQ ID NO:22 represented by SEQ LD NO:22. Nucleic acid sequence SEQ ED NO:22 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaVβ54417, the production of which is disclosed in the Examples. Nucleic acid molecule nCaVβ54417 comprises the coding region for the V, D and J regions of TCRVβ54 (also referred to herein as dtb54).
En another embodiment, a TCR Vβ gene of the present invention includes the nucleic acid sequence SEQ LD NO:25, as well as the complement of SEQ LD NO:25 represented by SEQ ID NO:25. Nucleic acid sequence SEQ ID NO:25 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as nucleic acid molecule nCaVβl82423, the production of which is disclosed in the Examples. Nucleic acid molecule nCaVβl82423 comprises the coding region for the V, D and J regions of TCRVβl82 (also referred to herein as dtbl82).
In another embodiment, a TCR Vβ gene or nucleic acid molecule can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 1 , SEQ LD NO:3, SEQ LD NO:4, SEQ ED NO:6, SEQ ID NO:9, SEQ ED NO: 11, SEQ LD NO:98, SEQ ID NO: 100, or any other TCR Vβ nucleic acid sequence cited herein. An allelic variant of a TCR Vβ nucleic acid molecule including SEQ ID NO: 1, SEQ LD NO:3, SEQ LD NO:4, SEQ LD NO:6, SEQ ID NO:9, SEQ ID NO: 11, SEQ LD NO:98 and SEQ ID NO: 100, is a nucleic acid molecule that occurs at essentially the same locus (or loci) in the genome as the nucleic acid molecule including SEQ ED NO: 1, SEQ LD NO:3, SEQ LD NO:4, SEQ ID NO:6, SEQ ED NO:9, SEQ ID NO: 11, SEQ ED NO:98 and SEQ LD NO: 100, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Because natural selection typically selects against alterations that affect function, allelic variants usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants of genes or nucleic acid molecules can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions), or can involve alternative splicing of a nascent transcript, thereby bringing alternative exons into juxtaposition. Allelic variants are well known to those skilled in the art and would be expected to be found within a given genome, since the respective genomes are diploid, and sexual reproduction will result in the reassortment of alleles.
In one embodiment of the present invention, an isolated TCR Vβ protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a gene encoding a TCR Vβ protein (i.e., to a TCR Vβ gene). The minimal size of a TCR Vβ protein of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridize under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. The size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the TCR Vβ nucleic acid molecule and the complementary nucleic acid sequence. It can easily be understood that the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered (i.e., localized) in distinct regions on a given nucleic acid molecule. Stringent hybridization conditions are determined based on defined physical properties of the gene to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al, 1984, Anal Biochem. 138, 267-284. As explained in detail in the cited references, the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (expressed as molarity (M), in moles/liter), the hybridization temperature (°C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybridized. For nucleic acid molecules longer than about 50 nucleotides, these variables are inserted into a standard mathematical formula to calculate the melting temperature, or Tm, of a given nucleic acid molecule. As defined in the formula below, Tm is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands:
Tm=81.5°C + 16.6 log M + 0.41(% G + C) - 500/n - 0.61(% formamide). For nucleic acid molecules smaller than about 50 nucleotides, hybrid stability is defined by the dissociation temperature (Td), which is defined as the temperature at which 50% of the duplexes dissociate. For these smaller molecules, the stability at a standard ionic strength is defined by the following equation:
Td = 4(G + C) + 2(A + T). A temperature of 5°C below Td is used to detect hybridization between perfectly matched molecules.
Also well known to those skilled in the art is how base-pair mismatch, i.e. differences between two nucleic acid molecules being compared, including non- complementarity of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared, will affect Tm or Td for nucleic acid molecules of different sizes. For example, Tm decreases about 1°C for each 1% of mismatched base-pairs for hybrids greater than about 150 bp, and Td decreases about 5°C for each mismatched base-pair for hybrids below about 50 bp. Conditions for hybrids between about 50 and about 150 base-pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art. These simple procedures allow one skilled in the art to set the hybridization conditions (by altering, for example, the salt concentration, the formamide concentration or the temperature) so that only nucleic acid hybrids with greater than a specified % base-pair mismatch will hybridize. Stringent hybridization conditions are commonly understood by those skilled in the art to be those experimental conditions that will allow about 30% base-pair mismatch (i.e., about 70% identity). Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropriate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybridize with a given nucleic acid molecule under stringent hybridization conditions and similarly whether the nucleic acid molecule will hybridize under conditions designed to allow a desired amount of base pair mismatch.
Hybridization reactions are often carried out by attaching the nucleic acid molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled probe suspended in a hybridization solution. Examples of common hybridization reaction techniques include the well-known Southern and northern blotting procedures. Typically, the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.
For example, if the skilled artisan wished to identify a nucleic acid molecule that hybridizes under stringent hybridization conditions with a specific or known canine nucleic acid molecule of about 150 bp in length, the following conditions could preferably be used. The average G + C content of canine DNA includes about 35%, about 36%, about 37%, about 38%, about 39%, about 41%, about 42%, about 43%, about 44%, about 45%, with about 40% being preferred. The unknown nucleic acid molecules would be attached to a support membrane, and the specified 150 bp nucleic acid molecule would be labeled, e.g. with a radioactive tag. The hybridization reaction could be carried out in a solution comprising 2X SSC and 0% formamide, at a temperature of about 37°C (low stringency conditions). Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20X SSC (175.3 gram NaCI and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC. In order to achieve high stringency hybridization, the skilled artisan would calculate the washing conditions required to allow up to 30%) base-pair mismatch. For example, in a wash solution comprising IX SSC and 0% formamide, the Tm of perfect hybrids would be about 80.8°C:
81.5°C + 16.6 log (.15M) + (0.41 x 40) - (500/150) - (0.61 x 0) = 80.8°C. Thus, to achieve hybridization with nucleic acid molecules having about 30%> base-pair mismatch, hybridization washes would be carried out at a temperature of about 50.8°C. It is within the skill of one in the art to calculate the hybridization temperature based on the formulae and G/C content disclosed herein.
In one embodiment of the present invention, a preferred TCR Vβ nucleic acid molecule includes a nucleic acid molecule which has greater than about 50 base pairs and which hybridizes under conditions which preferably allow about 20% base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10% base pair mismatch and even more preferably under conditions which allow about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ED NO:3, SEQ LD NO:6, SEQ ID NO:8, SEQ ID NO:l 1, SEQ LD NO:13, SEQ LD NO:100, SEQ ED NO: 18, SEQ ID NO:30, SEQ LD NO:33, SEQ ED NO:36, SEQ ED NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ED NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ LD NO:65, SEQ ED NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ LD NO:70 or SEQ ID NO:71.
Another preferred TCR Vβ nucleic acid molecule of the present invention includes a nucleic acid molecule which has greater than about 150 base pairs and which hybridizes under conditions which preferably allow about 30% base pair mismatch, more preferably under conditions which allow about 25% base pair mismatch, more preferably under conditions which allow about 20%) base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10% base pair mismatch and even more preferably under conditions which allow about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ LD NO:6, SEQ ID NO:8, SEQ ID NO:l l, SEQ LD NO:13, SEQ LD NO:100, SEQ ID NO:18, SEQ LD NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ LD NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ED NO:63, SEQ ED NO:64, SEQ LD NO:65, SEQ ED NO:66, SEQ ED NO:67, SEQ LD NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ LD NO:71.
Another preferred TCR Vβ nucleic acid molecule of the present invention includes a nucleic acid molecule which has greater than about 200 base pairs and which hybridizes under conditions which preferably allow about 30%) base pair mismatch, more preferably under conditions which allow about 25% base pair mismatch, more preferably under conditions which allow about 20% base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10%> base pair mismatch and even more preferably under conditions which allow about 5%> base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ LD NO:8, SEQ ID NO: 11, SEQ LD NO: 13, SEQ ID NO: 100, SEQ ID NO:18, SEQ LD NO:21, SEQ ID NO:30, SEQ ID NO:33, SEQ LD NO:36, SEQ LD NO:39, SEQ ID NO:42, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ LD NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ED NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ED NO:73 or SEQ ID NO:74.
Another embodiment of the present invention includes a nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 49°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ED NO:3, SEQ LD NO:30, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61 or SEQ LD NO:62.
Another embodiment of the present invention includes a nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 56°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ED NO:5, SEQ LD NO:8, SEQ ID NO:33, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ED NO: 63, SEQ ID NO: 64 or SEQ ED NO:65. Another embodiment of the present invention includes a nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 53°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 11 , SEQ LD NO: 13, SEQ ED NO:36, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:66, SEQ ED NO:67 or SEQ ID NO: 68. Another embodiment of the present invention includes a nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0%o formamide, at a temperature of about 41°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 100, SEQ LD NO: 18, SEQ ID NO:39, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:69, SEQ ED NO: 70 or SEQ LD NO:71. Another embodiment of the present invention includes a nucleic acid molecule comprising at least about 150 base-pairs, wherein said molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 29°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:21, SEQ ED NO:23, SEQ LD NO:42, the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:72, SEQ ED NO:73 or SEQ ED NO:74.
Another embodiment of the present invention includes TCR Vβ proteins. A preferred TCR Vβ protein includes a protein encoded by a nucleic acid molecule which has greater than about 50 base pairs and which hybridizes under conditions which preferably allow about 20%> base pair mismatch, more preferably under conditions which allow about 15%) base pair mismatch, more preferably under conditions which allow about 10%) base pair mismatch and even more preferably under conditions which allow about 5%o base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ LD NO:2, SEQ LD NO:5, SEQ ID NO:10, SEQ LD NO:15, SEQ LD NO:20, SEQ LD NO:23, SEQ ID NO:26, SEQ ED NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ LD NO:38, SEQ LD NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ LD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ LD NO:64, SEQ ED NO:65, SEQ ED NO:66, SEQ ID NO:67, SEQ LD NO:68, SEQ LD NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ LD NO:74, SEQ LD NO:75, SEQ LD NO:76, SEQ LD NO:77, SEQ ID NO:78, SEQ LD NO:79 or SEQ LD NO:80.
Another preferred TCR Vβ protein of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 150 base pairs and which hybridizes under conditions which preferably allow about 30%> base pair mismatch, more preferably under conditions which allow about 25% base pair mismatch, more preferably under conditions which allow about 20%> base pair mismatch, more preferably under conditions which allow about 15% base pair mismatch, more preferably under conditions which allow about 10% base pair mismatch and even more preferably under conditions which allow about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ LD NO:2, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ LD NO:20, SEQ ED NO:23, SEQ ID NO:26, SEQ LD NO:29, SEQ ID NO:32, SEQ ED NO:35, SEQ ID NO:38, SEQ LD NO:41, SEQ ED NO:44, SEQ ID NO:47, SEQ LD NO:60, SEQ ED NO:61, SEQ LD NO:62, SEQ LD NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ LD NO:67, SEQ ID NO:68, SEQ LD NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ LD NO:72, SEQ LD NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 or SEQ ID NO:80.
Another preferred TCR Vβ protein of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 50 base pairs which is preferably about 80%> identical, more preferably about 85% identical, more preferably about 90%) identical, and even more preferably about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ LD NO:3, SEQ LD NO:6, SEQ LD NO:8, SEQ LD NO:l l, SEQ LD NO:13, SEQ ED NO:100, SEQ ID NO:18, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ LD NO:66, SEQ LD NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ LD NO:70 or SEQ LD NO:71.
Yet another preferred TCR Vβ of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 150 base pairs which is preferably about 70% identical, more preferably about 75% identical, more preferably about 80%) identical, more preferably about 85%o identical, more preferably about 90%> identical and even more preferably about 95%> identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ED NO:13, SEQ LD NO:100, SEQ ED NO:18, SEQ ID NO:30, SEQ LD NO:33, SEQ ID NO:36, SEQ ID NO:39, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61, SEQ ED NO:62, SEQ ED NO:63, SEQ LD NO:64, SEQ ID NO:65, SEQ LD NO:66, SEQ LD NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ LD NO:71.
Yet another preferred TCR Vβ of the present invention includes a protein encoded by a nucleic acid molecule which has greater than about 200 base pairs which is preferably about 10% identical, more preferably about 75% identical, more preferably about 80%) identical, more preferably about 85%> identical, more preferably about 90%> identical and even more preferably about 95%o identical to a nucleic acid molecule having the nucleic acid sequence SEQ ED NO:3, SEQ ID NO:6, SEQ LD NO:8, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ED NO:100, SEQ ID NO:18, SEQ ID NO:21, SEQ ED NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ LD NO:64, SEQ LD NO:65, SEQ ID NO:66, SEQ LD NO:67, SEQ LD NO:68, SEQ LD NO:69, SEQ ID NO:70, SEQ LD NO:71, SEQ LD NO:72, SEQ ID NO:73 or SEQ LD NO:74.
The minimal size of such a nucleic acid molecule is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if the nucleic acid molecule is AT-rich. The minimal size of a nucleic acid molecule used to encode a TCR Vβ protein homolog of the present invention is from about 12 to about 18 nucleotides in length. Thus, the minimal size of a TCR Vβ protein homolog of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule encoding a TCR Vβ protein or protein homolog because a nucleic acid molecule of the present invention can include a portion of a gene, an entire gene, or multiple genes. The preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired. As used herein, "fragments thereof and "portions thereof are intended to be used interchangeably, and have a minimal size as disclosed herein.
The minimal size of a protein or nucleic acid molecule of the present invention also can include a portion of a protein or nucleic acid molecule that is less than 100%) identical to another protein or nucleic acid molecule, when determined using hybridization or computer alignment methods disclosed herein. For example, a fragment of a hcVβ3 protein of the present invention is at least about 15 residues, preferably 20 residues and more preferably 25 residues in length; a fragment of a hcVβ4 protein of the present invention is at least about 10 residues, preferably 15 residues and more preferably 20 residues in length; a fragment of a hcVβl2 protein of the present invention is at least about 11 residues, preferably 15 residues and more preferably 20 residues in length; a fragment of a hcVβ72 protein of the present invention is at least about 18 residues, preferably 25 residues and more preferably 30 residues in length; or a fragment of a hcVβ21 protein of the present invention is at least about 13 residues, preferably 20 residues and more preferably 25 residues in length. In addition, a nucleic acid molecule fragment of a hcVβ3 nucleic acid molecule of the present invention is at least about 33 nucleotides, preferably 35 nucleotides and more preferably 40 nucleotides in length; a fragment of a hcVβ4 nucleic acid molecule of the present invention is at least about 21 nucleotides, preferably 25 nucleotides and more preferably 30 nucleotides in length; a fragment of a hcVβl2 nucleic acid molecule of the present invention is at least about 19 nucleotides, preferably 25 nucleotides and more preferably 30 nucleotides in length; a fragment of a hcVβ72 nucleic acid molecule of the present invention is at least about 27 nucleotides, preferably 30 nucleotides and more preferably 35 nucleotides in length; or a fragment of a hcVβ21 nucleic acid molecule of the present invention is at least about 176 nucleotides, preferably 180 nucleotides and more preferably 185 nucleotides in length. Suitable protein fragments of the present invention include functional portions of a TCR Vβ protein of the present invention including, but not limited to, epitopes, MHC and/or peptide recognition sequences, antigen recognition sequences, superantigen recognition sequences, framework V regions and hypervariable V regions. Preferred functional portions of a TCR Vβ protein include the V, D or J regions. More preferred functional portions of a TCR Vβ protein include: the putative signal peptide encoded by about nucleotide 1 to nucleotide 51, the V region encoded by about nucleotide 52 to about nucleotide 333, and the D/J region encoded by about nucleotide 334 to about nucleotide 381 of SEQ LD NO: 1 ; the putative signal peptide encoded by nucleotide 25 to nucleotide 69, the V region encoded by nucleotide 70 to about nucleotide 351, and the D/J region encoded by about nucleotide 352 to about nucleotide 408 of SEQ LD NO:4; the putative signal peptide encoded by nucleotide 7 to nucleotide 63, the V region encoded by nucleotide 64 to about nucleotide 339, and the D/J region encoded by about nucleotide 340 to about nucleotide 408 of SEQ ED NO:9; the putative signal peptide encoded by nucleotide 85 to nucleotide 141, the V region encoded by nucleotide 142 to about nucleotide 423, and the D/J region encoded by about nucleotide 424 to about nucleotide 438 of SEQ ID NO:98; the putative signal peptide encoded by nucleotide 73 to nucleotide 114, the V region encoded by nucleotide 115 to about nucleotide 396, and the D/J region encoded by about nucleotide 397 to about nucleotide 462 of SEQ LD NO:19; the putative signal peptide encoded by nucleotide 13 to nucleotide 69, the V region encoded by nucleotide 70 to about nucleotide 354, and the D/J region encoded by about nucleotide 355 to about nucleotide 417 of SEQ ED NO:22; and the putative signal peptide encoded by nucleotide 40 to nucleotide 96, the V region encoded by nucleotide 97 to about nucleotide 369, and the D/J region encoded by about nucleotide 370 to about nucleotide 423 of SEQ LD NO:25.
It is known to those of skill in the art that the junction between the V and D region can vary but that typically the carboxyl end of the V region contains one or more of the amino acid residues alanine or serine following a cysteine. For example, one of skill in the art would know that a conserved carboxyl V region sequence comprises the amino acids CASS. Thus, a V region of the present invention can include the amino acid sequence SEQ ED NO:60, SEQ LD NO:61, SEQ ED NO:62, SEQ ED NO:63, SEQ ID NO:64, SEQ LD NO:65, SEQ LD NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ LD NO:71, SEQ ID NO: 72, SEQ LD NO: 73, SEQ ED NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ LD NO:77, SEQ LD NO:78, SEQ LD NO: 79 or SEQ LD NO: 80. The present invention includes nucleic acid molecules having nucleic acid sequences encoding such proteins, which can be identified using the nucleic acid sequences disclosed herein and standard codon usage known to those of skill in the art; such as disclosed, for example, in Lehninger, Biochemistry, Worth Publishers, Inc., 1975, which is incorporated herein by this reference in its entirety. For example, a skilled artisan would know that codons encoding serine include ACA, ACC, AGT, AGC or ACG, and codons that encode alanine include GCA, GCC, GCT or GCG. One embodiment of a TCR Vβ protein of the present invention is a fusion protein that includes a TCR Vβ protein-containing domain attached to one or more fusion segments. It is to be noted that the term "a fusion protein" refers to one or more or at least one fusion protein. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; deliver a TCR Vβ protein or portion thereof to a desired target; act as an immunopotentiator to enhance an immune response against a TCR Vβ protein; and/or assist in purification of a TCR Vβ protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts increased immunogenicity to a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the TCR Vβ-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a TCR Vβ protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a TCR Vβ -containing domain. Preferred fusion segments include a metal binding domain (e.g., a poly- histidine segment); an immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); and/or a "tag" domain (e.g., at least a portion of -galactosidase, a strep tag peptide, a T7 tag peptide, a Flag™ peptide, or other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, FL; and an SIO peptide. A preferred fusion protein of the present invention includes a TCR Vβ protein of the present invention linked to a TCR alpha chain in such a manner that the beta chain and alpha chain fold correctly to form a functional dimer. Another preferred fusion protein includes a TCR Vβ protein of the present invention linked to at least a portion of the constant region of an immunoglobulin in such a manner that crystallization of the V beta protein is enhanced by the presence of the immunoglobulin sequence. Preferably a TCR Vβ protein of the present invention is isolated (including isolation of the natural protein or production of the protein by recombinant or synthetic techniques) from canids.
A preferred isolated protein of the present invention is an isolated protein that is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence including SEQ ED NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 100, SEQ ID NO:18, SEQ LD NO:21, SEQ ID NO:24, SEQ ED NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ LD NO:39, SEQ ED NO:42, SEQ ED NO:45, SEQ ED NO:48, a complement of SEQ ID NO:50, SEQ ID NO:51, SEQ LD NO:52, SEQ ID NO:53, SEQ LD NO:54, SEQ ID NO:55, SEQ ED NO:56, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:60, SEQ LD NO:61, SEQ ED NO:62, SEQ LD NO:63, SEQ LD NO:64, SEQ LD NO:65, SEQ ED NO:66, SEQ LD NO:67, SEQ LD NO:68, SEQ LD NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ LD NO:72, SEQ LD NO:73, SEQ LD NO:74, SEQ LD NO:75, SEQ ED NO:76, SEQ ED NO:77, SEQ ID NO:78, SEQ LD NO:79 or SEQ LD NO:80.
A preferred isolated protein of the present invention is a protein encoded by at least one of the following nucleic acid molecules:nCaVβ33g], nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405,nCaVβ54354, nCaVβ54348, nCaVβl82423, nCaVβl82384, nCaVβl82369 and/or nCaVβl82327; fragments thereof; or allelic variants of any of these nucleic acid molecules. Another preferred isolated protein is encoded by a nucleic acid molecule having nucleic acid sequence SEQ ID NO:l, SEQ ED NO:4, SEQ ID NO:7, SEQ LD NO:9, SEQ ID NO: 12, SEQ ID NO:98, SEQ ID NO: 17, SEQ LD NO: 19, SEQ LD NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ D NO:31, SEQ LD NO:34, SEQ ED NO:37, SEQ LD NO:40, SEQ ED NO:43, SEQ LD NO:46, SEQ LD NO:50, SEQ LD NO:51, SEQ LD NO:52, SEQ ED NO:53, SEQ LD NO:54, SEQ ID NO:55, SEQ ED NO:56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO: 60, SEQ ID NO:61, SEQ YD NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ED NO:67, SEQ LD NO:68, SEQ LD NO:69, SEQ LD NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ LD NO:73, SEQ LD NO:74, SEQ ID NO:75, SEQ LD NO:76, SEQ LD NO:77, SEQ ID NO:78, SEQ ED NO:79 or SEQ LD NO:80; fragments thereof or an allelic variant of such a nucleic acid molecule. Translation of SEQ ID NO:l, the coding strand of nCaVβ3381, yields a TCR Vβ protein of about 127 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaVβ3127, the amino acid sequence of which is presented in SEQ ID NO:2, assuming an open reading frame having a first codon spanning from nucleotide 1 through nucleotide 3 of SEQ LD NO:l and a last codon spanning from nucleotide 379 through nucleotide 381 of SEQ LD NO : 1. The partial putative signal sequence extends from nucleotide 1 to nucleotide 51 of SEQ ID NO:l. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ3110, contains about 110 amino acids, extending from residue 18 through residue 127 of SEQ LD NO:2. The nucleic acid molecule encoding PCaVβ3no is denoted herein as nCaVβ3330, extending from nucleotide 52 through nucleotide 381 of SEQ ID NO: 1.
Translation of SEQ ID NO:4, the coding strand of nCaVβ4408, yields a protein of about 128 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaVβ4128, the amino acid sequence of which is presented in SEQ ED NO:5, assuming an open reading frame having an initiation codon spanning from nucleotide 25 through nucleotide 27 of SEQ ID NO:4 and a last codon spanning from nucleotide 406 through nucleotide 408 of SEQ ID NO:4. The coding region encoding PCaVβ4]2g is presented herein as nCaVβ4384, which has the nucleotide sequence SEQ LD NO: 7 (the coding strand) and SEQ ID NO: 8 (the complementary strand). The putative signal sequence extends from nucleotide 25 to nucleotide 69 of SEQ ED NO:4. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ4U3, contains about 113 amino acids, extending from residue 60 through residue 128 of SEQ ID NO:5. The nucleic acid molecule encoding PCaVβ4113 is denoted herein as nCaVβ4339, extending from nucleotide 70 through nucleotide 408 of SEQ ID NO:4. Translation of SEQ LD NO: 9, the coding strand of nCaVβl2408, yields a protein of about 134 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaVβl2134, the amino acid sequence of which is presented in SEQ ID NO: 10, assuming an open reading frame having an initiation codon spanning from nucleotide 7 through nucleotide 9 of SEQ ID NO:9 and a last codon spanning from nucleotide 406 through nucleotide 408 of SEQ LD NO:9. The coding region encoding PCaVβ 12134 is presented herein as nCaVβl2402, which has the nucleotide sequence SEQ ID NO: 12 (the coding strand) and SEQ ID NO: 13 (the complementary strand). The putative signal sequence extends from nucleotide 7 to nucleotide 63 of SEQ ID NO:9. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβl2115, contains about 115 amino acids, extending from residue 20 through residue 134 of SEQ ID NO: 10. The nucleic acid molecule encoding PCaVβl2] 15 is denoted herein as nCaVβl2345, extending from nucleotide 64 through nucleotide 408 of SEQ ID NO:9.
Translation of SEQ ID NO:98, the coding strand of nCaVβ72438, yields a protein of about 133 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaVβ72133, the amino acid sequence of which is presented in SEQ ED NO: 15, assuming an open reading frame having an initiation codon spanning from nucleotide 85 through nucleotide 87 of SEQ ED NO:98 and a last codon spanning from nucleotide 481 through nucleotide 438 of SEQ ED NO:98. The coding region encoding PCaVβ72133 is presented herein as nCaVβ72399, which has the nucleotide sequence SEQ ED NO: 17 (the coding strand) and SEQ LD NO: 18 (the complementary strand). The putative signal sequence extends from nucleotide 85 to nucleotide 141 of SEQ ID NO:98. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ72114, contains about 114 amino acids, extending from residue 20 through residue 133 of SEQ ID NO:98. The nucleic acid molecule encoding PCaVβ721 ]4 is denoted herein as nCaVβ72342, extending from nucleotide 142 through nucleotide 438 of SEQ ID NO: 19.
Translation of SEQ ID NO: 19, the coding strand of nCaVβ21462, yields a protein of about 130 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaVβ21]30, the amino acid sequence of which is presented in SEQ ID NO:20, assuming an open reading frame having an initiation codon spanning from nucleotide 73 through nucleotide 75 of SEQ ID NO: 19 and a last codon spanning from nucleotide 460 through nucleotide 462 of SEQ ED NO: 19. The coding region encoding PCaVβ21 !30 is presented herein as nCaVβ21390, which extends from nucleotide 73 to nucleotide 462 of SEQ ID NO: 19. The putative signal sequence extends from nucleotide 73 to nucleotide 114 of SEQ LD NO: 19. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ21116, contains about 116 amino acids, extending from residue 15 through residue 130 of SEQ ID NO: 19. The nucleic acid molecule encoding PCaVβ21116 is denoted herein as nCaVβ21348, extending from nucleotide 115 through nucleotide 462 of SEQ ID NO: 19. Translation of SEQ ID NO:22, the coding strand of nCaVβ54417, yields a protein of about 135 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaVβ54135, the amino acid sequence of which is presented in SEQ ID NO:23, assuming an open reading frame having an initiation codon spanning from nucleotide 13 through nucleotide 15 of SEQ ID NO:22 and a last codon spanning from nucleotide 415 through nucleotide 417 of SEQ ID NO:22. The coding region encoding PCaVβ54!35 is presented herein as nCaVβ54405, which extends from nucleotide 13 to nucleotide 417 of SEQ ID NO:22. The putative signal sequence extends from nucleotide 13 to nucleotide 69 of SEQ ID NO:22. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ54116, contains about 116 amino acids, extending from residue 20 through residue 135 of SEQ LD NO:22. The nucleic acid molecule encoding PCaVβ54116 is denoted herein as nCaVβ54348, extending from nucleotide 70 through nucleotide 417 of SEQ ID NO:22.
Translation of SEQ LD NO:25, the coding strand of nCaVβl82423, yields a protein of about 128 amino acids containing the beta chain V, D, and J regions, denoted herein as PCaVβ 182]28, the amino acid sequence of which is presented in SEQ LD NO:26, assuming an open reading frame having an initiation codon spanning from nucleotide 40 through nucleotide 43 of SEQ ID NO:25 and a last codon spanning from nucleotide 421 through nucleotide 423 of SEQ ED NO:25. The coding region encoding PCaVβ 182128 is presented herein as nCaVβl82384, which extends from nucleotide 40 to nucleotide 423 of SEQ ED NO:25. The putative signal sequence extends from nucleotide 40 to nucleotide 96 of SEQ ED NO:25. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβl82109, contains about 109 amino acids, extending from residue 20 through residue 128 of SEQ LD NO:25. The nucleic acid molecule encoding PCaVβ 182]09 is denoted herein as nCaVβl82327, extending from nucleotide 97 through nucleotide 423 of SEQ ID NO:25.
Translation of SEQ ID NO:28, the coding strand of nCaVβ3333, yields a protein of about 111 amino acids containing the beta chain V region, denoted herein as PCaVβ3ι n, the amino acid sequence of which is presented in SEQ DD NO:29, assuming an open reading frame having a first codon spanning from nucleotide 1 through nucleotide 3 of SEQ ID NO:28 and a last codon spanning from nucleotide 331 through nucleotide 333 of SEQ ED NO:28. The complement of SEQ DD NO:28 is SEQ DD NO:30.
Translation of SEQ ED NO:31, the coding strand of nCaVβ4351, yields a protein of about 109 amino acids containing the beta chain V region, denoted herein as PCaVβ4109, the amino acid sequence of which is presented in SEQ ED NO:32, assuming an open reading frame having a first codon spanning from nucleotide 25 through nucleotide 27 of SEQ ID NO:31 and a last codon spanning from nucleotide 349 through nucleotide 351 of SEQ TD NO:31. The complement of SEQ TD NO:31 is SEQ TD NO:33. Translation of SEQ ID NO: 34, the coding strand of nCaVβ 12339, yields a protein of about 111 amino acids containing the beta chain V region, denoted herein as PCaVβl2lu, the amino acid sequence of which is presented in SEQ LD NO:35, assuming an open reading frame having a first codon spanning from nucleotide 7 through nucleotide 9 of SEQ LD NO:34 and a last codon spanning from nucleotide 337 through nucleotide 339 of SEQ ED NO:34. The complement of SEQ ID NO:34 is SEQ LD NO:35.
Translation of SEQ ED NO:37, the coding strand of nCaVβ72423, yields a protein of about 113 amino acids containing the beta chain V region, denoted herein as PCaVβ72113, the amino acid sequence of which is presented in SEQ ID NO:38, assuming an open reading frame having a first codon spanning from nucleotide 85 through nucleotide 87 of SEQ ED NO:37 and a last codon spanning from nucleotide 421 through nucleotide 423 of SEQ ID NO:37. The complement of SEQ LD NO:37 is SEQ TD NO:39.
Translation of SEQ TD NO:40, the coding strand of nCaVβ21396, yields a protein of about 108 amino acids containing the beta chain V region, denoted herein as
PCaVβ21)08, the amino acid sequence of which is presented in SEQ ED NO:41, assuming an open reading frame having a first codon spanning from nucleotide 73 through nucleotide 75 of SEQ LD NO: 40 and a last codon spanning from nucleotide 394 through nucleotide 396 of SEQ ED NO:40. The complement of SEQ TD NO:40 is SEQ LD NO:42. Translation of SEQ ID NO:43, the coding strand of nCaVβ54354, yields a protein of about 114 amino acids containing the beta chain V region, denoted herein as PCaVβ54] ]4, the amino acid sequence of which is presented in SEQ LD NO:44, assuming an open reading frame having a first codon spanning from nucleotide 13 through nucleotide 15 of SEQ ID NO:43 and a last codon spanning from nucleotide 352 through nucleotide 354 of SEQ ID NO:43. The complement of SEQ LD NO:43 is SEQ LD NO:45. Translation of SEQ LD NO:46, the coding strand of nCaVβl82369, yields a protein of about 110 amino acids containing the beta chain V region, denoted herein as PCaVβ 182, ,0, the amino acid sequence of which is presented in SEQ LD NO:47, assuming an open reading frame having a first codon spanning from nucleotide 40 through nucleotide 42 of SEQ ED NO:46 and a last codon spanning from nucleotide 367 through nucleotide 369 of SEQ ID NO:46. The complement of SEQ LD NO:46 is SEQ LD NO:48.
Preferred TCR Vβ proteins of the present invention include proteins that are at least about 65%), preferably at least about 70%>, even more preferably at least about 75%, and even more preferably at least about 80% identical to PCaVβ3127; are at least about 69%), preferably at least about 75%, even more preferably at least about 80%, and even more preferably at least about 85% identical to PCaVβ4128; are at least about 57%, preferably at least about 60%, even more preferably at least about 65%, and even more preferably at least about 70% identical to PCaVβl2134; are at least about 65%, preferably at least about 70%o, even more preferably at least about 75%, and even more preferably at least about 80% identical to PCaVβ72133; or are at least about 60%, preferably at least about 65%), even more preferably at least about 70%, and even more preferably at least about 75%) identical to PCaVβ21130. More preferred are TCR Vβ proteins comprising PCaVβ3127, PCaVβ3ι n, PCaVβ3U0, PCaVβ4128, PCaVβ4113, PCaVβ4109, PCaVβl2134, PCaVβl2 , PCaVβl2115, PCaVβ72133, PCaVβ72113, PCaVβ72U4, PCaVβ21130, PCaVβ21108, PCaVβ21116, PCaVβ54135, PCaVβ54114, PCaVβ54116, PCaVβ 182128, PCaVβ 182, ]0, PCaVβ 182109 and fragments thereof; and proteins encoded by allelic variants of nucleic acid molecules encoding proteins PCaVβ3127, PCaVβ3l u, PCaVβ3] 10, PCaVβ4128, PCaVβ4„3, PCaVβ4109, PCaVβl2134, PCaVβl2u l, PCaVβl2115,
PCaVβ72133, PCaVβ72113, PCaVβ72H4, PCaVβ21130, PCaVβ21108, PCaVβ21116, PCaVβ54135, PCaVβ54] 14, PCaVβ54116, PCaVβ 182128, PCaVβ 182, 10 and/or PCaVβ 182,09,and fragments thereof.
Other preferred TCR Vβ proteins of the present invention include proteins having amino acid sequences that are at least about 65%), preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to SEQ ED NO:2; at least about 69%, preferably at least about 75%, even more preferably at least about 80%>, and even more preferably at least about 85 > identical to SEQ ED NO:5; are at least about 51%, preferably at least about 60%, even more preferably at least about 65%, and even more preferably at least about 70% identical to SEQ LD NO: 10; are at least about 65%, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to SEQ ID NO: 15; or are at least about 60%, preferably at least about 65%, even more preferably at least about 70%, and even more preferably at least about 75% identical to SEQ LD NO:20. More preferred are TCR Vβ proteins comprising amino acid sequences SEQ LD NO:2, SEQ ED NO:5, SEQ LD NO:10, SEQ TD NO:15, SEQ ED NO:20, SEQ DD NO:23, SEQ DD NO:26, SEQ ED NO:29, SEQ DD NO:32, SEQ DD NO:35, SEQ ID NO:38, SEQ LD NO:41, SEQ LD NO:44, SEQ ID NO:47, SEQ ID NO:60, SEQ LD NO:61, SEQ ID NO:62, SEQ ED NO:63, SEQ ED NO:64, SEQ ID NO:65, SEQ DD NO:66, SEQ DD NO:67, SEQ DD NO:68, SEQ DD NO:69, SEQ DD NO:70, SEQ DD NO:71, SEQ DD NO:72, SEQ DD NO:73, SEQ DD NO:74, SEQ DD NO:75, SEQ DD
NO:76, SEQ DD NO:77, SEQ DD NO:78, SEQ ED NO:79, SEQ DD NO:80 and/or proteins encoded by the complement of a nucleic acid sequence including SEQ ID NO:50, SEQ ID NO:51, SEQ ED NO:52, SEQ ID NO:53, SEQ DD NO:54, SEQ TD NO:55, SEQ TD NO:56; and TCR Vβ proteins encoded by allelic variants of nucleic acid molecules encoding TCR Vβ proteins having amino acid sequences SEQ TD NO:2, SEQ TD NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ LD NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ LD NO:32, SEQ ED NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ LD NO:44, SEQ ED NO:47, SEQ LD NO:60, SEQ LD NO:61, SEQ ID NO:62, SEQ LD NO:63, SEQ LD NO:64, SEQ ED NO:65, SEQ DD NO:66, SEQ ED NO:67, SEQ LD NO:68, SEQ LD NO:69, SEQ ID NO:70, SEQ LD NO:71, SEQ LD NO:72, SEQ LD NO:73, SEQ LD NO:74, SEQ LD NO:75, SEQ ID NO:76, SEQ LD NO:77, SEQ LD NO:78, SEQ TD NO:79, SEQ TD NO:80 and/or proteins encoded by the complement of a nucleic acid sequence including SEQ ED NO:50, SEQ ID NO:51, SEQ LD NO:52, SEQ ID NO:53, SEQ LD NO:54, SEQ LD NO:55, SEQ LD NO:56; and fragments of any of such amino acid sequences. A preferred isolated protein of the present invention comprises a protein selected from the group consisting of: (a) an isolated protein having an amino acid sequence that is at least about 55 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:29, SEQ ID NO:60, SEQ LD NO:61, and SEQ ID NO: 62, or a fragment thereof that is at least about 25 amino acids in length; (b) an isolated protein having an amino acid sequence that is at least about 75 percent identical to an amino acid sequence selected from the group consisting of SEQ ED NO:5, SEQ LD NO:32, SEQ DD NO:63, SEQ ED NO:64, and SEQ TD NO:65, or a fragment thereof that is at least about 15 amino acids in length; (c) an isolated protein having an amino acid sequence that is at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ TD NO: 10, SEQ LD NO:35, SEQ ED NO:66, SEQ LD NO:67, and SEQ ED NO:68, or a fragment thereof that is at least about 25 amino acids in length; and (d) an isolated protein having an amino acid sequence that is at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ED NO:38, SEQ DD NO:69, SEQ DD NO:70, and SEQ DD NO:71, or a fragment thereof that is at least about 35 amino acids in length.
A preferred isolated protein of the present invention comprises a protein selected from the group consisting of:(a) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ DD NO:28, SEQ ID NO:50, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ DD NO: 61, and SEQ ID NO: 62, or a fragment thereof that is at least about 25 nucleotides in length; (b) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:4, SEQ DD NO:7, SEQ ID NO:31, SEQ DD NO:51, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:63, SEQ TD NO:64, and SEQ DD NO:65, or a fragment that is at least about 30 nucleotides in length; (c) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, SEQ TD NO: 12, SEQ DD NO:34, SEQ DD NO:52, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ ID NO:67, and SEQ TD NO:68, or a fragment thereof that is at least about 40 nucleotides in length; and (d) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:98, SEQ TD NO: 17, SEQ TD NO:37, SEQ TD NO:53, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ TD NO:69, SEQ TD NO:70, and SEQ TD NO:71, or a fragment thereof that is at least about 75 nucleotides in length.
It is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules. Preferred methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, GCG™ (available from Genetics Computer Group, Madison, WI), DNAsis™ (available from Hitachi Software, San Bruno, CA) and Mac Vector™ (available from the Eastman Kodak Company, New Haven, CT). A preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Compare function by maximum matching within the program DNAsis Version 2.1. Additional preferred TCR Vβ proteins of the present invention include proteins encoded by nucleic acid molecules comprising at least a portion of nCaVβ3381, nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405,nCaVβ54354, nCaVβ54348, nCaVβl82423, nCaVβl823g4, nCaVβl82369 and/or nCaVβl82327, fragments thereof, as well as TCR Vβ proteins encoded by allelic variants of such nucleic acid molecules.
Also preferred are TCR Vβ proteins encoded by nucleic acid molecules having nucleic acid sequences comprising at least a portion of SEQ ID NO: 1, SEQ ED NO:4, SEQ ID NO:7, SEQ LD NO:9, SEQ ED NO: 12, SEQ LD NO:98, SEQ DD NO: 17, SEQ DD NO: 19, SEQ ID NO:22, SEQ ED NO:25, SEQ ED NO:28, SEQ DD NO:31, SEQ DD NO:34, SEQ DD NO:37, SEQ DD NO:40, SEQ DD NO:43, SEQ DD NO:46, SEQ DD NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ DD NO:54, SEQ ID NO:55, SEQ DD NO: 56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ DD NO:60, SEQ ED NO:61, SEQ ID NO:62, SEQ LD NO:63, SEQ ED NO:64, SEQ LD NO:65, SEQ ED NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ LD NO:69, SEQ LD NO:70, SEQ LD NO:71, SEQ LD NO:72, SEQ LD NO:73, SEQ LD NO:74, SEQ TD NO:75, SEQ TD NO:76, SEQ DD NO:77, SEQ DD NO:78, SEQ DD NO:79 or SEQ DD NO:80, fragments thereof, as well as allelic variants of these nucleic acid molecules.
The present invention also includes mimetopes of TCR Vβ proteins of the present invention. As used herein, a mimetope of a TCR Vβ protein of the present invention refers to any compound that is able to mimic the activity of a TCR Vβ protein of the present invention, often because the mimetope has a structure that mimics the particular TCR Vβ protein. It is to be noted that the term "a mimetope" refers to one or more or at least one mimetope. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation such as all-D retro peptides; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non- proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides, RNA or other organic molecules, non- organic molecules and screening such samples by affinity chromatography techniques using the corresponding binding partner. Another embodiment of the present invention includes a TCR Vβ nucleic acid molecule. It is to be noted that the term "a nucleic acid molecule homolog" refers to one or more or at least one nucleic acid molecule homologs. In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated TCR Vβ nucleic acid molecule of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis. Isolated TCR Vβ nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a TCR Vβ protein of the present invention. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof. The minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybrid with the complementary sequence of another nucleic acid molecule. As such, the minimal size of a TCR Vβ nucleic acid molecule of the present invention is from about 12 to about 18 nucleotides in length.
A TCR Vβ nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, ibid.. For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologs can be selected by hybridization with a TCR Vβ nucleic acid molecule or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least one epitope of a TCR Vβ protein).
An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one TCR Vβ protein of the present invention, examples of such proteins being disclosed herein. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a TCR Vβ protein.
A preferred nucleic acid molecule of the present invention, when administered to an animal, is capable of regulating an immune response in an animal. As will be disclosed in more detail below, such a nucleic acid molecule can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In additional embodiments, a nucleic acid molecule of the present invention can encode an immunoregulatory protein (e.g., a cell-bound or soluble TCR Vβ protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection (i.e, as a genetic vaccine) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine. One embodiment of the present invention is a TCR Vβ nucleic acid molecule comprising all or part of nucleic acid molecules nCaVβ33gl, nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405,nCaVβ54354, nCaVβ54348, nCaVβ 182423, nCaVβ 182384, nCaVβ 182369 and/or nCaVβ 182327, or allelic variants of these nucleic acid molecules. Another preferred nucleic acid molecule comprises SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ED NO:7, SEQ ED NO:8, SEQ LD NO:9, SEQ ED NO: 11, SEQ LD NO: 12, SEQ TD NO: 13, SEQ ED NO:98, SEQ DD NO:100, SEQ DD NO:17, SEQ DD NO:18, SEQ ID NO:19, SEQ TD NO:21, SEQ TD NO:22, SEQ DD NO:24, SEQ ID NO:25, SEQ DD NO:27,SEQ DD NO:28, SEQ DD NO:30, SEQ DD NO:31, SEQ DD NO:33, SEQ DD NO:34, SEQ DD NO:36, SEQ ID NO:37, SEQ DD NO:39, SEQ ED NO:40, SEQ TD NO:42, SEQ DD NO:43, SEQ TD NO:45, SEQ TD NO:46, SEQ TD NO:48, SEQ TD NO:50, SEQ TD NO:51, SEQ ED NO:52, SEQ ID NO:53, SEQ DD NO:54, SEQ DD NO:55, SEQ DD NO:56, a complement of SEQ DD NO:50, SEQ DD NO:51, SEQ ID NO:52, SEQ DD NO:53, SEQ ID NO:54, SEQ DD NO:55, SEQ DD NO:56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ TD NO: 60, SEQ DD
NO:61, SEQ ID NO:62, SEQ DD NO: :63, SEQ ID NO: 64, SEQ DD NO:65, SEQ DD
NO:66, SEQ TD NO:67, SEQ TD NO: :68, SEQ ID NO: 69, SEQ DD NO:70, SEQ DD
NO:71, SEQ DD NO:72, SEQ DD NO: :73, SEQ TD NO: 74, SEQ DD NO:75, SEQ TD NO:76, SEQ ED NO:77, SEQ π NO: :78, SEQ DD NO: 79 or SEQ DD NO: 80, and complements thereof; as well as fragments thereof; as well as allelic variants of nucleic acid molecules having these nucleic acid sequences. Such nucleic acid molecules can include nucleotides in addition to those included in the SEQ DD NOs, such as, but not limited to, a full-length gene, a full-length coding region, a nucleic acid molecule encoding a fusion protein, or a nucleic acid molecule encoding a multivalent therapeutic compound.
In one embodiment, a TCR Vβ nucleic acid molecule of the present invention encodes a protein that is at least about 65%, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to PCaVβ3127; are at least about 69%, preferably at least about 75%, even more preferably at least about 80%, and even more preferably at least about 85%> identical to PCaVβ4128; are at least about 57%, preferably at least about 60%, even more preferably at least about 65%), and even more preferably at least about 70%> identical to PCaVβl2134; are at least about 65%o, preferably at least about 70%, even more preferably at least about 15%, and even more preferably at least about 80% identical to PCaVβ72133; or are at least about 60%), preferably at least about 65%>, even more preferably at least about 70%, and even more preferably at least about 75% identical to PCaVβ21130. Even more preferred is a nucleic acid molecule encoding PCaVβ3]27, PCaVβ3ln, PCaVβ31]0, PCaVβ4]2g, PCaVβ4113, PCaVβ4109, PCaVβl2134, PCaVβl2m, PCaVβl2115, PCaVβ72133, PCaVβ72113, PCaVβ72U4, PCaVβ21130, PCaVβ2110g, PCaVβ21U6, PCaVβ54135, PCaVβ54114, PCaVβ54116, PCaVβ 182128, PCaVβ 182πo and/or PCaVβ 182109, fragments thereof, complements thereof, and/or an allelic variant of such a nucleic acid molecule.
In another embodiment, a TCR Vβ nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least about 65%>, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80%) identical to SEQ ID NO:2; are at least about 69%>, preferably at least about 75%>, even more preferably at least about 80%>, and even more preferably at least about 85% identical to SEQ ID NO:5; are at least about 57%>, preferably at least about 60%, even more preferably at least about 65%>, and even more preferably at least about 70% identical to SEQ LD NO: 10; are at least about 65%>, preferably at least about 70%, even more preferably at least about 75%, and even more preferably at least about 80% identical to SEQ ED NO: 15; or at least about 60%, preferably at least about 65%o, even more preferably at least about 70%>, and even more preferably at least about 75% identical to SEQ LD NO:20. The present invention also includes a TCR Vβ nucleic acid molecule encoding a protein having at least a portion of SEQ LD NO:2, SEQ TD NO:5, SEQ ID NO: 10, SEQ ED NO: 15, SEQ LD NO:20, SEQ LD NO:23, SEQ LD NO:26, SEQ ED NO:29, SEQ LD NO:32, SEQ ID NO:35, SEQ ED NO:38, SEQ DD NO:41, SEQ DD NO:44, SEQ DD NO:47, SEQ DD NO:60, SEQ DD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ DD NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ DD NO:69, SEQ DD NO:70, SEQ DD NO:71, SEQ DD NO:72, SEQ ID NO:73, SEQ DD NO:74, SEQ DD NO:75, SEQ DD NO:76, SEQ DD NO:77, SEQ ID NO:78, SEQ DD NO:79, SEQ DD NO:80 and/or proteins encoded by the complement of a nucleic acid sequence including SEQ ID NO:50, SEQ DD NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ DD NO:54, SEQ ED NO:55, SEQ TD NO:56; fragments thereof; complements thereof, as well as allelic variants of a TCR Vβ nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
In one embodiment, a TCR Vβ nucleic acid molecule of the present invention is at least about 69%, preferably at least about 75%, even more preferably at least about 80%), and even more preferably at least about 85%> identical to nCaVβ3381; is at least about 15%, preferably at least about 80%>, even more preferably at least about 85%, and even more preferably at least about 90%> identical to nCaVβ4408; is at least about 72%>, preferably at least about 80%>, even more preferably at least about 85%, and even more preferably at least about 90%> identical to nCaVβl2408; is at least about 60%>, preferably at least about 65%>, even more preferably at least about 70%>, and even more preferably at least about 75% identical to nCaVβ7243g; or is at least about 3 %>, preferably at least about 45%o, even more preferably at least about 50%>, and even more preferably at least about 55%) identical to nCaVβ21462. Even more preferred is a nucleic acid molecule comprising nCaVβ3381, nCaVβ3333, nCaVβ3330, nCaVβ440g, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405,nCaVβ54354, nCaVβ5434g, nCaVβl82423, nCaVβl82384, nCaVβl82369 and/or nCaVβl82327; fragments thereof; complements thereof; as well as an allelic variant of such a nucleic acid molecule. In another embodiment, a TCR Vβ nucleic acid molecule of the present invention comprises a nucleic acid sequence that is at least about 69%, preferably at least about 75%), even more preferably at least about 80%>, and even more preferably at least about 85%o identical to SEQ ED NO:l; is at least about 75%, preferably at least about 80%), even more preferably at least about 85%, and even more preferably at least about 90% identical to SEQ ED NO:4; is at least about 72%, preferably at least about 80%, even more preferably at least about 85%, and even more preferably at least about 90% identical to SEQ ED NO:9; is at least about 60%o, preferably at least about 65%, even more preferably at least about 70%, and even more preferably at least about 75% identical to SEQ ID NO:98; or is at least about 38%, preferably at least about 45%, even more preferably at least about 50%, and even more preferably at least about 55% identical to SEQ ID NO: 19. The present invention also includes a TCR Vβ nucleic acid molecule comprising at least a portion of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ HD NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ DD NO:9, SEQ ID NO: 11, SEQ DD NO:12, SEQ DD NO:13, SEQ DD NO:98, SEQ DD NO:100, SEQ ID NO:17, SEQ TD NO:18, SEQ HD NO:19, SEQ ID NO:21, SEQ TD NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ DD NO:27,SEQ DD NO:28, SEQ DD NO:30, SEQ ID NO:31, SEQ LD NO:33, SEQ HD NO:34, SEQ HD NO:36, SEQ TD NO:37, SEQ TD NO:39, SEQ TD NO.40, SEQ H) NO:42, SEQ TD NO:43, SEQ TD NO:45, SEQ ID NO:46, SEQ H NO:48, SEQ HD NO:50, SEQ HD NO:51, SEQ TD NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55, SEQ HD NO:56, a complement of SEQ TD NO:50, SEQ HD NO:51, SEQ HD NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55, SEQ DD NO: 56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:60, SEQ DD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ DD NO:68, SEQ ID NO:69, SEQ ED NO:70, SEQ LD NO:71, SEQ ED NO:72, SEQ LD NO:73, SEQ DD NO:74, SEQ DD NO:75, SEQ DD NO:76, SEQ ED NO:77, SEQ DD NO:78, SEQ DD NO:79 or SEQ ED NO:80, and complements thereof; fragments thereof; complements thereof; as well as allelic variants of such TCR Vβ nucleic acid molecules, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed. A preferred isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ DD NO:3, SEQ DD NO:6, SEQ DD NO:8, SEQ DD NO:l l, SEQ DD NO: 13, SEQ ID NO: 100, SEQ TD NO: 18, SEQ LD NO:21, SEQ ID NO:24, SEQ ED NO:27, SEQ LD NO:30, SEQ ID NO:33, SEQ LD NO:36, SEQ LD NO:39, SEQ LD NO:42, SEQ HD NO:45, SEQ LD NO:48, a complement of SEQ ID NO:50, SEQ ID NO:51, SEQ HD NO:52, SEQ TD NO:53, SEQ TD NO:54, SEQ ED NO:55, SEQ LD NO:56, and/or the complement of a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:60, SEQ LD NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ TD NO:65, SEQ TD NO:66, SEQ ID NO:67, SEQ DD NO:68, SEQ ID NO:69, SEQ DD NO:70, SEQ DD NO:71, SEQ ID NO:72, SEQ DD NO:73, SEQ DD NO:74, SEQ TD NO:75, SEQ TD NO:76, SEQ HD NO:77, SEQ ID NO:78, SEQ DD NO:79 or SEQ ID NO:80; or fragments thereof.
A preferred isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence that is any of the following: (a) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:l, SEQ DD NO:3, SEQ DD NO:28, SEQ DD NO:30 and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:60, SEQ ED NO:61, SEQ DD NO:62, and the complement of a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:60, SEQ TD NO:61 , and SEQ TD NO:62, or a fragment thereof that is at least about 25 nucleotides in length; (b) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ HD NO:6, SEQ DD NO:7, SEQ DD NO:8, SEQ DD NO:31, SEQ DD NO:33, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ED NO:63, SEQ DD NO:64, and SEQ DD NO:65, and the complement of a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ LD NO:63, SEQ TD NO:64, and SEQ ED NO:65, or a fragment thereof that is at least about 30 nucleotides in length; (c) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ LD NO:9, SEQ ID NO: 11, SEQ LD NO: 12, SEQ ID NO: 13, SEQ LD NO:34, SEQ ED NO:36, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ LD NO:66, SEQ DD NO:67, and SEQ DD NO:68, and the complement of a nucleic acid sequence that encodes an amino acid sequence selecte from the group consisting of SEQ DD NO:66, SEQ DD NO:67, and SEQ DD NO:67, or a fragment thereof that is at least about 40 nucleotides in length; (d) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:98, SEQ DD NO: 100, SEQ DD NO: 17, SEQ TD NO: 18, SEQ TD NO:37, SEQ ID NO:39, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ LD NO:69, SEQ ID NO:70, and SEQ LD NO:71, and the complement of a nucleic acid sequence that encodes an amino acid sequence selecte from the group consisting of SEQ ID NO:69, SEQ LD NO:70, and SEQ ED NO:71, or a fragment thereof that is at least about 75 nucleotides in length; and (e) a nucleic acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ED NO:51, SEQ LD NO:52, SEQ LD NO:53, SEQ LD NO:54, SEQ LD NO:55, and SEQ ED NO:56. A preferred isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding a protein selected from the group consisting of: (a) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ DD NO:28, SEQ DD NO:50, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:60, SEQ LD NO:61, and SEQ DD NO:62, or a fragment thereof that is at least about 25 nucleotides in length; (b) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ HD NO:7, SEQ ID NO:31, SEQ DD NO: 51, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 63, SEQ HD NO:64, and SEQ ID NO:65, or a fragment thereof that is at least about 30 nucleotides in length; (c) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:9, SEQ DD NO: 12, SEQ HD NO:34, SEQ ID NO:52, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ DD NO:67, and SEQ DD NO:68, or a fragment thereof that is at least about 40 nucleotides in length; and (d) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ LD NO:98, SEQ ID NO:17, SEQ LD NO:37, SEQ LD NO:53, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ LD NO:69, SEQ LD NO:70, and SEQ ID NO:71, or a fragment thereof that is at least about 75 nucleotides in length.
Another embodiment of the present invention is an isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ DD NO: 1 , SEQ ID NO:3, SEQ ID NO:28, and SEQ LD NO:30, or a fragment thereof, wherein said fragment has an at least a 20 contiguous nucleotide region identical in sequence to a 20 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ DD NO:3, SEQ ID NO:28, and SEQ ID NO:30; (b) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ DD NO:4, SEQ DD NO:6, SEQ DD NO:7, SEQ TD NO:8, SEQ ID NO:31, and SEQ DD NO:33, or a fragment thereof, wherein said fragment has an at least a 25 contiguous nucleotide region identical in sequence to a 25 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ DD NO:4, SEQ DD NO:6, SEQ DD NO:7, SEQ DD NO:8, SEQ DD NO:31 and SEQ DD NO:33; (c) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ DD NO:9, SEQ TD NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:34, and SEQ TD NO:36, or a fragment thereof, wherein said fragment has an at least a 30 contiguous nucleotide region identical in sequence to a 30 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 11, SEQ TD NO: 12, SEQ DD NO: 13, SEQ DD NO:34, and SEQ TD NO:36; and (d) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:98, SEQ TD NO:100, SEQ TD NO:17, SEQ HD NO:18, SEQ HD NO:37, and SEQ TD NO:39, or a fragment thereof, wherein said fragment has an at least a 60 contiguous nucleotide region identical in sequence to a 60 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO:17, SEQ ID NO:18, SEQ ED NO:37, and SEQ DD NO:39. The phrase, a homolog having an at least "x" contiguous nucleotide region identical in sequence to an "x" contiguous nucleotide region of a nucleic acid molecule selected from the group consisting of SEQ DD NO:"y", refers to an "x"-nucleotide in length nucleic acid molecule that is identical in sequence to an "x"-nucleotide portion of SEQ ID NO:"y", as well as to nucleic acid molecules that are longer in length than "x". The additional length may be in the form of nucleotides that extend from either the 5 ' or the 3' end(s) of the contiguous identical "x"-nucleotide portion. The 5' and/or 3' extensions can include one or more extensions that have no identity to an immunoregulatory molecule of the present invention, as well as extensions that show similarity or identity to cited nucleic acids sequences or portions thereof.
Knowing the nucleic acid sequences of certain TCR Vβ nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain other TCR Vβ nucleic acid molecules. Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecules include mammalian cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources from which to amplify nucleic acid molecules include mammalian cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.
The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising TCR Vβ nucleic acid molecules. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. A preferred oligonucleotide of the present invention has a maximum size of about 100 nucleotides. A preferred oligonucleotide of the present invention has a minimum size of about 12 nucleotides. Preferably, an oligonucleotide of the present invention has a size from about 12 nucleotides to about 30 nucleotides and more preferably from about 15 nucleotides to about 25 nucleotides. A preferred isolated oligonucleotide of the present invention comprises a unique nucleic acid sequence within a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ HD NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ED NO:7, SEQ ID NO: 8, SEQ ED NO:9, SEQ DD NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, SEQ LD NO:98, SEQ LD NO: 100, SEQ LD NO: 17, SEQ ED NO: 18, SEQ LD NO: 19, SEQ LD NO:21, SEQ LD NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ DD NO:27,SEQ DD NO:28, SEQ ID NO:30, SEQ TD NO:31, SEQ DD NO:33, SEQ DD NO:34, SEQ ID NO:36, SEQ TD NO:37, SEQ ΩD NO:39, SEQ HD NO:40, SEQ TD NO:42, SEQ ID NO:43, SEQ TD NO:45, SEQ DD NO:46, SEQ DD NO:48, SEQ DD NO:50, SEQ ID NO:51, SEQ HD NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55, SEQ ID NO:56, a complement of SEQ DD NO:50, SEQ DD NO:51, SEQ DD NO:52, SEQ ED NO:53, SEQ TD NO:54, SEQ DD NO:55, SEQ DD NO:56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO:60, SEQ ED NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ ID NO:64, SEQ ED NO:65, SEQ ID NO:66, SEQ DD NO:67, SEQ TD NO:68, SEQ DD NO:69, SEQ DD NO:70, SEQ ID NO:71, SEQ DD NO:72, SEQ ID NO:73, SEQ ED NO:74, SEQ DD NO:75, SEQ HD NO:76, SEQ TD NO:77, SEQ πD NO:78, SEQ DD NO:79 or SEQ ID NO:80, and complements thereof; and a homolog thereof.
The present invention includes oligonucleotides that can be used, for example, as probes to identify nucleic acid molecules, primers to produce nucleic acid molecules, or therapeutic reagents to inhibit TCR Vβ protein production or activity (e.g., as antisense-, triplex formation-, ribozyme- and/or RNA drug-based reagents). The present invention also includes the use of such oligonucleotides to protect animals from disease using one or more of such technologies. Appropriate oligonucleotide-containing therapeutic compositions can be administered to an animal using techniques known to those skilled in the art. Preferred oligonucleotides of the present invention include oligonucleotides comprising a unique nucleic acid sequence, as defined herein, within a nucleic acid molecule of the present invention or homologs thereof. Preferred homologs of an oligonucleotide are capable of priming a nucleic acid sequence.
One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of TCR Vβ nucleic acid molecules of the present invention.
One type of recombinant vector, referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells, and more preferably in the cell types disclosed herein
En particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, helminth or other endoparasite, or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, Υllac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as immediate early promoter), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with mammals, such as canine or feline transcription control sequences.
Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein. Preferred nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules, include nCaVβ3381, nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ43g4, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405, nCaVβ54354, nCaVβ54348, nCaVβ 182423, nCaVβ 1823g4, nCaVβl82369 and/or nCaVβl82327.
Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed TCR Vβ protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments. Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Eukaryotic recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred nucleic acid molecules with which to transform a cell include TCR Vβ nucleic acid molecules disclosed herein. Particularly preferred nucleic acid molecules with which to transform a cell include nCaVβ3381, nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl240g, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405, nCaVβ54354, nCaVβ54348, nCaVβl82423, nCaVβl82384, nCaVβl82369 and/or nCaVβ 182327.
Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines). Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing TCR Vβ proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), other insect, other animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, helminth, insect and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Pichia, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CN-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, Chinese hamster ovary (CHO) cells, Ltk cells and Nero cells. Particularly preferred host cells are Escherichia coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 03987 and SR-11 04072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CN-1 cells; COS cells; Nero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse ΝLH/3T3 cells, LMTK31 cells and/or HeLa cells. In one embodiment, the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters. A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences, examples of which are disclosed herein.
A recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein.
It is to be noted that the term "a recombinant molecule", "a host cell" or "a recombinant cell" refers to one or more or at least one recombinant molecule, host cell or recombinant cell, respectively. Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including TCR Vβ nucleic acid molecules encoding one or more proteins of the present invention and one or more other nucleic acid molecules encoding other therapeutic compounds, as disclosed herein (e.g., to produce multivalent vaccines).
Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein. Isolated TCR Vβ proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective, medium refers to any medium in which a cell is cultured to produce a TCR Vβ protein of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
The phrase "recovering the protein", as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
The present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to a TCR Vβ protein of the present invention or a mimetope thereof (e.g., anti-TCR Vβ antibodies). As used herein, the term "selectively binds to" a TCR Vβ protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid., and Harlow, et al, 1988, Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid. An anti- TCR Vβ antibody of the present invention preferably selectively binds to a TCR Vβ protein in such a way as to inhibit the function of that protein.
Isolated antibodies of the present invention can include antibodies in serum, or antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to one or more epitopes.
A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce TCR Vβ proteins of the present invention. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition. Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used (a) as reagents in assays to detect TCR Vβ protein, (b) as reagents in assays to modulate cellular activity through a TCR Vβ protein (e.g., mimicking ligand binding to TCR Vβ protein), and/or (c) as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present invention can be used to target compounds (e.g., nucleic acid molecules, drugs or proteins) to antigen presenting cells. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the compounds using techniques known to those skilled in the art. Suitable compounds are known to those skilled in the art. One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is capable of regulating an immune response in an animal. Therapeutic compositions of the present invention include at least one of the following therapeutic compounds: an isolated TCR Vβ protein of the present invention or a mimetope thereof, an isolated TCR Vβ nucleic acid molecule of the present invention, an isolated antibody that selectively binds to a TCR Vβ protein of the present invention, an inhibitor of TCR Vβ function identified by its ability to bind to a TCR Vβ protein of the present invention and inhibit binding of a TCR Vβ protein to MHC, and a mixture thereof (i.e., combination of at least two of the compounds). As used herein, a therapeutic compound refers to a compound that, when administered to an animal in an effective manner, is able to treat, ameliorate, and/or prevent a disease. Examples of proteins, nucleic acid molecules, antibodies and inhibitors of the present invention are disclosed herein.
The present invention also includes a therapeutic composition comprising at least one TCR Vβ-based compound of the present invention in combination with at least one additional therapeutic compound. Examples of such compounds are disclosed herein.
Therapeutic compositions of the present invention can be administered to any animal susceptible to such therapy, preferably to mammals, and more preferably to dogs. A therapeutic composition of the present invention is administered to an animal in an effective manner such that the composition is capable of regulating an immune response in that animal. Therapeutic compositions of the present invention can be administered to animals prior to onset of a disease (i.e., as a preventative vaccine) and/or can be administered to animals after onset of a disease in order to treat the disease (i.e., as a therapeutic vaccine). Preferred diseases to prevent or treat include autoimmune diseases, allergic reactions, infectious diseases and cancer.
Therapeutic compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration. In one embodiment of the present invention, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., Flt-3 ligand, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (EL-3), interleukin 4 (LL-4), interleukin 5 (E -5), interleukin 6 (LL-6), interleukin 7 (LL-7), interleukin 8 (LL-8), interleukin 10 (LL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGLF), transforming growth factor beta, RANTES (regulated upon activation, normal T cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEEF)); bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc. Norcross, GA), Ribi adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives (e.g., Quil A (Superfos Biosector A/S, Denmark). Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein. A therapeutic composition can contain one or more adjuvants. In one embodiment of the present invention, a therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols. A therapeutic composition can contain one or more carriers.
One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition to regulate an immune response in an animal. The therapeutic composition is preferably released over a period of time ranging from about 1 to about 12 months. A controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
Therapeutic compositions of the present invention can be administered to animals prior to and/or after onset of disease. Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of regulating the immune response in an animal when administered one or more times over a suitable time period. For example, a preferred single dose of a protein, mimetope or antibody therapeutic composition is from about 1 microgram (g) to about 10 milligrams (mg) of the therapeutic composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 10 g to about 1 mg of the therapeutic composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, intraoccular, oral, transdermal and intramuscular routes.
According to one embodiment, a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a therapeutic protein or therapeutic RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNA drug) in the animal. Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a) administering a naked (i.e., not packaged in a viral coat or cellular membrane) nucleic acid as a genetic vaccine (e.g., as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or (b) administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle). One or more nucleic acid molecules can be delivered to an animal.
A genetic (i.e., naked nucleic acid) vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent. A genetic vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule. Preferred genetic vaccines include at least a portion of a viral genome (i.e., a viral vector). Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses, with those based on alphaviruses (such as sindbis or Semliki forest virus), species- specific herpesviruses and poxviruses being particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production. Particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron-A), Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a "strong" polyadenylation signal is also preferred.
Genetic vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, intranasal and oral routes of administration being preferred. A preferred single dose of a genetic vaccine ranges from about 1 nanogram (ng) to about 600 g, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically. Genetic vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or in a carrier (e.g., lipid-based vehicles).
A recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging- or replication-deficient and/or encodes an attenuated virus. A number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses. Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus vaccines are disclosed in PCT Publication No. WO 94/17813, by Xiong et al., published August 18, 1994.
When administered to an animal, a recombinant virus vaccine of the present invention infects cells within the immunized animal and directs the production of a therapeutic protein or RNA nucleic acid molecule that is capable of protecting the animal from disease caused by a parasitic helminth as disclosed herein. For example, a recombinant virus vaccine comprising a TCR Vβ nucleic acid molecule of the present invention is administered according to a protocol that results in the regulation of an immune response in an animal. A preferred single dose of a recombinant virus vaccine of the present invention is from about 1 x 104 to about 1 x 108 virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines, with subcutaneous, intramuscular, intranasal, intraoccular and oral administration routes being preferred. One or more recombinant virus vaccines can be delivered to an animal. A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention. Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae andPichia pastoris), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells. Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 108 to about 1012 cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
The efficacy of a therapeutic composition of the present invention to regulate the immune response in an animal can be tested in a variety of ways including, but not limited to, detection of cellular immunity within the treated animal, determination of T cell activity (helper or cytotoxic T cell activity), identification of T cell repertoire, detection of immunoregulatory cytokines, e.g., E -2, IL-4, EL- 10, LL-12, levels, detection of antibody levels, determine tumor development or challenge of the treated animal with an infectious agent to determine whether the treated animal is resistant to disease. En one embodiment, therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art. According to the present invention, a therapeutic composition is used to treat a disease requiring immunological regulation, such as cancer, infectious diseases, autoimmune diseases or allergy. Suitable cancers to treat include lymphomas or leukemias. Suitable infectious diseases to treat include diseases caused by viral, bacterial, yeast, fungal or parasitic infection. Suitable autoimmune diseases to treat include: autoimmune skin diseases, e.g. pemphigus foliaceous, pemphigus vulgaris, pemphigus vegetans, pemphigus erythematosus, bollous pemphigoid, discoid lupus, dermatomyositis or subcorneal pustular dermatosis; blood disorders, e.g. autoimmune hemolytic anemia, immune-mediated thrombocytopenia, aplstic anemia, pure red cell aplasia or immune mediated neutropenia; endocrine dysfuntion, e.g. lymphocytic thyroditis, diabetes, hypoadrenocorticism or hypoparathyroidism; multi-system dysfunction, e.g. systemic lupus erythematosus or Sjogren's syndrome; neurologic dysfunction, e.g. myasthenia gravis, distemper and rabies post vaccinal encephalopathy or acute polyradiculoneuritis; or musculoskeletal disease, e.g. rheumatoid arthritis, idiopathic polyarthritis, plasmacytic-lymphocytic arthritis or polymyosutus. Suitable allergies to treat include allergic dermatitis, atopic dermatitis, allergic rhinitis or allergic bronchitis.
One therapeutic composition of the present invention includes a TCR Vβ protein of the present invention, or a portion of such TCR Vβ protein that elicits a cytotoxic T cell response against a T cell bearing the TCR Vβ protein. A preferred TCR Vβ protein comprises a soluble form of a TCR Vβ protein of the present invention, with a peptide of a TCR Vβ protein being more preferred. A preferred TCR Vβ peptide is from about 5 to about 50 residues, more preferably from about 10 to about 40 residues and even more preferably from about 12 to about 30 residue in length. One or more TCR Vβ proteins of the present invention, or portions of such TCR Vβ proteins can be used in a therapeutic composition. According to the present invention, a therapeutic composition comprising a TCR
Vβ peptide, e.g. a portion of a polypeptide, can be delivered to an animal as a peptide or in the form of DNA encoding such peptide. A TCR Vβ peptide of the present invention can be linked to another molecule to assist in the delivery of the peptide to an animal. Preferably, a TCR Vβ peptide of the present invention is administered systemically to an animal. One or more peptides can be used in a therapeutic composition. Another therapeutic composition of the present invention includes an inhibitory compound that inhibits a TCR Vβ protein from binding to MHC. An inhibitory compound is capable of substantially interfering with the function of a TCR Vβ protein susceptible to inhibition. For example, an inhibitory compound is administered in an amount and manner that inhibits an immune response that is sufficient to treat an animal for a disease that requires downregulation of an immune response. One or more inhibitors can be used in a therapeutic composition.
Suitable inhibitory compounds include compounds that prevent the activation of an immunoregulatory cell through TCR Vβ by, for example, interfering with the binding of TCR Vβ protein to MHC by binding to either the TCR Vβ protein or MHC. An example of an inhibitory compound is an antibody of the present invention, administered to an animal in an effective manner; i.e., an antibody of the present invention, is administered in an amount so as to be present in the animal at a titer that is sufficient, upon interaction of that antibody with a native TCR Vβ protein, to decrease TCR Vβ activity in an animal, at least temporarily. Oligonucleotide nucleic acid molecules of the present invention can also be administered in an effective manner, thereby reducing expression of TCR Vβ proteins in order to interfere with TCR Vβ activity targeted in accordance with the present invention. Peptides of TCR Vβ proteins of the present invention can also be administered in an effective manner, thereby reducing binding of TCR Vβ proteins to MHC in order to interfere with TCR Vβ activity targeted in accordance with the present invention. Preferably, an inhibitory compound is derived from a TCR Vβ protein of the present invention.
An inhibitory compound of TCR Vβ function can be identified using TCR Vβ proteins of the present invention. One embodiment of the present invention is a method to identify a compound capable of inhibiting TCR Vβ function. Such a method includes the steps of: (a) contacting (e.g., combining, mixing) an isolated TCR Vβ protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein binds to a protein including MHC, and (b) determining if the putative inhibitory compound inhibits the binding of TCR Vβ to MHC. Putative inhibitory compounds to screen include small organic molecules, antibodies (including mimetopes thereof), and ligand analogs. Such compounds can also be screened to identify those compounds that are substantially not toxic to a recipient animal.
One embodiment of the present invention is a method to detect expansion of T cells in an animal. The present method utilizes the discovery by the inventors of unique sequences in a TCR Vβ and more particularly in the V region of a TCR Vβ that function, or can be used, as markers for T cells. As used herein, the term "unique sequences" refers to nucleic acid or amino acid sequences that are present in one TCR Vβ nucleic acid molecule or TCR Vβ protein, but not in another TCR Vβ nucleic acid molecule or TCR Vβ protein, respectively. Thus, a unique sequence differentiates one TCR Vβ nucleic acid molecule or TCR Vβ protein of the present invention from another TCR Vβ nucleic acid molecule or TCR Vβ protein, respectively. For example, a unique sequence within hcVβ3, is not found within hcVβ4, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82, and a unique sequence within hcVβ4, is not found within hcVβ3, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82, and so on. A unique sequence of the present invention can be used to detect expansion of T cells by, for example, determining the presence or absence of any one or more unique sequences, determining increased or decreased levels of molecules carrying one or more unique sequences compared with other unique sequences in the same animal and/or a different animal, or comparing levels of different TCR Vβ proteins in an animal. It is within the scope of the present invention that any unique sequence present in a TCR Vβ sequence of the present invention can be used in the practice of the present method, including those identified herein and those which will be identified based on the sequences of nucleic acid molecules or proteins disclosed herein. Preferably, unique sequences of the present invention include those sequences located within about the first 200 nucleotides or about the first 70 residues of the 5' or amino terminus of a nucleic acid molecule or a protein. The suitable length of a unique sequence depends upon the reagent used to detect the presence of the unique sequence. For example, a suitable length of a unique sequence is from about 15 to about 30 nucleotides if the detection reagent is a DNA primer. Alternatively, a suitable length of a unique sequence is from about 50 to about 300 nucleotides if the detection reagent is a DNA hybridization probe. Alternatively, a suitable length of a unique sequence is at least about 5 amino acids if the detection reagent is an antibody. En addition, one or more unique sequences can be identified, and one or more reagents can be used to identify such sequences, using a method of the present invention.
Any method of the present invention can be used to determine the presence, absence, amount or ratio of TCR Vβ's of the present invention in an animal to determine T cell expansion and/or diagnose an abnormal state or a specific disease. Typically, numbers of T cells in an animal are regulated. Expansion of total T cell numbers, or of a particular T cell clone, can represent an abnormal state or disease. For example, a particular T cell clone is expanded in a T cell lymphoma or leukemia. Thus, information derived using the present methods is particularly useful because the inventors have discovered the seven different TCR Vβ proteins disclosed herein which can comprise at least about 95%> of the TCR Vβ repertoire in canids. The discovery of the existence of a small repertoire of TCR Vβ proteins enables the production of appropriate reagents that detect a substantial amount of T cell receptors in an animal. The reagents can be used to correlate T cell expansion with a specific disease by comparing results obtained using samples from normal animals compared with animals having or suspected of having a disease. Thus, any diagnostic method of the present invention is useful for determining if an animal is susceptible to, has or is in remission from a disease.
According to the present invention, T cell expansion is determined by detecting increased levels of one or more specific TCR Vβ molecules in a tissue sample isolated from an animal. Increased levels of TCR Vβ molecules can be determined by comparing levels of two or more different TCR Vβ molecules in a tissue sample isolated from one animal, or levels of one or more TCR Vβ molecules in tissue samples from two or more different animals. Total amounts or ratios of particular TCR Vβ molecules can be determined by one of skill in the art depending upon the method used to detect the presence of a TCR Vβ molecule in a sample. Preferably, a ratio illustrative of increased levels of a particular TCR Vβ molecule is from about 2-fold to about 5-fold, more preferably from about 5-fold to about 25, and more preferably 10-fold more of one particular TCR Vβ molecule compared with another sample of the same TCR Vβ molecule or a different TCR Vβ molecule. Methods to determine ratios are known to those in the art and include, for example, densitometry, spectrophotometry or determining counts per minute of isotopes.
One embodiment of the present invention is a method to detect expansion of T cells in an animal comprising detecting the presence of one or more T cell receptors having unique nucleic acid sequences within hcVβ3, hcVβ4, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82 nucleic acid molecules or hcVβ3, hcVβ4, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82 proteins by forming detectable products, wherein the increased level of a detectable product compared with another detectable product indicates expansion of the T cells. According to the present method, a suitable sample containing T cells is isolated from an animal. Samples containing T cell receptors can be isolated from the same or different animals. Control samples can be obtained from the same or different animals. The sample is contacted under appropriate conditions with one or more reagents capable of identifying one or more unique nucleic acid or amino acid sequences, respectively. Preferably, a reagent distinguishes one member of the group comprising hcVβ3, hcVβ4, hcVβl2, hcVβ72, hcVβ21, hcVβ54 or hcVβl82 nucleic acid molecules or hcVβ3, hcVβ4, hcVβl2, hcVβ72, hcVβ21, hcVβ54 or hcVβl82 proteins, preferably nCaVβ33gl, nCaVβ4408, nCaVβl2408, nCaVβ72438, nCaVβ21462, nCaVβ54417, nCaVβl82423, PCaVβ3127, PCaVβ4128, PCaVβl2134, PCaVβ72133, PCaVβ21130, PCaVβ54135 or PCaVβl82128, from another member of that group. By determining increased production of one of the detectable products, one can detect T cell expansion. Preferably, expansion of a T cell is determined by comparing formation of one detectable product with formation of one or more other detectable products and looking for increased production of at least one of the products compared to another. According to the present invention, a detectable product can comprise a nucleic acid molecule, a peptide, a protein or an antibody. Preferred detectable products and methods to form detectable products are disclosed herein. It is within the skill of one in the art that methods to identify detectable products based on the product being detected.
The invention also provides novel reagents useful in the methods which have been described herein above. Thus, the invention includes reagents capable of binding to unique nucleic acid or unique amino acid sequences contained within a TCR Vβ. Preferred reagents include those which can differentiate one TCR V beta protein, or nucleic acid molecule, from another. Preferred reagents can distinguish TCR V beta proteins hcVβ2, hcVβ3, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82, or the nucleic acid molecules that encode them, from each other. More preferred reagents can distinguish proteins comprising PCaVβ3127, PCaVβ3u„ PCaVβ3U0, PCaVβ4128, PCaVβ4113, PCaVβ4109, PCaVβl2134, PCaVβl2] U, PCaVβl2115, PCaVβ72133, PCaVβ72113, PCaVβ72114, PCaVβ21130, PCaVβ21108, PCaVβ21116, PCaVβ54I35, PCaVβ54114, PCaVβ54116, PCaVβl82128, PCaVβl82110 anαVorPCaVβl82109 from each other, or nucleic acid molecules comprising nCaVβ3381, nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405,nCaVβ54354, nCaVβ54348, nCaVβ 182423, nCaVβ 182384, nCaVβ 182369 and or nCaVβ 182327 from each other. Even more preferred reagents can distinguish TCR V beta proteins comprising amino acid sequence SEQ TD NO:29, SEQ ED NO:32, SEQ ED NO:35, SEQ LD NO:38, SEQ ID NO:41, SEQ LD NO:44, SEQ TD NO:47 from each other.
In particular, a method of the present invention comprises: (a) contacting a sample, i.e., one or more samples, containing DNA from T cells with a reagent, i.e., one or more reagents, having specificity for a unique nucleic acid sequence; and (b) determining the presence of DNA carrying the unique nucleic acid sequences. Methods to determine the presence of the DNA are disclosed herein.
In one embodiment, identifying the presence of a T cell receptor having unique sequence can be achieved by polymerase chain reaction (PCR) amplification techniques known to those of skill in the art. The PCR amplification forms a detectable product comprising DNA. An example of a suitable reagent to use in PCR amplification techniques is a DNA primer complementary to all or a portion of a unique nucleic acid sequence, referred to herein as a unique sequence primer. Preferably, another primer is used in conjunction with the unique sequence primer in order o effect amplification. This second primer can be complementary to a unique sequence or to a common sequence (i.e., a sequence shared by different TCR beta chain sequences). Typically, the second primer is complementary to a common sequence and is chosen based on its distance from the unique sequence primer, i.e. based on ease of detection of PCR amplification product. One of skill in the art understands that a preferred product of a PCR reaction is from about 100 to about 500 nucleotides, preferably from about 150 to about 450 nucleotides and more preferably from about 200 to about 400 nucleotides. Thus, it is within the skill of one in the art to design and create a second primer located from about 100 to about 500 nucleotides from the site of a unique sequence. For example, a suitable second primer includes a DNA primer complementary to a sequence in the constant region of a beta chain. Preferred second primers are described herein in the Examples section. Methods to resolve PCR products are well known to those of skill in the art. In addition, methods to quantitate the amount of PCR product produced in a PCR reaction are well known to those of skill in the art. Examples of preferred unique nucleic acid sequences to be identified by PCR include unique sequences contained within SEQ LD NO:l, SEQ ED NO:3, SEQ ID NO:4, SEQ TD NO:6, SEQ HD NO:7, SEQ TD NO:8, SEQ HD NO:9, SEQ TD NO:l 1, SEQ ID NO: 12, SEQ TD NO: 13, SEQ ID NO:98, SEQ DD NO: 100, SEQ ID NO: 17, SEQ DD NO: 18, SEQ DD NO: 19, SEQ DD NO:21, SEQ DD NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ DD NO:27 and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ DD NO:60, SEQ DD NO:61, SEQ DD NO:62, SEQ ID NO:63, SEQ HD NO:64, SEQ ID NO:65, SEQ DD NO:66, SEQ ID NO:67, SEQ DD NO:68, SEQ DD NO:69, SEQ ID NO:70, SEQ TD NO:71, SEQ DD NO:72, SEQ ID NO:73, SEQ TD NO:74, SEQ HD NO:75, SEQ DD NO:76, SEQ DD NO:77, SEQ DD NO:78, SEQ TD NO:79 or SEQ HD NO:80. More preferred unique nucleic acid sequences to be identified by PCR include SEQ TD NO:50, SEQ HD NO:51, SEQ TD NO:52, SEQ HD NO:53, SEQ TD NO:54, SEQ ID NO:55 and/or SEQ ID NO:56, or complements thereof. Preferred second primers include primers located in the constant region sequence of a beta chain. More preferred second primers include SEQ ID NO:58 and SEQ ID NO:59, or complements thereof. In another embodiment, identifying the presence of a T cell receptor having a unique nucleic acid sequence can be achieved by nucleic acid, e.g., DNA, RNA, modified DNA or modified RNA, hybridization techniques using a nucleic acid probe. The hybridization forms a detectable product comprising a hybrid between the nucleic acid and the reagent. A suitable reagent for use with hybridization techniques include nucleic acid probes which are complementary to nucleic acid sequences that include all or a portion of a unique sequence. The hybridization forms a detectable product comprising a hybrid between the nucleic acid and the reagent. It is within the skill of one in the art to design and produce suitable probes based on sequences of the present invention. The presence of a unique sequence in a sample from an animal is determined by detecting the hybridization of a "unique sequence" probe to that unique sequence in the TCR Vβ nucleic acid molecule. Methods to detect hybridization of a probe are well known to those of skill in the art and include those that allow one to distinguish one Vβ nucleic acid from another. En addition, methods to quantitate the extent of hybridization are well known to those of skill in the art. Preferred unique nucleic acid sequences to be identified by nucleic acid hybridization include unique sequences contained within SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ DD NO:9, SEQ DD NO: 11, SEQ ED NO: 12, SEQ LD NO: 13, SEQ ID NO:98, SEQ ED NO:100, SEQ ID NO:17, SEQ LD NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ LD NO:22, SEQ LD NO:24, SEQ ED NO:25, SEQ DD NO:27 and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ ID NO: 60, SEQ DD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ DD NO:65, SEQ DD NO:66, SEQ DD NO:67, SEQ DD NO:68, SEQ DD NO:69, SEQ TD NO:70, SEQ ED NO:71, SEQ DD NO:72, SEQ DD NO:73, SEQ DD NO:74, SEQ ID NO:75, SEQ HD NO:76, SEQ ED NO:77, SEQ DD NO:78, SEQ ID NO:79 or SEQ ID NO:80. More preferred unique nucleic acid sequences to be identified by nucleic acid hybridization include unique sequences SEQ ID NO:50, SEQ ED NO:51, SEQ ID NO:52, SEQ LD NO:53, SEQ LD NO:54, SEQ ED NO:55 and/or SEQ LD NO:56.
A unique amino acid sequence of the present invention can be used to produce antibodies that bind specifically to the portion of a TCR Vβ protein that contains the unique amino acid sequence. Such antibodies can be monoclonal or polyclonal, and produced using methods described herein. The antibodies can be used to detect T cells having T cell receptors containing such unique sequences by contacting T cells isolated from an animal with the antibody under appropriate conditions known in the art that enable formation of a complex between an antibody and a T cell receptor in a specific manner, i.e., such that the antibody only binds specifically a particular TCR Vβ. The complex between an antibody and a T cell receptor is a detectable product. Methods to detect such complex formation are known to those of skill in the art and include, for example, using a detectable moiety such as a radioisotope, an enzyme or a fluorescent dye, to detect complex formation. Preferred unique amino acid sequences to be identified using antibodies include antibodies that bind specifically to a unique amino acid sequence contained within SEQ ED NO:2, SEQ ID NO:5, SEQ HD NO: 10, SEQ ID NO: 15, SEQ ED NO:20, SEQ ID NO:23, SEQ TD NO:25, SEQ ID NO:29, SEQ TD NO:32, SEQ HD NO:35, SEQ ID NO:38, SEQ TD NO:41, SEQ ID NO:44, SEQ DD NO:47, SEQ DD NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ DD NO:65, SEQ DD NO:66, SEQ TD NO:67, SEQ TD NO:68, SEQ TD NO:69, SEQ ID NO:70, SEQ TD NO:71, SEQ DD NO:72, SEQ DD NO:73, SEQ ED NO:74, SEQ TD NO:75, SEQ ID NO:76, SEQ ED NO:77, SEQ DD NO:78, SEQ DD NO:79 or SEQ ID NO:80. Particularly preferred unique amino acid sequences to be identified using antibodies include antibodies that bind specifically to unique amino acid sequences encoded by a nucleic acid sequence SEQ ID NO: 50, SEQ ED NO:51, SEQ LD NO:52, SEQ ED NO:53, SEQ LD NO:54, SEQ LD NO:55, SEQ ED NO:56.
In another embodiment, identifying the presence of a T cell receptor having a unique nucleic acid sequence can be achieved by PCR amplification and nucleic acid sequencing techniques. Suitable reagents for use with such techniques include DNA primers which are complementary to common sequences (i.e., sequences shared by two or more TCR Vβ molecules) that flank all or a portion of a unique sequence. It is within the skill of one in the art to design and produce suitable primers based on sequences of the present invention. The presence of a unique sequence in a sample from an animal is determined by sequencing the PCR product produced using the common sequence primers and identifying whether one or more nucleic acid sequences are present in the PCR product. An example of such method is described in Example 4 herein. Methods to produce and sequence PCR products are well known to those of skill in the art. T cell expansion is determined by identifying the heterogeneity or homogeneity of nucleic acid sequence displayed in a DNA fingerprint profile of a given sequence in a given sample of PCR products. Preferred reagents include, but are not limited to, DNA primers or probes complementary to unique nucleic acid sequences contained in SEQ LD NO: 1, SEQ ED NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ DD NO:7, SEQ DD NO:8, SEQ DD NO:9, SEQ DD NO:l l, SEQ DD NO:12, SEQ ID NO:13, SEQ TD NO:98, SEQ ΩD NO:100, SEQ TD NO:17, SEQ TD NO:18, SEQ TD NO:19, SEQ TD NO:21, SEQ ID NO:22, SEQ TD NO:24, SEQ ED NO:25, SEQ ED NO:27 and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ DD NO: 60, SEQ DD NO:61, SEQ LD NO:62, SEQ LD NO:63, SEQ ED NO:64, SEQ ED NO:65, SEQ ED NO:66, SEQ ID NO:67, SEQ LD NO:68, SEQ LD NO:69, SEQ LD NO:70, SEQ LD NO:71, SEQ LD NO:72, SEQ ID NO:73, SEQ LD NO:74, SEQ LD NO:75, SEQ LD NO:76, SEQ LD NO:77, SEQ LD NO:78, SEQ ID NO:79 or SEQ LD NO:80, including DNA primers or probes comprising SEQ ED NO:50, SEQ DD NO:51, SEQ DD NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55, SEQ ID NO:56, and/or complements of SEQ ID NO:50, SEQ TD NO:51, SEQ TD NO:52, SEQ TD NO:53, SEQ ID NO:54, SEQ TD NO:55, SEQ DD NO:56; or antibodies that bind specifically to an amino acid sequence encoded by a comprising SEQ TD NO:50, SEQ ID NO:51, SEQ HD NO:52, SEQ HD NO:53, SEQ ED NO:54, SEQ TD NO:55 and/or SEQ ID NO:56, or to other unique amino acid sequences in larger proteins.
One embodiment of the present invention is a method of detecting T cell expansion in an animal comprising detecting the expansion of a T cell receptor having a unique amino acid sequence within a protein selected from the group consisting of hcVβ3, hcVβ4, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82 proteins by forming detectable products, wherein the increased level of a detectable product compared with another detectable product indicates expansion of the T cells. Prefereed Vβ proteins containing unique amino acid sequence include PCaVβ3127, PCaVβ3l u, PCaVβ3U0, PCaVβ4128, PCaVβ4113, PCaVβ4109, PCaVβl2134, PCaVβl2H 1, PCaVβl2115, PCaVβ72133, PCaVβ72113, PCaVβ72114, PCaVβ21130, PCaVβ21108, PCaVβ21116, PCaVβ54I35, PCaVβ54114, PCaVβ54U6, PCaVβl82128, PCaVβ 182, ]0 and/or PCaVβ 182109.
Examples of samples useful a method of the present invention include samples from an animal that contains T cells. Preferably, a sample to be tested using a method of the present invention comprises blood, synovial fluid, lung lavage, saliva, spleen, thymus, tumors, granulomas, abscesses, edematous fluid, central nervous system fluid. Preferred animals from which to isolate a sample of the present invention includes a mammal, more preferably a canid and more preferably a dog.
Any method of the present invention can be used to determine the disease state of an animal. Such methods can be used to determine the presence or absence of disease in an animal, including an animal susceptible to disease, an animal suspected of having disease, an animal having disease or an animal being or having been treated for a disease. Examples of specific diseases that can be diagnosed using a method of the present invention include: various forms of cancer, e.g., lymphoma and leukemia; various autoimmune diseases, e.g., rheumatoid arthritis or diabetes; various infectious diseases, such as those caused by viruses, by a yeast, e.g. of the genus Candida, by a parasite, e.g., Trichinella, Leishmania, Toxoplasma, a filariid, a mycobacterium, a protozoan, and by a bacterium; and allergies involving T cells, e.g. allergic dermatitis, atopic dermatitis, allergic bronchitis or allergic rhinitis.
A prefened embodiment of the present invention is a method to diagnose T cell cancer comprising: (a) contacting a sample containing DNA from an animal with a DNA primer that is complementary to a unique nucleic acid sequence within a nucleic acid molecule including nCaVβ338], nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405, nCaVβ54354, nCaVβ54348, nCaVβl82423, nCaVβl82384, nCaVβl82369 or nCaVβl82327; and (b) diagnosing the cancer by determining the amount of DNA containing the unique nucleic acid sequence by comparing the amount so determined with the amount of DNA containing the unique sequence from a normal animal. According to the present invention, "a DNA primer" refers to one or more primers and "a unique nucleic acid sequence" refers to one or more unique nucleic acid sequences.
Another preferred embodiment of the present invention is a method to diagnose T cell cancer comprising: (a) contacting a sample containing DNA from an animal with a DNA primer that is complementary to a unique nucleic acid sequence within nucleic acid molecule selected from the group consisting of nCaVβ3381, nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405,nCaVβ54354, nCaVβ54348, nCaVβl82423, nCaVβl82384, nCaVβl82369 or nCaVβl82327; and (b) diagnosing cancer by quantitatively determining the ratio of DNA containing each of the unique nucleic acid sequences and by comparing the amount of DNA containing each of the unique sequence with each other.
The present invention also includes a kit comprising one or more reagents of the present invention. Preferred reagents include those which can differentiate TCR V beta proteins or nucleic acid molecules that encode such proteins. Preferred TCR V beta proteins include hcVβ2, hcVβ3, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82, with PCaVβ3127, PCaVβ3 , PCaVβ3110, PCaVβ4128, PCaVβ4113, PCaVβ4109, PCaVβl2,34, PCaVβl2 , PCaVβl2115, PCaVβ72133, PCaVβ72113, PCaVβ72114, PCaVβ21130, PCaVβ21108, PCaVβ21116, PCaVβ54135, PCaVβ54114, PCaVβ54116, PCaVβ 182128, PCaVβl82110and/orPCaVβl82109 being more preferred and proteins comprising SEQ ED NO:2, SEQ TD NO:5, SEQ TD NO: 10, SEQ ED NO: 15, SEQ ID NO:20, SEQ ED NO:23, SEQ LD NO:26, SEQ TD NO:29, SEQ ED NO:32, SEQ ED NO:35, SEQ LD NO:38, SEQ ED NO:41, SEQ LD NO:44, SEQ DD NO:47, SEQ TD NO:60, SEQ DD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ ED NO:64, SEQ ID NO:65, SEQ TD NO:66, SEQ TD NO:67, SEQ ED NO:68, SEQ DD NO:69, SEQ DD NO:70, SEQ ID NO:71, SEQ DD NO:72, SEQ DD NO:73, SEQ DD NO:74, SEQ DD NO:75, SEQ ID NO:76, SEQ HD
NO:77, SEQ TD NO:78, SEQ HD NO:79, SEQ TD NO: 80 and/or proteins encoded by the complement of a nucleic acid sequence including SEQ DD NO:50, SEQ ED NO:51, SEQ LD NO:52, SEQ HD NO:53, SEQ TD NO:54, SEQ TD NO:55, SEQ TD NO:56 being even more preferred. Prefened TCR V beta nucleic acid molecules include hcVβ2, hcVβ3, hcVβl2, hcVβ72, hcVβ21, dtb54 or dtbl82, with nCaVβ3381, nCaVβ3333, nCaVβ3330, nCaVβ4408, nCaVβ4384, nCaVβ4351, nCaVβ4339, nCaVβl2408, nCaVβl2402, nCaVβl2339, nCaVβl2345, nCaVβ72438, nCaVβ72399, nCaVβ72423, nCaVβ72342, nCaVβ21462, nCaVβ21390, nCaVβ21396, nCaVβ21348, nCaVβ54417, nCaVβ54405,nCaVβ54354, nCaVβ54348, nCaVβl82423, nCaVβl82384, nCaVβl82369 and/or nCaVβl82327 being more preferred and nucleic acid molecules comprising sequences SEQ TD NO: 1, SEQ ID
NO:3, SEQ DD NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ DD NO:9, SEQ DD NO:l l, SEQ DD NO:12, SEQ DD NO:13, SEQ DD NO:98, SEQ DD NO:100, SEQ DD NO: 17, SEQ ID NO: 18, SEQ DD NO: 19, SEQ DD NO:21, SEQ ID NO:22, SEQ DD NO:24, SEQ DD NO:25, SEQ TD NO:27,SEQ ΩD NO:28, SEQ TD NO:30, SEQ TD NO:31, SEQ DD NO:33, SEQ DD NO:34, SEQ ED NO:36, SEQ ID NO:37, SEQ TD NO:39, SEQ πD NO:40, SEQ DD NO:42, SEQ πD NO:43, SEQ TD NO:45, SEQ TD NO:46, SEQ DD NO:48, SEQ DD NO:50, SEQ DD NO:51, SEQ DD NO:52, SEQ ID NO:53, SEQ π> NO:54, SEQ DD NO:55, SEQ LD NO:56, a complement of SEQ LD NO:50, SEQ TD NO:51, SEQ ED NO:52, SEQ ED NO:53, SEQ LD NO:54, SEQ LD NO:55, SEQ LD NO:56, and/or a nucleic acid molecule that encodes a protein having an amino acid sequence including SEQ LD NO:60, SEQ ED NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ LD NO:64, SEQ LD NO:65, SEQ ID NO:66, SEQ DD NO:67, SEQ ID NO:68, SEQ ΩD NO:69, SEQ ID NO:70, SEQ TD NO:71, SEQ DD NO:72, SEQ DD NO:73, SEQ πD NO:74, SEQ TD NO:75, SEQ π NO:76, SEQ ID NO:77, SEQ DD NO:78, SEQ ID NO:79 or SEQ ID NO:80, and complements thereof, being even more preferred. A kit of the present invention can include mixtures of reagents disclosed herein.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
EXAMPLES It is to be noted that the examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be familiar to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al., ibid, and Ausubel, et al., 1993, Current Protocols in Molecular Biology, Greene/Wiley Interscience, New York, NY, and related references. It should also be noted that since nucleic acid sequencing technology, and in particular the sequencing of PCR products, is not entirely enor-free, that the nucleic acid and deduced protein sequences presented herein represent apparent nucleic acid sequences of the nucleic acid molecules encoding TCR Vβ proteins of the present invention. Example 1 This example describes the isolation and sequencing of canine T cell receptor
(TCR) Vβ nucleic acid molecules. Canine TCR Vβ nucleic acid molecules were produced as follows. RNA was purified from mitogen activated canine peripheral blood lymphocytes and resuspended in water at about 2.5 X 105 cell equivalent/microliter (μl). About 5 μl of RNA and random hexanucleotide primers were used to synthesize cDNA using the First Strand cDNA Synthesis™ kit (available from Pharmacia, Uppsala, Sweden). TCR Vβ genes were selectively PCR amplified from the cDNA using degenerate oligonucleotides designed using the conserved sequence motifs WYRQ and Y(Y/F)CA of T and B cell antigen receptors (Rast and Litman, Proc. Natl. Acad. Sci. USA, vol. 91, p. 9248, 1994). The degenerate primer FR2, having the nucleic acid sequence 5' CCG AAT TCT GGT A(TC) C(GA) NCA 3' (SEQ LD NO:81) was used in combination with either the FR3A primer, having the nucleic acid sequence 5' CGG ATC CGC (GA)CA (GA)TA (GA)T A 3' (SEQ ID NO:82) or the primer FR3B, having the nucleic acid sequence 5' CGG ATC CGC (GA)CA (GA)A A(GA)T A 3' (SEQ ID NO:83). First round PCR reactions were performed in about 50μl of 50 mM KCI, 10 mM Tris-HCl, 0.01% gelatin (pH 8.3), 3.5 mM MgCl2, 0.2 mM of each of the four deoxyribonucleotide triphosphates (dNTP), about lμM of each primer and about 2.0 units Taq polymerase (available from Perkin- Elmer). This reaction mixture was created after attempts using published methods failed. Moreover, 20 additional cycles of PCR using about 5μl of the first round PCR sample as template under identical conditions were needed to obtain useful second round PCR product. No PCR products were identified after 30 cycles. Second round PCR products were resolved by loading about 15 μl of the second round PCR product onto a 1.0% agarose gel in TBE buffer and staining the gel with ethidium bromide.
No PCR product was obtained using the FR2 primer combined with the FR3A primer. Several bands of DNA were obtained using the FR2 primer combined with the FR3B primer, one of which migrated at a predicted size of about 190 base pair (bp). This band was excised, and the DNA was purified using Qiaquick gel extraction kit (available from Qiagen, Chatsworth, CA) according to the manufacturer's instructions. The purified DNA was cloned into the pCR 2.1 vector (available from Invitrogen, San Diego, CA), and used to transform DH5α E. coli cells (available from Invitrogen). The transformed colonies were grown overnight in about 2 ml LB media containing about 100 μg/ml ampicillin. Plasmid DNA was purified using BioRad's Quantum Prep™ mini-prep kit (available from BioRad, Hercules, CA). Inserted sequence in 11 different plasmid samples were subjected to DNA sequence analysis using standard sequencing methods. Three different nucleic acid sequences were obtained. The 3 different nucleic acid molecules are referred to herein as hcvb3, hcvb4 and hcvbl2. The sequences obtained above indicated that incomplete nucleic acid molecules were obtained using the foregoing PCR procedure. To obtain more complete clones of the PCR products, primers were designed using the nucleic acid sequences obtained above, including primer Phcvb3, having the nucleic acid sequence 5' CCA GAC CTG GGT CTT GTC G 3' (SEQ TD NO:84), primer Phcvb4, having the nucleic acid sequence 5' CTC TGT CCT GGG AGC TGA C 3' (SEQ ID NO:85), and primer Phcvbl2, having the nucleic acid sequence 5' TTG TTT GAT CTA GAG ACT GTG 3' (SEQ ID NO:86). Each primer was then used in combination with the 5' vector primer T3 (available from available from Stratagene Cloning Systems, La Jolla, CA) to amplify PCR products representing Vβ genes from a canine PBL cDNA library generated in the λ Zap II vector (available from Stratagene Cloning Systems, La Jolla, CA). The C. familiaris mitogen activated PBMC cDNA library was constructed in the Uni-ZAP® XR vector (available from Stratagene Cloning Systems), using Stratagene' s ZAP-cDNA® Synthesis Kit and the manufacturer's protocol. The mRNA was isolated from C. familiaris peripheral blood mononuclear cells 4 hours after they were activated by a polyclonal activating agent in culture. PCR reaction were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min, about 94°C for about 30 sec, about 58 °C for about 30 sec, and about 72 °C for about 1 min. for about 35 cycles, then clonal extension was performed at about 72°C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 25 mM MgCl2, about 1 unit Amplitaq Gold™ (available from Perkin-Elmer Cetus), about 200 μM dNTP's and about 1 μM primer.
The resulting PCR products were gel purified, cloned and sequenced using standard methods. The resulting nucleic acid sequences were aligned with the nucleic acid sequences obtained from the first set of PCR products to obtain a more complete sequence of hcvb3, hcvb4 and hcvbl2. Since the PCR primers used to generate the first set of PCR products were degenerate primers, the nucleic acid sequences of hcvb3, hcvb4 and hcvbl2 at the degenerate primer sites were ambiguous. To determine the sequence in the area of the degenerate primers, PCR primers corresponding to the newly derived extreme 5' ends of the hcvb3, hcvb4 and hcvbl2 nucleic acid molecules were designed. Primer 5'Phcvb3, having the nucleic acid sequence 5' ATC GGA CTC CTC TGT GGT GT 3' (SEQ ID NO:87), primer 5'hcvb4, having the nucleic acid sequence 5' ACG GTG AAG GGC TAG CAC CT 3' (SEQ ID NO:88) and primer 5'hcvbl2, having the nucleic acid sequence 5' GCT GAA ATG GCC ACC GGC GT 3' (SEQ ID NO:89), each were used in combination with a primer specific for a sequence in the constant region of a TCR beta chain (SED ID NO: 57) in PCR reactions using the cDNA library described above. The resulting PCR products were purified, cloned into PCR2.1 vector (available from Invitrogen) and sequenced using standard methods.
A. A first clone (hcVβ3) was isolated, referred to herein as nCaVβ338], the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO: 1. SEQ ID NO: 1 includes the V, D and J regions of the sequenced PCR product. The complement of SEQ TD NO: 1 is represented herein by SEQ ID NO:3. Translation of SEQ ID NO:l suggests that nucleic acid molecule nCaVβ3381 encodes a TCR Vβ protein of about 127 amino acids, denoted herein as PCaVβ3127, the amino acid sequence of which is presented in SEQ ID NO:2, assuming an open reading frame having a first codon spanning from nucleotide 1 through nucleotide 3 of SEQ TD NO:l and a last codon spanning from nucleotide 379 through nucleotide 381 of SEQ LD NO: 1. The putative signal sequence extends from nucleotide 1 to nucleotide 51 of SEQ LD NO:l. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ3110, contains about 110 amino acids, extending from residue 18 through residue 127 of SEQ ID NO:2. The nucleic acid molecule encoding PCaVβ3U0 is denoted herein as nCaVβ3330, extending from nucleotide 52 through nucleotide 381 of SEQ ID NO:l.
Comparison of nucleic acid sequence SEQ DD NO:l with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 1 showed the most homology, i.e., about 69% identity, between SEQ ID NO: 1 and a human TCR β chain gene (Genbank Accession No. Z223040). Comparison of amino acid sequence SEQ ID NO:2 with amino acid sequences reported in GenBank indicates that SEQ ID NO:2 showed the most homology, i.e., about 65% identity, between SEQ ID NO:2 and an Ovis aries TCR Vβ chain protein (Genbank Accession No. gi 2665554).
B. A second clone (hcVβ4) was isolated, refened to herein as nCaVβ4408, the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO:4. SEQ TD NO:4 includes the V, D and J regions of the sequenced PCR product. The complement of SEQ ID NO:4 is represented herein by SEQ ID NO:6. Translation of SEQ ID NO:4 suggests that nucleic acid molecule nCaVβ4408 encodes a TCR Vβ protein of about 128 amino acids, denoted herein as PCaVβ4128, the amino acid sequence of which is presented in SEQ ID NO:5, assuming an open reading frame having an initiation codon spanning from nucleotide 24 through nucleotide 26 of SEQ ID NO:4 and a last codon spanning from nucleotide 405 through nucleotide 407 of SEQ ID NO:4. The coding region encoding PCaVβ4128 is presented herein as nCaVβ4384, which has the nucleotide sequence SEQ ID NO:6 (the coding strand) and SEQ DD NO:7 (the complementary strand). The putative signal sequence extends from nucleotide 25 to nucleotide 69 of SEQ TD NO:4. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ4113, contains about 113 amino acids, extending from residue 60 through residue 128 of SEQ ID NO:5. The nucleic acid molecule encoding PCaVβ41 ]3 is denoted herein as nCaVβ4339, extending from nucleotide 70 through nucleotide 408 of SEQ TD NO:4. Comparison of nucleic acid sequence SEQ ID NO:4 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:4 showed the most homology, i.e., about 75% identity, between SEQ ID NO:4 and a human TCR β chain gene (Genbank Accession No. M97713). Comparison of amino acid sequence SEQ ID NO:5 with amino acid sequences reported in GenBank indicates that SEQ ID NO: 5 showed the most homology, i.e., about 69% identity, between SEQ ID NO:5 and an Ovis aries TCR Vβ chain protein (Genbank Accession No. gi 2665558).
C. A third clone (hcVβl2) was isolated, referred to herein as nCaVβl2408, the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO:9. SEQ ID NO:9 includes the V, D and J regions of the sequenced PCR product. The complement of SEQ ID NO:9 is represented herein by SEQ ID NO: 11. Translation of SEQ ID NO:9 suggests that nucleic acid molecule nCaVβl2408 encodes a TCR Vβ protein of about 134 amino acids, denoted herein as PCaVβl2134, the amino acid sequence of which is presented in SEQ ID NO: 10, assuming an open reading frame having an initiation codon spanning from nucleotide 7 through nucleotide 9 of SEQ ID NO:9 and a last codon spanning from nucleotide 406 through nucleotide 408 of SEQ ED NO:9. The coding region encoding PCaVβl2134 is presented herein as nCaVβl2402, which has the nucleotide sequence SEQ ID NO: 12 (the coding strand) and SEQ ID NO: 13 (the complementary strand). The putative signal sequence extends from nucleotide 7 to nucleotide 63 of SEQ ID NO:9. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβl2H5, contains about 115 amino acids, extending from residue 20 through residue 134 of SEQ ID NO: 10. The nucleic acid molecule encoding PCaVβl2115 is denoted herein as nCaVβl2345, extending from nucleotide 64 through nucleotide 408 of SEQ ID NO:9.
Comparison of nucleic acid sequence SEQ ID NO:9 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:9 showed the most homology, i.e., about 72% identity, between SEQ LD NO:9 and a Macaca mulatta TCR β chain mRNA (Genbank Accession No. U04578). Comparison of amino acid sequence SEQ ID NO: 10 with amino acid sequences reported in GenBank indicates that SEQ ID NO: 10 showed the most homology, i.e., about 57% identity, between SEQ ED NO: 10 and a human TCR Vβ protein (Genbank Accession No. 138312). Example 2
This example describes the isolation and sequencing of two additional canine TCR Vβ nucleic acid molecules.
Two canine TCR Vβ nucleic acid molecules were PCR amplified from the canine PBL cDNA library described above in Example 1. A pair of primers was used to amplify DNA from the cDNA library. The 5' vector primer T3, described in Example 1, was used in combination with primer Phcvb21, having the nucleic acid sequence 5' CTG TTG CCC ACG TTA GAG G 3' (SEQ DD NO:90) or primer Phcvb72, having the nucleic acid sequence 5' TTA CTG AAC TGC TGC ACT G 3' (SEQ ID NO:91). PCR reaction were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min., about 94°C for about 30 sec, about 60°C for about 30 sec, about 72 °C for about 1 min. for about 35 cycles; then clonal extension was performed at about 72 °C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl2, about 1 unit Amplitaq Gold, about 200 μM dNTP's and about 1 μM primer. The resultant PCR products obtained using standard PCR conditions (e.g., Sambrook et al., ibid.), were gel purified, cloned and sequenced. The PCR products are refened to herein as hcvb21 or hcvb72, respectively.
The sequences obtained were compared with sequences disclosed in Ito et al., Immuno genetics, vol. 38, p. 60, 1993 or Takano et al., Immunogenetics, vol. 40, p. 246, 1994. The PCR products hcvb21 or hcvb72 were found to contain more 5' nucleic acid sequence than that disclosed in the above-referenced publications. To obtain more complete nucleic acid molecules containing the V, D and J regions, PCR amplification was performed using primers designed from the 5' sequence obtained from hcvb21 or hcvb72 nucleic acid molecules. Primer 5'Phcvb21, having the nucleic acid sequence 5' GCT GCA GGA TTC GGC ACG AG 3' (SEQ ID NO:92) or primer 5'Phcvb72, having the nucleic acid sequence 5' TAC GAC TGT CAG CTT GGT CC 3' (SEQ ID NO:93), each were used in conjunction with a TCR beta constant region primer (SEQ ID NO:57) to amplify these sequences from mRNA prepared from canine concavalin A (ConA) activated PBMC. The PCR products were cloned and sequenced using standard methods.
A. The clone hcVβ21 was isolated, referred to herein as nCaVβ21462, the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO: 19. SEQ ID NO: 19 includes the V, D and J regions of the sequenced PCR product. The complement of SEQ DD NO: 19 is represented herein by SEQ ID NO:21. Translation of SEQ ID NO: 19 suggests that nucleic acid molecule nCaVβ21462 encodes a TCR Vβ protein of about 130 amino acids, denoted herein as PCaVβ21130, the amino acid sequence of which is presented in SEQ ID NO:20, assuming an open reading frame having an initiation codon spanning from nucleotide 73 through nucleotide 75 of SEQ ID NO: 19 and a last codon spanning from nucleotide 460 through nucleotide 462 of SEQ ID NO: 19. The coding region encoding PCaVβ21130 is presented herein as nCaVβ21390, which extends from nucleotide 73 to nucleotide 462 of SEQ ID NO: 19. The putative signal sequence extends from nucleotide 73 to nucleotide 114 of SEQ ID NO: 19. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ21] 16, contains about 116 amino acids, extending from residue 15 through residue 130 of SEQ ID NO: 19. The nucleic acid molecule encoding PCaVβ211 ]6 is denoted herein as nCaVβ2134g, extending from nucleotide 115 through nucleotide 462 of SEQ ID NO: 19.
A comparison of SEQ ID NO: 19 with the DNA sequence DTCRB21 described in Ito et al., ibid, indicated that SEQ ID NO: 19 is 221 nucleotides longer than at the 5'end of DTCRB21. The sequences of SEQ ID NO: 19 and DTCR21 overlap by 176 nucleotides with 100% identity, corresponding to nucleotides 222 through 398 of SEQ LD NO:19. An alignment of SEQ LD NO:19 with DTCR21 is shown in Table 1. TABLE 1. Nucleotide Sequence Comparison of hcvb21 and DTCRB21
DTCRB21
SEQ ID NO: 19 GCTGCAGGAT TCGGCACGAG GCGTGGTCAT ATCTATCTTG AGAGAGGTAT
DTCRB21
SEQ ID NO: 19 GGTATGAGGC CATCACCTGA AGATGCTGAT GCTTCTGCTG CTCCTGGGGC
DTCRB21
SEQ ID NO: 19 CCAGCTCTGG ACTCGGTGCC CTCGTCTTCC AGGCGCCCAG CACAATGATC DTCRB21
SEQ ID NO: 19 TGTAAGAGCG GAGCCACCGT GCAGATCCAG TGTCAAACAG TGGACCTTCA
DTCRB21 TCGCCAGCT CCCGAAGCAG GGCCTTACCC
SEQ ID NO: 19 AGCCACAACC GTGTTTTGGT ATCGCCAGCT CCCGAAGCAG GGCCTTACCC
DTCRB21 TTATGGTGAC CTCTAACGTG GGCAACAGTG CTACACACGA GCAGGGGTTC SEQ ID NO: 19 TTATGGTGAC CTCTAACGTG GGCAACAGTG CTACACACGA GCAGGGGTTC
DTCRB21 CCTGCAGCCA AGTTCCCTGT TAACCACCCA AACCTCACGT TTTCCTCCCT
SEQ ID NO: 19 CCTGCAGCCA AGTTCCCTGT TAACCACCCA AACCTCACGT TTTCCTCCCT
DTCRB21 GATGGTGACG AGTTCAGGTC CTGGAGACAG CGGCCTCTAC TTCTGTGGCT
SEQ ID NO: 19 GATGGTGACG AGTTCAGGTC CTGGAGACAG CGGCCTCTAC TTCTGTGGTG DTCRB21 ACC ... TACA GGGCGCGCGC TACGAGCAGT ATTTCGGCGC CGGCACCAGG
SEQ ID NO: 19 TTCGGGCGTA TGGTGGGAAC TCGCCCCTCT ACTTTGGAAC AGGCACCAGG
DTCRB21 CTCACGGTCC TC
SEQ ID NO: 19 CTCACCGTGA CA The amino acid sequence SEQ HD NO:20 is 50 amino acids longer at the 5'end than the amino acid sequence encoded by DTCR21. An alignment of SEQ ED NO:20 with the amino acid sequence encoded by DTCR21 is shown in Table 2. TABLE 2. Amino Acid Sequence Comparison of hcvb21 and DTCR21. SEQ ID NO: 20 MLMLLLLLGP SSGLGALVFQ APSTMICKSG ATVQIQCQTV DLQATTVFWY DTCRB21
SEQ ID NO: 20 RQLPKQGLTL MVTSNVGNSA THEQGFPAAK FPVNHPNLTF SSLMVTSSGP DTCRB21 RQLPKQGLTL MVTSNVGNSA THEQGFPAAK FPVNHPNLTF SSLMVTSSGP
SEQ ID NO: 20 GDSGLYFCGV RAYGGNSPLY FGTGTRLTVT DTCRB21 GDSGLYFCGY . QGARYEQY FGAGTRLTVL
The amino acid sequence SEQ ID NO:20 is 50 amino acids longer at the 5'end than the amino acid sequence encoded by DTCR21. An alignment of SEQ LD NO:20 with the amino acid sequence encoded by DTCR21 is shown in Table 2.
Comparison of nucleic acid sequence SEQ ID NO: 19 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 19 showed the most homology, i.e., about 38% identity, between SEQ HD NO: 19 and a dog TCR β chain gene (Genbank Accession No. M97510). Comparison of amino acid sequence SEQ ID NO:20 with amino acid sequences reported in GenBank indicates that SEQ ID NO:20 showed the most homology, i.e., about 60% identity, between SEQ ID NO:20 and DTCR21 protein.
B. The clone hcVβ72 was isolated, referred to herein as nCaVβ72438, the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO:98. SEQ ED NO:98 includes the V, D and J regions of the sequenced PCR product. The complement of SEQ ID NO:98 is represented herein by SEQ ID NO: 100. Translation of SEQ ID NO:98 suggests that nucleic acid molecule nCaVβ72438 encodes a TCR Vβ protein of about 133 amino acids, denoted herein as PCaVβ72133, the amino acid sequence of which is presented in SEQ ID NO: 15, assuming an open reading frame having an initiation codon spanning from nucleotide 40 through nucleotide 42 of SEQ ID NO:98 and a last codon spanning from nucleotide 436 through nucleotide 438 of SEQ ID NO:98. The coding region encoding PCaVβ72133 is presented herein as nCaVβ72399, which has the nucleotide sequence SEQ ID NO: 17 (the coding strand) and SEQ LD NO: 18 (the complementary strand). The putative signal sequence extends from nucleotide 40 to nucleotide 96 of SEQ TD NO:98. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ72]]4, contains about 114 amino acids, extending from residue 20 through residue 133 of SEQ HD NO: 15. The nucleic acid molecule encoding
PCaVβ72u4 is denoted herein as nCaVβ72342, extending from nucleotide 142 through nucleotide 438 of SEQ ID NO: 19.
A comparison of SEQ LD NO:98 with the DNA sequence DTB72 described in Takano et al., ibid, indicated that SEQ ID NO:98 differs substantially from the published canine TCR Vβ sequence. A comparison between SEQ ID NO:98 and DTB72 is shown in Table 3. TABLE 3. Nucleotide Sequence Comparison of hcvb72 and DTB72
DTB72
SEQ ID NO: 98 CACGA DTB72 CAGC TTCCCAGGGC TGCCATGGGC TCCAGGCTTC
SEQ ID NO: 98 GGAGCGGGGA GGCTATCAGC TTCCCAGGGC TGCCATGGGC TCCAGGCTTC
DTB72 TCTGCTGTGT GGCCCTTTTC TCCTGGGAGC CGGCCCCCGT GGAGTCTGAG SEQ ID NO: 98 TCTGCTGTGT GGCCCTTTGT CTCCTGGGAG CCGGCCCCGT GGAGTCTGAG
DTB72 GTCATCCAAA CTCCAAGACA CATGATCAAA GTCAAGAGGA CAGACAGTGA SEQ ID NO: 98 GTCATCCAAA CTCCAAGACA CATGATCAAA G . CAAGAGGA CAGACAGTGA
DTB72 CC...TGAGA TGTCCTTATC TCTGGACA.C TATCTGTGTA CTGGTACCAA SEQ ID NO: 98 CCCTGAGATG TTCCCTTATC TCTGGACACC TATCTGTGTA CTGGTACCAA
DTB72 CAGGCCTTGA TGGTCCGTTT ACCGGTTTCT CATTCAGT C
SEQ ID NO: 98 CAGGCCCTG. GGCCAGGGTC CCCGGTTTCT CATTCAGTAT TACAATAGGG DTB72 ATCATAGTCA AAAAAGAAAC ATCCGGTCAA GATTCTCAGT GCAGCAGTTC SEQ ID NO: 98 AAGAGAGAGA CAAAGGAGAC ATCCCGGCAA GATTCTCAGT GCAGCAGTTC
DTB72 AGTAACTACA GCATCCCAGC TTGAGATGAA CTCCCTGGAG CCAGGAGACT SEQ ID NO: 98 AGTAACTACA GC.TCCCAGC TGGAGATGAA CTCCCTGGAG CCAGGAGACT
DTB72 CAGCCCTATA TCTCTGTGCC AGCAGC ... G GGTACAGTGA GAGCTACGAG SEQ ID NO: 98 CAGCCCTATA TCTCTGTGCC AGCAGCTTAG ATGCGTTCGA CGCGGGGCAG
DTB72 CGGTATTTCG GAGCCGGCAC CAGGCTCACG GTCCTC SEQ ID NO: 98 CTGTACTTCG GGGCCGGTTC CAAGCTGGCC GTGCTG
Comparison of SEQ LD NO:98 with DTB72 indicates that the identity between the two sequences is about 90%, when determined using the Compare function by maximum matching within the program DNAsis™ Version 2.1. A comparison of the amino acid re¬
sequence SEQ LD NO: 15 and the amino acid sequence encoded by DTB72 indicates that the identity between the two sequences is only 57%, when determined using the Compare function by maximum matching within the program DNAsis™ Version 2.1. An alignment of the two sequences is shown in Table 4. TABLE 4. Amino Acid Sequence Comparison of hcvb72 and DTB72
SEQ ID NO: 15 MGSRLLCCVA LCLLGAGPVE SEVIQTPRHM IKARGQTVTL RCSLISGHLS DTB 2 MGSRLLCCVA LFSWEPAPVE SEVIQTPRHM IKVKRTDSDL RCPYL.WTLS
SEQ ID NO: 15 VYWYQQAL.G QGPRFLIQYY NREERDKGDI PARFSVQQFS NYSSQLEMNS DTB72 VYWYQQALMV RLPVSHSVII VKKETSGQDS QCSSSV.... TTASQLEMNS
SEQ ID NO: 15 LEPGDSALYL CASSLDAFDA GQLYFGAGSK LAVL DTB72 LEPGDSALYL CASS.GYSES YERYFGAGTR LTVL
The comparison of the two sequences indicates that SEQ ID NO:98 and DTB72 encode different proteins .
Comparison of nucleic acid sequence SEQ ID NO: 19 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 19 showed the most homology, i.e., about 60% identity, between SEQ ID NO: 19 and a feline leukemia virus transduced TCR β chain gene (Genbank Accession No. X05155). Comparison of amino acid sequence SEQ HD NO:20 with amino acid sequences reported in GenBank indicates that SEQ ID NO:20 showed the most homology, i.e., about 65% identity, between SEQ TD NO:20 and a a feline leukemia virus transduced TCR β chain protein (Genbank Accession No. RWMVTV). Example 3 This example describes the production of two canine TCR Vβ nucleic acid molecules.
Two canine TCR Vβ nucleic acid molecules were PCR amplified from the canine PBL cDNA library described above in Example 1. A pair of primers was used to amplify DNA from the cDNA library. The 5' vector primer T3, described in Example 1, was used in combination with primer Pdtb54, having the nucleic acid sequence 5'
CTT TTG CTG GGA TCT GCT GA 3' (SEQ ID NO:94) or primer Pdtbl82, having the nucleic acid sequence 5' CAG TTG CTT AG GTC TTG CT 3' (SEQ ID NO:95). The resultant PCR products obtained using standard PCR conditions (e.g., Sambrook et al., ibid.), were gel purified, cloned and sequenced. The PCR products are referred to herein as dtb54 or dtbl 82, respectively.
The sequences obtained were compared with sequences disclosed in Takano et al., ibid. The PCR products dtb54 or dtbl 82 were found to contain more 5' nucleic acid sequence than that disclosed in the above-referenced publication. To obtain more complete nucleic acid molecules containing the V, D and J regions, PCR amplification was performed using primers designed from the 5' sequence obtained from dtb54 or dtbl 82 nucleic acid molecules. Primer 5'Pdtb54, having the nucleic acid sequence 5' CAC GAG CCT GCC ATG TGC CC 3' (SEQ ID NO:96) or primer 5'Pdtbl82, having the nucleic acid sequence 5' GGC ACG AGC ACT GAG GAC CA 3' (SEQ TD NO:97), each were used in conjunction with a TCR beta constant region primer (SEQ DD NO:57) to amplify these sequences from mRNA prepared from canine concavalin A (ConA) activated PBMC. The PCR products were cloned and sequenced using standard methods. A. The clone dtb54 was isolated, referred to herein as nCaVβ54417, the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO:22. SEQ LD NO:22 includes the V, D and J regions of the sequenced PCR product. The complement of SEQ ID NO:22 is represented herein by SEQ ID NO:24. Translation of SEQ ID NO:22 suggests that nucleic acid molecule nCaVβ54417 encodes a TCR Vβ protein of about 135 amino acids, denoted herein as PCaVβ54135, the amino acid sequence of which is presented in SEQ ID NO:23, assuming an open reading frame having an initiation codon spanning from nucleotide 13 through nucleotide 15 of SEQ DD NO:22 and a last codon spanning from nucleotide 415 through nucleotide 417 of SEQ ID NO:22. The coding region encoding PCaVβ54]35 is presented herein as nCaVβ54405, which extends from nucleotide 13 to nucleotide 417 of SEQ ID NO:22. The putative signal sequence extends from nucleotide 13 to nucleotide 69 of SEQ ID NO:22. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ54]16, contains about 116 amino acids, extending from residue 20 through residue 135 of SEQ ID NO:22. The nucleic acid molecule encoding PCaVβ541]6 is denoted herein as nCaVβ54348, extending from nucleotide 70 through nucleotide 417 of SEQ LD NO:22. A comparison of SEQ ED NO:22 with the DNA sequence DTB54 described in Takano et al., ibid, indicated that the sequences are substantially similar except for 12 additional nucleotides at the 5'end and an additional amino acid at residue 55 in SEQ TD NO:22. B. The clone dtbl 82 was isolated, refened to herein as nCaVβl82423, the coding strand of which was shown to have a nucleic acid sequence denoted herein as SEQ ID NO:25. SEQ ID NO:25 includes the V, D and J regions of the sequenced PCR product. The complement of SEQ TD NO:25 is represented herein by SEQ ID NO:27. Translation of SEQ ID NO:25 suggests that nucleic acid molecule nCaVβl82423 encodes a TCR Vβ protein of about 128 amino acids, denoted herein as PCaVβl 82128, the amino acid sequence of which is presented in SEQ ED NO: 26, assuming an open reading frame having an initiation codon spanning from nucleotide 40 through nucleotide 43 of SEQ ID NO:25 and a last codon spanning from nucleotide 421 through nucleotide 423 of SEQ ID NO:25. The coding region encoding PCaVβl 82128 is presented herein as nCaVβl82384, which extends from nucleotide 40 to nucleotide 423 of SEQ ID NO:25. The putative signal sequence extends from nucleotide 40 to nucleotide 96 of SEQ ID NO:25. The proposed mature protein (i.e., canine TCR Vβ protein from which the signal sequence has been cleaved), denoted herein as PCaVβ 182]09, contains about 109 amino acids, extending from residue 20 through residue 128 of SEQ ID NO:25. The nucleic acid molecule encoding PCaVβ 182109 is denoted herein as nCaVβ 182327, extending from nucleotide 97 through nucleotide 423 of SEQ ED NO:25.
A comparison of SEQ ID NO:25 with the DNA sequence DTB182 described in Takano et al., ibid, indicated that the sequences are substantially similar except for 84 additional nucleotides at the 5'end of SEQ ID NO:25. Example 4
This example describes the identification of unique TCR Vβ sequences by designing PCR primers that distinguish between different (TCR) Vβ nucleic acid molecules.
Seven different primers for PCR reactions were designed to amplify DNA from seven different TCR Vβ nucleic acid molecules. The primers were designed based on the nucleic acid sequences SEQ ID NO:l, SEQ LD NO:4, SEQ ID NO:9, SEQ LD NO:98, SEQ TD NO: 19, SEQ ID NO:22 or SEQ ID NO:25. The inventors discovered unique sequences for each of the foregoing nucleic acid molecules that are not shared between each of the different molecules. In addition, the inventors discovered which primers designed based on those unique sequences, and under what specific conditions, did not cross-prime between the different nucleic acid molecules. For some Vβ genes, several different Vβ primers to unique sequences had to be tested in order to find one that was specific to only one Vβ gene. Some of the primers that were designed include: primer hcVβ3 unique, having the nucleic acid sequence 5' CGA CAA GAC CCA GGT CTG G 3' (SEQ ID NO:50), complementary to nucleotides 157 to 175 of SEQ ID NO: 1 ; primer hcVβ4 unique, having the nucleic acid sequence 5' GTC AGC TCC CAG GAC AGA G
3' (SEQ ID NO:51), complementary to nucleotides 176 to 194 of SEQ ID NO:4; primer hcVβl2 unique, having the nucleic acid sequence 5' CAT GAC CTG GGA CAT GGG C 3' (SEQ ID NO:52), complementary to nucleotides 172 to 190 of SEQ LD NO:9; primer hcVβ72 unique, having the nucleic acid sequence 5' GAG ATG TTC CCT TAT CT CTGG 3' (SEQ ID NO:53), complementary to nucleotides 201 to 226 of SEQ ED NO:98; primer hcVβ21 unique, having the nucleic acid sequence 5' CCT CTA ACG TGG GCA ACA G 3' (SEQ TD NO:54), complementary to nucleotides 260 to 278 of SEQ ID NO: 19; primer dtb54 unique, having the nucleic acid sequence 5' TCA GCA GAT CCC AGC AAA AG 3' (SEQ ID NO:55), complementary to nucleotides 174 to 193 of SEQ ED NO:22; and primer dtbl82 unique, having the nucleic acid sequence 5' AGC AAG ACC TCA AGC AAC TG 3' (SEQ ID NO:56), complementary to nucleotides 203 to 222 of SEQ ID NO:25. The ability of each primer to specifically prime a specific TCR Vβ gene was tested by performing PCR reactions using each of the above primers in combination with a Vβ constant region primer including: primer Cβl, having the nucleic acid sequence 5' GTG ACC TTC TGC AGA TCC TC 3' (SEQ ED NO:57); primer Cβ2, having the nucleic acid sequence 5' AGC TCA GCT CCA CGT GGT C 3' (SEQ ID NO:58); or primer Cβ3, having the nucleic acid sequence 5' TGC TGA ACC CAC TCG TGA C 3' (SEQ DD NO:59).
The specificity of these primers was first tested using 7 different DNA plasmids that contained nucleic acid molecules comprising either SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:9, SEQ LD NO:98, SEQ LD NO: 19, SEQ LD NO:22 or SEQ LD NO:25 as templates for different PCR reaction. Each unique Vβ primer was used to amplify DNA from each template. PCR reactions were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min., about 94°C for about 30 sec, about 60 °C for about 30 sec, about 72 °C for about 1 min. for about 35 cycles; then clonal extension was performed at about 72 °C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl2, about 1 unit Amplitaq Gold, about 0.1 μM dNTP's and about 0.4 μM primer. The results indicated that PCR products were only present when the conect template and primer combination was used. As such, only PCR products were present when primer hcVβ3 unique was used with a plasmid containing SEQ ID NO: 1 , primer hcVβ4 unique was used with a plasmid containing SEQ ID NO:4, primer hcVβl2 unique was used with a plasmid containing SEQ ID NO:9, primer hcVβ72 unique was used with a plasmid containing SEQ ID NO:98, primer hcVβ21 unique was used with a plasmid containing SEQ ID NO: 19, primer dtb54 unique was used with a plasmid containing SEQ ID NO:22 or primer dtbl82 unique was used with a plasmid containing SEQ ID NO:25.
The ability of the Vβ unique primers to prime DNA of the predicted size for different Vβ genes using cDNA from a canine ConA activated T cell population as template material was tested. The same primers and PCR amplification conditions described immediately above were used in these experiments. The resulting PCR products were resolved by electrophoresis on an about 1.2% LE agarose gel in TBE buffer and stained with ethidium bromide. The results shown in Fig. IA indicated that all seven Vβ unique primers were able to prime DNA fragments of the correct size. To confirm that only a specific Vβ gene was amplified by each Vβ primer, the DNA bands shown in Fig. 1 A were extracted from the gel and cloned. Different clones containing DNA from the different bands were sequenced. The sequencing results indicated that each Vβ unique primer conectly primed only one Vβ gene containing sequence complementary to that primer. Example 5 This example describes a method for identifying T cell expansion.
About 105 lymph node cells or PBMC were isolated from 2 dogs known to have lymphoma (Haynes and Stoll) and a control dog. cDNA samples from the cells were used as templates in separate PCR reactions using primer hcVβ3 unique, primer hcVβ4 unique, primer hcVβl2 unique, primer hcVβ72 unique, primer hcVβ21 unique, primer dtb54 unique or primer dtbl 82 unique, in combination with Cβ3 primer (SEQ DD
NO:59). PCR reactions were performed using the following conditions: Taq activation was performed at about 95°C for about 10 min., about 94°C for about 30 sec, about 60°C for about 30 sec, about 72°C for about 1 min. for about 35 cycles; then clonal extension was performed at about 72 °C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl2, about 1 unit Amplitaq Gold, about 0.1 μM dNTP's and about 0.4 μM primer. PCR products were resolved by electrophoresis on an about 1.2% LE agarose gel in TBE buffer and stained with ethidium bromide.
The results shown in Fig. IA indicated that PCR products using cells isolated from the control dog were generated using all 7 Vβ specific primers. In addition, the levels of PCR products were substantially the same using the 7 Vβ specific primers. The results shown in Fig. IB and IC indicated that the Vβ profiles of PCR products using cells isolated from the lymphoma dog Haynes, or the lymphoma dog Stoll, were different from the Vβ profile of the control dog. In particular, T cells expressing hcVβ21 genes have been expanded in both patients, while T cells expressing the other 6 Vβ genes have decreased in proportion.
Taken together, the results disclosed in Examples 3 and 4 indicate that primers complementary to unique Vβ sequences of the present invention can be used to: (1) detect the presence of specific Vβ genes in a population of cells; (2) identify clonal expansion of cells expressing a particular Vβ gene; and (3) associate clonal T cell expansion with an abnormal state or disease; or (4) distinguish a general lymphoproliferative state involving polyclonal T cell activation from clonal T cell expansion. Example 6 This example describes a method for DNA fingerprinting the junctional regions of rearranged Vβ genes to identify clonal expansion of T cells.
DNA sequence analysis of the junction between the V, D and J regions of a Vβ gene can determine whether a mRNA population that translates into a Vβ protein is homogenous or heterogeneous by looking at the fluorescent DNA fingerprint. Fluorescent DNA fingerprints of Vβ mRNA populations were determined as follows. cDNA was prepared using standard methods from mitogen stimulated canine PBL cells isolated from a control dog and from lymph node cells isolated from the lymphoma dog Haynes or. The cDNA samples from either Haynes or the control dog were used as templates for PCR reactions using variable region primers in combination with constant region primers. Use of such primers were designed so that the resulting PCR products span the D/J junction of a Vβ cDNA. Primer hcVβ3 unique, primer hcVβ4 unique, primer hcVβl2 unique, primer hcVβ72 unique, primer hcVβ21 unique, primer dtb54 unique or primer dtbl 82 unique, was used in combination with the Cβ3 primer (SEQ LD NO:59). PCR reactions were performed using the following conditions: Taq activation was performed at about 95 °C for about 10 min., about 94°C for about 30 sec, about 60°C for about 30 sec, about 72°C for about 1 min. for about 35 cycles; then clonal extension was performed at about 72°C for about 5 min.; PCR was performed in a reaction buffer containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin, pH 3, 3 mM MgCl2, about 1 unit Amplitaq Gold, about 0.1 μM dNTP's and about 0.4 μM primer.
About 20-25 μl of each resulting PCR product was resolved by electrophoresis on an about 1.2% LE agarose gel in TBE buffer and stained with ethidium bromide. DNA bands of about 400 bp identified on the gel were excised and the DNA purified. The purified DNA was sequenced using the Cβ2 primer (SEQ ID NO:58) using standard fluorescent dyes and an automated DNA sequencer. Fluorescent electrophoretic histograms were generated for sequence obtained using each primer and each template. Fig. 2 illustrates 7 histograms generated using cDNA from the control dog and each of the 4 Vβ primers. The histograms show the typical heterogeneity of a T cell population in a normal dog. These histograms indicate that the DNA sequences of a particular amplified Vβ PCR product can be determined through the first about 120 bp of the Vβ constant region, but become ambiguous as the sequence profile enters the heterogenous J/D junctional regions.
Fig. 3 illustrates 4 histograms generated using cDNA from the lymphoma dog Haynes and Vβ primers hcVβl2 unique, hcVβ72 unique, hcVβ21 unique, dtb54 unique or dtbl 82 unique. The histograms show that 3 of the 4 Vβ genes amplified have fingerprints similar to those seen with a heterogeneous population of T cells, such as shown in Fig. 2. The histogram generated using the hcvb21 unique primer has a fingerprint which allows the unambiguous determination of the DNA sequence throughout the entire junctional region between the V, D, J and C regions. This result indicates that the sequence recognized by the hcvb21 unique primer was dominant among the canine PBL cell population from the lymphoma dog.
A comparison of the histogram generated using the hcvb21 unique primer with cDNA from the normal dog with the histogram generated using the hcvb21 unique primer with cDNA from the lymphoma dog is shown in Fig. 4. The comparison illustrates that the difference between the two histograms can be used to determine clonal expansion of a single T cell and association of such expansion to an abnormal state or disease. Example 7
This example describes the generation of T cell clones reactive to flea saliva allergens, and the characterization of the TCR Vβ genes used by the T cell receptors of the T cell clones.
It is to be noted that this example includes a number of cellular immunology techniques considered to be familiar to those skilled in the art. Disclosure of such techniques can be found, for example, in Coligan et al., Current Protocol in Immunology, Wiley Interscience, New York. T cell clones reactive to flea saliva antigens were generated from peripheral blood mononuclear cells (PBMC) isolated from the experimentally-induced flea allergic dog CPO2 (described in U.S. Patent No. 5,646,115, issued on July 8, 1997) as follows. Blood samples were isolated from experimentally-induced flea allergic dog CPO2. Peripheral blood lymphocytes (PBL) were harvested from each sample by centrifugation using a ficoll gradient (available from Pharmacia). The PBL cells were cultured in about 5 ml cultures in culture medium containing LMDM and 10% fetal calf serum (available from Gibco, Gaithersburg, MD) at about 5 X 106 cells/ml in the presence of about 5 μg/ml flea saliva protein (prepared according to the methods described in U.S. Patent No. 5,646,115) for about 14 days, at about 37 °C, in about 5% CO2. The incubated cells were harvested and the number of viable cells determined. The viable cells were plated in 96 well round bottom plates at about 102 or about 103 cells per well per 200 μl of culture medium in the presence of about 105 autologous irradiated PBL (prepared according to the methods generally described in Coligan, ibid.) as antigen presenting cells (APC), about 5 μg/ml of flea saliva protein, and about 10 units/ml of recombinant hLL-2 for another 14 days. Wells that contain growing cells were restimulated in situ by replacing about 150 μl of spent culture medium (i.e., medium in which cells had been grown) with about 150 μl of fresh culture medium, flea saliva protein and APC as described above. About 10-14 days later, cultures in which the T cells were actively proliferating were transferred into 48 well plates, and tested for antigen specificity by comparing growth of the cells in the presence of flea saliva protein and APC, with growth of cells in the presence of APC alone. Cells that required flea saliva protein and APC to grow were selected and expanded in the presence of APC and flea saliva protein. These expanded cells are refened to as T cell clones. Seven different T cell clones were derived from cells isolated from CPO2. mRNA was prepared from about 106 cells from each of the 7 clones using standard methods. cDNA was prepared from the mRNA using methods described above in Example 1. The cDNA samples from the 7 clones were then used as templates in PCR reactions to determine the presence of particular TCR Vβ molecules using the methods described above in Example 4. Analysis of the resulting PCR products indicated that 6 of the 7 clones expressed TCR using the dtbl 82 V beta chain, thereby indicating a bias in TCR Vβ usage in the T cell reactivity of CPO2 to flea saliva protein.
While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed is:
1. An isolated protein comprising a protein selected from the group consisting of:
(a) an isolated protein having an amino acid sequence that is at least about 55 percent identical to an amino acid sequence selected from the group consisting of SEQ TD NO:2, SEQ HD NO:29, SEQ TD NO:60, SEQ TD NO:61, and SEQ HD NO:62, or a fragment thereof that is at least about 25 amino acids in length;
(b) an isolated protein having an amino acid sequence that is at least about 75 percent identical to an amino acid sequence selected from the group consisting of SEQ LD NO:5, SEQ ED NO:32, SEQ ID NO:63, SEQ LD NO:64, and SEQ ED NO:65, or a fragment thereof that is at least about 15 amino acids in length;
(c) an isolated protein having an amino acid sequence that is at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ TD NO:35, SEQ ED NO:66, SEQ LD NO:67, and SEQ LD NO:68, or a fragment thereof that is at least about 25 amino acids in length; and
(d) an isolated protein having an amino acid sequence that is at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ TD NO:38, SEQ TD NO:69, SEQ ED NO:70, and SEQ ED NO:71, or a fragment thereof that is at least about 35 amino acids in length.
2. An isolated protein comprising a protein selected from the group consisting of:
(a) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOT, SEQ LD NO:28, SEQ ED NO:50, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ LD NO:60, SEQ LD NO:61, and SEQ TD NO:62, or a fragment thereof that is at least about 25 nucleotides in length;
(b) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ HD NO:7, SEQ HD NO:31, SEQ TD NO:51, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 63, SEQ DD NO: 64, and SEQ DD NO: 65, or a fragment that is at least about 30 nucleotides in length;
(c) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ED NO:9, SEQ DD NO: 12, SEQ DD NO:34, SEQ ED NO:52, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ TD NO:66, SEQ DD NO:67, and SEQ DD NO:68, or a fragment thereof that is at least about 40 nucleotides in length; and
(d) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of
SEQ ED NO:98, SEQ LD NO: 17, SEQ DD NO:37, SEQ DD NO:53, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ TD NO:69, SEQ ID NO:70, and SEQ DD NO:71, or a fragment thereof that is at least about 75 nucleotides in length.
3. An isolated nucleic acid molecule having a nucleic acid sequence that is selected from the group consisting of:
(a) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:l, SEQ ED NO:3, SEQ ID NO:28, SEQ DD NO:30 and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ TD NO: 60, SEQ DD
NO:61, SEQ DD NO:62, and the complement of a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ED NO:60, SEQ LD NO:61, and SEQ LD NO:62, or a fragment thereof that is at least about 25 nucleotides in length; (b) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ED NO:6, SEQ LD NO:7, SEQ LD NO:8, SEQ LD NO:31, SEQ ED NO:33, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:63, SEQ LD NO:64, and SEQ ID NO:65, and the complement of a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:63, SEQ DD NO:64, and SEQ DD NO:65, or a fragment thereof that is at least about 30 nucleotides in length;
(c) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ TD NO: 9, SEQ DD NO:l l, SEQ DD NO:12, SEQ ID NO:13, SEQ DD NO:34, SEQ DD NO:36, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ TD NO:67, and SEQ ED NO:68, and the complement of a nucleic acid sequence that encodes an amino acid sequence selecte from the group consisting of SEQ ID NO:66, SEQ TD NO:67, and SEQ ID NO:67, or a fragment thereof that is at least about 40 nucleotides in length;
(d) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ LD NO:98, SEQ LD NO: 100, SEQ LD NO: 17, SEQ LD NO: 18, SEQ TD NO:37, SEQ TD NO:39, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ED NO:69, SEQ ID NO:70, and SEQ LD NO:71, and the complement of a nucleic acid sequence that encodes an amino acid sequence selecte from the group consisting of SEQ HD NO:69, SEQ TD NO:70, and SEQ TD NO:71, or a fragment thereof that is at least about 75 nucleotides in length; and
(e) a nucleic acid sequence selected from the group consisting of SEQ ID NO:50, SEQ TD NO:51, SEQ DD NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ HD
NO:55, and SEQ DD NO:56.
4. An isolated nucleic acid molecule selected from the group consisting of:
(a) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:28, and SEQ DD NO:30, or a fragment thereof, wherein said fragment has an at least a 20 contiguous nucleotide region identical in sequence to a 20 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ TD NO:l, SEQ ID NO:3, SEQ LD NO:28, and SEQ ED NO:30;
(b) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ DD NO:7, SEQ DD NO:8, SEQ TD NO:31, and SEQ TD NO:33, or a fragment thereof, wherein said fragment has an at least a 25 contiguous nucleotide region identical in sequence to a 25 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ TD NO:6, SEQ TD NO:7, SEQ TD NO:8, SEQ DD NO:31 and SEQ ID NO:33; (c) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ HD NO: 11, SEQ TD NO: 12, SEQ DD NO: 13, SEQ DD NO:34, and SEQ DD NO:36, or a fragment thereof, wherein said fragment has an at least a 30 contiguous nucleotide region identical in sequence to a 30 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, SEQ DD NO:l l, SEQ DD NO:12, SEQ ID NO:13, SEQ DD NO:34, and SEQ DD NO:36; and
(d) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ DD NO:98, SEQ ID NO: 100, SEQ ID NO: 17, SEQ HD NO: 18, SEQ ED NO:37, and SEQ LD NO:39, or a fragment thereof, wherein said fragment has an at least a 60 contiguous nucleotide region identical in sequence to a 60 contiguous nucleotide region of a nucleic acid sequence selected from the group consisting of SEQ TD NO:98, SEQ DD NO: 100, SEQ ID NO: 17, SEQ HD NO: 18, SEQ TD NO:37, and SEQ DD NO:39.
5. An isolated nucleic acid molecule having a nucleic acid sequence encoding a protein selected from the group consisting of:
(a) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ TD NO:l, SEQ LD NO:28, SEQ LD NO:50, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO: 60, SEQ DD NO : 61 , and SEQ ID NO : 62, or a fragment thereof that is at least about 25 nucleotides in length;
(b) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:4, SEQ ID NO:7, SEQ ID NO:31, SEQ ID NO:51, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ED NO:63, SEQ DD NO:64, and SEQ DD NO:65, or a fragment thereof that is at least about 30 nucleotides in length;
(c) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ HD NO:9, SEQ DD NO: 12, SEQ DD NO:34, SEQ DD NO:52, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ DD NO:67, and SEQ HD NO:68, or a fragment thereof that is at least about 40 nucleotides in length; and
(d) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of
SEQ DD NO:98, SEQ DD NO: 17, SEQ TD NO:37, SEQ TD NO:53, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:69, SEQ DD NO:70, and SEQ ED NO:71, or a fragment thereof that is at least about 75 nucleotides in length.
6. An isolated oligonucleotide comprising a unique nucleic acid sequence within a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ED NO: 1, SEQ ED NO:3, SEQ LD NO:4, SEQ LD NO:6, SEQ LD NO:7, SEQ ED NO:8, SEQ DD NO:9, SEQ ID NO: 11, SEQ DD NO:12, SEQ ED NO: 13, SEQ DD NO:98, SEQ ID NO:100, SEQ ED NO:17, SEQ TD NO:18, SEQ TD NO:19, SEQ ID NO:21, SEQ TD NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ LD NO:27, SEQ ID NO:28, SEQ ED NO:30, SEQ LD NO:31, SEQ LD NO:33, SEQ LD NO:34, SEQ LD NO:36, SEQ LD NO:37, SEQ LD NO:39, SEQ ID NO:40, SEQ LD NO:42, SEQ LD NO:43, SEQ LD NO:45, SEQ ED NO:46, SEQ LD NO:48, SEQ LD NO:50, SEQ LD NO:51, SEQ LD NO:52, SEQ TD NO:53, SEQ ED NO:54, SEQ ID NO:55, SEQ LD NO:56, complements of SEQ TD NO:50, SEQ LD NO:51, SEQ LD NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ED NO:55, SEQ DD NO:56, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:60, SEQ DD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ DD NO:65, SEQ DD NO:66, SEQ ED NO:67, SEQ ID NO:68, SEQ DD NO:69, SEQ ID NO:70, SEQ TD NO:71, SEQ HD NO:72, SEQ ID NO:73, SEQ TD NO:74, SEQ TD NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ DD NO:78, SEQ DD NO:79, and SEQ DD NO:80; and a homolog thereof.
7. A reagent that is unique to a nucleic acid molecule selected from the group consisting of nCaV╬▓3381, nCaV╬▓4408, nCaV╬▓4384, nCaV╬▓l2408, nCaV╬▓l2402, nCaV╬▓7243g, nCaV╬▓72399, nCaV╬▓3333, nCaV╬▓435!, nCaV╬▓l2339, nCaV╬▓72423, nCaV╬▓21396, nCaV╬▓54354 and nCaV╬▓l82369, wherein said reagent can distinguish one member of said group from another member of said group.
8. A method to detect expansion of T cells in an animal comprising: (a) identifying the presence of one or more T cell receptor nucleic acid molecule(s) having unique nucleic acid sequences within variable regions of beta chain nucleic acid molecules selected from the group consisting of nCaV╬▓3381, nCaV╬▓440g, nCaV╬▓4384, nCaV╬▓l2408, nCaV╬▓l2402, nCaV╬▓72438, nCaV╬▓72399, nCaV╬▓3333, nCaV╬▓4351, nCaV╬▓l2339, nCaV╬▓72423, nCaV╬▓21396, nCaV╬▓54354 and nCaV╬▓l82369 by forming detectable products; and (b) detecting the expansion of said T cells by determining production of said product.
9. A therapeutic composition that, when administered to an animal, regulates an immune response in said animal, said therapeutic composition comprising a therapeutic compound selected from the group consisting of: (i) an isolated protein comprising a TCR V╬▓ protein selected from the group consisting of:
(a) an isolated protein having an amino acid sequence that is at least about 55 percent identical to an amino acid sequence selected from the group consisting of SEQ TD NO:2, SEQ DD NO:29, SEQ DD NO:60, SEQ TD NO:61, and SEQ ID NO:62, or a fragment thereof that is at least about 25 amino acids in length;
(b) an isolated protein having an amino acid sequence that is at least about 75 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ TD NO:32, SEQ ID NO:63, SEQ TD NO:64, and SEQ ID NO:65, or a fragment thereof that is at least about 15 amino acids in length; (c) an isolated protein having an amino acid sequence that is at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 10, SEQ DD NO:35, SEQ ID NO:66, SEQ ID NO:67, and SEQ DD NO:68, or a fragment thereof that is at least about 25 amino acids in length; and
(d) an isolated protein having an amino acid sequence that is at least about 50% identical to an amino acid sequence selected from the group consisting of SEQ DD NO: 15, SEQ DD NO:38, SEQ TD NO:69, SEQ TD NO:70, and SEQ DD NO:71, or a fragment thereof that is at least about 35 amino acids in length; (ii) a mimetope of any of said TCR V╬▓ proteins; (iii) a chimeric form of any of said TCR V╬▓ proteins; (iv) an isolated nucleic acid molecule selected from the group consisting of:
(a) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ TD NO:l, SEQ DD NO:3, SEQ DD NO:28, SEQ ID NO:30 and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:60, SEQ DD NO:61, SEQ DD NO: 62, and the complement of a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:60, SEQ ED NO:61, and SEQ DD NO: 62, or a fragment thereof that is at least about 25 nucleotides in length;
(b) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ TD NO:4,
SEQ LD NO:6, SEQ ED NO:7, SEQ LD NO:8, SEQ DD NO:31, SEQ HD NO:33, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO:63, SEQ DD NO:64, and SEQ ED NO:65, and the complement of a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ TD NO:63, SEQ ED NO:64, and SEQ LD NO:65, or a fragment thereof that is at least about 30 nucleotides in length;
(c) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 11, SEQ LD NO: 12, SEQ LD NO: 13, SEQ ED NO:34, SEQ LD NO:36, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ TD NO:66, SEQ ID NO:67, and SEQ TD NO:68, and the complement of a nucleic acid sequence that encodes an amino acid sequence selecte from the group consisting of SEQ ID NO:66, SEQ TD NO:67, and SEQ ED NO:67, or a fragment thereof that is at least about 40 nucleotides in length;
(d) a nucleic acid sequence that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ LD
NO:98, SEQ DD NO: 100, SEQ ED NO: 17, SEQ ED NO: 18, SEQ TD NO:37, SEQ ID NO:39, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, and SEQ TD NO:71, and the complement of a nucleic acid sequence that encodes an amino acid sequence selecte from the group consisting of SEQ TD NO: 69, SEQ TD NO: 70, and SEQ HD NO: 71, or a fragment thereof that is at least about 75 nucleotides in length; and
(e) a nucleic acid sequence selected from the group consisting of SEQ TD NO:50, SEQ TD NO:51, SEQ DD NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55, and SEQ DD NO:56; (v) an isolated antibody that selectively binds to any of said TCR V╬▓ proteins; and
(vi) an inhibitor of TCR V╬▓ protein activity identified by its ability to inhibit the activity of said TCR V╬▓ proteins.
10. A method to produce a TCR V╬▓ protein, said method comprising culturing a cell capable of expressing said protein, said protein selected from the group consisting of:
(a) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:l, SEQ DD NO:28, SEQ ID NO:50, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ TD NO: 60, SEQ ID NO: 61, and SEQ ED NO: 62, or a fragment thereof that is at least about 25 nucleotides in length;
(b) a protein encoded by a nucleic acid molecule that is at least about 75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ DD NO:7, SEQ DD NO:31, SEQ ID NO:51, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:63, SEQ TD NO:64, and SEQ TD NO:65, or a fragment that is at least about 30 nucleotides in length;
(c) a protein encoded by a nucleic acid molecule that is at least about 70 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, SEQ TD NO: 12, SEQ HD NO:34, SEQ TD NO:52, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ TD NO:67, and SEQ ED NO:68, or a fragment thereof that is at least about 40 nucleotides in length; and (d) a protein encoded by a nucleic acid molecule that is at least about
75 percent identical to a nucleic acid sequence selected from the group consisting of SEQ DD NO:98, SEQ DD NO: 17, SEQ DD NO:37, SEQ DD NO:53, and a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ DD NO: 69, SEQ ED NO: 70, and SEQ ED NO: 71, or a fragment thereof that is at least about 75 nucleotides in length.
11. The invention of Claim 1 , 2, 9 or 10, wherein said protein, when administered to an animal, can perform a function selected from the group consisting of eliciting an immune response against a TCR V╬▓ protein and binding to a MHC molecule that binds to a TCR V╬▓ protein.
12. The invention of Claim 1, 2, 9 or 10, wherein said protein is selected from the group consisting of: a protein comprising an amino acid sequence selected from the group consisting of SEQ LD NO:2, SEQ ED NO:5, SEQ LD NO:10, SEQ LD NO:15, SEQ ID NO:29, SEQ LD NO:32, SEQ ED NO:35, SEQ DD NO:38, SEQ DD NO:60, SEQ DD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ ID NO:64, SEQ TD NO:65, SEQ ID NO:66, SEQ HD NO:67, SEQ TD NO:68, SEQ TD NO:69, SEQ ID NO:70 and SEQ LD NO:71; and a protein encoded by an allelic variant of a nucleic acid molecule encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ TD NO:29, SEQ ID NO:32, SEQ TD NO:35, SEQ TD NO:38, SEQ TD NO:60, SEQ TD NO:61, SEQ HD NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ ED NO:65, SEQ DD NO:66, SEQ DD NO:67, SEQ ID NO:68, SEQ ED NO:69, SEQ ID NO:70 and SEQ DD NO:71.
13. An isolated antibody that selectively binds to a protein as set forth in the invention of Claim 1, 2, 9 or 10.
14. The nucleic acid molecule of the invention of Claim 3-10, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a TCR V╬▓ protein.
15. The nucleic acid molecule of the invention of Claim 3-10, wherein said nucleic acid molecule encodes a protein that elicits an immune response against a naturally-occurring TCR V╬▓ protein.
16. The nucleic acid molecule of the invention of Claim 3-10, wherein said nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of nCaV╬▓3381, nCaV╬▓4408, nCaV╬▓4384, nCaV╬▓l2408, nCaV╬▓l2402, nCaV╬▓72438, nCaV╬▓72399, nCaV╬▓3333, nCaV╬▓435], nCaV╬▓l2339 and nCaV╬▓72423.
17. The nucleic acid molecule of the invention of Claim 3-10, wherein said nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence that encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ED NO:5, SEQ DD NO: 10, SEQ DD NO: 15, SEQ DD NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ HD NO:38, SEQ ID NO:60, SEQ TD NO:61, SEQ DD NO:62, SEQ DD NO:63, SEQ DD NO:64, SEQ ED NO:65, SEQ DD NO:66, SEQ DD NO:67, SEQ DD NO:68, SEQ DD NO:69, SEQ DD NO:70 and SEQ DD NO:71; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule encoding a protein having any of said amino acid sequences.
18. The nucleic acid molecule of the invention of Claim 3-10, wherein said nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ DD NO:l, SEQ ID NO:3, SEQ HD NO:4, SEQ ID NO:6, SEQ ED NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ED NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ TD NO:98, SEQ HD NO: 100, SEQ DD NO: 17 and SEQ ID NO: 18; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule comprising any of said nucleic acid sequences.
19. The nucleic acid molecule of the invention of Claim 3-10, wherein said nucleic acid molecule comprises an oligonucleotide.
20. A recombinant molecule comprising a nucleic acid molecule as set forth in the invention of Claim 3-10 operatively linked to a transcription control sequence.
21. A recombinant virus comprising a nucleic acid molecule as set forth in the invention of Claim 3-10.
22. A recombinant cell comprising a nucleic acid molecule as set forth in the invention of Claim 3-10.
23. The invention of Claim 3-10, wherein said nucleic acid molecule comprises an oligonucleotide from about 15 nucleotides to about 25 nucleotides in length.
24. The invention of Claim 3-10, wherein said nucleic acid molecule comprises an oligonucleotide selected from the group consisting of SEQ ID NO:50, SEQ ID NO:51, SEQ TD NO:52, SEQ ID NO:53, SEQ TD NO:54, SEQ ID NO:55, SEQ HD NO:56, and complements of SEQ TD NO:50, SEQ ED NO:51, SEQ ED NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55, and SEQ DD NO:56.
25. The invention of Claim 3-10, wherein said nucleic acid molecule has a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ TD NO:3, SEQ TD NO:4, SEQ TD NO:6, SEQ DD NO:7, SEQ DD NO:8, SEQ DD NO:9, SEQ ID NO:l l, SEQ TD NO:12, SEQ ID NO:13, SEQ TD NO:98, SEQ DD NO:100, SEQ DD NO:17, SEQ ED NO:18, SEQ DD NO:28, SEQ DD NO:30, SEQ DD NO:31, SEQ DD NO:33, SEQ ED NO:34, SEQ ED NO:36, SEQ DD NO:37, SEQ ED NO:39, SEQ DD NO:40, SEQ DD NO:42, SEQ DD NO:43, SEQ DD NO:45, SEQ DD NO:46, SEQ ID NO:48, SEQ ED NO:50, SEQ ID NO:51, SEQ DD NO:52, SEQ ID NO:53, SEQ LD NO:54, SEQ LD NO:55, SEQ ED NO:56, and complements of SEQ ED NO:50, SEQ LD NO:51, SEQ LD NO:52, SEQ ED NO:53, SEQ LD NO:54, SEQ ID NO:55, and SEQ LD NO:56.
26. The invention of Claim 3-10, wherein said invention comprises a reagent which identifies the presence of a T cell receptor having a unique nucleic acid sequence within said nucleic acid molecule.
27. The invention of Claim 3-10, wherein said invention comprises a reagent which is a DNA primer complementary to said unique nucleic acid sequence.
28. The invention of Claim 3-10, wherein said invention comprises a unique nucleic acid sequence that is selected from the group consisting of SEQ DD NO:50, SEQ ID NO:51, SEQ TD NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55 and SEQ ID O:56.
29. A kit comprising said reagent of Claim 7, wherein said kit comprises one or more of said reagents and a means for detecting said reagents.
30. A kit comprising said reagent of Claim 7, wherein said kit comprises seven of said reagents, wherein each of said seven reagents identifies the presence of a different beta chain V region selected from the group consisting of nCaV╬▓3381, nCaV╬▓4408, nCaV╬▓4384, nCaV╬▓l2408, nCaV╬▓l2402, nCaV╬▓72438, nCaV╬▓72399, nCaV╬▓3333, nCaV╬▓4351, nCaV╬▓l2339, nCaV╬▓72423, nCaV╬▓21396, nCaV╬▓54354 and nCaV╬▓l82369.
31. The kit of Claim 30, wherein said kit further comprises a DNA primer complementary to a nucleic acid sequence in the constant region of a T cell receptor beta chain.
32. The kit of Claim 30, wherein said constant region nucleic acid sequence is selected from the group consisting of SEQ TD NO:58 and SEQ HD NO:59.
33. The kit of Claim 30, wherein said kit comprises a composition comprising a mixture of said reagents and said DNA primer complementary to a nucleic acid sequence in the constant region of a T cell receptor beta chain.
34. The method of Claim 8, wherein said step of detecting comprises comparing formation of one detectable product with one or more other detectable products.
35. The method of Claim 8, wherein said variable region has a nucleic acid sequence selected from the group consisting of SEQ ED NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ LD NO:33, SEQ TD NO:34, SEQ ID NO:36, SEQ DD NO:37, SEQ DD NO:39, SEQ DD NO:40, SEQ DD NO:42, SEQ TD NO:43, SEQ HD NO:45, SEQ ED NO:46, SEQ DD NO:48, SEQ DD NO:50, SEQ ID NO:51, SEQ DD NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ ID NO:55, SEQ DD NO:56, and complements of SEQ DD NO:50, SEQ DD NO:51, SEQ TD NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ TD NO:55, and SEQ DD NO:56.
36. The method of Claim 8, wherein said step of identification comprises:
(a) contacting a sample containing DNA from T cells with a reagent having specificity for one of said unique nucleic acid sequences; and
(b) determining the presence of DNA carrying said unique nucleic acid sequence.
37. The method of Claim 8, wherein said reagent is a DNA primer that is complementary to said unique nucleic acid sequence.
38. The method of Claim 8, wherein said step of identification is performed using polymerase chain reaction amplification.
39. The method of Claim 8, wherein said method detects a disease that is selected from the group consisting of cancer, an autoimmune disease, an infectious disease and allergy.
40. The invention of Claim 8 or 9, wherein said animal is selected from the group consisting of an animal suspected of having a disease, an animal having a disease and an animal being treated for a disease, wherein said disease is selected from the group consisting of lymphoma, leukemia, rheumatoid arthritis, diabetes, viral infections, bacterial infections, yeast infections, parasite infections, dermatitis, and asthma.
41. The method of Claim 40, wherein said disease is selected from the group consisting of T cell lymphoma and T cell leukemia.
42. A composition comprising the invention of Claim 1, 2, 3, 4, 5, 6, or 9, wherein said composition further comprises a component selected from the group consisting of an excipient, an adjuvant and a carrier.
43. The composition of Claim 9, wherein said therapeutic compound is selected from the group consisting of a peptide, a naked nucleic acid vaccine and a recombinant cell vaccine.
44. A method to regulate an immune response in an animal comprising administering to the animal a therapeutic composition comprising the invention of Claim 1, 2, 3, 4, 5, 6, or 9.
45. A method for prescribing treatment for specific disease, comprising: (a) identifying the presence of a T cell receptor nucleic acid molecule having a unique nucleic acid sequence within a variable region of a beta chain nucleic acid molecule selected from the group consisting of nCaV╬▓3333, nCaV╬▓435], nCaV╬▓l2339, nCaV╬▓72423, nCaV╬▓21396, nCaV╬▓54354, nCaV╬▓l82369, nCaV╬▓3381, nCaV╬▓440g, nCaV╬▓l2408, nCaV╬▓72438, nCaV╬▓21462, nCaV╬▓54417 and nCaV╬▓l82423; and
(b) prescribing a treatment comprising administering to said animal a therapeutic composition comprising the invention of Claim 1, 2, 3, 4, 5, 6, or 9.
46. The reagent of Claim 7, wherein said reagent identifies the presence of a T cell receptor having a unique nucleic acid sequence within said nucleic acid molecule.
47. The reagent of Claim 7, wherein said reagent is a DNA primer complementary to said unique nucleic acid sequence.
48. The reagent of Claim 7, wherein said unique nucleic acid sequence is selected from the group consisting of SEQ DD NO:50, SEQ DD NO:51, SEQ ID NO:52, SEQ DD NO:53, SEQ DD NO:54, SEQ DD NO:55 and SEQ ID NO:56.
PCT/US1999/017284 1998-07-31 1999-07-30 Polynucleotide encoding an autoantigen associated with endometriosis WO2000006732A2 (en)

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AU37460/00A AU3746000A (en) 1999-07-22 2000-03-10 Polynucleotide encoding autoantigens associated with endometriosis
PCT/US2000/006742 WO2001007616A1 (en) 1999-07-22 2000-03-10 Polynucleotide encoding autoantigens associated with endometriosis

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WO2013156560A1 (en) 2012-04-20 2013-10-24 Bayer Cropscience Ag N-cycloalkyl-n-[(trisubstitutedsilylphenyl)methylene]-(thio)carboxamide derivatives
WO2019002444A1 (en) * 2017-06-30 2019-01-03 Immatics Biotechnologies Gmbh Novel t cell receptors and immune therapy using the same

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2013156560A1 (en) 2012-04-20 2013-10-24 Bayer Cropscience Ag N-cycloalkyl-n-[(trisubstitutedsilylphenyl)methylene]-(thio)carboxamide derivatives
WO2019002444A1 (en) * 2017-06-30 2019-01-03 Immatics Biotechnologies Gmbh Novel t cell receptors and immune therapy using the same

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