HK40005833B - T cell receptors recognizing hla-a1- or hla-cw7-restricted mage - Google Patents

Description

T-cell receptors recognizing HLA-A1-or HLA-CW 7-restricted MAGE

The application is a divisional application of a patent application named as 'T cell receptor for recognizing HLA-a 1-or HLA-CW 7-restricted MAGE' international application No. PCT/US2012/054623, international application No. 2012, 9/11/2012, chinese application No. 201280055972. X.

The invention was made with government support under national cancer research institute project number ZIABC010984, national institutes of health, usa. The government has certain rights in the invention.

Cross reference to related applications

This application claims priority to U.S. patent application No. 61/535,086, filed on 9/15/2011, which is incorporated by reference in its entirety.

Incorporation by reference of electronically submitted materials

Incorporated herein by reference in its entirety are computer-readable nucleotide/amino acid sequence listings filed concurrently herewith, identified as follows: a 52, 162 byte ASCII (text) file with a date of 2012, 8, 22 months and a name of "710922 st25. txt.

Background

Adoptive Cell Therapy (ACT) involves the transfer of reactive T cells into patients, including the transfer of tumor-reactive T cells into cancer patients. Adoptive cell therapy using T cells targeting Human Leukocyte Antigen (HLA) -a2 restricted T cell epitopes has been successful in causing regression of tumors in some patients. However, patients lacking HLA-A2 expression cannot be treated with T cells targeting HLA-A2 restricted T cell epitopes. This limitation creates a barrier to the widespread use of adoptive cell therapy. Accordingly, there is a need for improved immunological compositions and methods for treating cancer.

Brief summary of the invention

The invention provides isolated or purified T Cell Receptors (TCRs) having antigenic specificity for melanoma antigen family A (MAGE A) -3 in the case of a) HLA-A1 or for MAGE-A12 in the case of HLA-Cw 7. The invention further provides related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and cell populations. Further provided by the invention are antibodies, or antigen-binding portions thereof, and pharmaceutical compositions related to the inventive TCRs.

Further provided by the invention are methods of detecting the presence of cancer in a host and methods of treating or preventing cancer in a host. The inventive method of detecting the presence of cancer in a host comprises (i) contacting a sample comprising cancer cells with any of the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, host cell populations, or antibodies, or antigen binding portions thereof, described herein, thereby forming a complex, and (ii) detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the host.

The methods of the invention for treating or preventing cancer in a host comprise administering to the host any of the TCRs, polypeptides, or proteins described herein, any nucleic acid comprising a nucleotide sequence or recombinant expression vector encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of host cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in a host.

Brief description of several views of the drawings

FIG. 1A is a line graph showing Interferon (IFN) - γ secretion (pg/ml) in response to Untransduced (UT) cells (black line) co-cultured with different tumor cell lines or cells transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46) (unshaded line) or anti-MAGE-A3 TCR 13-18(SEQ ID NO: 48) (grey line).

FIG. 1B is a line graph showing IFN- γ secretion (pg/ml) in response to UT cells co-cultured with HLA-A1+/MAGE-A3+ fresh tumor FrTu 2767 (black line), FrTu 3178 (grey line), FrTu 2823 (unshaded line) or FrTu 3068 (diagonal line) or HLA-A0201 +/MART-1+ fresh tumor FrTu 2851 (horizontal line) or FrTu3242 (vertical line) or cells transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46) or anti-MART-1 TCR DMF 5. The bars in the squares indicate cells that were not co-cultured with tumor cells.

FIGS. 2A and 2B are line graphs showing IFN- γ secretion (pg/ml) in response to cells from the first (FIG. 2A) and second (FIG. 2B) donors transduced with a control construct (black line) encoding a truncated human low affinity Nerve Growth Factor Receptor (NGFR), anti-MAGE-A12 TCR502(SEQ ID NO: 47) (unshaded line), or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (grey line) co-cultured with different tumor cell lines.

FIG. 3A illustrates a line graph of the cumulative percentage of normal Caucasian populations expressing HLA-A1 (unshaded portion of line), HLA-A2 (grey portion of line), and/or HLA-Cw7 (black portion of line).

FIGS. 3B and 3C illustrate that HLA-A2 and NY-ESO-1 (diagonal portions of lines) would be expected to be expressed; HLA-A1 and MAGE-A3 (unshaded part of the line); HLA-A2, MAGE-A3, and MAGE-A12 (grey part of line); and/or HLA-Cw7 and MAGE-A12 (black portion of line) of human melanoma (FIG. 3B) and synovial cell sarcoma (FIG. 3C) patient populations.

FIG. 4 is a line graph showing secretion of IFN- γ (pg/ml) by cells transduced with NGFR (black bar), anti-MAGE-A12 TCR502(SEQ ID NO: 47) (unshaded bar), or anti-MAGE-A12 TCR 8(SEQ ID NO: 49) (grey bar) in response to co-culture with HLA-Cw0701 and HLA-Cw0702 target cells stimulated by peptides derived from MAGE-A12 (VRIGHLYIL; SEQ ID NO: 4), MAGE-A2 (VPISHLYIL; SEQ ID NO: 50), MAGE-A3 (DPIGHLYIF; SEQ ID NO: 51), MAGE-A6 (DPIGHVYIF; SEQ ID NO: 52), or a control peptide (EDGCPAAEK; SEQ ID NO: 53).

FIGS. 5A-5D show the effect of non-transduced (closed circles) or transduced anti-MAGE-A12 TCR502(SEQ ID NO: 47) at the indicated effector to target (E: T) ratioLine graphs of% lysis of cells caused by PBMCs against MAGE-A12TCR FM8(SEQ ID NO: 49) (diamonds), against MAGE-A3TCR A10(SEQ ID NO: 46) (squares), against MAGE-A3TCR 13-18(SEQ ID NO: 48) (. tangle-solidup.), or against MAGE-A3TCR 112 and 120 (open circles) (397 mel (A), 624mel (B)), 2984mel (C), and 2661RCC (D). Representative results from one of two independent experiments are provided.

FIG. 6A is a line graph showing IFN- γ secretion (pg/ml) in response to untransduced (control) cells (striped lines) co-cultured with different tumor cell lines or cells transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46) (shaded lines) or anti-MAGE-A3 TCR 13-18(SEQ ID NO: 48) (checkered lines). Representative results from two of three independent experiments evaluating responses of T cells transduced with these TCRs are provided.

FIG. 6B is a line graph showing the estimated relative copies of vector DNA measured on cells transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46), anti-MAGE-A3 TCR 13-18(SEQ ID NO: 48), anti-MAGE-A12 TCR502(SEQ ID NO: 47), or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49).

FIG. 6C is a line graph showing the amount of IFN- γ secreted by cells transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46) (circles) or anti-MAGE-A3 TCR 13-18(SEQ ID NO: 48) (squares) in response to co-culture with target cells incubated with different concentrations of MAGE-A3168-176 peptide.

FIG. 6D is a line graph showing IFN- γ secretion (pg/ml) in response to cells transduced with anti-MAGE-A12 TCR502(SEQ ID NO: 47) (shaded line) or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (square line) co-cultured with different tumor cell lines. Representative results from two of three independent experiments evaluating the response of T cells transduced with these TCRs are provided.

FIG. 6E is a graph showing the response versus concentration of MAGE-A12: co-culture of 170-plus 178 peptide incubated target cells transduced cells with anti-MAGE-A12 TCR502(SEQ ID NO: 47) (circles) or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (squares) secreted IFN- γ in a line graph.

FIG. 6F is a line graph showing IFN- γ secretion (pg/ml) in response to untransduced (control) (striped line) or cells transduced with anti-MAGE-A12 TCR502(SEQ ID NO: 47) (shaded line) or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (checkered line) co-cultured with different tumor cell lines. Representative results from two of three independent experiments evaluating the response of T cells transduced with these TCRs are provided.

FIG. 7A is a line graph showing IFN- γ secretion (pg/ml) in response to untransduced cells (control) (striped line) co-cultured with different fresh, uncultured tumors or cells transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46) (shaded line) or anti-MAGE-A3 TCR 13-18 (checkered line). Representative results from one of three independent experiments evaluating the response of T cells transduced with these TCRs are provided.

FIG. 7B is a line graph showing IFN- γ secretion (pg/ml) in response to untransduced cells (control) (striped line) co-cultured with different fresh uncultured tumors or cells transduced with anti-MAGE-A12 TCR502(SEQ ID NO: 47) (shaded line) or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (checkered line). Representative results from one of three independent experiments evaluating responses of T cells transduced with these TCRs are provided.

FIG. 8A is a bar graph showing IFN- γ secretion (pg/ml) from untransduced cells (control) (striped bars) or cells transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46) (shaded bars) or anti-MAGE-A3 TCR 13-18 (striped bars) co-cultured overnight with target cells transfected with HLA-A01 plus one of MAGE-A3, A1, A2, A4, A6, A9, A10 or A12.

Fig. 8B shows the results obtained with HLA-C07: 02 plus one of MAGE-A3, A1, A2, A4, A6, A9, A10 or A12 transfected target cells were co-cultured overnight in untransduced cells (control) (striped line) or in cells transduced with anti-MAGE-A12 TCR502(SEQ ID NO: 47) (shaded line) or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (checkered line) for IFN- γ secretion (pg/ml).

Fig. 8C is a graph showing the correlation between the expression of HLA-C07: 01 plus one of MAGE-A3, A1, A2, A4, A6, A9, A10 or A12 were co-cultured overnight in untransduced cells (control) (striped line) or in cells transduced with anti-MAGE-A12 TCR502(SEQ ID NO: 47) (shaded line) or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (checkered line) IFN- γ secretion (pg/ml).

FIGS. 9A and 9B are line graphs showing IFN- γ secretion from CD8+ (FIG. 9A) or CD4+ cells (FIG. 9B) co-cultured with different tumor targets, either untransduced (control) (striped line) or transduced with anti-MAGE-A3 TCR A10(SEQ ID NO: 46) (shaded line) or anti-MAGE-A3 TCR 13-18 (checkered line). Representative results from one of two independent experiments are provided.

FIG. 9C is a line graph showing IFN- γ secretion from CD8+ cells co-cultured with different tumor targets, either untransduced (control) (striped line) or transduced with anti-MAGE-A12 TCR502(SEQ ID NO: 47) (shaded line) or anti-MAGE-A12 TCR FM8(SEQ ID NO: 49) (checkered line). Representative results from one of two independent experiments are provided.

Detailed Description

One embodiment of the invention provides a T Cell Receptor (TCR) having antigenic specificity for either a) melanoma antigen family a (MAGE a) -3 (also known as MAGE-3) in the case of HLA-a1 or b) MAGE-a12 (also known as MAGE-12) in the case of HLA-Cw 7.

MAGE-A3 and MAGE-A12 are members of the MAGE-A family of 12 homologous proteins, which also include MAGE-A1, MAGE-A2, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, and MAGE-A11. The MAGE-a protein is a Cancer Testis Antigen (CTA) that is expressed only in tumor cells and MHC-free expressing germ cells of the testis and placenta. MAGE-a protein is expressed in a variety of human cancers including, but not limited to, melanoma, breast cancer, leukemia, thyroid cancer, gastric cancer, pancreatic cancer, liver cancer (e.g., hepatocellular cancer), lung cancer (e.g., non-small cell lung cancer), ovarian cancer, multiple myeloma, esophageal cancer, kidney cancer, head cancer (e.g., squamous cell cancer), neck cancer (e.g., squamous cell cancer), prostate cancer, and urothelial cancer.

The TCRs of the invention provide a number of advantages, including when used for adoptive cell transfer. For example, by targeting a) MAGE-A3 presented in the context of HLA-A1 or b) MAGE-A12 presented in the context of HLA-Cw7, the inventive TCRs are likely to treat patients who cannot be treated with TCRs that target MAGE antigens presented in the context of other HLA molecules (e.g., HLA-A2). Because HLA-A1 and HLA-Cw7 are highly common alleles, the inventive TCRs advantageously greatly expand the patient population that can be treated. Furthermore, without being bound by a particular theory, it is believed that because MAGE-A3 and/or MAGE-a12 are expressed by cells of multiple cancer types, the inventive TCRs advantageously provide the ability to destroy cells of multiple types of cancer and, thus, treat or prevent multiple types of cancer. Furthermore, without being bound by a particular theory, it is believed that because the MAGE-a protein is a cancer testis antigen, which is expressed only in tumor cells and MHC-free expressed germ cells of the testis and placenta, the inventive TCR advantageously targets the destruction of cancer cells while minimizing or eliminating the destruction of normal, non-cancer cells, thereby reducing toxicity, e.g., by minimizing or eliminating.

The phrase "antigen-specific" as used herein means that the TCR can specifically bind and immunorerecognise MAGE-A3 or MAGE-a12 with high affinity. For example, if T cells expressing the TCR secrete at least about 200pg/ml or more (e.g., 200pg/ml or more, 300pg/ml or more, 400pg/ml or more, 500pg/ml or more, 600pg/ml or more, 700pg/ml or more, 1000pg/ml or more, 5,000pg/ml or more, 7,000pg/ml or more, 10,000pg/ml or more) IFN- γ when co-cultured with antigen-negative HLA-A1+ target cells or HLA-Cw7+ target cells stimulated with a low concentration of MAGE-A3 peptide or MAGE-A12 peptide (e.g., about 0.05ng/ml to about 5ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, or 5ng/ml), respectively, the TCR may be considered to have "antigen specificity" for MAGE-A3 or MAGE-A12. Alternatively or additionally, a TCR may be considered to have "antigen specificity" for MAGE-A3 or MAGE-a12 if T cells expressing said TCR secrete at least twice the amount of IFN- γ as the background level of untransduced PBL when co-cultured with antigen-negative HLA-a1+ target cells or HLA-Cw7+ target cells, respectively, stimulated with a low concentration of MAGE-A3 peptide or MAGE-a12 peptide, respectively. The inventive TCRs also secrete IFN- γ when co-cultured with antigen-negative HLA-a1+ target cells or HLA-Cw7+ target cells stimulated with higher concentrations of MAGE-A3 peptide or MAGE-a12 peptide, respectively.

One embodiment of the invention provides a TCR with antigenic specificity for any MAGE-a3 protein, polypeptide or peptide. The inventive TCR may have antigenic specificity for a MAGE-A3 protein, which MAGE-A3 protein comprises the amino acid sequence of SEQ ID NO: 1, consisting of SEQ ID NO: 1, or consists essentially of SEQ ID NO: 1. In a preferred embodiment of the invention, the TCR has antigenic specificity for a MAGE-A3168-176 peptide comprising, consisting of, or consisting essentially of EVDPIGHLY (SEQ ID NO: 2).

The inventive TCRs can recognize MAGE-A3 in a Human Leukocyte Antigen (HLA) -a 1-dependent manner. By "HLA-a 1-dependent manner" as used herein is meant that the TCR induces an immune response when bound to MAGE-A3 cancer antigen in the context of HLA-a1 molecules. The inventive TCR recognizes MAGE-A3 presented by the HLA-a1 molecule and can bind to HLA-a1 molecules other than MAGE-A3. Typical HLA-a1 molecules, in this case the inventive TCRs, recognize MAGE-A3, including those encoded by HLA-a 0101, HLA-a 0102, and/or HLA-a 0103 alleles.

One embodiment of the invention provides a TCR with antigenic specificity for any MAGE-a12 protein, polypeptide or peptide. The inventive TCR may have antigenic specificity for a MAGE-a12 protein, said MAGE-a12 protein comprising the amino acid sequence of SEQ ID NO: 3, consisting of SEQ ID NO: 3, or consists essentially of SEQ ID NO: 3, and (3). In a preferred embodiment of the invention, the TCR has antigenic specificity for the MAGE-A12170-178 peptide which comprises VRIGHLYIL (SEQ ID NO: 4), consists of VRIGHLYIL (SEQ ID NO: 4), or consists essentially of VRIGHLYIL (SEQ ID NO: 4).

The inventive TCR can recognize MAGE-a12 in an HLA-Cw 7-dependent manner. By "HLA-Cw 7-dependent manner" as used herein is meant that the TCR induces an immune response when bound to MAGE-a12 cancer antigen in the context of HLA-Cw7 molecules. The inventive TCR can recognise MAGE-a12 presented by HLA-Cw7 molecules and can bind to HLA-Cw7 molecules other than MAGE-a 12. Typical HLA-Cw7 molecules, in this case the inventive TCRs recognize MAGE-a12, including those encoded by HLA-Cw0701 and/or HLA-Cw0702 alleles.

The present invention provides TCRs comprising two polypeptides (i.e., polypeptide chains), such as an alpha chain of a TCR, a beta chain of a TCR, a gamma chain of a TCR, a delta chain of a TCR, or a combination thereof. Such polypeptide chains of TCRs are known in the art. The polypeptide of the inventive TCR may comprise any amino acid sequence, provided that the TCR has antigenic specificity for a) MAGE-A3 in the case of HLA-a1 or b) MAGE-a12 in the case of HLA-Cw 7.

In one embodiment of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising the Complementarity Determining Region (CDR)1, CDR2, and CDR3 of the TCR. In one embodiment of the invention the TCR has antigenic specificity for MAGE-A3168-176 and comprises a first polypeptide chain comprising CDR1 (comprising the amino acid sequence of SEQ ID NO: 5 or 16 (CDR 1 of the alpha chain), CDR2 (comprising the amino acid sequence of SEQ ID NO: 6 or 17 (CDR 2 of the alpha chain) and CDR3 (comprising the amino acid sequence of SEQ ID NO: 7 or 18 (CDR 3 of the alpha chain) and a second polypeptide chain comprising CDR1 (comprising the amino acid sequence of SEQ ID NO: 8 or 19 (CDR 1 of the beta chain)), CDR2 (comprising the amino acid sequence of SEQ ID NO: 9 or 20 (CDR 2 of the beta chain) and CDR3 (comprising the amino acid sequence of SEQ ID NO: 10 or 21 (CDR 3 of the beta chain)). in another embodiment of the invention the TCR has antigenic specificity for MAGE-A12170-178, and comprises a first polypeptide chain comprising CDR1 (comprising the amino acid sequence of SEQ ID NO: 26 or 36 (CDR 1 of the alpha chain)), CDR2 (comprising the amino acid sequence of SEQ ID NO: 27 or 37 (CDR 2 of the alpha chain), and CDR3 (comprising the amino acid sequence of SEQ ID NO: 28 or 38 (CDR 3 of the alpha chain)), and a second polypeptide chain comprising CDR1 (comprising the amino acid sequence of SEQ ID NO: 29 or 39 (CDR 1 of the beta chain)), CDR2 (comprising the amino acid sequence of SEQ ID NO: 30 or 40 (CDR 2 of the beta chain)), and CDR3 (comprising the amino acid sequence of SEQ ID NO: 31 or 41 (CDR 3 of the beta chain)). In this regard, the inventive TCR may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7, 8-10, 16-18, 19-21, 26-28, 29-31, 36-38, and 39-41. Preferably, the TCR comprises SEQ ID NO: 5-10, 16-21, 26-31, or 36-41. More preferably, the TCR comprises SEQ ID NO: 5-10 or 26-31.

Alternatively or additionally, the TCR may comprise the amino acid sequence of the variable region of the TCR comprising the CDRs listed above. In this regard, the TCR having antigenic specificity for MAGE-a3168-176 may comprise the amino acid sequence of SEQ ID NO: 11 or 22 (variable region of α chain) or 12 or 23 (variable region of β chain), SEQ ID NO: 11 and 12 or SEQ ID NO: 22 and 23. In another embodiment of the invention, the TCR has antigenic specificity for MAGE-a12170-178 and comprises the amino acid sequence of SEQ ID NO: 32 or 42 (variable region of α chain) or 33 or 43 (variable region of β chain), SEQ ID NO: 32 and 33, or SEQ ID NO: 42 and 43. Preferably, the inventive TCR comprises SEQ ID NO: 11 and 12 or SEQ ID NO: 32 and 33, respectively.

Alternatively or additionally, the TCR may comprise the α chain of the TCR and the β chain of the TCR. Each of the α and β chains of the inventive TCR may independently comprise any amino acid sequence. Preferably, the alpha chain comprises the variable region of the alpha chain as listed above. In this regard, the inventive TCR having antigenic specificity for MAGE-a3168-176 may comprise the amino acid sequence of SEQ ID NO: 13 or 24 and the inventive TCR with antigenic specificity for MAGE-a12170-178 may comprise the amino acid sequence of SEQ ID NO: 34 or 44. This type of inventive TCR can be paired with the β chain of any TCR. Preferably, the β chain of the inventive TCR comprises the variable region of the β chain as listed above. In this regard, the inventive TCR with antigenic specificity for MAGE-a3168-176 may comprise the amino acid sequence of SEQ ID NO: 14 or 25 and the inventive TCR with antigenic specificity for MAGE-a12170-178 may comprise the amino acid sequence of SEQ ID NO: 35 or 45. The inventive TCR, therefore, may comprise SEQ ID NO: 13, 14, 24, 25, 34, 35, 44, or 45, SEQ ID NO: 13 and 14, SEQ ID NO: 24 and 25, SEQ ID NO: 34 and 35, or SEQ ID NO: 44 and 45, respectively. Preferably, the inventive TCR comprises SEQ ID NO: 13 and 14 or SEQ ID NO: 34 and 35.

Also provided by the invention are polypeptides comprising a functional portion of any of the TCRs described herein. The term "polypeptide" as used herein includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

With respect to the inventive polypeptide, the functional moiety can be any moiety comprising contiguous amino acids of the TCR which is part of the TCR, provided that the functional moiety specifically binds MAGE-A3 or MAGE-a 12. The term "functional portion" as used in reference to a TCR refers to any portion or fragment of the TCR of the invention which retains the biological activity of the TCR of which it is a part, (the parent TCR). Functional moieties include, for example, those TCR portions that retain the ability to specifically bind to MAGE-A3 (e.g., in an HLA-A1-dependent manner) or MAGE-A12 (e.g., in an HLA-Cw 7-dependent manner), or to detect, treat, or prevent cancer to a similar extent, to the same extent, or to a greater extent, as the parent TCR. With respect to the parent TCR, the functional moiety can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent TCR.

The functional moiety may comprise an additional amino acid at the amino or carboxy terminus of the moiety, or both, which is not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acid does not interfere with the biological function of the functional moiety, for example, specifically binds to MAGE-A3 or MAGE-a 12; and/or has the functions of detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acid enhances biological activity when compared to the biological activity of the parent TCR.

The polypeptide may comprise a functional portion of one or both of the α and β chains of a TCR of the invention, such as one or more of the CDR1, CDR2, and CDR3 of the variable region of the α chain and/or β chain of a TCR of the invention. In this regard, the polypeptide may comprise a polypeptide comprising SEQ ID NO: 5, 16, 26, or 36 (CDR 1 of the α chain), 6, 17, 27, or 37 (CDR 2 of the α chain), 7, 18, 28, or 38 (CDR 3 of the α chain), 8, 19, 29, or 39 (CDR 1 of the β chain), 9, 20, 30, or 40 (CDR 2 of the β chain), 10, 21, 31, or 41 (CDR 3 of the β chain), or a combination thereof. Preferably, the polypeptide of the invention comprises a polypeptide comprising SEQ ID NO: 5-7; 8-10; 16-18; 19-21; 26-28; 29-31; 36-38; 39-41; SEQ ID NO: 5-10; SEQ ID NO: 16-21; SEQ ID NO: 26-31; or SEQ ID NO: 36-41. More preferably, the polypeptide comprises a polypeptide comprising SEQ ID NO: 5-10 or SEQ ID NO: 26-31, or a functional portion of the amino acid sequence of all of them.

Alternatively or in addition, the inventive polypeptide may comprise, for example, the variable region of a TCR of the combined invention comprising the CDR regions listed above. In this regard, the polypeptide may comprise SEQ ID NO: 11, 22, 32, or 42 (variable region of α chain), SEQ ID NO: 12, 23, 33, or 43 (variable region of β chain), SEQ ID NO: 11 and 12, SEQ ID NO: 22 and 23, SEQ ID NO: 32 and 33, or SEQ ID NO: 42 and 43. Preferably, the polypeptide comprises SEQ ID NO: 11 and 12 or SEQ ID NO: 32 and 33, respectively.

Alternatively or additionally, the inventive polypeptide may comprise the full length of the α or β chain of one of the TCRs described herein. In this regard, the polypeptide of the invention may comprise SEQ ID NO: 13, 14, 24, 25, 34, 35, 44, or 45. Alternatively, the polypeptides of the invention may comprise the α and β chains of the TCRs described herein. For example, the polypeptide of the invention may comprise SEQ ID NO: 13 and 14; the amino acid sequence of SEQ ID NO: both 24 and 25; the amino acid sequence of SEQ ID NO: 34 and 35; or SEQ ID NO: 44 and 45, respectively. Preferably, the polypeptide comprises SEQ ID NO: 13 and 14 or SEQ ID NO: 34 and 35.

The invention further provides a protein comprising at least one of the polypeptides described herein. By "protein" is meant a molecule comprising one or more polypeptide chains.

In one embodiment, the protein of the invention may comprise a polypeptide comprising SEQ ID NO: 5-7; the amino acid sequence of SEQ ID NO: 16-18; the amino acid sequence of SEQ ID NO: 26-28; or SEQ ID NO: 36-38 and a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 8-10; the amino acid sequence of SEQ ID NO: 19-21; SEQ ID NO: 29-31; or SEQ ID NO: 39-41 amino acid sequence second polypeptide chain. Alternatively or additionally, the protein of the invention may comprise a polypeptide comprising SEQ ID NO: 11, 22, 32, or 42 and a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 12, 23, 33, or 43. The protein of the invention may, for example, comprise a polypeptide comprising SEQ ID NO: 13, 24, 34, or 44 and a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 14, 25, 35, or 45. In this case, the protein of the present invention may be a TCR. Alternatively, if, for example, the protein comprises a polypeptide comprising SEQ ID NO: 13, 24, 34, or 44 and SEQ ID NO: 14, 25, 35, or 45, or if the first and/or second polypeptide chain of said protein further comprises other amino acid sequences, for example, amino acid sequences encoding an immunoglobulin or a portion thereof, the protein of the invention may be a fusion protein. In this regard, the invention also provides fusion proteins comprising at least one of the polypeptides of the invention described herein together with at least one other polypeptide. The other polypeptide may be present as an isolated polypeptide of a fusion protein, or may be present as a polypeptide expressed in-frame (in tandem) with one of the polypeptides of the invention described herein. Other polypeptides may encode any peptide or protein molecule, or portion thereof, including, but not limited to, immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules, for example, CDla, CD1b, CD1c, CD1d, and the like.

The fusion protein may comprise one or more copies of a polypeptide of the invention and/or one or more copies of other polypeptides. For example, the fusion protein may comprise 1, 2, 3, 4, 5, or more copies of the polypeptide of the invention and/or the additional polypeptide. Suitable methods for preparing fusion proteins are known in the art and include, for example, recombinant methods. See, e.g., Choi et al, mol.biotechnol.31: 193-202(2005).

In some embodiments of the invention, the TCRs, polypeptides, and proteins of the invention may be expressed as a single protein comprising a linker peptide linking the alpha chain and the beta chain. In this regard, a polypeptide comprising SEQ ID NO: 13, 24, 34, or 44 and SEQ ID NO: 14, 25, 35, or 45 of the invention may further comprise a TCR, polypeptide, and protein comprising SEQ ID NO: 15 or 54. The linker peptide can advantageously facilitate expression of the recombinant TCR, polypeptide, and/or protein in a host cell. When the construct comprising the linker peptide is expressed by a host cell, the linker peptide may be cleaved, producing isolated alpha and beta chains.

The protein of the invention may be a recombinant antibody comprising at least one of the polypeptides of the invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptide chains of the polypeptides and antibodies of the invention, or portions thereof. The polypeptide of an antibody, or portion thereof, can be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or f (ab) 2' fragment of an antibody, or the like. Said polypeptide chain of the antibody, or a portion thereof, may be present as an isolated polypeptide of a recombinant antibody. Alternatively, said polypeptide chain of an antibody, or a portion thereof, may be present as a polypeptide expressed in frame (in tandem) with said polypeptide of the invention. The polypeptide of an antibody, or portion thereof, can be a polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.

Included within the scope of the invention are functional variants of the TCRs, polypeptides, and proteins of the invention described herein. The term "functional variant" as used herein refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, which functional variant retains the biological activity of the TCR, polypeptide, or protein, which is a variant of the TCR, polypeptide, or protein. Functional variants include, for example, those variants of the TCRs, polypeptides, or proteins described herein (the parent TCRs, polypeptides, or proteins) that retain the ability to specifically bind to MAGE-A3 or MAGE-a12, the parent TCR having antigenic specificity for the MAGE-A3 or MAGE-a12 or the parent polypeptide or protein being specific to bind to the MAGE-A3 or MAGE-a12 to a similar degree, to the same degree, or to a greater degree, as the parent TCR, polypeptide, or protein. With respect to the parent TCR, polypeptide, or protein, the functional variant can, for example, be at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acid sequence to the parent TCR, polypeptide, or protein.

The functional variant may, for example, comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, the conservative amino acid substitution can be an acidic amino acid in place of another acidic amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain in place of another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid in place of another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain in place of another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), and the like.

Alternatively or additionally, the functional variant may comprise an amino acid sequence of the parent TCR, polypeptide, or protein having at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased when compared to the parent TCR, polypeptide, or protein.

The TCR, polypeptide, or protein can consist essentially of one or more of the particular amino acid sequences described herein, such that other components of the functional variant, e.g., other amino acids, do not materially alter the biological activity of the functional variant. In this regard, an inventive TCR, polypeptide, or protein can, for example, consist essentially of SEQ ID NO: 13, 14, 24, 25, 34, 35, 44, or 45, SEQ ID NO: 13 and 14, SEQ ID NO: 24 and 25, SEQ ID NO: 34 and 35, or SEQ ID NO: 44 and 45, respectively. Also, for example, an inventive TCR, polypeptide, or protein can consist essentially of SEQ ID NO: 11, 12, 22, 23, 32, 33, 42, or 43, SEQ ID NO: 11 and 12, SEQ ID NO: 22 and 23, SEQ ID NO: 32 and 33, or SEQ ID NO: 42 and 43 in sequence. Moreover, an inventive TCR, polypeptide, or protein can consist essentially of SEQ ID NO: 5, 16, 26, or 36 (CDR 1 of α chain), SEQ ID NO: 6, 17, 27, or 37 (CDR 2 of α chain), SEQ ID NO: 7, 18, 28, or 38 (CDR 3 of α chain), SEQ ID NO: 8, 19, 29, or 39 (CDR 1 of β chain), SEQ ID NO: 9, 20, 30, or 40 (CDR 2 of β chain), SEQ ID NO: 10, 21, 31, or 41 (CDR 3 of the β chain), or any combination thereof, for example, SEQ ID NO: 5-7; 8-10; 5-10; 16-18; 19-21; 16-21; 26-28; 29-31; 26-31; 36-38; 39-41; or 36-41.

The TCRs, polypeptides, proteins (including functional portions and functional variants) of the invention can be of any length, i.e., can comprise any number of amino acids, provided that the TCR, polypeptide, or protein (or functional portion or functional variant thereof) retains its biological activity, e.g., specifically binds MAGE-A3 or MAGE-a 12; detecting cancer in the host; or the ability to treat or prevent cancer in a host, and the like. For example, the polypeptide can be in the range of about 50 to about 5000 amino acids in length, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length. In this regard, the polypeptides of the invention also include oligopeptides.

The TCRs, polypeptides, and proteins (including functional portions and functional variants) of the invention can comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α -amino N-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine β -hydroxyphenylalanine, phenylglycine, α -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N '-benzyl-N' -methyl-lysine, n ', N' -dibenzyl-lysine, 6-hydroxylysine, ornithine, α -aminocyclopentanecarboxylic acid, α -aminocyclohexanecarboxylic acid, α -aminocycloheptanecarboxylic acid, α - (2-amino-2-norbornane) -carboxylic acid, α, γ -diaminobutyric acid, α, β -diaminopropionic acid, homophenylalanine, and α -tert-butylglycine.

The TCRs, polypeptides, and proteins (including functional moieties and functional variants) of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, by, for example, disulfide bridging, or conversion to an acid addition salt and/or optional dimerization or multimerization, or coupling.

When the TCRs, polypeptides, and proteins (including functional portions and functional variants) of the invention are in the form of a salt, preferably the polypeptide is in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulfuric acids, and organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulfonic acids, e.g., p-toluenesulfonic acid.

The TCRs, polypeptides, and/or proteins of the invention (including functional portions and functional variants thereof) may be obtained by methods known in the art. Suitable methods for de novo Synthesis of polypeptides and proteins are described in references such as Chan et al, Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; peptide and Protein Drug Analysis, edit Reid, r., Marcel Dekker, inc., 2000; epitope Mapping, editors Westwood et al, Oxford University Press, Oxford, United Kingdom, 2000; and U.S. patent No. 5,449,752. Also, polypeptides and proteins can be recombinantly produced using nucleic acids described herein using standard recombinant methods. See, e.g., Sambrook et al, Molecular Cloning: a Laboratory Manual, third edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Further, some of the TCRs, polypeptides, and proteins of the invention (including functional portions and functional variants thereof) can be isolated and/or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans, and the like. Methods of isolation and purification are well known in the art. Alternatively, the TCRs, polypeptides, and/or proteins described herein (including functional portions and functional variants thereof) may be commercially synthesized by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this regard, the inventive TCRs, polypeptides, and proteins can be synthetic, recombinant, isolated, and/or purified.

Included within the scope of the invention are conjugates, for example, bioconjugates, comprising any inventive TCR, polypeptide, or protein (including any such functional portion or variant thereof), nucleic acid, recombinant expression vector, host cell population, or antibody, or antigen-binding portion thereof. In general, conjugates, and methods for synthesizing conjugates, are known in the art (see, e.g., Hudecz, F., Method mol. biol. 298: 209-54223 (2005) and Kirin et al, Inorg chem.44 (15): 5405-5415 (2005)).

By "nucleic acid" as used herein includes "polynucleotide," oligonucleotide, "" and "nucleic acid molecule," and generally means a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or obtained from a natural source (e.g., isolated and/or purified), that may contain natural, non-natural or altered nucleotides, and that may contain natural, non-natural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages in place of the phosphodiester found between unmodified oligonucleotides. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions and/or substitutions. However, as discussed herein, in some cases it may be appropriate for the nucleic acid to comprise one or more insertions, deletions, inversions and/or substitutions.

Preferably, the nucleic acid of the invention is recombinant. As used herein, the term "recombinant" refers to a molecule that is either (i) constructed outside a living cell by linking a natural or synthetic nucleic acid fragment to a nucleic acid molecule that can replicate in a living cell, or (ii) produced by replication such as those described in (i) above. For the purposes herein, the replication may be in vitro or in vivo.

The nucleic acid may be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al, supra, and Ausubel et al, supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or differently modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridinium nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, β -D-galactosylqueosine, inosine, N ⁶ -isopentenyl adenine1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N ⁶ -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β -D-mannosylqueosine, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N ⁶ Isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, 3- (3-amino-3-N-2-carboxypropyl) uracil, and 2, 6-diaminopurine alternatively, one or more nucleic acids of the invention may be purchased from companies such as Macromolecular Resources (Forr Collins, CO) and Syngen (Houston, TX).

The nucleic acid may comprise any nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or a functional portion or functional variant thereof. For example, the nucleic acid may comprise the nucleotide sequence SEQ ID NO: 46-49 consisting of the nucleotide sequence of SEQ ID NO: 46-49, or consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 46-49.

The invention also provides a nucleic acid comprising a nucleotide sequence that is complementary to a nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence that hybridizes under stringent conditions preferably hybridizes under highly stringent conditions. By "highly stringent conditions" is meant that the nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in a detectable amount that is stronger than non-specific hybridization. Highly stringent conditions include those that distinguish a polynucleotide from the exact complementary sequence, or that distinguish a sequence that contains only a few discrete mismatches from a random sequence that occasionally has a small region of some matching nucleotide sequence (e.g., 3-10 bases). Such small regions of complementarity are more readily incorporated than full-length complements of 14-17 or more bases, and highly stringent conditions make them readily distinguishable. In contrast, highly stringent conditions will include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1M NaCl or equivalent amounts at temperatures of about 50-70 ℃. Such highly stringent conditions allow for little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any inventive TCR. It is generally understood that the conditions can be made more stringent by adding increased amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, for example, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.

The nucleic acids of the invention may be incorporated into recombinant expression vectors. In this regard, the invention provides a recombinant expression vector comprising any of the nucleic acids of the invention. For the purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows for the expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct comprising the nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient for expression of the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the present invention are not naturally occurring in general. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the invention may comprise any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources and which may comprise natural, non-natural or altered nucleotides. The recombinant expression vector may comprise naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

The recombinant expression vector of the invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. The vector may be selected from the group consisting of: the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λ GT10, λ GT11, λ ZapII (Stratagene), λ EMBL4, and λ NM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, for example, a retroviral vector.

Recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al, supra, and Ausubel et al, supra. Constructs of circular or linear expression vectors can be prepared to contain replication systems that function in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific for the type of host (e.g., bacterial, fungal, plant, or animal), as appropriate and taking into account whether the vector is DNA or RNA based for integration of the vector into the host.

The recombinant expression vector may include one or more marker genes that allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, for example, to antibiotics, heavy metals, and the like, supplementation in auxotrophic hosts to provide prototrophy, and the like. Suitable marker genes for use in the expression vectors of the invention include, for example, the neomycin/G418 resistance gene, the hygromycin resistance gene, the histamine alcohol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding the TCR, polypeptide, or protein (including functional portions and functional variants thereof), or a nucleotide sequence complementary or hybridized to a nucleotide sequence encoding the TCR, polypeptide, or protein. The choice of promoter, for example, strong, weak, inducible, tissue-specific and development-specific, is within the ordinary skill of the artisan. Similarly, combinations of nucleotide sequences and promoters are also within the skill of the artisan. The promoter may be a non-viral promoter or a viral promoter, for example, a Cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long terminal repeat of murine stem cell virus.

The recombinant expression vectors of the invention may be designed for either transient expression, stable expression, or both. Likewise, the recombinant expression vector may be prepared for constitutive expression or inducible expression. Further, the recombinant expression vector may be prepared to include a suicide gene.

As used herein, the term "suicide gene" refers to a gene that causes the death of a cell that expresses the suicide gene. The suicide gene may be a gene that confers sensitivity to an agent (e.g., a drug) on a cell expressing the gene and causes cell death when the cell is contacted with or exposed to the agent. Suicide genes are known in the art (see, e.g., suide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and include, e.g., the Herpes Simplex Virus (HSV) Thymidine Kinase (TK) Gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

Another embodiment of the invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that may contain a recombinant expression vector of the invention. The host cell may be a eukaryotic cell, for example, a plant, an animal, a fungus, or an algae, or may be a prokaryotic cell, for example, a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, i.e., isolated directly from an organism, for example, a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5 α escherichia coli cells, chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the amplification or replication of the recombinant expression vector, the host cell is preferably a prokaryotic cell, for example, a DH 5a cell. For the production of recombinant TCRs, polypeptides, or proteins, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. Whilst the host cell may be of any cell type, may be derived from any type of tissue, and may be of any developmental stage, the host cell is preferably a Peripheral Blood Lymphocyte (PBL) or Peripheral Blood Mononuclear Cell (PBMC). More preferably, the host cell is a T cell.

For the purposes herein, the T cell may be any T cell, such as a cultured T cell, for example, a primary T cell, or a T cell derived from a cultured T cell line, for example, Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human. The T cells may be any type of T cell and may be at any developmental stage, including, but not limited to, CD4 ⁺ /CD8 ⁺ Double positive T cell, CD4 ⁺ Helper T cells, e.g. Th ₁ And Th ₂ Cell, CD8 ⁺ T cells (e.g., cytotoxic T cells), Tumor Infiltrating Lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), natural T cells, and the like. Preferably, the T cell is CD8 ⁺ T cells or CD4 ⁺ T cells.

Also provided by the invention is a cell population comprising at least one host cell described herein. The cell population can be a heterogeneous population of host cells comprising any described recombinant expression vector in addition to at least one other cell (e.g., host cells that do not comprise any recombinant expression vector (e.g., T cells), or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc.). Alternatively, the population of cells can be a substantially homogeneous population, wherein the population comprises (e.g., consists essentially of) host cells comprising the recombinant expression vector. The population may also be a clonal population of cells, wherein all cells of the population are clones of a single host cell comprising the recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the cell population is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

The invention further provides antibodies, or antigen-binding portions thereof, that specifically bind to a functional portion of any of the TCRs described herein. Preferably, the functional moiety specifically binds to the cancer antigen, for example, the functional moiety comprises the amino acid sequence SEQ ID NO: 5, 16, 26, or 36 (CDR 1 of α chain), 6, 17, 27, or 37 (CDR 2 of α chain), 7, 18, 28, or 38 (CDR 3 of α chain), 8, 19, 29, or 39 (CDR 1 of β chain), 9, 20, 30, or 40 (CDR 2 of β chain), 10, 21, 31, or 41 (CDR 3 of β chain), SEQ ID NO: 11, 22, 32, or 42 (variable region of α chain), SEQ ID NO: 12, 23, 33, or 43 (variable region of beta strand), or a combination thereof, for example, 5-7; 8-10; 5-10; 16-18, 19-21; 16-21; 26-28; 29-31; 26-31; 36-38; 39-41; or 36-41. More preferably, said functional portion comprises SEQ ID NO: 5-10 or SEQ ID NO: 26-31. In a preferred embodiment, the antibody, or antigen binding portion thereof, binds to an epitope consisting of all 6 CDRs (CDR 1-3 of the alpha chain and CDR1-3 of the beta chain). The antibody may be any type of immunoglobulin known in the art. For example, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, and the like. The antibody may be monoclonal or polyclonal. The antibody can be a naturally occurring antibody, for example, an antibody isolated and/or purified from a mammal (e.g., a mouse, rabbit, goat, horse, chicken, hamster, human, etc.). Alternatively, the antibody may be a genetically engineered antibody, for example, a humanized antibody or a chimeric antibody. The antibodies can be in monomeric or multimeric form. Likewise, the antibody may have any level of affinity (affinity) or avidity (avidity) for the functional portion of the inventive TCR. Ideally, the antibody is specific for the functional portion of the inventive TCR such that there is minimal cross-reactivity with other peptides or proteins.

Methods of detecting the ability of an antibody to bind to any functional portion of the inventive TCR are known in the art and include any antibody-antigen binding assay, such as, for example, Radioimmunoassays (RIA), ELISA, western blots, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al, infra, and U.S. patent application publication No. 2002/0197266a 1).

Suitable methods for preparing antibodies are known in the art. For example, standard hybridoma methods are described, for example,and Milstein, eur.j.immunol., 5, 511-: a Laboratory Manual, CSH Press (1988), and C.A. Janeway et al (eds.), immunology, fifth edition, Garland Publishing, New York, NY (2001). Alternatively, other Methods, such as the EBV-hybridoma method (Haskard and Archer, J.Immunol. Methods, 74(2), 361-67(1984), and Roder et al, Methods Enzymol., 121, 140-67(1986)), and phage vector expression systems (see, e.g., Huse et al, Science, 246, 1275-81(1989)) are known in the art. Further, methods for producing antibodies in non-human animalsMethods are described, for example, in U.S. Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. patent application publication No. 2002/0197266A 1.

Phage display can additionally be used to produce antibodies of the invention. In this regard, phage libraries encoding the antigen-binding variable (V) domains of antibodies can be generated using standard Molecular biology and recombinant DNA techniques (see, for example, Sambrook et al (eds.), Molecular Cloning, A Laboratory Manual, third edition, Cold Spring Harbor Laboratory Press, New York (2001)). Phage encoding variable regions with the desired specificity are selected for specific binding to the desired antigen, and the recombinant full or partial antibody is one that comprises the selected variable domain. Nucleic acid sequences encoding the recombinant antibodies are introduced into a suitable cell line, such as a myeloma cell for hybridoma production, such that antibodies having the characteristics of a monoclonal antibody are secreted by the cell (see, for example, Janeway et al, supra, hue et al, supra, and U.S. patent 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and are described, for example, in U.S. Pat. Nos. 5,545,806 and 5,569,825, and Janeway et al, supra.

Methods for producing humanized antibodies are well known in the art and are described in detail, for example, in Janeway et al, previously, U.S. Pat. nos. 5,225,539, 5,585,089 and 5,693,761, european patent No. 0239400B 1, and british patent No. 2188638. Humanized antibodies can also be generated using antibody resurfacing techniques described, for example, in U.S. Pat. No. 5,639,641 and Pedersen et al, J.mol.biol., 235, 959-973 (1994).

The invention also provides an antigen binding portion of any of the antibodies described herein. The antigen binding portion can be a peptide having at least one antigen binding site (e.g., Fab, F (ab') ₂ dsFv, sFv, diabodies, and triabodies.

Single chain variable fragment (sFv) antibody fragments, which consist of truncated Fab fragments comprising the variable (V) domain of the antibody heavy chain linked to the V domain of the light antibody chain by a synthetic peptide, can be produced using conventional recombinant DNA processing techniques (see, e.g., Janeway et al, supra). Similarly, disulfide-stabilized variable region fragments (dsFvs) can be prepared by recombinant DNA techniques (see, e.g., Reiter et al, Protein Engineering, 7, 697-. However, the antibody fragment of the present invention is not limited to these typical types of antibody fragments.

Also, the antibody, or antigen-binding portion thereof, may be modified to include a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., a gold particle).

Inventive TCRs, polypeptides, proteins, (including functional portions and functional variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof), may be isolated and/or purified. The term "isolated" as used herein means having been removed from its natural environment. The term "purified" as used herein means having been increased in purity, wherein "purity" is a relative term and is not necessarily to be understood as absolute purity. For example, the purity can be at least about 50%, can be greater than 60%, 70%, 80%, 90%, 95%, or can be 100%.

The inventive TCRs, polypeptides, proteins (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof), collectively referred to hereinafter collectively as "inventive TCR materials," can be formulated as compositions, such as pharmaceutical compositions. An inventive pharmaceutical composition comprising any inventive TCR material may comprise more than one inventive TCR material, for example, a polypeptide and a nucleic acid, or two or more different TCRs. Alternatively, the pharmaceutical composition may comprise the inventive TCR material in combination with other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier may be any that is convenient to use and is limited only by chemical-physical considerations (such as solubility and lack of reactivity with the active compound) and by the mode of administration. The pharmaceutically acceptable carriers, e.g., carriers (vehicles), adjuvants, excipients, and diluents described herein are well known to those skilled in the art and readily available to the public. Preferably, the pharmaceutically acceptable carrier is one that chemically intercalates the active agent and one that does not have deleterious side effects or toxicity under the conditions of use.

The choice of vector will be determined in part by the particular inventive TCR material and by the particular method used to administer the inventive TCR material. Thus, there are a variety of suitable formulations of the pharmaceutical compositions of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal use are typical and in no way limiting. More than one route may be used to administer the inventive TCR materials, and in some instances, a particular route may provide a more direct and more effective response than other routes.

Topical formulations are well known to those skilled in the art. Such formulations are particularly suitable within the scope of the present invention for application to the skin.

Formulations suitable for oral administration may consist of (a) a liquid solution, such as an effective amount of the inventive TCR material dissolved in a diluent, such as water, saline, or orange juice; (b) capsules, sachets, tablets, dragees (lozenes), and troches (troches), each containing a predetermined amount of active ingredient in solid or granulated form; (c) a powder; (d) a suspension in a suitable liquid; and (e) a suitable emulsion. Liquid formulations may include diluents, such as water and alcohols, e.g., ethanol, benzyl alcohol, and polyvinyl alcohol, with or without the addition of pharmaceutically acceptable surfactants. The capsule form may be of the conventional hard or soft shell gel type, and contains, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms may include one or more of lactose, sucrose, mannose, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, wetting agents, preservatives, flavoring agents, and other pharmaceutically acceptable excipients. Lozenge (Lozenge) forms may comprise the inventive TCR material in a flavourant, typically sucrose, and acacia or tragacanth, and pastilles (pastilles) comprise the inventive TCR material in an inert matrix, such as gelatin and glycerol, or sucrose and acacia, emulsions, gums and the like in addition to such excipients as are known in the art.

The inventive TCR materials, alone or in combination with other suitable components, can be prepared as an aerosol formulation for administration by inhalation. These aerosol formulations may be placed in a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like. It may also be formulated as a medicament for atmospheric preparation, such as in a nebulizer (nebulizer) or nebulizer (atomizer). Such spray formulations are also used for spraying mucus.

Formulations suitable for parenteral administration include aqueous or non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The inventive TCR materials may be administered as a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or a mixture of liquids, including water, saline, aqueous glucose, and related sugar solutions, alcohols, such as ethanol or cetyl alcohol, glycols, such as propylene glycol or polyvinyl alcohol, dimethyl sulfoxide, glycerol, ketals, such as 2, 2-dimethyl-1, 3-dioxolane-4-methanol, ethers, poly (vinyl alcohol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of a pharmaceutically acceptable surfactant, such as fatty acid salts or detergents, suspending agents, if gum, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils that may be used in the parenteral formulation include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, paraffin oil and mineral oil. Suitable fatty acids for use in the parenteral formulation include oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable fatty acid salts for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolammonium salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyldihydrocarbylammonium halides, and haloalkylpyridines, (b) anionic detergents such as, for example, alkyl, aryl, and alkene sulfonates, alkyl, alkene, ether, and monoglyceride glyceryl sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, oxidized fatty amines, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

Typically, the parenteral formulation will comprise from about 0.5% to about 25%, or more, by weight of the inventive TCR material in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may comprise one or more nonionic surfactants having a lipophilic balance (HLB) of from about 12 to about 17. The mass of surfactant in such formulations typically ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Injectable formulations are consistent with the present invention. The need for effective pharmaceutical carriers for Injectable compositions is well known to those of ordinary skill in the art (see, for example, pharmaceuticals and pharmaceutical Practice, J.B. Lippincott Company, Philadelphia, PA, Bank and Chalmers, eds., pp.238-250 (1982), and ASHP Handbook on Inable Drugs, Toissel, fourth edition, pp.622-630 (1986)). Preferably, when cells, for example, T cells, are administered, the cells are administered by injection.

One skilled in the art will appreciate that the inventive TCR materials of the present invention can be configured to comprise complexes, such as cyclodextrin inclusion complexes, or liposomes, in addition to the pharmaceutical compositions described above.

For purposes of the present invention, the amount or dose of the inventive TCR materials administered should be sufficient to produce an effect, for example, a therapeutic or prophylactic response in a subject or animal over a suitable time frame. For example, the dose of the inventive TCR materials should be sufficient to bind to a cancer antigen, or to detect, treat or prevent cancer over a period of from about 2 hours or more, e.g., 12 to 24 or more hours, from the time of administration. In some embodiments, the period of time may be even longer. The dosage will be determined by the efficacy of the particular inventive TCR material and the condition of the animal (e.g., human), as well as the weight of the treated animal (e.g., human).

Many assays for determining the dosage administered are known in the art. For purposes of the present invention, the initial dose administered to a mammal can be determined using an assay comprising comparing the extent of target cell lysis or the extent of secretion of IFN- γ by T cells expressing the invention when a given dose of such T cells is administered to a mammal in a group of mammals each of which is administered a different dose of T cells. When a dose is administered, the extent to which the target cells lyse and/or secrete IFN- γ can be tested by methods known in the art.

The dosage of the inventive TCR materials will also be determined by the presence, nature and extent of any deleterious side effects that may accompany the administration of a particular inventive TCR material. Typically, the attending physician will determine the dosage of the inventive TCR material for use in treating an individual patient, taking into account a number of factors (such as age, body weight, general health, diet, sex, TCR material of the invention to be administered, route of administration and severity of the condition being treated). For example, and not intended to limit the invention, dosages of the inventive TCR materials can range from about 0.001 to about 1000mg/kg body weight/day or more of the subject being treated, from about 0.01 to about 10mg/kg body weight/day or more, or from about 0.01mg to about 1mg/kg body weight/day or more. In one embodiment, where the inventive TCR material is a population of cells, the number of cells administered can vary, for example, from about 1x10 ⁶ To about 1x10 ¹¹ Cells or more.

One of ordinary skill in the art will readily appreciate that the inventive TCR materials of the present invention can be modified in a number of ways so as to increase the therapeutic or prophylactic efficacy of the inventive TCR materials by modification. For example, the inventive TCR materials can be coupled to a targeting moiety directly or indirectly through a bridge. The practice of coupling compounds (e.g., inventive TCR materials versus targeting moieties) is known in the art. See, e.g., Wadwa et al, j.drug Targeting 3: 111(1995) and us patent 5,087,616. The term "targeting moiety", as used herein, refers to any molecule or agent that specifically recognizes and binds to a cell surface receptor such that the targeting moiety directs the delivery of the inventive TCR material to a population of cells that surface express the receptor. Targeting moieties include, but are not limited to, antibodies, or fragments thereof, that bind to cell surface receptors (e.g., Epithelial Growth Factor Receptor (EGFR), T Cell Receptor (TCR), B Cell Receptor (BCR), CD28, platelet-derived growth factor receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.), peptides, hormones, growth factors, cytokines, and any other natural or non-natural ligand. The term "bridge," as used herein, refers to any agent or molecule that links the inventive TCR material to a targeting moiety. One of ordinary skill in the art recognizes that sites on the inventive TCR material that are not necessary for the function of the inventive TCR material are ideal sites for attachment of a bridge and/or targeting moiety, provided that the attachment of the bridge and/or targeting moiety to the inventive TCR material does not affect the function of the inventive TCR material, i.e., binding to MAGE-A3 or MAGE-a 12; or the ability to detect, treat, or prevent cancer.

Alternatively, the inventive TCR materials can be modified into a stored form such that the manner in which the inventive TCR materials are released into the body to which they are administered is controllable relative to the time and location in the body (see, e.g., U.S. patent 4,450,150). A stock form of the inventive TCR material can be, for example, an implantable composition comprising the inventive TCR material and a porous or non-porous material (e.g., a polymer), wherein the inventive TCR material is encapsulated by the material or dispersed throughout the material and/or degradation products of the non-porous material. The depot is then implanted at a desired site in the body and the TCR material of the invention is released from the implant at a predetermined rate.

It is contemplated that the inventive pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations can be used in methods of treating or preventing cancer. Without being bound by a particular theory, it is believed that the inventive TCR specifically binds to MAGE-A3, MAGE-a12, such that the TCR (or related inventive polypeptide or protein), when expressed by a cell, is capable of mediating an immune response against a target cell expressing MAGE-A3 or MAGE-a 12. In this regard, the invention provides a method of treating or preventing cancer in a host, comprising administering to the host any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the host.

The terms "treatment," treating, "" and "prevention" and words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there is a varying degree of treatment or prevention that one of ordinary skill in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the inventive methods can provide any level of therapeutic or prophylactic amount of cancer in the host. In addition, the treatment or prevention provided by the methods of the invention may include the treatment or prevention of one or more conditions or symptoms of a disease (e.g., a cancer being treated or prevented). Also, for purposes herein, "preventing" may include delaying the onset of the disease or symptoms or conditions thereof.

Also provided are methods of detecting the presence of cancer in a host. The method comprises (i) contacting a sample comprising cancer cells with any of the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or antibodies, or antigen binding portions thereof, described herein, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the host.

With respect to the inventive method of detecting cancer in a host, the sample of cancer cells can be a sample comprising whole cells, a lysate thereof, or a portion of a whole cell lysate (e.g., a nuclear or cytoplasmic portion, a whole protein portion, or a nucleic acid portion).

For the detection method of the invention, the contacting may occur in vitro or in vivo with respect to the host. Preferably, the contacting is in vitro.

Furthermore, complex detection can occur in many ways known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or antigen-binding portions thereof of the invention described herein can be labeled with detectable labels such as, for example, radioisotopes, fluorophores (e.g., Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

For the methods of the invention, wherein a host cell or population of cells is administered, the cells may be heterologous or autologous to the host. Preferably, the cells are autologous to the host.

As for the inventive method, the cancer may be any cancer, including sarcomas (e.g., synovial sarcoma, osteogenic sarcoma, uterine leiomyosarcoma, and alveolar rhabdomyosarcoma), lymphomas (e.g., hodgkin lymphoma and non-hodgkin lymphoma), hepatocellular carcinoma, glioma, head cancer (e.g., squamous cell carcinoma), neck cancer (e.g., squamous cell carcinoma), acute lymphoma, leukemia (e.g., acute myeloid leukemia and chronic lymphocytic leukemia), bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joint, cancer of the neck, gall bladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic myeloid cancer, colon cancer (e.g., colon malignancy), esophageal cancer, cervical cancer, gastric and intestinal carcinoid tumors, hypopharyngeal cancer, laryngeal cancer, liver cancer (e.g., hepatocellular cancer), lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancers, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma), small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, and urothelial cancer (e.g., ureteral cancer and bladder cancer).

The host referred to in the method of the invention may be any host. Preferably, the host is a mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order rodentia, such as mice and hamsters, and mammals of the order lagomorphis, such as rabbits. Preferably, the mammal is derived from the order carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is derived from the order artiodactyla, including bovines (cows) and porcines (pigs) or perssodactyla, including equines (horses). Most preferably, the mammal is primate, Ceboids, or Simoids (monkey) or of human-like order (human and ape). A particularly preferred mammal is a human.

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the cloning of TCR genes and the generation of TCR constructs derived from T cell clones.

Initially, four T cell clones were identified that recognized epitopes of the MAGE-a gene family in the case of the dominant class I (class I) alleles HLA-a 01 and C07. About 30% of melanoma patient populations express HLA-A01, and more than 95% HLA-A01 ⁺ Individuals express HLA-a 0101 subtypes, while more than 50% of melanoma patients express two dominant HLA-C07 subtypes, C07: 01 and C07: 02.

The expressed TCR alpha and beta chains were isolated from two clones, A10 and 13-18, which recognized residues 168-176 of the protein MAGE-A3 in the case of HLA-A01 (MAGE-A3: 168-176). In addition, HLA-C07 restricted TCRs recognizing peptides corresponding to residues 170-178 (MAGE-A12: 170-178) of the MAGE-A12 protein were isolated from clone 502 and FM 8.

The α and β chains encoding functional TCRs were isolated from two MAGE-A12-reactive, HLA-C07-reactive T cell clones, PHIN LB831-501D/19, denoted "502" (Heidecker et al, J.Immunol., 164: 6041-6045(2000)) and "FM 8" (Panelli et al, J.Immunol., 164: 4382-4392(2000)), and two MAGE-A3-reactive, HLA-A01-restricted T cell clones, LAU147CTL1 or 810/A10, denoted "A10" (parameterier et al, Nat.Immunol., 11: 449-454(2010)) and AV1000P-113-18, denoted "13-18". Briefly, total RNA isolated from T cell clones was reverse transcribed as cDNA using oligo-dT using the SMART RACE cDNA amplification kit (Clontech, Mountain View, Calif.). The TCR alpha and beta chains expressed by the T cell clones were identified by performing a5 ' -RACE reaction using primer 5'-CACTGTTGCTCTTGAA GTCC-3' (SEQ ID NO: 55) complementary to the TCR alpha chain constant region and 5'-CAGGCAGTAT CTGGAGTCATTGAG-3' (SEQ ID NO: 56) complementary to the TCR beta chain constant region in combination with a linker primer derived from the SMART RNA synthesis kit. After sequencing the 5' -RACE product, the full-length gene product is amplified using specific primers designed to amplify the appropriate full-length TCR α and β chains. A10TCR expresses AV12-1/BV24-1, 13-18 expresses AV12-3/BV15, 502TCR expresses AV13-1/BV25-1, and FM8 expresses AV38-2/BV 4-3.

Transcripts encoding paired alpha and beta chains for each of the four T cell clones were inserted into the MSGV1 retroviral expression vector.

Example 2

This example shows the reactivity of cells expressing anti-MAGE-A3 TCR-A10(SEQ ID NOS: 13 and 14) and anti-MAGE-A3 TCR 13-18(SEQ ID NOS: 24 and 25) in response to HLA-A1+/MAGE-A3+ cells.

anti-CD 3 stimulated T cells transduced with TCR-A10(SEQ ID NO: 46) and TCR 13-18(SEQ ID NO: 48) were evaluated for their ability to recognize a panel of MAGE-A3 expressing HLA-A01 + melanoma cell lines. Untransduced (UT) and transduced cells were co-cultured overnight with different tumor cell lines (Table 1A, 1B and FIG. 6A) and interferon-gamma (IFN-. gamma.) (pg/ml) was measured.

TABLE 1A

TABLE 1B

Tumor(s)	HLA-A*01	MAGE-A3
			2984mel	+	+
397mel	+	+
			2630mel	+	+
2556mel	+	+
			526mel	-	+
624mel	-	+
			2359mel	-	+
2661RCC	+	-

The result shows evaluationSix of the eight HLA-a 01+/MAGE-A3+ melanoma cell lines stimulated higher levels of IFN- γ release from TCR a10 than from TCR 13-18-transduced T cells (fig. 1A). After co-culture of TCR-transduced T cells with two HLA-A1+ melanoma cell lines expressing relatively low levels of MAGE-A3, A375mel and 537mel, lower levels of IFN- γ were released, while TCR A10-transduced T cells released higher levels of IFN- γ in response to these target cells than TCR 13-18 transduced T cells. These responses were restricted by HLA-a1, since 1300mel, lacking HLA-a1 expression, failed to stimulate IFN- γ release from TCR a10 and TCR 13-18-transduced cells, whereas the cell line generated by transfection of the parental 1300mel cell line with HLA-a 01 (designated 1300-a1) stimulated IFN- γ release from TCR a10 and TCR 13-18 transduced T cells. HLA-a 01+ renal cancer cell line, 2661RCC, lacking MAGE-A3 expression failed to stimulate significant IFN- γ release from TCR a10 and TCR 13-18 transduced T cells. These results indicate that when combined with MAGE-A3 ⁺ /HLA-A1 ⁺ When the target cells were co-cultured, the cells expressing TCRA10 released higher levels of IFN-. gamma.than the cells expressing TCR-13-18. These results also indicate that TCR A10 and TCR-13-18 are stimulated in the presence of MAGE-A3+/HLA-A1+ target cells.

The results of co-culture assays with transduced PBMCs indicated that TCRA 10-transduced T cells produced high levels of IFN- γ in response to HLA-a 01+/MAGE-A3+ tumor cell lines 397mel 2984mel, and 2556 mel. Cytokine levels were between five to ten times those generated from TCR 13-18 transduced T cells (fig. 6A). The MAGE-A3+ but HLA-a 01 negative cell lines 562, 624 and 2359mel, and the MAGE-A3 negative but HLA-a 01+ kidney cancer cell line 2661RCC failed to stimulate significant cytokine levels in one of the T cells transduced from TCR a10 or 13-18 (fig. 6A).

The level of transduction of the TCR was assessed using a quantitative PCR assay performed using genomic DNA with forward (SEQ ID NO: 58) and reverse (SEQ ID NO: 59) primers and probes (SEQ ID NO: 60) designed to specifically detect the MSGV1 retroviral LTR but not human endogenous retroviral sequences. The level of amplification product was normalized to a positive control sample of PBMC transduced with a TCR directed against the NY-ESO-1: 157-165 epitope estimated to contain about 80% transduced T cells by staining with the NY-ESO-1 tetramer.

The difference in activity of T cells transduced with a10 or 13-18 TCRs did not appear to be due to the difference in frequency of transduction with the two TCRs, as they appeared to be comparable (fig. 6B). Furthermore, T cells transduced with a10TCR recognized target cells incubated with the MAGE-a3168-176 peptide at a minimum concentration of 0.5nM, which is 10-fold lower than that required for recognition by cells transduced with 13-18TCR (fig. 6C), indicating that a10TCR has a higher functional affinity than 13-18 TCR.

Fresh, uncultured tumor cells were evaluated for their ability to stimulate T cells transduced with one of TCR A10(SEQ ID NO: 46) or DMF5 (TCR against HLA-A0201/MART-1: 27-35T cell epitope). Untransduced (UT) and transduced cells were co-cultured with different fresh tumors and IFN- γ (pg/ml) was measured.

The results showed that TCR a 10-transduced T cells recognized four of the four tested HLA-a 01+/MAGE-A3+ fresh tumors (FrTu 2767, FrTu 3178, FrTu 2823 and FrTu 3068), and DMF 5-transduced T cells recognized both of the tested HLA-a 0201+/MART-1+ fresh tumor cells (FrTu 2851 and FrTu 3242) (fig. 1B). The TCR a10 transduced T cells failed to recognize HLA-a 0201+ fresh tumors, while DMF5 transduced T cells failed to recognize HLA-a 01+ fresh tumors, indicating that IFN- γ secretion by TCR a10 is an HLA-a1+/MAGE-A3+ -specific reaction.

T cells transduced with TCR A10 and 13-18 recognized six strains of MAGE-A3+ and HLA-A01 + fresh tumor (FrTu), five of FrTu 3178, 2767, 2823, 2830 and 3068, but not or FrTu 2685 (HLA-A01 lacking MAGE-A3 expression) ⁺ Fresh tumor) or three strains of MAGE-A3 deficient in HLA-A01 expression ⁺ Fresh tumors, FrTu 2181, 3242 and 2803 (FIG. 7A; Table 1C).

TABLE 1C

Fresh tumor	HLA-A*01	MAGE-A3
			3178	+	+
2767	+	+
			2823	+	+
2830	+	+
			3068	+	+
2268	+	+
			2685	+	-
2181	-	+
			3242	-	+
2803	-	+

Example 3

This example shows the reactivity of cells expressing anti-MAGE-A12 TCR502(SEQ ID NOS: 34 and 35) or anti-MAGE-A12 TCR FM8(SEQ ID NOS: 44 and 45) in response to co-culture with HLA-Cw07 +/MAGE-A12+ cells.

anti-CD 3-stimulated CD8 isolated from two patient PBMC samples ⁺ T cells were transduced with control constructs encoding truncated human low affinity Nerve Growth Factor Receptor (NGFR) and the ability of TCR502(SEQ ID NO: 47), or TCR FM8(SEQ ID NO: 49), to recognize a panel of CW07 + melanoma cell lines expressing MAGE-A12 was evaluated.

Expression of the MAGE-A12 gene product was assessed by Q-PCR using two primers (SEQ ID NOS: 61 and 62) designed to specifically amplify the MAGE-A12 gene product but not other members of the MAGE-A gene family, and a probe specific for MAGE-A12 (SEQ ID NO: 63). Antigen expression was determined using a plasmid control as a standard to assess copy number and using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for normalization. Expression of greater than 1, 000 copies of MAGE-A12 Per 10 ⁶ Tumor cell lines of GAPDH and fresh tumors were shown to be positive for MAGE-a12 expression.

The transduced cells were co-cultured overnight with different tumor cell lines (Table 2A; FIGS.6D and 6F) and IFN- γ (pg/m1) was measured.

TABLE 2A

Irritant substance	HLA-C allelesGene	MAGE-A12
			1910mel	0701，0303	+
586mel	0701	+
			2359mel	0701，16	+
F002mel	0701，1203	+
			1300mel	0702	+
624mel	0702，0802	+
			SK23mel	0701，0702	+
1909mel	0701，0702	+
			1011mel	0702	-
397mel	0701	+
			526mel	-	+
2556mel	-	+
			2984mel	-	+
2630mel	0701	-

The results showed that T cells transduced with TCR502 recognized eight of the MAGE-a12+ melanoma cell lines expressing one of HLA-Cw0701 or 0702, whereas T cells transduced with TCR FM8 only recognized melanoma cell lines expressing HLA-Cw0702 (fig. 2A and 2B; see also fig. 6F). Furthermore, TCR502 transduced T cells released higher levels of IFN- γ (figs.6d and 6F) in response to HLA-Cw0702 + target SK23mel, 1300mel and 624mel when compared to TCR FM8 transduced T cells. 1011mel cells (which express HLA-Cw0702 but lack expression of MAGE-a 12) did not stimulate significant cytokine release from cells transduced with TCR502 or TCR FM 8. T cells transduced with the control construct encoding NGFR failed to respond significantly to any of the targets tested. These results indicate that the 502 cells expressing the TCR released higher levels of IFN- γ than the cells expressing TCR FM8 when co-cultured with MAGE-A12+/HLA-Cw7 target cells, and that TCR502 recognized MAGE-A12 in the case of either HLA-Cw0701 or HLA-Cw 0702. These results also indicate that TCRA502 and TCR FM8 were stimulated in the presence of MAGE-A12+ cells.

The TCR502 transduced T cells recognized HLA-C0702 +, MAGE-a12+ tumor 624mel, and two HLA-C07: 01+, MAGE-A12+ tumors, 397 and 2359mel, whereas FM8 transduced T cells recognized HLA-C07: the 02+ tumor cell line 624mel failed to recognize 397 and 2359mel (fig. 6D). None of the transduced T cell populations identified 526, 2556, or 2984mel (a MAGE-A3+ melanoma cell line lacking HLA-C07 expression), or 2630mel (an HLA-C07: 01+ tumor cell line lacking MAGE-a12 expression) (fig. 6D). These differences do not appear to be due to differences in the transduction frequencies of the two TCRs (as measured as described in example 2), which are similar in cells transduced with either TCR (fig. 6B). In addition, cells transduced with 502TCR recognized the presence of a minimal concentration of 2.5nM MAGE-a 12: 170-.

T cells transduced with MAGE-A12-reactive TCRs were then evaluated for their response to enzymatic digestion of fresh, uncultured tumor cells. T cells transduced with TCR502 recognized four HLA-C0701-expressing MAGE-A12 ⁺ One of the fresh tumors, FrTu 3068, and TCR502 and FM8 transduced T cells recognized two HLA-C07 expressing: 02 MAGE-a12 ⁺ One of the tumors, FrTu 2181 (FIG. 7B; Table 2B). None of the TCR-transduced T cell populations recognized HLA-C07: 01 ^- And 07: 02 ^- Fresh tumors 2767 or 2823, or MAGE-A12 lacking MAGE-A12 expression ^- Tumors 2685, 3242 and 2803.

TABLE 2B

Example 4

This example demonstrates a population that can be treated using the inventive TCR.

About 28% of the patient population expressed HLA-a 01, and about 54% of the patient population expressed HLA-Cw 07. Two predominant subtypes of HLA-Cw07, Cw0701 and Cw0702 are expressed by about 27% and about 31% of the patient population, respectively. FIG. 3A illustrates the cumulative percentage of populations expected to be treatable by using TCRs restricted by HLA-A1, HLA-A2, and/or HLA-Cw7 (based on the percentage of these alleles in the general Caucasian population).

Because about 30% of patients express high levels of MAGE-A3 and MAGE-a12, the use of the inventive TCR will render a significantly higher percentage of patients suitable for TCR-based adoptive immunotherapy. FIGS. 3B and 3C illustrate the cumulative percentage of patient populations expected to be human melanoma (FIG. 3B) and synovial cell sarcoma (FIG. 3C) treatable with TCRs that recognize NY-ESO-1 in the case of HLA-A2; MAGE-A3 in the case of HLA-A1; MAGE-A3 and MAGE-A12 in the case of HLA-A2; and/or MAGE-a12 in the case of HLA-Cw 7.

Example 5

This example shows the reactivity of cells expressing TCR502 or TCR FM8 in response to co-culture with target cells expressing HLA-Cw0701 or HLA-Cw0702 stimulated with peptides derived from different proteins of the MAGE family.

Cells transduced with NGFR, TCR502(SEQ ID NO: 47), or TCR FM8(SEQ ID NO: 49) were co-cultured with cells expressing HLA-Cw0701 or HLA-Cw 0702. IFN-gamma (pg/ml) secretion was measured.

The results show that cells transduced with TCR502 recognized HLA-Cw0701 or HLA-Cw0702 cells when stimulated with MAGE-A12 (VRIGHLYIL; SEQ ID NO: 4) and cells transduced with TCR FM8 recognized HLA-Cw0702 cells when stimulated with MAGE-A12 (VRIGHLYIL; SEQ ID NO: 4) (FIG. 4). Cells transduced with NGFR did not show significant reactivity.

Example 6

This example demonstrates the specificity of anti-MAGE-A3 and anti-MAGE-A12 TCR.

PMBC derived from a single donor were either untransduced or transfected following stimulation with anti-CD 3 antibodyTransduction of PBMCs with anti-MAGE-A12 TCR502(SEQ ID NO: 47), anti-MAGE-A12 TCR FM8(SEQ ID NO: 49), anti-MAGE-A3 TCR A10(SEQ ID NO: 46), anti-MAGE-A3 TCR 13-18(SEQ ID NO: 48), or anti-MAGE-A3 TCR 112-120. Thirteen days after stimulation, 4 hours at standard ⁵¹ In the Cr release assay, transduced T cells were incubated with tumor targets listed in table 3.

TABLE 3

	MAGE-A3	MAGE-A12	HLA-A	HLA-C
					397mel	+	+	01/02	0401/0701
624mel	+	+	02/03	0702/0802
					2984mel	+	+	01/02	06
2661RCC	-	-	01/02	07

As shown in FIGS. 5(A) -5(D), anti-MAGE-A12 TCR502 specifically lyses tumor cells expressing MAGE-A12 and HLA-Cw7 and does not lyse tumor cells lacking MAGE-A12 or HLA-Cw7 expression. anti-MAGE-A3 TCR A10 specifically lyses tumor cells expressing MAGE-A3 and HLA-A1 and does not lyse tumor cells lacking MAGE-A3 or HLA-A1 expression.

Example 7

This example demonstrates the specificity of anti-MAGE-A3 and anti-MAGE-A12 TCR.

Monkey kidney cell line COS-7 was purified using HLA-a 01, C07: 01 or C07: 02 plus one of MAGE-A3, A1, A2, A4, A6, A9, A10 or A12. The following day T cells were added, either transduced with TCR A10, 13-18 or untransduced control cells or TCR502, FM8 or untransduced control cells, and the release of soluble IFN- γ was assessed by ELISA after overnight co-culture.

T cells transduced with MAGE-A3-reactive TCR A10 recognize HLA-A1 transfected with MAGE-A3 ⁺ Target cells, but failed to recognize targets transfected with MAGE-a1, a2, a4, a6, a9, a10 or a12 constructs (fig. 8A) encoding proteins similar to MAGE-A3: the 170-178 epitope differs between 1-3 sites. T cells transduced with one of TCR502 or FM8 recognized HLA-C07 transfected with MAGE-a 12: 02 ⁺ Target but not MAGE-A3, a1, a2, A4, A6, a9, a10 (fig. 8B), whereas T cells transduced with TCR502 but not FM8 recognized HLA-C07: 01 ⁺ Target, but not additional MAGE-a family members tested (fig. 8C).

Example 8

This example demonstrates the reactivity of transduced T cells.

Purified CD8+ and CD4+ T cells were isolated by negative screening using CD8 and CD4T lymphocyte enrichment kits (Becton/Dickinson, Franklin Lakes, n.j.), followed by positive screening using CD8 and CD4 magnetic beads (Becton/Dickinson). The isolated CD8+ and CD4+ cells were evaluated by Fluorescence Activated Cell Screening (FACS) analysis to contain less than 1% contaminating CD4+ and CD8+ T cells, respectively.

Isolated populations of CD8+ and CD4+ T cells transduced with the TCR were subsequently evaluated for response to tumor cell targets. Highly purified TCR a10 transduced CD4+ T cells containing less than 1% contaminating CD8+ T cells released low but significant levels of IFN- γ in response to MAGE-A3+ tumor cell line 397mel stably transfected with HLA-a 01 and MAGE-A3+ tumor cell line 1300Almel (table 4A; fig. 9B). CD8+ T cells transduced with TCR A10 released interferon- γ in response to 397mel or 1300-Al mel (Table 4A; FIG. 9A). CD8+ T cells transduced with TCR502 or TCR FM8 released interferon- γ in response to 624mel, and TCR502 released interferon- γ in response to 397mel (fig. 9C and table 4B).

TABLE 4A

TABLE 4B

Cell lines	HLA-C*07	MAGE-A12
			397mel	01	+
624mel	02	+
			2359mel	01	+
526mel	-	+
			2661RCC	-	-

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The term "at least one" followed by a list of one or more items (e.g., "at least one of a and B") will be understood to mean one item selected from the listed items (a or B) or a combination of two or more of the listed items (a and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," "containing," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary terminology (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims (13)

1. An isolated or purified T Cell Receptor (TCR), which

(a) Has antigen specificity for melanoma antigen family a (mage a) -3 in the case of HLA-a1, and comprises:

the alpha chain Complementarity Determining Region (CDR)1 amino acid sequence of SEQ ID NO. 5,

the alpha chain CDR2 amino acid sequence of SEQ ID NO. 6,

the amino acid sequence of the alpha chain CDR3 of SEQ ID NO. 7,

the beta chain CDR1 amino acid sequence of SEQ ID NO. 8,

the beta chain CDR2 amino acid sequence of SEQ ID NO. 9, and

10, the beta chain CDR3 amino acid sequence of SEQ ID NO;

(b) has antigenic specificity for MAGE A-3 in the case of HLA-A1, and comprises

The amino acid sequence of the alpha chain CDR1 of SEQ ID NO. 16,

17, the amino acid sequence of the alpha chain CDR2 of SEQ ID NO,

18, the amino acid sequence of the alpha chain CDR3 of SEQ ID NO,

19, the beta chain CDR1 amino acid sequence of SEQ ID NO,

20 beta chain CDR2 amino acid sequence, and

21 beta chain CDR3 amino acid sequence;

(c) has antigenic specificity to MAGE-A12 in the case of HLA-Cw7, and comprises

26, the amino acid sequence of the alpha chain CDRL of SEQ ID NO,

the amino acid sequence of the alpha chain CDR2 of SEQ ID NO. 27,

28, the amino acid sequence of the alpha chain CDR3 of SEQ ID NO,

the beta chain CDR1 amino acid sequence of SEQ ID NO. 29,

30 of the amino acid sequence of the beta chain CDR2, and

31, the beta chain CDR3 amino acid sequence of SEQ ID NO; or

(d) Having antigenic specificity for MAGE-A12 in the context of HLA-Cw7 and comprising

The alpha chain CDR1 amino acid sequence of SEQ ID NO. 36,

37, the amino acid sequence of the alpha chain CDR2 of SEQ ID NO,

38, the amino acid sequence of the alpha chain CDR3 of SEQ ID NO,

the beta chain CDRL amino acid sequence of SEQ ID NO: 39,

the beta chain CDR2 amino acid sequence of SEQ ID NO 40, and

41 beta chain CDR3 amino acid sequence.

2. The isolated or purified TCR of claim 1, having a substitution for a) a peptide comprising EVDPIGHLY (SEQ ID NO: 2) or b) comprises VRIGHLYIL (SEQ ID NO: 4) the antigen specificity of the MAGE-A12 epitope of (A).

3. An isolated or purified polypeptide comprising a functional portion of a TCR as claimed in any one of claims 1 to 2, wherein

The amino acid sequences of the functional parts are respectively shown as SEQ ID NO: 5-10;

the amino acid sequences of the functional parts are respectively shown as SEQ ID NO: 16-21;

the amino acid sequences of the functional parts are respectively shown as SEQ ID NO: 26-31; or

The amino acid sequences of the functional parts are respectively shown as SEQ ID NO: 36-41.

4. The isolated or purified TCR of any one of claims 1-2, or the isolated or purified polypeptide of claim 3, comprising SEQ ID NOs 11-12, 22-23, 32-33, or 42-43; or SEQ ID NO 13-14, 24-25, 34-35 or 44-45.

5. An isolated or purified protein comprising:

a first polypeptide chain comprising a sequence as set forth in SEQ ID NO: 5-7, or an alpha chain CDR1-3 comprising SEQ ID NO: 11, or the alpha chain as shown in SEQ ID NO 13; and

a second polypeptide chain comprising the beta chain CDR1-3 shown in SEQ ID NOS: 8-10, and the amino acid sequence shown in SEQ ID NO: 12, or the beta chain as shown in SEQ ID NO. 14;

alternatively, the isolated or purified protein comprises:

a first polypeptide chain comprising a sequence set forth as SEQ ID NO: 16-18, as shown in SEQ ID NO: 22, or the alpha chain as shown in SEQ ID NO: 24; and

a second polypeptide chain comprising the beta chain CDR1-3 shown in SEQ ID NOS: 19-21, and the amino acid sequence shown in SEQ ID NO: 23, or the beta-chain as shown in SEQ ID No. 25;

alternatively, the isolated or purified protein comprises:

a first polypeptide chain comprising a sequence as set forth in SEQ ID NO: 26-28, as shown in SEQ ID NO: 32, or the alpha chain as shown in SEQ ID NO: 34; and

a second polypeptide chain comprising the beta chain CDR1-3 shown in SEQ ID NOS: 29-31, and the amino acid sequence shown in SEQ ID NO: 33, or the beta chain as set forth in SEQ ID NO: 35;

alternatively, the isolated or purified protein comprises:

a first polypeptide chain comprising a sequence set forth as SEQ ID NO: 36-38, as shown in SEQ ID NO: 42, or the alpha chain as shown in SEQ ID NO: 44; and

a second polypeptide chain comprising a beta chain CDR1-3 as set forth in SEQ ID NOS: 39-41, as set forth in SEQ ID NOS: 43 or the beta chain as shown in SEQ ID NO: 45.

6. The protein of claim 5, wherein the protein is a fusion protein or a recombinant antibody.

7. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR of any one of claims 1-2, a polypeptide of claim 3, or a protein of claim 5.

8. A recombinant expression vector comprising the nucleic acid of claim 7.

9. An isolated host cell comprising the recombinant expression vector of claim 8.

10. A cell population comprising the isolated host cell of claim 9.

11. A pharmaceutical composition comprising a TCR according to any one of claims 1 to 2, a polypeptide according to claim 3, a protein according to claim 5, a nucleic acid according to claim 7, a recombinant expression vector according to claim 8, an isolated host cell according to claim 9, or a population of cells according to claim 10, and a pharmaceutically acceptable carrier.

12. Use of a TCR according to any one of claims 1 to 2 in the manufacture of a detection agent for detecting the presence of melanoma cancer in a host, wherein detection of a complex formed by contacting a sample comprising cancer cells with the TCR indicates the presence of cancer in the host.

13. Use of a TCR according to any one of claims 1 to 2, a polypeptide according to claim 3, a protein according to claim 5, a nucleic acid according to claim 7, a recombinant expression vector according to claim 8, an isolated host cell according to claim 9, or a population of cells according to claim 10 in the manufacture of a medicament for the treatment or prophylaxis of melanoma cancer in a host.