MAGE A4 T cell receptor

Abstract

To provide an isolated T cell receptor (TCR) specific to MAGE A4 T cell receptor.SOLUTION: The invention relates to an isolated TCR specific to MAGE-A4 and a polypeptide comprising the functional moiety of the TCR. The invention also relates to multivalent TCR complex, nucleic acids encoding the TCRs, cells expressing the TCRs, and pharmaceutical compositions comprising the TCRs. The invention also relates to TCRs for use as a medicament, in particular for use in the treatment of cancer.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of European Patent Application No. 19 165 387.2, filed on March 27, 2019, which is hereby incorporated by reference in its entirety.

[0002] Statement regarding the sequence listing The sequence listing related to this application is provided in text format instead of a hard copy, and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is MED16702PCT_ST25. This text file is 146 KB, was created on March 24, 2020, and was electronically submitted via EFS - Web simultaneously with the filing of this application.

[0003] The present invention relates to an isolated T - cell receptor (TCR) specific for MAGE - A4, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex, a nucleic acid encoding the TCR, a cell expressing the TCR, and compositions and pharmaceutical compositions containing them. The present invention also relates to methods of using the foregoing in a medical treatment or for a formulation, and / or use as a medicament, particularly for use in the treatment of cancer.

Background Art

[0004] Description of related technologies T lymphocytes (i.e., T cells), which constitute part of the cell-mediated immune system, play a major role in the eradication of pathogens. T cells develop in the thymus and express TCR molecules on their surface that enable the recognition of peptides (known as antigen presentation) presented on major histocompatibility complex (MHC) molecules expressed on nucleated cells. Antigens derived from pathogens, i.e., foreign antigens presented by MHC molecules, elicit a strong T cell response, while self-antigens usually do not lead to a T cell response due to the negative selection of self-antigen-specific T cells in the thymus during the development of such T cells. The immune system can thus distinguish between nucleated cells presenting foreign antigens and nucleated cells presenting self-antigens, and through the potent cytokine release and cytotoxic mechanisms of T cells, it can specifically target and eradicate infected cells.

[0005] The capabilities of the immune system are recognized as a promising tool for future cancer treatments. Over the past few decades, research has begun to leverage the unique properties of T cells by using adoptive cell therapy (ACT), which involves administering patient-derived lymphocytes grown ex vivo. ACT is an attractive idea for cancer treatment because it does not require the patient's immune function and the specificity of the transplanted lymphocytes can be directed towards tumor antigens that are non-mutant and therefore have low immunogenicity and generally cannot effectively trigger the autologous T cell response. While ACT has shown promise as a treatment for various types of cancer, its broad application as a clinical treatment has been hindered by the need to custom isolate and characterize tumor-specific T cells from each patient, a process that is not only difficult and time-consuming but also often fails to yield high-avidity T cells (Xue et al. Clin. Exp. Immunol. 2005 Feb;139(2):167-172, Schmitt et al., Hum. Gene Ther. 2009 Nov;20(11):1240-1248).

[0006] Genetic transfer of tumor antigen-specific TCRs into primary T cells can overcome some of the limitations of current ACTs by enabling the rapid generation of tumor-reactive T lymphocytes with predetermined antigen specificity, even in immunocompromised patients. However, the identification of suitable T cell clones that specifically recognize tumor antigens and possess TCRs that exhibit the desired antitumor effect in vivo remains an ongoing subject of research. Given that there were approximately 14.1 million new cancer cases worldwide in 2012, and that cancer now accounts for approximately 14.6% of all deaths worldwide, there is an urgent need for efficient and novel treatment options. The object of this invention is to satisfy the above-mentioned needs.

[0007] MAGE-A4 belongs to the melanoma antigen (MAGE) family. The MAGE family is expressed in various types of malignant tumors, including melanoma, colorectal, lung, breast, and other tumors. Specifically, the MAGE family is classified into two groups based on its tissue expression pattern, and the MAGE-A subfamily is expressed in testicular germ cells and ectopically re-expressed in malignant tumors. This is also true for MAGE-A4.

[0008] Tumor tissue expression studies have revealed that MAGE-A4 is (over)expressed in 19-35% of non-small cell lung cancer (NSCLC) cases, 13% of breast cancer cases, 47% of ovarian epithelial carcinoma cases, and 22% of colorectal cancer cases (Tajima et al. Lung Cancer 2003;42:23-33, Gure et al. Clin Cancer Res 2005,11:8055-8062, Kim et al. Int J Mol Med 2012,29:656-662, Otte et al. Cancer Res 2001,61:6682-668, Daudi et al. PLoS One 2014,9:e104099, Li et al. Clin Cancer Res 2005,11:1809-1814). [Overview of the project]

[0009] overview The objective of this invention is to provide an isolated T cell receptor (TCR) specific to MAGE-A4.

[0010] In particular, this TCR specifically recognizes the amino acid sequence or fragment of SEQ ID NO:1.

[0011] In a specific embodiment, the TCR specifically recognizes the amino acid sequence of SEQ ID NO:1 in a form bound to HLA-A2, and more specifically, the TCR recognizes the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01.

[0012] In some embodiments, the TCR includes a variable TCRα region comprising CDR1 having the amino acid sequence of SEQ ID NO:2, CDR2 having the amino acid sequence of SEQ ID NO:3, and CDR3 having the amino acid sequence of SEQ ID NO:4, and a variable TCRβ region comprising CDR1 having the amino acid sequence of SEQ ID NO:5, CDR2 having the amino acid sequence of SEQ ID NO:6, and CDR3 having the amino acid sequence of SEQ ID NO:7; or a variable TCRα region comprising CDR1 having the amino acid sequence of SEQ ID NO:12, CDR2 having the amino acid sequence of SEQ ID NO:13, and CDR3 having the amino acid sequence of SEQ ID NO:14, and a variable TCRβ region comprising CDR1 having the amino acid sequence of SEQ ID NO:15, CDR2 having the amino acid sequence of SEQ ID NO:16, and CDR3 having the amino acid sequence of SEQ ID NO:17; or CDR1 having the amino acid sequence of SEQ ID NO:22, CDR2 having the amino acid sequence of SEQ ID NO:23, and SEQ ID It includes a variable TCRα region containing CDR3 with amino acid sequence NO:24, and a variable TCRβ region containing CDR1 with amino acid sequence SEQ ID NO:25, CDR2 with amino acid sequence SEQ ID NO:26, and CDR3 with amino acid sequence SEQ ID NO:27. These TCRs are described in more detail below.

[0013] In a particular embodiment, the TCR includes a variable TCRα region having the amino acid sequence of SEQ ID NO:8 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:9; or a variable TCRα region having the amino acid sequence of SEQ ID NO:18 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:19; or a variable TCRα region having the amino acid sequence of SEQ ID NO:28 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:29.

[0014] In certain embodiments, the TCR comprises a TCRα chain having the amino acid sequence of SEQ ID NO:10 and a TCRβ chain having the amino acid sequence of SEQ ID NO:11; a TCRα chain having the amino acid sequence of SEQ ID NO:20 and a TCRβ chain having the amino acid sequence of SEQ ID NO:21; or a TCRα chain having the amino acid sequence of SEQ ID NO:30 and a TCRβ chain having the amino acid sequence of SEQ ID NO:31.

[0015] In another embodiment, the TCR comprises a TCRα chain having the amino acid sequence of SEQ ID NO:87 and a TCRβ chain having the amino acid sequence of SEQ ID NO:88; a TCRα chain having the amino acid sequence of SEQ ID NO:89 and a TCRβ chain having the amino acid sequence of SEQ ID NO:90; or a TCRα chain having the amino acid sequence of SEQ ID NO:91 and a TCRβ chain having the amino acid sequence of SEQ ID NO:92.

[0016] In some embodiments, the TCR comprises a TCRα chain having the amino acid sequence of SEQ ID NO:102 and a TCRβ chain having the amino acid sequence of SEQ ID NO:103; a TCRα chain having the amino acid sequence of SEQ ID NO:108 and a TCRβ chain having the amino acid sequence of SEQ ID NO:109; or a TCRα chain having the amino acid sequence of SEQ ID NO:114 and a TCRβ chain having the amino acid sequence of SEQ ID NO:115.

[0017] The TCRs intended herein are isolated and / or purified and may be soluble or membrane-bound.

[0018] In some embodiments, the present invention relates to an isolated TCR described herein, wherein IFN-γ secretion induced by the binding of a TCR expressed on effector cells to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01 can be more than 100 times, preferably more than 500 times, and more preferably more than 2000 times higher when bound to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01 compared to binding to an unrelated peptide presented by the molecule encoded by HLA-A*02:01.

[0019] In some embodiments, the amino acid sequence of the TCR may include one or more phenotypically silent substitutions. In addition, the TCR may be labeled. Useful labels are known in the art and can be coupled to the TCR or TCR variant by conventional methods, optionally via linkers of varying lengths. The terms “label” or “labeling group” refer to any detectable label. In addition to or instead of the foregoing, the amino acid sequence may be modified to include a therapeutic agent or a pharmacokinetic modifier. The therapeutic agent may be selected from the group consisting of immunoeffector molecules, cytotoxic agents, and radionuclides. An immunoeffector molecule may be, for example, a cytokine. The pharmacokinetic modifier may be at least one polyethylene glycol repeating unit, at least one glycol group, at least one sialyl group, or a combination thereof.

[0020] TCRs, particularly soluble TCRs as intended herein, can be modified by attaching functional parts to, for example, reduce immunogenicity, increase hydrodynamic size (size in soluble state), solubility and / or stability (e.g., by enhanced protection from proteolysis), and / or extend serum half-life. Other useful functional parts and modifications include “suicide switches” or “safety switches” that can be used to deactivate or activate effector host cells holding the TCRs of the present invention in a patient’s body. TCRs with altered glycosylation patterns are also envisioned herein.

[0021] It is also conceivable to add a drug or therapeutic entity, such as a small molecule compound, to the TCR, particularly the soluble TCR of the present invention. The TCR, particularly the soluble TCR of the present invention, can be further modified to introduce additional domains (tags) that aid in the identification, tracking, purification, and / or isolation of each molecule.

[0022] In some embodiments, the TCR is a single-chain type in which a TCRα chain and a TCRβ chain are linked by a linker sequence, and the linker sequence is optionally cleavable.

[0023] Another aspect relates to polypeptides comprising the functional portion of the TCR described herein, wherein the functional portion comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 4, 7, 14, 17, 24, and 27.

[0024] In a specific embodiment, the functional portion includes a TCRα variable region and / or a TCRβ variable region.

[0025] A specific embodiment relates to a polyvalent TCR complex comprising at least two TCRs described herein. In a more specific embodiment, the therapeutic agent is conjugated to at least one of the TCRs.

[0026] A particular embodiment relates to a fusion protein comprising a TCRα chain and a TCRβ chain, and containing an amino acid sequence represented by any one of SEQ ID NO: 94, 96, 98, 104, 110, and 116.

[0027] In specific contexts, the fusion protein further includes furin cleavage sites and / or ribosome skipping sequences.

[0028] Another aspect relates to nucleic acids encoding TCRs as described herein or nucleic acids encoding the polypeptides or fusion proteins described above.

[0029] In one plane, the nucleic acid sequence encoding the TCRα chain is represented by one of SEQ ID NO: 69, 77, 85, 99, 105, and 111. In another plane, the nucleic acid sequence encoding the TCRβ chain is represented by one of SEQ ID NO: 70, 78, 86, 100, 106, and 112. In another context, the TCR includes an α chain encoded by SEQ ID NO:69 and a β chain encoded by SEQ ID NO:70; an α chain encoded by SEQ ID NO:77 and a β chain encoded by SEQ ID NO:78; an α chain encoded by SEQ ID NO:85 and a β chain encoded by SEQ ID NO:86; an α chain encoded by SEQ ID NO:99 and a β chain encoded by SEQ ID NO:100; an α chain encoded by SEQ ID NO:105 and a β chain encoded by SEQ ID NO:106; or an α chain encoded by SEQ ID NO:111 and a β chain encoded by SEQ ID NO:112.

[0030] A further aspect concerns fusion proteins encoded by nucleic acid sequences represented by any one of the following SEQ ID NOs: 93, 95, 97, 101, 107, and 113.

[0031] A further aspect relates to plasmids or vectors containing the nucleic acids of the present application as described above. Another aspect relates to plasmids or vectors containing nucleic acids encoding polypeptide sequences shown in SEQ ID NO:87 and SEQ ID NO:88; polypeptide sequences shown in SEQ ID NO:89 and SEQ ID NO:90; polypeptide sequences shown in SEQ ID NO:91 and SEQ ID NO:92; polypeptide sequences shown in SEQ ID NO:102 and SEQ ID NO:103; polypeptide sequences shown in SEQ ID NO:108 and SEQ ID NO:109; or polypeptide sequences shown in SEQ ID NO:114 and SEQ ID NO:115. Preferably, the vector is an expression vector or a vector suitable for transduction or transfection of cells, particularly eukaryotic cells. The vector may be, for example, a retroviral vector, such as a gamma retroviral vector or a lentiviral vector.

[0032] Another aspect relates to cells expressing the TCR described herein. The cells may be isolated or non-natural.

[0033] Another aspect relates to cells containing the nucleic acids or plasmids or vectors described above. More specifically, a cell may contain an expression vector containing one or more of the nucleic acids described above, or a first expression vector containing a nucleic acid encoding the α-chain of the TCR described herein and a second expression vector containing a nucleic acid encoding the beta-chain of the TCR described herein.

[0034] The cells can be peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). Typically, the cells are immune effector cells, particularly T cells. Other suitable cell types include gamma-delta T cells, natural killer (NK) cells, and NK-like T (NKT) cells.

[0035] Another aspect relates to antibodies or antigen-binding fragments that specifically bind to the portion of the TCR described herein that mediates specificity to MAGE-A4.

[0036] Another aspect relates to compositions comprising the TCR described herein, the polypeptide described herein, the fusion protein described herein, the polyvalent TCR complex described herein, the nucleic acid described herein, the vector described herein, the cell described herein, or the antibody described herein.

[0037] Another aspect relates to a pharmaceutical composition comprising a TCR as described herein, a polypeptide as described herein, a fusion protein as described herein, a polyvalent TCR complex as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, or an antibody as described herein.

[0038] Typically, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

[0039] Another aspect relates to the TCRs, polypeptides, polyvalent TCR complexes, nucleic acids, vectors, cells, antibodies, compositions, or pharmaceutical compositions described herein for use as pharmaceuticals, particularly for use in the treatment of cancer. Cancer may be a hematological cancer or a solid tumor. Cancer may be selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia. Preferably, cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer or colorectal cancer, sarcoma, and osteosarcoma. [Invention 1001] a) A variable TCRα region comprising CDR1 having the amino acid sequence of SEQ ID NO:2, CDR2 having the amino acid sequence of SEQ ID NO:3, and CDR3 having the amino acid sequence of SEQ ID NO:4. A variable TCRβ region comprising CDR1 having the amino acid sequence of SEQ ID NO:5, CDR2 having the amino acid sequence of SEQ ID NO:6, and CDR3 having the amino acid sequence of SEQ ID NO:7; or b) A variable TCRα region comprising CDR1 having the amino acid sequence of SEQ ID NO:12, CDR2 having the amino acid sequence of SEQ ID NO:13, and CDR3 having the amino acid sequence of SEQ ID NO:14, A variable TCRβ region comprising CDR1 having the amino acid sequence of SEQ ID NO:15, CDR2 having the amino acid sequence of SEQ ID NO:16, and CDR3 having the amino acid sequence of SEQ ID NO:17; or c) A variable TCRα region comprising CDR1 having the amino acid sequence of SEQ ID NO:22, CDR2 having the amino acid sequence of SEQ ID NO:23, and CDR3 having the amino acid sequence of SEQ ID NO:24. A variable TCRβ region containing CDR1 having the amino acid sequence of SEQ ID NO:25, CDR2 having the amino acid sequence of SEQ ID NO:26, and CDR3 having the amino acid sequence of SEQ ID NO:27. A MAGE-A4-specific isolated T cell receptor (TCR), including [specific component]. [Invention 1002] An isolated TCR of the present invention 1001, which specifically recognizes the amino acid sequence or fragment of SEQ ID NO:1, preferably the amino acid sequence of SEQ ID NO:1 in a form bound to HLA-A2, and more preferably the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01. [Invention 1003] a) A variable TCRα region having the amino acid sequence of SEQ ID NO:8 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:9; or b) A variable TCRα region having the amino acid sequence of SEQ ID NO:18 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:19; or c) Variable TCRα region having amino acid sequence SEQ ID NO:28 and variable TCRβ region having amino acid sequence SEQ ID NO:29 An isolated TCR, including any of the above-described inventions. [Invention 1004] An isolated TCR according to the present invention, comprising a functional moiety containing at least one of the amino acid sequences of SEQ ID NO: 4, 7, 14, 17, 24, and 27. [Invention 1005] a) TCRα chain having the amino acid sequence of SEQ ID NO:10 and TCRβ chain having the amino acid sequence of SEQ ID NO:11; b) A TCRα chain having the amino acid sequence of SEQ ID NO:20 and a TCRβ chain having the amino acid sequence of SEQ ID NO:21; or c) TCRα chain having amino acid sequence SEQ ID NO:30 and TCRβ chain having amino acid sequence SEQ ID NO:31 An isolated TCR comprising any of the present invention 1001 to 1004. [Invention 1006] a) TCRα chain having the amino acid sequence of SEQ ID NO:87 and TCRβ chain having the amino acid sequence of SEQ ID NO:88; b) A TCRα chain having the amino acid sequence of SEQ ID NO:89 and a TCRβ chain having the amino acid sequence of SEQ ID NO:90; or c) TCRα chain having amino acid sequence SEQ ID NO:91 and TCRβ chain having amino acid sequence SEQ ID NO:92 An isolated TCR comprising any of the present invention 1001 to 1004. [Invention 1007] a) TCRα chain having the amino acid sequence of SEQ ID NO:102 and TCRβ chain having the amino acid sequence of SEQ ID NO:103; b) A TCRα chain having the amino acid sequence of SEQ ID NO:108 and a TCRβ chain having the amino acid sequence of SEQ ID NO:109; or c) TCRα chain having amino acid sequence SEQ ID NO:114 and TCRβ chain having amino acid sequence SEQ ID NO:115 An isolated TCR comprising any of the present invention 1001 to 1004. [Invention 1008] An isolated TCR of any of Invention 1001-1007 or a polyvalent TCR complex of Invention 1008, wherein IFN-γ secretion is induced by binding to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01. [Invention 1009] An isolated TCR according to any of invention 1001 to 1007, wherein IFN-γ secretion induced by the binding of a TCR expressed on effector cells to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01 can be more than 100 times, preferably more than 500 times, and more preferably more than 2000 times higher when bound to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01 compared to binding to an unrelated peptide presented by the molecule encoded by HLA-A*02:01. [Invention 1010] A polyvalent TCR complex comprising at least two TCRs according to any of the present invention 1001 to 1007. [Invention 1011] A fusion protein comprising a TCRα chain and a TCRβ chain, and containing an amino acid sequence represented by any one of SEQ ID NO: 94, 96, 98, 104, 110, and 116. [Invention 1012] A nucleic acid encoding a TCR according to any of inventions 1001 to 1007 or a fusion protein according to invention 1011. [Invention 1013] The nucleic acid of the present invention 1012, wherein the nucleic acid sequence encoding the TCRα chain is represented by one of SEQ ID NO: 69, 77, 85, 99, 105, and 111. [Invention 1014] The nucleic acid of the present invention 1012, wherein the nucleic acid sequence encoding the TCRβ chain is represented by one of SEQ ID NO: 70, 78, 86, 100, 106, and 112. [Invention 1015] TCR a) TCRα chain encoded by SEQ ID NO:69 and TCRβ chain encoded by SEQ ID NO:70; b) TCRα chain encoded by SEQ ID NO:77 and TCRβ chain encoded by SEQ ID NO:78; c) TCRα chain encoded by SEQ ID NO:85 and TCRβ chain encoded by SEQ ID NO:86; d) TCRα chain encoded by SEQ ID NO:99 and TCRβ chain encoded by SEQ ID NO:100; e) TCRα chain encoded by SEQ ID NO:105 and TCRβ chain encoded by SEQ ID NO:106; or f) TCRα chain encoded by SEQ ID NO:111 and TCRβ chain encoded by SEQ ID NO:112 The nucleic acid of the present invention 1010, which includes the above. [Invention 1016] The nucleic acid of the present invention 1012, wherein the fusion protein is encoded by a nucleic acid sequence shown in any one of SEQ ID NO: 93, 95, 97, 101, 107, and 113. [Invention 1017] A vector comprising any nucleic acid of the present invention 1012 to 1016, wherein the vector is preferably an expression vector, more preferably a retroviral vector, and even more preferably a lentiviral vector. [Invention 1018] a) Polypeptide sequences shown in SEQ ID NO:87 and SEQ ID NO:88; b) Polypeptide sequences shown in SEQ ID NO:89 and SEQ ID NO:90; c) Polypeptide sequences shown in SEQ ID NO:91 and SEQ ID NO:92; d) Polypeptide sequences shown in SEQ ID NO:102 and SEQ ID NO:103; e) Polypeptide sequences shown in SEQ ID NO:108 and SEQ ID NO:109; or f) Polypeptide sequences shown in SEQ ID NO:114 and SEQ ID NO:115 A vector comprising a nucleic acid encoding a, preferably an expression vector, more preferably a retroviral vector, and even more preferably a lentiviral vector. [Invention 1019] A cell expressing any of the TCRs described in invention 1001 to 1047. [Invention 1020] A cell comprising the vector of the present invention 1017 or 1018. [Invention 1021] A cell according to invention 1019 or 1020, which is an immune effector cell. [Invention 1022] A cell according to any of the invention 1019 to 1021, wherein the immune effector cell is a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. [Invention 1023] An antibody or its antigen-binding fragment that specifically binds to a portion of any of the TCRs 1001 to 1007 of the present invention that mediates specificity to MAGE-A4, preferably the portion of the TCR that mediates specificity to MAGE-A4 a) CDR3 of the alpha strand of SEQ ID NO:4 and / or CDR3 of the beta strand of SEQ ID NO:7, b) CDR3 of the alpha strand of SEQ ID NO:14 and / or CDR3 of the beta strand of SEQ ID NO:17, c) CDR3 of the alpha strand of SEQ ID NO:24 and / or CDR3 of the beta strand of SEQ ID NO:27 The antibody or its antigen-binding fragment, comprising the antibody or its antigen-binding fragment. [Invention 1024] A composition comprising any TCR of Invention 1001 to 1007, a polyvalent TCR complex of Invention 1008, a fusion protein of Invention 1011, a nucleic acid of any of Invention 1012 to 1016, a vector of Invention 1017 or 1018, a cell of any of Invention 1019 to 1022, or an antibody of Invention 1023. [Invention 1025] A pharmaceutical composition comprising a pharmaceutically acceptable carrier, a TCR of any of Invention 1001 to 1007, a polyvalent TCR complex of Invention 1008, a fusion protein of Invention 1011, a nucleic acid of any of Invention 1012 to 1016, a vector of Invention 1017 or 1018, a cell of any of Invention 1019 to 1022, or an antibody of Invention 1023. [Invention 1026] A TCR of any of Invention 1001 to 1007, a polyvalent TCR complex of Invention 1008, a fusion protein of Invention 1011, a nucleic acid of any of Invention 1012 to 1016, a vector of Invention 1017 or 1018, a cell of any of Invention 1019 to 1022, an antibody of Invention 1023, a composition of Invention 1024, or a pharmaceutical composition of Invention 1025, for use as a pharmaceutical. [Invention 1027] Cancer, preferably hematological cancer or solid tumor cancer, more preferably sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia, most preferably NSCLC, SCLC, breast cancer, ovarian cancer A TCR of any of Invention 1001 to 1007, a polyvalent TCR complex of Invention 1008, a fusion protein of Invention 1011, a nucleic acid of any of Invention 1012 to 1016, a vector of Invention 1017 or 1018, a cell of any of Invention 1019 to 1022, an antibody of Invention 1023, a composition of Invention 1024, or a pharmaceutical composition of Invention 1025 for use in the treatment of cancer selected from the group consisting of colorectal cancer, sarcoma, or osteosarcoma. [Brief explanation of the drawing]

[0040] [Figure 1] Figure 1 shows MAGE-A4GVY-MHC multimer binding in CD8+ T cells transduced with different MAGE-A4-responsive TCRs. CD8+ T cells were isolated from healthy donor PBMCs and transduced with three different MAGE-A4-TCRs and one control TCR that does not recognize MAGE-A4. Transduced CD8+ T cells were enriched by FACS using the mouse constant beta region as a marker for transduction. After proliferation of these cells, they were stained with the MAGE-A4GVY-MHC multimer, as well as antibodies against CD8 and the mouse constant beta region (mmCb), and analyzed by flow cytometry. The population was gated with viable CD8+ / mCb+ cells. Multimer / CD8 staining is shown. [Figure 2]Figure 2 shows that MAGE-A4-TCR transgenic T cells recognize the MAGE-A4GVY peptide presented on HLA-A2. Transgenic T cells were co-cultured with T2 cells externally loaded with the MAGE-A4GVY peptide or K562 / HLA-A2 cells into which the MAGE-A4 gene had been introduced. Negative controls included T2 cells loaded with a control peptide and untransduced K562 / HLA-A2 cells, respectively. Recognition of target cells was analyzed by measuring the IFN-γ concentration in the co-culture supernatant using standard ELISA. [Figure 3] Figure 3 shows the functional avidity of MAGE-A4-TCR transgenic T cells. Transgenic T cells were co-cultured with T2 cells treated with MAGE-A4GVY peptide at stepwise concentrations (10⁻¹² M to 10⁻⁴ M). The IFN-γ concentration in the co-culture supernatant was measured by standard ELISA. [Figure 4A] Figures 4A-C show the ability of MAGE-A4-TCR transgenic T cells (TCR-1, TCR-2, and TCR-3) to lyse MAGE-A4-positive tumor cell lines in an HLA-A2-dependent manner. Transgenic T cells were co-cultured with different MAGE-A4-positive HLA-A2-positive tumor cell lines (NCI-H1703, NCI-H1755), MAGE-A4-negative HLA-A2-positive tumor cell line (Saos-2), and MAGE-A4-negative HLA-A2-negative tumor cell line (A549). Tumor cells loaded with the MAGE-A4GVY peptide were used as a positive control. The cytotoxicity of the fluorescent marker against stably transduced tumor cell lines was measured by taking photographs every two hours using an IncuCyte® ZOOM device (Essen Bioscience). To analyze cytokine release, the co-culture supernatant was harvested after 24 hours, and IFN-γ concentration was analyzed using a standard sandwich ELISA (BD Human IFN-γ ELISA set). [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5A]Figures 5A-C show that MAGE-A4-TCR transgenic T cells (TCR-1, TCR-2, and TCR-3) do not recognize normal human cells. Transgenic T cells were co-cultured with different primary cells and induced pluripotent stem cell (iPS)-derived cells representing basic tissues or organs. Normal cells loaded with the MAGE-A4GVY peptide were used as positive controls. In neurons, HLA-A2 expression was induced by pre-incubation with IFN-γ. HLA-A2-negative NHBE cells were electroporated with HLA-A2-ivtRNA, and HLA-A2 expression in all cells was confirmed by flow cytometry. To analyze cytokine release, the co-culture supernatant was harvested after 24 hours, and IFN-γ and IL-2 concentrations were analyzed using a standard sandwich ELISA (BD Human IFN-γ or IL-2 ELISA set). [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 6] Figure 6 shows MAGE-A4GVY-MHC multimer binding to CD3+ T cells transduced with different MAGE-A4-responsive fully human TCRs. CD3+ T cells were isolated from PBMCs of healthy donors and transduced with three different MAGE-A4-TCRs and one control TCR that does not recognize MAGE-A4. After proliferation of these cells, they were stained with antibodies against MAGE-A4GVY-MHC multimers and CD3 and analyzed by flow cytometry. The population was gated for viable CD3+ cells and multimer staining. [Figure 7]Figure 7 shows that MAGE-A4 fully human TCR transgenic T cells recognize the MAGE-A4GVY peptide presented on HLA-A2. Transgenic T cells were co-cultured with T2 cells externally loaded with the MAGE-A4GVY peptide or with A549 / HLA-A2 cells into which the MAGE-A4 gene had been introduced. Negative controls included T2 cells loaded with a control peptide and untransduced A549 / HLA-A2 cells, respectively. Recognition of target cells was analyzed by measuring the IFN-γ concentration in the co-culture supernatant using the Luminex assay. [Figure 8] Figure 8 shows the ability of MAGE-A4 fully human TCR transgenic T cells to respond specifically to MAGE-A4-positive tumor cell lines in an HLA-A2-dependent manner. Transgenic T cells were co-cultured with different MAGE-A4-positive HLA-A2-positive tumor cell lines (A375, NCI-H1703, NCI-H1755), MAGE-A4-positive HLA-A2-negative tumor cell line (NCI-H520), and MAGE-A4-negative HLA-A2-positive tumor cell line (A549). To analyze cytokine release, the co-culture supernatant was harvested after 24 hours, and IFN-γ concentrations were analyzed by Luminex assay. [Figure 9] Figure 9 shows the ability of MAGE-A4 fully human TCR transgenic T cells to lyse MAGE-A4-positive tumor cell lines in an HLA-A2-dependent manner. Transgenic T cells were co-cultured with different MAGE-A4-positive HLA-A2-positive tumor cell lines (A375, NCI-H1755, A549-HLA-A2-MAGE-A4) and MAGE-A4-negative HLA-A2-positive tumor cell line (A549-HLA-A2). Cytotoxicity against tumor cell lines was measured by impedance assay initiated 6 hours after the start of co-culture. [Figure 10] Figure 10 shows the ability of MAGE-A4 fully human TCR transgenic T cells to suppress MAGE-A4-positive tumors transplanted into NSG mice. [Figure 11]Figures 11A-C show the vector copy number (VCN) and expression of MAGE-A4 TCR. Peripheral blood mononuclear cells (PBMCs) were transduced with lentiviral vectors encoding either the fully human MAGE-A4 TCR (TCR-5) or the enhanced variant (TCR-8). A) VCN measurements were similar in TCR-5 T cells and TCR-8 T cells. B) TCR expression on the T cell surface was evaluated using GVY-specific tetramer detection by flow cytometry and is shown as a percentage of total CD3+ T cells. TCR-8 expression was increased compared to TCR-5 expression. C) The density of TCR molecules on the T cell surface was evaluated using GVY-specific tetramer detection by flow cytometry and is shown as the geometric mean fluorescence intensity (gMFI) of total tetramer + TCR T cells. TCR-8 expression density was increased compared to TCR-5 expression density. [Figure 12]Figures 12A-B show that T cells expressing the fully human MAGE-A4 TCR (TCR-5) or its enhanced variant (TCR-8) specifically kill MAGE-A4-expressing target cells in vitro. A) TCR-5 T cells, TCR-8 T cells, or untransduced (UTD) T cells were co-cultured with A549.A2 cells (A2+, MAGE-A4(-)), NCI-H2023 cells (A2+, MAGE-A4(+)), A375 cells (A2+, MAGE-A4(+)), or A549.A2.MAGEA4 cells (A2+, MAGE-A4(+)) in a 1:1 E:T ratio. After 24 hours, IFNγ release was evaluated as a biomarker of T cell activity. TCR-5 T cells and TCR-8 T cells secreted INFγ when co-cultured with MAGE-A4-expressing target cells, but not in the presence of MAGE-A4-negative cells. B) TCR-5 T cells, TCR-8 T cells, or untransduced (UTD) T cells were co-cultured with A375 cells (A2+, MAGE-A4(+)), A549.A2.MAGEA4 cells (A2+, MAGE-A4(+)), or U2OS cells (A2+, MAGE-A4(low)) at E:T ratios of 10:1, 5:1, and 2.5:1. Cytotoxicity was measured using impedance at 6 hours as a standardized percentage against tumor cells only. TCR-5 T cells and TCR-8 T cells mediated comparable cytotoxicity against the three MAGE-A4-expressing cell lines. [Figure 13]Figures 13A-C show that T cells expressing the fully human MAGE-A4 TCR (TCR-5) or its enhanced variant (TCR-8) mediate regression in mice with MAGE-A4-expressing tumors. MAGEA4(+)A375 tumor cells were subcutaneously injected into five NSG mice (each condition) and treated with either a medium, UTD T cells, or T cells expressing the fully human MAGE-A4 TCR (TCR-5) or its enhanced variant (TCR-8). Tumor growth was measured twice weekly, and the antitumor activity of TCR T cells was evaluated compared to mice given UTD controls and medium controls. A-B) Mice with 50 mm3 A375 tumors were given 5 × 10⁶ cells (A) or 1.5 × 10⁶ cells (B) of UTD T cells, TCR-5 T cells, or TCR-8 T cells. Both TCR-5 T cells and TCR-8 T cells suppressed tumors at a T-cell dose of 5 × 10⁶ cells, but TCR-8 T cells showed increased tumor suppression at a lower dose of 1.5 × 10⁶ T cells. C) Mice with 100 mm³ A375 tumors were given 10 × 10⁶ UTD T cells, TCR-5 T cells, or TCR-8 T cells. TCR-8 T cells mediated increased tumor regression compared to TCR-5 T cells or UTD T cells. [Modes for carrying out the invention]

[0041] Detailed explanation A. Overview Due to its expression pattern, MAGE-A4 is a suitable tumor-specific target for ACT. MAGE-A4 contains an epitope in the form of a decapeptide with the amino acid sequence GVYDGREHTV (SEQ ID NO:1) presented by the HLA-A2 molecule (Duffour et al. Eur J Immunol. 1999 10:3329-37). Overall, MAGE-A4 is a suitable tumor-specific target for ACT. There is a need for novel, safe, and effective TCR effectors that target the MAGE-A antigen for cancer immunotherapy. This disclosure aims to address this unmet medical need with respect to a T cell receptor that efficiently targets the MAGE-A4 antigen on cancer cells.

[0042] B. Definition Before describing some of the preferred embodiments of the present invention in detail, the following general definitions are given.

[0043] The inventions described below illustratively may be successfully implemented even without one or more elements or one or more limitations not specifically disclosed herein.

[0044] The present invention will be described with reference to certain drawings in relation to specific embodiments, but the present invention is not limited thereto and is limited only by the claims.

[0045] Where the term “comprising” is used in this specification and in the claims, it does not exclude other elements. In the present invention, the term “consisting of” is considered a preferred embodiment of the term “comprising of.” Where a group is defined below as comprising at least a certain number of embodiments, it should be understood that a group comprising only those embodiments is also disclosed.

[0046] In the present invention, the term “obtained” is considered a preferred embodiment of the term “obtainable.” For example, if it is stated below that an antibody can be obtained from a specific source, it should be understood that antibodies obtained from that source are also disclosed.

[0047] When an indefinite or definite article, such as "a," "an," or "the," is used with a singular noun, it encompasses the plural of that noun unless otherwise specified. The terms "about" or "approximately" in this invention mean an interval of accuracy that a person skilled in the art would understand to still be guaranteed in terms of the technical effect of the feature in question. This term typically indicates a deviation of ±10%, preferably ±5%, more preferably ±2%, and most preferably ±1% from the stated value.

[0048] Unless otherwise indicated, the term “at least” preceding a set of elements should be understood to refer to all of those elements. The term “at least one” refers to one or more, e.g., two, three, four, five, six, seven, eight, nine, ten or more. Numerous equivalents of specific embodiments of the inventions described herein will be apparent to those skilled in the art, or at best, can be verified by routine experimentation. Such equivalents shall be incorporated herein.

[0049] Whenever the terms “and / or” are used herein, they encompass the meanings of “and,” “or,” and “all or any other combination of the elements connected by the terms.”

[0050] The terms "less than" and "more than" do not include a specific number. For example, "less than 20" means less than the number indicated. Similarly, "more than" or "greater than" means more than or greater than the number indicated; for example, "more than 80%" means more than or greater than the indicated number of 80%.

[0051] "Including" means "including but not limited to." "Including" and "including but not limited to" are interchangeable.

[0052] Throughout this specification and the claims, the singular form includes the plural form unless otherwise specified in the context. In particular, where the indefinite article is used, this specification should be understood to intend both the singular and the plural unless otherwise specified in the context.

[0053] Technical terms are used in their respective common sense or meaning to those skilled in the art. Where a special meaning is assigned to a particular term, its definition is given below in the context in which it is used.

[0054] Additional definitions are provided throughout this disclosure.

[0055] All publications cited throughout this specification (including all patents, patent applications, scientific publications, and manuals), both those mentioned above and those mentioned below, are incorporated herein in their entirety by reference. Nothing in this specification should be considered an admission that the present invention does not qualify as prior to any prior invention or disclosure. If any material incorporated by reference is inconsistent with or contradicts this specification, this specification shall prevail over any such material.

[0056] All documents and patent documents referenced herein are incorporated herein by reference in their entirety.

[0057] C. Basic Information about TCR The TCR consists of two distinct protein chains: the TCR alpha (α) chain and the TCR beta (β) chain. The TCR α chain contains a variable (V) region, a joining (J) region, and a constant (C) region. The TCR β chain contains a variable (V) region, a diversity (D) region, and a constant (C) region. Both the reconstituted V(D)J regions of the TCR α and TCR β chains contain a hypervariable region (CDR, complementarity-determining region), the CDR3 region in particular, which determines specific epitope recognition. Both the TCR α and TCR β chains contain a hydrophobic transmembrane domain in their C-terminal region and terminate with a short cytoplasmic tail.

[0058] Typically, a TCR is a heterodimer consisting of one α-chain and one β-chain. This heterodimer can bind to MHC molecules that present peptides.

[0059] In this context, the terms "variable TCRα region," "TCRα variable chain," or "variable domain" refer to the variable region of the TCRα chain. In this context, the terms "variable TCRβ region" or "TCRβ variable chain" refer to the variable region of the TCRβ chain.

[0060] TCR loci and TCR genes are named using the International Immunogenetics (IMGT) TCR nomenclature (IMGT database, www.IMGT.org; Giudicelli et al. Nucl. Acids Res., 34, D781-D784 (2006); Lefranc and Lefranc, Academic Press 2001).

[0061] D. MAGE-A4 The first phase concerns isolated T cell receptors (TCRs) specific to MAGE-A4.

[0062] MAGE-A4 belongs to the group of so-called cancer / testicular antigens. Cancer / testicular antigens are expressed in various malignant tumors and germ cells, but not in other adult tissues. Therefore, MAGE-A4 is an interesting immunotherapy target antigen. The human gene encoding MAGE-A4 is called MAGEA4 (ENSG00000147381).

[0063] In particular, the TCR intended in this specification is amino acids 230-239 of MAGE-A4, i.e., amino acid sequence SEQ ID NO:1 (GVYDGREHTV, referred to as MAGE-A4 in this specification). GVY It also refers to the specific recognition of epitopes containing fragments of or other such fragments.

[0064] Typically, the TCR recognizes an antigen when its peptide fragment is presented by a major histocompatibility complex (MHC) molecule.

[0065] The human leukocyte antigen (HLA) system, or HLA complex, is a gene complex that codes for major histocompatibility complex (MHC) proteins in humans. HLA-A*02 (HLA-A2) is a specific group of class I major histocompatibility complex (MHC) alleles at the HLA-A locus. HLA-A*02:01 is a specific HLA-A*02 allele.

[0066] Therefore, in one specific embodiment, the TCR recognizes the amino acid sequence of SEQ ID NO:1 in a form bound to HLA-A2. In a more specific embodiment, the TCR specifically recognizes the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01.

[0067] This TCR is highly specific to MAGE-A4 and does not exhibit cross-reactivity to other peptides. This means that this TCR does not recognize normal human cell lines, including cardiomyocytes, endothelial cells, pulmonary fibroblasts, hepatocytes, renal cortical epithelial cells, astrocytes, bronchial epithelial cells, and neurons, which do not express MAGE-A4. Cross-reactivity can be measured by IFN-γ secretion as described herein.

[0068] In this context, the term "specific to" means that the TCR specifically binds to its target. In a particular embodiment, the TCR is specific to MAGE-A4 and specifically binds to the amino acid sequence shown in SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01.

[0069] E. TCR-specific sequence The CDR3 of the TCRα chain of the TCR may have an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:14, and SEQ ID NO:24.

[0070] The CDR3 of the TCRβ chain of the TCR may have an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, and SEQ ID NO:27.

[0071] Some embodiments relate to an isolated TCR comprising a TCRα chain and a TCRβ chain, wherein a) the TCRα chain comprises a complementation-determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 4, and the TCRβ chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 7; or b) the TCRα chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 14, and the TCRβ chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 17; or c) the TCRα chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 24, and the TCRβ chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 27.

[0072] More specific embodiments include: a) a TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO:2, CDR2 having the amino acid sequence of SEQ ID NO:3, and CDR3 having the amino acid sequence of SEQ ID NO:4, and a TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO:5, CDR2 having the amino acid sequence of SEQ ID NO:6, and CDR3 having the amino acid sequence of SEQ ID NO:7; or b) a TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO:12, CDR2 having the amino acid sequence of SEQ ID NO:13, and CDR3 having the amino acid sequence of SEQ ID NO:14, and a TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO:15, CDR2 having the amino acid sequence of SEQ ID NO:16, and CDR3 having the amino acid sequence of SEQ ID NO:17; or c) a TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO:22, CDR2 having the amino acid sequence of SEQ ID NO:23, and SEQ This relates to an isolated TCR comprising a TCRα chain containing CDR3 having the amino acid sequence ID NO:24, a TCRβ chain containing CDR1 having the amino acid sequence SEQ ID NO:25, CDR2 having the amino acid sequence SEQ ID NO:26, and CDR3 having the amino acid sequence SEQ ID NO:27.

[0073] A preferred embodiment relates to an isolated TCR defined by the CDRs of the TCRα and TCRβ chains described above, particularly by CDR3, wherein the recombinant TCR sequence is modified to include murinized Cα and Cβ regions, preferably minimal murinized Cα and Cβ regions.

[0074] In a particularly preferred embodiment, the isolated TCR is defined by the CDRs of the TCRα and TCRβ chains described above, particularly by CDR3, and the recombinant TCR sequence is modified to include minimally mouse-like Cα and Cβ regions and hydrophobic amino acid mutations in the Cα transmembrane domain. In a particular embodiment, these TCRs exhibit increased expression and functional avidity compared to TCRs that are not minimally mouse-like and do not contain hydrophobic displacements in the Cα transmembrane domain.

[0075] In a more preferred embodiment, the isolated TCR is defined by the CDRs of the TCRα and TCRβ chains described above, particularly by CDR3, and the recombinant TCR sequence is not modified to include murinized Cα and Cβ regions or minimally murinized Cα and Cβ regions.

[0076] Some embodiments relate to isolated TCRs including a) a variable TCRα region having an amino acid sequence at least 80% identical to SEQ ID NO:8 and a variable TCRβ region having an amino acid sequence at least 80% identical to SEQ ID NO:9; or b) a variable TCRα region having an amino acid sequence at least 80% identical to SEQ ID NO:18 and a variable TCRβ region having an amino acid sequence at least 80% identical to SEQ ID NO:19; or c) a variable TCRα region having an amino acid sequence at least 80% identical to SEQ ID NO:28 and a variable TCRβ region having an amino acid sequence at least 80% identical to SEQ ID NO:29.

[0077] As used herein, “at least 80% identical,” and in particular “having at least 80% identical amino acid sequences,” encompasses that the amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the described amino acid sequence.

[0078] In some embodiments, the TCR comprises a TCRα chain and a TCRβ chain, where a) the variable TCRα region has an amino acid sequence at least 80% identical to SEQ ID NO: 8 and includes a CDR3 having the amino acid sequence described in SEQ ID NO: 4, and the variable TCRβ region has an amino acid sequence at least 80% identical to SEQ ID NO: 9 and includes a CDR3 having the amino acid sequence described in SEQ ID NO: 7, or b) the variable TCRα region has an amino acid sequence at least 80% identical to SEQ ID NO: 18 and includes a CDR3 having the amino acid sequence described in SEQ ID NO: 14, and the variable TCRβ region has an amino acid sequence at least 80% identical to SEQ ID NO: 19 and includes a CDR3 having the amino acid sequence described in SEQ ID NO: 17; or c) the variable TCRα region has an amino acid sequence at least 80% identical to SEQ ID NO: 28 and includes a CDR3 having the amino acid sequence described in SEQ ID NO: 24, and the variable TCRβ region is SEQ ID It contains CDR3 having an amino acid sequence that is at least 80% identical to NO:29 and has the amino acid sequence described for SEQ ID NO:27.

[0079] Exemplary embodiments relate to isolated TCRs including a) a variable TCRα region having the amino acid sequence of SEQ ID NO:8 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:9; or b) a variable TCRα region having the amino acid sequence of SEQ ID NO:18 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:19; or c) a variable TCRα region having the amino acid sequence of SEQ ID NO:28 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:29.

[0080] The following table shows a summary of exemplary TCRs. TIFF0007877420000001.tif31165

[0081] As can be seen from the examples, the TCR intended herein is specific to MAGE-A4.

[0082] The determination of identity percentages between multiple sequences is preferably achieved using the AlignX application of the Vector NTI Advance™ 10 program (Invitrogen Corporation, Carlsbad, California, USA). This program uses a modified Clustal W algorithm (Thompson et al. Nucl Acids Res 1994;42:23-33, Invitrogen Corporation. User Manual 2004;389-662). Identity percentage determination is performed using the standard parameters of the AlignX application.

[0083] The TCRs referred to herein are isolated or purified. "Isolated" means that the TCR is not present in the conditions in which it originally existed in nature. "Purified" means, for example, that the TCR does not contain, or substantially contains, other proteins and non-protein portions of the cell from which it originally came.

[0084] In some embodiments, the amino acid sequence of the TCR may include one or more phenotypically silent substitutions.

[0085] "Phenotypically silent substitutions" are also called "conservative amino acid substitutions." The concept of "conservative amino acid substitutions" is understood by those skilled in the art and preferably means that codons encoding positively charged residues (H, K, and R) are substituted with codons encoding positively charged residues, codons encoding negatively charged residues (D and E) are substituted with codons encoding negatively charged residues, codons encoding neutral polar residues (C, G, N, Q, S, T, and Y) are substituted with codons encoding neutral polar residues, and codons encoding neutral nonpolar residues (A, F, I, L, M, P, V, and W) are substituted with codons encoding neutral nonpolar residues. These mutations may occur spontaneously, be introduced by random mutagenesis, or be introduced by targeted mutagenesis. These changes can be made without disrupting the essential characteristics of these polypeptides. Those skilled in the art can easily screen mutant amino acids and / or nucleic acids encoding them by conventional methods and determine, by means of methods known in the art, whether these mutations substantially reduce or disrupt ligand binding ability.

[0086] In some embodiments, the amino acid sequence of the TCR is modified to include a detectable label, therapeutic agent, or pharmacokinetic modifier.

[0087] Non-limiting examples of detectable labels include radioactive labels, fluorescent labels, nucleic acid probes, enzymes, and contrast reagents. Therapeutic agents that can be conjugated to TCRs include radioactive compounds, immunomodulators, enzymes, or chemotherapeutic agents. Therapeutic agents can be encapsulated in liposomes linked to the TCR so that the compound can be released slowly at the target site. This would avoid damage during transport in the body and ensure that the therapeutic agent, such as a toxin, exerts its maximum effect after binding of the TCR to the relevant antigen-presenting cell. Other examples of therapeutic agents are listed below: peptide cytotoxicities, i.e., proteins or peptides with the ability to kill mammalian cells, e.g., lysine, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase. Low molecular weight cytotoxic agents, i.e., compounds with the ability to kill mammalian cells and a molecular weight of less than 700 daltons. Such compounds may contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these low molecular weight cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release cytotoxic agents. Examples of such agents include docetaxel, gemcitabine, cisplatin, meitansine derivatives, rashelmycin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimethrexate glucuronate, mitoxantrone, auristatin E, vincristine, and doxorubicin; radionuclides, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225, and astatine-213. Binding of radionuclides to TCRs or their derivatives can be carried out, for example, by chelating agents; and immunostimulants, also known as immunostimulants, i.e., immune effector molecules that stimulate the immune response.Exemplary immunostimulants include cytokines such as IL-2 and IFN-γ; antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28, or anti-CD16); alternative protein scaffolds with antibody-like binding features; superantigens, i.e., antigens that induce nonspecific activation of T cells, resulting in polyclonal T cell activation and massive cytokine release, as well as their variants; chemokines such as IL-8, platelet factor 4, and melanoma growth-stimulating proteins; complement activators; heterologous protein domains, allologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides.

[0088] Antigen receptor molecules (T cell receptor molecules) on human T lymphocytes are non-covalently bound to CD3 (T3) molecular complexes on the cell surface. Perturbation of this complex by anti-CD3 monoclonal antibodies induces T cell activation. Therefore, several embodiments relate to the TCRs described herein to which an anti-CD3 antibody or a functional fragment or variant of said CD3 antibody is bound (usually by fusion to the N-terminus or C-terminus of the alpha or beta chain). Suitable antibody fragments and variants / analogs for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv and scFv fragments, molecules containing synthetic monoimmunoglobulin variable heavy chain domains derived from Nanobodies® (Ablynx (Belgium)), camelid (camel, llama, or alpaca) antibodies), and alternative protein scaffolds exhibiting antibody-like binding characteristics such as domain antibodies (containing affinity-mature monoimmunoglobulin variable heavy chain domains or immunoglobulin variable light chain domains (Domantis (Belgium)), or Affibody (containing an engineered protein A scaffold, Affibody (Sweden)) or antikalin (containing an engineered antikalin, Pieris (Germany)).

[0089] The therapeutic agent may preferably be selected from the group consisting of immunoeffector molecules, cytotoxic agents, and radionuclides. Preferably, the immunoeffector molecule is a cytokine.

[0090] The pharmacokinetic modifier may be, for example, at least one polyethylene glycol repeating unit, at least one glycol group, at least one sialyl group, or a combination thereof. The bonding of at least one polyethylene glycol repeating unit, at least one glycol group, and at least one sialyl group can be brought about by several methods known to those skilled in the art. In one preferred embodiment, the unit is covalently linked to the TCR. The TCR contemplated herein may be modified with one or more pharmacokinetic modifiers. In particular, soluble TCRs are modified with one or more pharmacokinetic modifiers. The pharmacokinetic modifiers may result in beneficial changes to the pharmacokinetic profile of the therapeutic agent, such as improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility.

[0091] The TCRs intended herein may be soluble or membrane-bound. The term "soluble" means that the TCR is soluble (i.e., does not have a transmembrane domain or cytoplasmic domain) for use as a targeting agent for delivering a therapeutic agent to antigen-presenting cells, for example. For stability, soluble αβ heterodimer TCRs preferably have disulfide bonds introduced between residues of each constant domain, as described, for example, in WO 03 / 020763. One or both of the constant domains present in the αβ heterodimer may have one or more C-terminuses cleaved by, for example, up to 15, up to 10, or up to 8 or fewer amino acids. For use in adoptive therapy, αβ heterodimer TCRs may be transfected as full-length chains having both a cytoplasmic domain and a transmembrane domain, for example. The TCR may contain disulfide bonds between each alpha-steady domain and beta-steady domain corresponding to those found naturally, and in addition to or instead of these, unnatural disulfide bonds may be present.

[0092] Thus, TCRs, particularly the soluble TCRs intended herein, can be modified by attaching additional functional parts, for example, to reduce immunogenicity, increase hydrodynamic size (size in soluble state), solubility and / or stability (e.g., by enhanced protection from proteolysis), and / or extend the serum half-life. Other useful functional parts and modifications include “suicide switches” or “safety switches” that can be used to deactivate effector host cells holding the TCRs of the present invention in a patient’s body. One example is the inducible caspase 9 (iCasp9) “safety switch” described in Gargett and Brown. Front Pharmacol 2014;5:235. Briefly, effector host cells are modified by known methods to express a caspase 9 domain, the dimerization of which depends on a small molecule dimerizer such as AP1903 / CIP, and the dimerization results in rapid induction of apoptosis in the modified effector cells. This system is described, for example, in EP2173869(A2). Other examples of “suicide switches” or “safety switches” are also known in the art, such as herpes simplex virus thymidine kinase (HSV-TK), CD20 expression followed by depletion using anti-CD20 antibodies, or myc tags (Kieback et al. Proc Natl Acad Sci USA 2008 15;105(2):623-628).

[0093] In this specification, TCRs with altered glycosylation patterns are also considered. As is well known in the art, the glycosylation pattern may depend on the amino acid sequence (e.g., the presence or absence of specific glycosylated amino acid residues, as described later) and / or the host cell or host organism in which the protein is produced. Polypeptide glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The addition of an N-linked glycosylation site to a binding molecule is conveniently achieved by altering the amino acid sequence so that it contains one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline). O-linked glycosylation sites can be introduced by adding or substituting one or more serine or threonine residues into the starting sequence.

[0094] Another means of glycosylation of TCRs is by chemical or enzymatic coupling of glycosides to proteins. Depending on the coupling method used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, e.g., those of cysteine, (d) free hydroxyl groups, e.g., those of serine, threonine, or hydroxyproline, (e) aromatic residues, e.g., those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. Similarly, deglycosylation (i.e., removal of the carbohydrate moiety present on the binding molecule) can also be achieved chemically, for example by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically, by using endoglycosidases and exoglycosidases.

[0095] It is also conceivable to add drugs such as low-molecular-weight compounds to the TCR, particularly the soluble TCR of the present invention. Linking can be achieved by covalent bonding or by non-covalent interactions, such as electrostatic forces. Various linkers known in the art can be used to form drug conjugates.

[0096] TCRs, particularly soluble TCRs of the present invention, can be further modified to introduce additional domains (tags) that aid in the identification, tracking, purification, and / or isolation of each molecule. Thus, in some embodiments, the TCRα chain or TCRβ chain can be modified to include an epitope tag.

[0097] Epitope tags are a useful example of tags that can be incorporated into TCRs. Epitope tags are short stretches of amino acids that allow binding of specific antibodies, enabling the identification and tracking of binding and migration of soluble TCRs or host cells in a patient's body or cultured (host) cells. Detection of epitope tags, and therefore tagged TCRs, can be achieved using several different techniques. Tags can also be used to stimulate and proliferate host cells that hold the TCRs of the present invention by culturing the cells in the presence of a binding molecule (antibody) specific to the tag.

[0098] In general, in some cases, TCRs can be modified with various mutations that alter their affinity and dissociation rate (off-rate) to target antigens. In particular, these mutations can increase affinity and / or decrease dissociation rate. Thus, mutations can be introduced into at least one of the CDR and variable domain framework regions of the TCR.

[0099] However, in a preferred embodiment, the CDR region of the TCR is not modified, and no in vitro affinity maturation occurs, as in the case of the TCR receptor in the examples. This means that the CDR region retains its native sequence. This may be advantageous because in vitro affinity maturation can impart immunogenicity to the TCR molecule, which can lead to the production of anti-drug antibodies that reduce or inactivate therapeutic effects and treatments, and / or induce adverse effects.

[0100] Mutations can consist of one or more substitutions, deletions, or insertions. These mutations can be introduced by any suitable method known in the art, such as DNA synthesis, polymerase chain reaction, restriction enzyme cloning, or ligation-independent cloning techniques, as described, for example, Sambrook, Cold Spring, Harbor Laboratory Press 2012.

[0101] Theoretically, mispairing between endogenous and exogenous TCR chains can lead to unpredictable TCR specificity with a risk of cross-reactivity. To avoid TCR sequence mispairing, recombinant TCR sequences can be modified to include minimally mouse-like Cα and Cβ regions. This is a technique that has been shown to efficiently enhance the correct pairing of several different transduced TCR chains. Mouse-like TCRs (i.e., exchanging the human constant regions in the alpha and beta chains with their respective mouse counterparts) are a commonly applied technique to improve the cell surface expression of TCRs in host cells. While we do not wish to be bound by any particular theory, it is thought that mouse-like TCRs bind more effectively to the CD3 co-receptor and / or preferentially pair with each other, and that on human T cells genetically modified ex vivo to express TCRs with desired antigen specificity, they are less likely to form mixed-type TCRs, but each of the "original" TCRs is still retained and expressed.

[0102] Nine amino acids responsible for the improved expression of the mouse-like TCR have been identified (Sommermeyer and Uckert, J Immunol. 2010;184(11):6223-6231), and it is hypothesized that one or all of these amino acid residues in the constant regions of the TCR alpha and / or beta chains will be replaced with their respective mouse-corresponding residues. This technique, also known as "minimal murinization," has the advantage of enhancing cell surface expression while simultaneously reducing the number of "foreign" amino acid residues in the amino acid sequence, thereby reducing the risk of immunogenicity.

[0103] In a preferred embodiment, the TCRs containing the minimally mouse-like Cα and Cβ regions are TCR-1, which includes the α chain of SEQ ID NO:10 and the β chain of SEQ ID NO:11; TCR-2, which includes the α chain of SEQ ID NO:20 and the β chain of SEQ ID NO:21; and TCR-3, which includes the α chain of SEQ ID NO:30 and the β chain of SEQ ID NO:31.

[0104] In a preferred embodiment, the TCR contains minimally moused Cα and Cβ regions and further includes hydrophobic amino acid mutations in the Cα transmembrane domain. The transmembrane domain of the TCRα chain has been shown to contribute to the lack of overall chain stability, consequently affecting the formation and surface expression of the entire TCR-CD3 complex. Substitution of three amino acids in the TCRα transmembrane domain with the hydrophobic amino acids leucine or valine increased TCR expression and functional avidity. Haga-Friedman et al. J.Immunology 2012;188:5538-5546.

[0105] In a preferred embodiment, TCRs containing minimally mouse-like Cα and Cβ regions and hydrophobic amino acid substitutions in the TCRα chain are TCR-7, which includes the α chain of SEQ ID NO:102 and the β chain of SEQ ID NO:103; TCR-8, which includes the α chain of SEQ ID NO:108 and the β chain of SEQ ID NO:109; and TCR-9, which includes the α chain of SEQ ID NO:114 and the β chain of SEQ ID NO:115.

[0106] Some embodiments relate to isolated TCRs described herein, which are of a single-chain type in which a TCRα chain and a TCRβ chain are linked by a linker sequence, and the linker is optionally cleavable.

[0107] A suitable single-chain TCR type includes a first segment encoded by an amino acid sequence corresponding to the variable TCRα region, a second segment consisting of an amino acid sequence corresponding to the variable TCRβ region fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the constant region of the TCRβ chain, and a linker sequence ligating the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may consist of an amino acid sequence corresponding to the variable region of the TCRβ chain, and the second segment may consist of an amino acid sequence corresponding to the variable region sequence of the TCRα chain fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the constant region of the TCRα chain. The single-chain TCR may further include a disulfide bond between the first and second chains, and the length of the linker sequence and the position of the disulfide bond are such that the variable domain sequences of the first and second segments are oriented toward each other essentially in the same way as in the innate T cell receptor. More specifically, the first segment may consist of an amino acid sequence corresponding to the TCRα chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the TCRα chain constant region, and the second segment may consist of an amino acid sequence corresponding to the TCRβ chain variable region fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the TCRβ chain constant region, and a disulfide bond may be provided between the first and second chains. The linker sequence can be any sequence that does not impair the function of the TCR.

[0108] A “functional” TCRα and / or β-chain fusion protein means a TCR or TCR variant modified, for example, by the addition, deletion, or substitution of amino acids, which maintains at least substantial biological activity. In the case of the α and / or β-chains of a TCR, this means that both chains (together with the unmodified α and / or β-chain, or together with the α and / or β-chain of another fusion protein of the present invention) are still capable of forming a T cell receptor that exerts its biological function, in particular binding to the specific peptide-MHC complex of the TCR and / or functional signaling during specific peptide:MHC interactions.

[0109] In a specific embodiment, the TCR may be modified to become a functional T cell receptor (TCR) α and / or β chain fusion protein in which the epitope tag has a length of 6 to 15 amino acids, preferably 9 to 11 amino acids. In another embodiment, the TCR may be modified to become a functional T cell receptor (TCR) α and / or β chain fusion protein containing two or more spaced or directly linked epitope tags. The embodiment of the fusion protein may contain two, three, four, five or more epitope tags, insofar as the fusion protein maintains its one or more biological activities ("functional").

[0110] The epitope tag is preferably a functional T cell receptor (TCR) α and / or β chain fusion protein of the present invention, selected from, but not limited to, CD20 or Her2 / neu tags, or other conventional tags such as myc tags, FLAG tags, T7 tags, HA (hemagglutinin) tags, His tags, S tags, GST tags, or GFP tags. The myc tag, T7 tag, GST tag, and GFP tag are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, for example, U.S. Patents 4,703,004 and 4,851,341). The myc tag is preferably used because high-quality reagents are available and can be used for its detection. The epitope tag can, of course, have one or more additional functions in addition to recognition by antibodies. The sequences of these tags are described in the literature and are well known to those skilled in the art.

[0111] In a more preferred embodiment, the isolated TCR is expressed as a fusion protein in which the TCRα chain and the TCRβ chain are separated by one or more polypeptide cleavage signals. In a particular embodiment, the fusion protein may comprise, from 5' to 3', the TCRα chain, one or more polypeptide cleavage signals, and the TCRβ chain. In a particular embodiment, the fusion protein may comprise, from 5' to 3', the TCRβ chain, one or more polypeptide cleavage signals, and the TCRα chain.

[0112] Polypeptide cleavage signals intended herein include, but are not limited to, protease cleavage sites and ribosome skip sequences. Polypeptide cleavage signals may be positioned between each of the polypeptide domains described herein, for example, between the TCRα and TCRβ chains. In addition, polypeptide cleavage signals may be incorporated into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides or ribosome skip sequences (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0113] Specific examples of protease cleavage sites suitable for use in a particular embodiment include furin (e.g., Arg-XX-Arg, e.g., Arg-X-Lys / Arg-Arg or Arg-Gln / Tyr-Lys / Arg-Arg; furin can also cleave sequences Arg-Ala-Arg-Tyr-Lys-Arg or Arg-Ala-Arg-Tyr-Lys-Arg-Ser); subtilisin (e.g., PC2, PC1 / PC3, PACE4, PC4, PC5 / PC6, LPC) Examples include, but are not limited to, / PC7IPC8 / SPC7 and SKI-I); enterokinases (e.g., Asp-Asp-Asp-Aps-Lys* and Asp / Glu-Arg-*Met); factor Xa (e.g., Glu-Gly-Arg*); thrombin (e.g., Leu-Val-Pro-Arg*Gly-Ser); granzyme B (e.g., Ile-Glu-Pro-Asp*); and caspase-3 (e.g., Asp-Glu-Val-Asp*).

[0114] Specific examples of self-cleaving viral peptides or ribosome skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). In a particular embodiment, the viral 2A peptide is aftvirus 2A peptide, potivirus 2A peptide, or cardiovirus 2A peptide. In a preferred embodiment, the viral 2A peptide is selected from the group consisting of foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), Thosea asigna virus 2A peptide (T2A), porcine rhinitis virus-1 2A peptide (P2A), tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0115] In one embodiment, the fusion protein comprises a TCRα chain, a proteolytic cleavage site and / or a ribosome skip sequence, and a TCRβ chain. In a preferred embodiment, the fusion protein comprises a TCRα chain, a furin cleavage site and / or a P2A ribosome skip sequence, and a TCRβ chain. In another preferred embodiment, the fusion protein comprises a TCRα chain, a P2A ribosome skip sequence, and a TCRβ chain.

[0116] In a particular embodiment, the fusion protein comprises a TCRβ chain, a proteolytic cleavage site and / or a ribosome skip sequence, and a TCRα chain. In a preferred embodiment, the fusion protein comprises a TCRβ chain, a furin cleavage site and / or a P2A ribosome skip sequence, and a TCRα chain. In another preferred embodiment, the fusion protein comprises a TCRβ chain, a P2A ribosome skip sequence, and a TCRα chain.

[0117] In a preferred embodiment, the fusion protein comprises an amino acid sequence represented by any one of SEQ ID NO: 94, 96, 98, 104, 110, and 116.

[0118] F. TCR fragments and variants Another aspect relates to polypeptides containing the functional portion of the TCR described herein. The functional portion may contain at least one amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:7, SEQ ID NO:17, and SEQ ID NO:27.

[0119] In specific embodiments, the polypeptide may consist only of the functional portion of the TCR, for example, in a soluble form. Alternatively, the polypeptide may be combined with other domains.

[0120] The functional portion can mediate the binding of the TCR to an antigen, particularly to an antigen-MHC complex. In one embodiment, the functional portion comprises the TCRα variable chain and / or TCRβ variable chain described herein.

[0121] TCR variant molecules, i.e., molecules possessing a polypeptide containing the functional portion of the TCR combined with other domains, may have TCR binding properties, but they may also combine with the signaling domains of effector cells (other than T cells), particularly the signaling domains of NK cells. Therefore, some embodiments relate to proteins containing the functional portion of the TCR described herein, combined with the signaling domains of immune effector cells such as NK cells.

[0122] "Binding" refers to the ability to bind, integrate, or bind to a target specifically and non-covalently.

[0123] Another aspect relates to a polyvalent TCR complex comprising at least two TCRs described herein. In one aspect of this aspect, at least two TCR molecules are linked by a linker moiety for forming a polyvalent complex. Preferably, since the complex is water-soluble, the linker moiety should be appropriately selected. Preferably, the linker moiety can be attached to a predetermined position on the TCR molecule so that the structural diversity of the complex formed is minimized. In one aspect of this aspect, the TCR complex is provided by a polymer chain or peptide linker sequence that spans between amino acid residues of each TCR that are not within the variable region sequence of the TCR. Since the complex may also be for medical use, the linker moiety should be appropriately selected with due consideration to its pharmaceutically appropriateness, such as its immunogenicity. Examples of linker moieties that meet the above desirable criteria are known in the art, for example, in the art of linking antibody fragments.

[0124] Examples of linkers include hydrophilic polymers and peptide linkers. An example of a hydrophilic polymer is polyalkylene glycol. The most commonly used of this type is based on polyethylene glycol, i.e., PEG. However, there are other suitable, possibly substituted, polyalkylene glycol-based linkers, including, for example, polypropylene glycol and copolymers of ethylene glycol and propylene glycol. Peptide linkers consist of chains of amino acids and function to produce a simple linker or polymerizing domain to which TCR molecules can be attached.

[0125] One embodiment relates to a polyvalent TCR complex in which a therapeutic agent is bound to at least one of the TCRs.

[0126] G. Release of cytokines and chemokines Some embodiments relate to the isolated TCR, polypeptide, and polyvalent TCR complex described herein, in which IFN-γ secretion is induced by the binding of the TCR expressed on effector cells to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01.

[0127] IFN-γ secretion induced by the binding of the TCR of the present invention, expressed on effector cells, to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01, can be more than 100 times, preferably more than 500 times, and more preferably more than 2000 times higher when bound to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01, compared to binding to unrelated peptides (ASTN1, SEQ ID NO:56, KLYGLDWAEL) presented by the molecule encoded by HLA-A*02:01. IFN-γ secretion can be, for example, more than 100 pg / ml, for example more than 500 pg / ml, or more than 2000 pg / ml.

[0128] The release of cytokines and chemokines, such as IFN-γ secretion, is achieved by transfecting K562 cells (Greiner et al. 2006, Blood. 2006 Dec 15;108(13):4109-17) with ivtRNA or by transduction, thereby expressing either the amino acid sequence of SEQ ID NO:1 or an unrelated peptide, respectively, and then analyzing these with CD8 cells expressing the TCR to be investigated. + Enriched PBMC and / or non-CD8 + This can be measured using an in vitro assay in which the cells are incubated with enriched PBMCs, or by using T2 cells externally loaded with SEQ ID NO:1 or an unrelated peptide, and then testing them for the CD8 expression of the TCR being examined. + Enriched PBMC and / or non-CD8 + It can be measured in an in vitro assay co-incubated with concentrated PBMCs.

[0129] Some embodiments relate to an isolated TCR, a polypeptide or a multivalent TCR complex described herein, wherein IFN-γ secretion induced by binding of the TCR of the present invention expressed on effector cells to the amino acid sequence of SEQ ID NO:1 or, in particular, to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01 is below a predetermined threshold. The threshold can be determined by using a specific effector-to-target ratio that is at least 2:1.

[0130] "Effector cells" can be peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). Typically, effector cells are immune effector cells, especially T cells. Other suitable cell types include gamma-delta T cells, natural killer (NK) cells, and NK-like T (NKT) cells.

[0131] IFN-γ secretion upon binding of the TCR of the present invention expressed on effector cells to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01 is at least 10 -7 [M], preferably at least 10 -8 [M], more preferably 10 -9 [M] of MAGE-A4 peptide concentration can be induced. In a specific embodiment, for example, when the ratio of TCR transgenic T cells to T2 cells is 2:1, IFN-γ secretion upon binding of the TCR of the present invention expressed on effector cells to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01 is at least 10 -7 [M], preferably at least 10 -8 [M], more preferably 10 -9 [M] of MAGE-A4 peptide concentration can be induced.

[0132] The present invention relates to a method for identifying a TCR or fragment thereof that binds to a target amino acid sequence SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01, the method comprising the steps of contacting a candidate TCR or fragment thereof with the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01, and determining whether the candidate TCR or fragment thereof binds to a target and / or mediates an immune response.

[0133] Whether a candidate TCR or its fragment mediates an immune response can be determined by measuring cytokine secretion, such as IFN-γ secretion. As mentioned above, cytokine secretion is measured by, for example, K562 cells (or other APCs) transfected with ivtRNA encoding the amino acid sequence of SEQ ID NO:1, and CD8 cells expressing the TCR under investigation or a molecule containing a fragment of that TCR. + It can be measured by an in vitro assay in which it is incubated with concentrated PBMCs.

[0134] H. Nucleic acid, vector Another aspect relates to nucleic acids encoding TCRs as described herein, or nucleic acids encoding polynucleotides encoding TCRs as described herein.

[0135] The following table shows the nucleotide sequences that encode each peptide sequence. TIFF0007877420000002.tif88128TIFF0007877420000003.tif212126TIFF0007877420000004.tif21128

[0136] "Nucleic acid molecule" and "nucleotide sequence" generally mean polymers of DNA or RNA, which may be single-stranded or double-stranded, synthesized, or obtained from natural sources (e.g., isolated and / or purified), and may contain natural nucleotides, unnatural or modified nucleotides, and may contain natural nucleotide bonds, or unnatural or modified nucleotide bonds, such as phosphoramidate bonds or phosphorothioate bonds, instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant nucleic acids. As used herein, the term "recombinant" means (i) a molecule constructed outside of a living cell by joining natural or synthetic nucleic acid segments to a nucleic acid molecule that can be replicated in a living cell, or (ii) a molecule resulting from the replication of the molecule described in (i) above. Replication herein may be in vitro or in vivo replication. Nucleic acids can be constructed using methods known in the art or commercially available methods (e.g., from companies such as Genscript and Thermo Fisher) based on chemical synthesis and / or enzymatic ligation reactions. For example, nucleic acids can be chemically synthesized using natural nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the double helix formed during hybridization (see Sambrook et al.). Nucleic acids can contain any nucleotide sequence encoding either a recombinant TCR, a recombinant polypeptide, or a recombinant protein, or a functional part or functional variant thereof.

[0137] This disclosure also provides isolated or purified nucleic acid variants that are at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences encoding the TCRs described herein. Such variant nucleotide sequences encode functional TCRs that specifically recognize MAGE-A4.

[0138] This disclosure also provides isolated or purified nucleic acids that include a nucleotide sequence complementary to any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes to any of the nucleotides of the nucleic acids described herein under stringent conditions.

[0139] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high-stringency conditions. “High-stringency conditions” means that the nucleotide sequence hybridizes to the target sequence (any nucleotide sequence of the nucleic acids described herein) in a detectably stronger amount than nonspecific hybridization. High-stringency conditions include those in which polynucleotides with strictly complementary sequences or containing only a few scattered mismatches are distinguished from random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small complementary regions are more easily melted than full-length complementary chains of 14-17 bases or more, and high-stringency hybridization makes them easily distinguishable. Relatively high-stringency conditions would include, for example, low-salt and / or high-temperature conditions, such as those given by about 0.02-0.1 M NaCl or its equivalent and a temperature of about 50-70°C. Such highly stringent conditions tolerate little to no mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any of the TCRs described herein. It is generally understood that the stringency of the conditions can be enhanced by increasing the amount of formamide added.

[0140] In certain embodiments, nucleic acids are codon-optimized. As used herein, the term “codon-optimized” refers to the substitution of codons in the polynucleotide encoding a polypeptide in order to increase its expression, stability, and / or activity. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variations in codon bias between two or more organisms or genes or between synthetically constructed bias tables; (ii) variations in the degree of codon bias within an organism, gene or gene set; (iii) systematic variations in codons including context; (iv) variations in codons corresponding to each decoding tRNA; (v) variations in codons corresponding to GC% in the entire triplet or at one position in the triplet; (vi) variations in similarity to a reference sequence, e.g., a natural sequence; (vii) variations in codon frequency cutoff; (viii) structural characteristics of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of codon substitution sets should be based; (x) systematic variations in the codon set for each amino acid; and / or (xi) the removal of a spurious translation initiation site.

[0141] Another embodiment relates to a vector comprising a nucleic acid encoding a TCR as described herein. The vector is preferably a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector, or particle and / or vector used in gene therapy.

[0142] A “vector” is any molecule or composition having the ability to deliver a nucleic acid sequence into a suitable host cell in which the synthesis of the encoded polypeptide can occur. Typically, and preferably, a vector is a nucleic acid that has been engineered using recombinant DNA techniques known in the art to incorporate a desired nucleic acid sequence. A vector may contain DNA or RNA and / or liposomes. A vector may be a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector, or particles and / or vectors used in gene therapy. A vector may contain a nucleic acid sequence, such as an origin of replication, which enables it to replicate in a host cell. A vector may also contain one or more selectable marker genes and other genetic elements known to those skilled in the art. Preferably, a vector is an expression vector containing the nucleic acid of the present invention functionally linked to a sequence that enables the expression of the nucleic acid of the present invention.

[0143] In a preferred embodiment, the vector comprises nucleic acids encoding a TCRβ chain having the amino acid sequence shown in SEQ ID NO:88 and a TCRα chain having the amino acid sequence shown in SEQ ID NO:87; a TCRβ chain having the amino acid sequence shown in SEQ ID NO:90 and a TCRα chain having the amino acid sequence shown in SEQ ID NO:89; a TCRβ chain having the amino acid sequence shown in SEQ ID NO:92 and a TCRα chain having the amino acid sequence shown in SEQ ID NO:91; a TCRβ chain having the amino acid sequence shown in SEQ ID NO:103 and a TCRα chain having the amino acid sequence shown in SEQ ID NO:102; a TCRβ chain having the amino acid sequence shown in SEQ ID NO:109 and a TCRα chain having the amino acid sequence shown in SEQ ID NO:108; or a TCRβ chain having the amino acid sequence shown in SEQ ID NO:115 and a TCRα chain having the amino acid sequence shown in SEQ ID NO:114.

[0144] Preferably, the vector is an expression vector. More preferably, the vector is a retroviral vector, more specifically a gamma retroviral vector or a lentiviral vector.

[0145] I. Cells, cell lines Another aspect relates to cells expressing the TCR described herein. In some embodiments, the cells are isolates or non-natural products. In specific embodiments, the cells may include a nucleic acid encoding the TCR described herein, or a vector containing such nucleic acid.

[0146] Cells may be introduced into the aforementioned vector containing the nucleic acid sequence encoding the TCR, or into the ivtRNA encoding the TCR. The cells may be peripheral blood lymphocytes such as T cells. Methods for cloning and exogenous expression of the TCR are described, for example, in Engels et al. Cancer Cell, 2013;23(4):516-526. Transduction of primary human T cells using lentiviral vectors is described, for example, in Cribbs et al. BMC Biotechnol. 2013;13:98.

[0147] The term "transfection" refers to a non-viral process in which an exogenous nucleic acid sequence is introduced into a host cell, such as a eukaryotic host cell. It should be noted that the introduction or transfer of nucleic acid sequences is not limited to the methods mentioned and can be achieved by many means, including electroporation, microinjection, gene gun delivery, lipofection, or superfection.

[0148] The term "transduction" refers to the introduction of exogenous nucleic acid sequences into host cells using viral vectors, such as adenoviruses, adeno-associated viruses (AAVs), vaccinia viruses, herpesviruses, retroviruses, or lentiviruses.

[0149] Some embodiments relate to a) a cell comprising an expression vector comprising at least one nucleic acid described herein, or b) a cell comprising a first expression vector comprising a nucleic acid encoding the α-chain of the TCR described herein and a second expression vector comprising a nucleic acid encoding the beta-chain of the TCR described herein.

[0150] In some embodiments, the cells are peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). The cells may be natural killer (NK) cells, natural killer-like T (NKT) cells, or T cells. Preferably, the cells are T cells. T cells are CD4 + T cells or CD8 + These cells may be T cells, or double-negative T cells, i.e., T cells that do not express either CD4 or CD8. In some embodiments, the cells are stem cell-like memory T cells.

[0151] In a preferred embodiment, the TCR functions independently of the co-receptor. That is, TCRs, for example, TCR-5 and TCR-8, function independently of CD8 + CD4 in cells + It also functions in cells.

[0152] Stem cell-like memory T cells (TSCMs) are CD8 +These are a subpopulation of T cells with a low degree of differentiation, characterized by their self-renewal ability and ability to persist for long periods. When these cells encounter their respective antigens in vivo, they further differentiate into central memory T cells (TCMs), effector memory T cells (TEMs), and terminally differentiated effector memory T cells (TEMRAs), although some TSCMs remain quiescent (Flynn et al., Clinical & Translational Immunology 2014;3(7):e20). These residual TSCM cells demonstrate the ability to establish durable immunological memory in vivo and are therefore considered an important T cell subpopulation for adoptive T cell therapy (Lugli et al., Nature Protocols 2013;8:33-42, Gattinoni et al., Nat. Med. 2011;Oct;17(10):1290-1297). Immunomagnetic selection can be used to limit the T cell pool to stem cell memory T cell subtypes (see Riddell et al. Cancer Journal 2014;20(2):141-144).

[0153] Antibodies targeting the TCR Another aspect relates to an antibody or its antigen-binding fragment that specifically binds to the portion of the TCR described herein that mediates specificity to MAGE-A4. In one embodiment, the portion of the TCR that mediates MAGE-A4 specificity includes a CDR3 of the TCR alpha chain selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 14, and SEQ ID NO: 24, and a CDR3 of the beta chain selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO: 27.

[0154] Antibodies or their antigen-binding fragments can modulate the activity of the TCR. This may or may not involve blocking the binding of the TCR to MAGE-A4. This can be used to modulate the therapeutic activity of the TCR or for diagnostic purposes.

[0155] K. Pharmaceutical compositions, medical procedures and kits Another aspect relates to a composition comprising a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a polyvalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of the TCR as described herein.

[0156] Another aspect relates to a pharmaceutical composition comprising a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a polyvalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of the TCR as described in the present invention.

[0157] These active components of the present invention are preferably mixed with an acceptable carrier or carrier material in a dose that can treat or at least alleviate a disease, and used in the above-described pharmaceutical composition. Such composition may include (in addition to the active components and the individual components) filling materials, salts, buffers, stabilizers, solubilizers, and other materials of the known art.

[0158] The term "pharmaceutically acceptable" refers to non-toxic materials that do not interfere with the effectiveness of the biological activity of the active components. The choice of individual components depends on the application.

[0159] A pharmaceutical composition may contain additional components that enhance the activity of the active component, or additional components that complement the treatment. Such additional components and / or factors may be part of the pharmaceutical composition to achieve a synergistic effect or to minimize adverse or unwanted effects.

[0160] The formulation or preparation and application / techniques for the active components of the present invention are published in Remington: The Science and Practice of Pharmacy, Volumes I and II, 22nd edition, edited by Loyd V. Allen Jr., Philadelphia, Pennsylvania, Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety. Suitable applications include parenteral administration, e.g., intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intravenous, intraintravenous, intranodal, intraperitoneal, or intratumoral injection. Intravenous injection or intravenous infusion is a preferred patient treatment.

[0161] In one preferred embodiment, the pharmaceutical composition is administered by infusion or injection. The injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, for example, a population of proliferated T cells expressing a TCR (e.g., autologous or allogeneic to the patient being treated). The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition may further contain trace amounts of one or more non-toxic adjuvants, such as emulsifiers, preservatives, and pH buffers. Such injectable compositions useful for use with the fusion proteins of this disclosure are conventional, and suitable formulations are well known to those skilled in the art.

[0162] Typically, a pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

[0163] Therefore, another aspect relates to a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a polyvalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, an antibody that specifically binds to a portion of the TCR, a composition comprising one or more cells expressing the TCR as described herein, or a pharmaceutical composition for use as a pharmaceutical.

[0164] Some embodiments relate to TCRs as described herein, polypeptides comprising a functional portion of the TCR, polyvalent TCR complexes as described herein, nucleic acids encoding the TCR, vectors comprising the nucleic acid, cells comprising the TCR, or compositions or pharmaceutical compositions comprising the same, for use in the treatment of cancer.

[0165] In one aspect, cancer is blood cancer or solid tumor. Blood cancer, also called cancer of the blood, does not form solid tumors and is therefore scattered throughout the body. Examples of blood cancers include leukemia, lymphoma, or multiple myeloma. Solid tumors are broadly divided into two types: sarcomas and carcinomas. Sarcomas are tumors of blood vessels, bones, adipose tissue, ligaments, lymphatic vessels, muscles, or tendons, for example.

[0166] In one embodiment, cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia. Preferably, cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer or colorectal cancer, or sarcoma. More preferably, cancer is selected from urothelial (bladder) cancer, melanoma, head and neck cancer, ovarian cancer, NSCLC, esophageal cancer, gastric cancer, synovial sarcoma, and myxoid round cell liposarcoma (MRCLS).

[0167] In one embodiment, the TCR recognizes lung cancer cell lines such as the NSCLC cell line NCI-H1703 and the NSCLC liver metastasis cell line NCI-H1755.

[0168] This specification also envisions pharmaceutical compositions and kits containing (i) isolated TCRs as described herein; (ii) viral particles containing nucleic acids encoding recombinant TCRs; (iii) immune cells, such as T cells or NK cells, modified to express recombinant TCRs as described herein; and (iv) one or more nucleic acids encoding recombinant TCRs as described herein. In some embodiments, this disclosure provides compositions comprising lentiviral vector particles containing nucleotide sequences encoding recombinant TCRs as described herein (or T cells modified using the vector particles described herein to express recombinant TCRs). Such compositions can be administered to a subject in the methods of this disclosure, as further described herein.

[0169] Compositions comprising modified T cells as described herein can be used in methods and compositions for adoptive immunotherapy by known techniques or by modifications thereof that would become apparent to those skilled in the art based on this disclosure.

[0170] In some embodiments, cells are first harvested from their culture medium and then formulated in a treatment-effective dose by washing and concentrating the cells in a suitable medium and container system ("pharmaceutically acceptable" carrier). Suitable infusion media include any isotonic medium formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose aqueous solution or lactated Ringer's solution may also be used. Human serum albumin may be supplemented in the infusion medium.

[0171] The number of cells in the composition for effective treatment is typically more than 10 cells, up to 10 6 cells, up to 10 8 or 10 9 It is smaller than a cell, 10 10The number of cells can be greater than 10. The number of cells will depend on the final use of the composition and the type of cells contained therein. For example, if cells specific to a particular antigen are desired, the population will contain more than 70%, generally more than 80%, more than 85%, and more than 90-95% of such cells. For the uses provided herein, the cells are generally in a volume of 1 liter or less, and can be 500 ml or less, even 250 ml or less, or 100 ml or less. Thus, the desired cell density is typically 10 6 It is more than cells / ml, and is generally 10 7 Cells / ml or more, generally 10 8 The number of cells / ml is greater than 10. A clinically reasonable number of immune cells is cumulatively 10 9 More than 10 cells 10 More than 10 cells 11 It can be divided into multiple injections, each involving more than a single cell.

[0172] The pharmaceutical compositions provided herein may take various forms, such as solids, liquids, powders, aqueous solutions, or lyophilized forms. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and / or additives can be formulated in conventional methods and administered to a target in an appropriate dose. Stabilizers such as lipids, nuclease inhibitors, polymers, and chelating agents can protect the composition from degradation in the body. Compositions intended for administration by injection may contain one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0173] Viral vector particles comprising a recombinant TCR described herein, or a nucleotide sequence encoding a recombinant TCR provided herein, can be packaged as a kit. The kit may optionally include one or more components, such as instructions for use, a device and additional reagents, and components such as tubes, containers and syringes for carrying out the method. An exemplary kit may include a nucleic acid encoding a recombinant TCR, a recombinant TCR polypeptide, or a virus provided herein, and may optionally include instructions for use, a device for detecting the virus in the subject, a device for administering the composition to the subject, and a device for administering the composition to the subject.

[0174] This specification also envisions kits comprising polynucleotides encoding a gene of interest (e.g., recombinant TCR). This specification also envisions kits comprising a viral vector encoding a sequence of interest (e.g., recombinant TCR) and, optionally, polynucleotide sequences encoding an immune checkpoint inhibitor.

[0175] Kits contemplated herein also include kits for performing methods to detect the presence of polynucleotides encoding any one or more of the TCRs disclosed herein. In particular, such diagnostic kits may include a suitable set of amplification and detection primers and other related reagents for performing deep sequencing to detect the polynucleotides encoding the TCRs disclosed herein. In a further embodiment, kits herein may include reagents for detecting the TCRs disclosed herein, such as antibodies or other binding molecules. Diagnostic kits may also include instructions for determining the presence of polynucleotides encoding the TCRs disclosed herein or instructions for determining the presence of the TCRs disclosed herein. Kits may also include instructions. Instructions typically include specific descriptions of the components included in the kit and methods for administration, including methods for determining the appropriate condition of the subject, the appropriate dosage, and the appropriate method of administration. Instructions may also include guidance for monitoring the subject during the course of treatment.

[0176] The kits provided herein may also include devices for administering the compositions described herein. Any of the various devices known in the art for administering pharmaceuticals or vaccines may be included in the kits provided herein. Exemplary devices include, but are not limited to, subcutaneous needles, intravenous needles, catheters, needleless injection devices, and liquid dispensers such as inhalers and eye drops. Typically, a device for administering the virus in the kit will be compatible with the virus in the kit. Needleless injection devices, such as high-pressure injection devices, may be included in the kit with viruses that are not damaged by high-pressure injection, but typically not with viruses that are damaged by high-pressure injection.

[0177] The kits provided herein may also include devices for administering compounds such as T cell activators or T cell stimulants, or TLR agonists, such as TLR4 agonists. Any of the various devices known in the art for administering pharmaceuticals may be included in the kits provided herein. Exemplary devices include, but are not limited to, subcutaneous needles, intravenous needles, catheters, needle-free injectors, subcutaneous needles, intravenous needles, catheters, needle-free injector devices, inhalers, and eye drop dispensers. Typically, the device for administering the compound in the kit will be compatible with the desired method of administration of that compound.

[0178] In certain embodiments, preparations of pharmaceutically acceptable carrier solutions are well known to those skilled in the art, as are the development of administration and treatment regimens suitable for use in various treatment regimens, including, for example, enteral and parenteral administration, intravascular, intravenous, intra-arterial, intraosseous, intracavitary, intracerebral, intracranial, intraspinal, intrathecal, and intrathecal administration and formulations. It will be understood to those skilled in the art that certain embodiments contemplated herein may include other formulations, for example, those well known in the field of pharmacy, described, for example, in Remington: The Science and Practice of Pharmacy, Volumes I and II, 22nd edition, edited by Loyd V. Allen Jr., Philadelphia, Pennsylvania, Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0179] All publications, patent applications, and granted patents referenced in this specification are incorporated herein by reference as if each individual publication, patent application, or granted patent were specifically and individually indicated as being incorporated herein by reference.

[0180] While the above embodiments have been described in some detail with examples and embodiments for clarity, it will be apparent to those skilled in the art, in light of the teachings intended herein, that certain changes and modifications can be made therein without departing from the essence or scope of the appended claims. The following embodiments are provided for illustrative purposes only and not as limitations. Various non-definitive parameters that can be changed or modified to produce essentially similar results will be readily apparent to those skilled in the art. [Examples]

[0181] Example 1 MAGE-A4-TCR transgenic T cells are MAGE-A4 GVY -Binds to MHC polymers An in vitro sensitization approach was used to isolate MAGE-A4-reactive T cell clones. In this sensitization system, mature dendritic cells (mDCs) from HLA-A*02:01-positive and HLA-A*02:01-negative donors were used as antigen-presenting cells, and autologous CD8 +Enriched T cells were used as response cells. In vitro transcribed RNA (ivtRNA) encoding the human MAGE-A4 gene was used as the source of the specific antigen. After electroporation to mDCs, the ivtRNA encoding MAGE-A4 was transcribed into a protein, subsequently processed, and presented as a peptide on the mDCs by a molecule encoding HLA-A*02:01. In the case of HLA-A*02:01-negative donors, in addition to MAGE-A4 ivtRNA, ivtRNA encoding HLA-A*02:01 was used to transgenically express each HLA allele in antigen-presenting cells (allogeneic approach). In vitro co-culture of T cells with ivtRNA-transfected mDCs from the same donor led to the induction of novel antigen-specific T cells, which were used as the source of the corresponding TCR. Antigen-specific T cells can be enriched in various ways and are cloned by limiting dilution or FACS-based single-cell sorting. The TCR alpha and TCR beta chain sequences of MAGE-A4-reactive T cell clones were identified by next-generation sequencing, and the constant TCR regions were exchanged with their mouse counterparts before cloning into the retroviral vector pES.12-6. PBMCs from healthy donors were isolated by Ficol gradient centrifugation. CD8 was separated by negative magnetic sorting (Miltenyi). + T cells were enriched and stimulated in 24-well non-tissue culture plates pre-coated with anti-CD3 mAb (5 μg / ml) and anti-CD28 mAb (1 μg / ml) (BD Pharmingen, Heidelberg, Germany). Amphotropic retroviral particles were produced by transfecting HEK293 T cells with each TCR-coding retroviral plasmid and two expression plasmids. CD8 + Transduced T cells were transduced, and on day 12, transduced CD8 was identified using FACS, with the mouse constant beta region used as a marker for transduction. +The cells were concentrated and then grown using a rapid expansion protocol (Riddell SR, Science, 1992 Jul 10;257(5067):238-41).

[0182] In the experiments described in Examples 2 to 5, TCRs containing mouse-like Cα and Cβ regions were tested (i.e., TCR-1 containing the α chain of SEQ ID NO: 57 and the β chain of SEQ ID NO: 58, TCR-2 containing the α chain of SEQ ID NO: 59 and the β chain of SEQ ID NO: 60, and TCR-3 containing the α chain of SEQ ID NO: 61 and the β chain of SEQ ID NO: 62). The same type of experiment as described in Examples 2 to 5 can also be performed with the TCRs containing the minimal mouse-like Cα and Cβ regions mentioned above.

[0183] result: CD8 + T cells were transduced with three different TCRs isolated from MAGE-A4-responsive T cell clones and one control TCR that does not recognize MAGE-A4. GVY -MHC polymer (MAGE-A4 230-239 The cells were stained with antibodies against GVYDGREHTV (immuneAware), CD8, and the mouse constant beta region. All MAGE-A4-TCR transgenic T cell populations were MAGE-A4 GVY - Binds to MHC polymers very efficiently (>70%). MAGE-A4 GVY -MHC multimer staining was not observed in the control TCR. These results indicate that TCRs isolated from MAGE-A4-responsive T cell clones can be transgenically expressed in T cells from healthy donors (Figure 1).

[0184] Example 2 MAGE-A4-TCR transgenic T cells are MAGE-A4 GVY Recognizes peptides The MAGE-A4 specificity of TCR transgenic T cells was confirmed according to the following protocol. As target cells, T2 cells (HLA-A*02 positive) are given a saturation dose (10 -5 M) MAGE-A4 GVY The cells were loaded with either the peptide (SEQ ID NO:1) or an unrelated control peptide. In addition, K562 cells were transduced with the HLA-A*02:01 and MAGE-A4 genes (K562 / A2 / MAGE-A4). K562 cells transduced with only HLA-A*02:01 were used as a control (K562 / A2). Each target cell line was incubated with TCR transgenic T cells in a 2:1 ratio using 20,000 T cells and 10,000 target cells. After 20-24 hours, the IFN-γ concentration in the co-culture supernatant was analyzed using a standard sandwich ELISA (BD Human IFN-γ ELISA set).

[0185] result: MAGE-A4-TCR transgenic T cells are MAGE-A4 GVY T2 cells loaded with MAGE-A4 and K562 cells transduced with MAGE-A4 recognized the control target cells. T cells transduced with TCR-3 showed recognition of the K562 / A2 control. These results indicate that TCRs isolated from MAGE-A4-responsive T cell clones are functional when transferred to T cells from healthy donors (Figure 2).

[0186] Example 3 MAGE-A4-TCR transgenic T cells exhibit high functional avidity. MAGE-A4 GVY We analyzed the differences in functional avidity of MAGE-A4-TCR transgenic T cells using T2 cells loaded with peptides.

[0187] In T2 cells, MAGE-A4 GVY Peptides in stepwise concentrations (10 -11 M~10 -5TCR transgenic T cells were externally loaded with M) and co-cultured with 10,000 T2 cells and 20,000 T cells in a 1:2 ratio. After 20-24 hours, the IFN-γ concentration in the co-culture supernatant was analyzed using a standard sandwich ELISA (BD Human IFN-γ ELISA set).

[0188] result: When multiple donors are combined, MAGE-A4 is loaded onto HLA-A*02. GVY The highest functional avidity for peptides was demonstrated by TCR-2. TCR-1 and TCR-3 showed slightly reduced functional avidity compared to TCR-2 (Figure 3).

[0189] Example 4 MAGE-A4-TCR transgenic T cells lyse MAGE-A4-positive tumor cell lines. MAGE-A4-positive HLA-A2-positive tumor cell lines (NCI-H1703, NCI-H1755), MAGE-A4-negative HLA-A2-positive tumor cell line (Saos-2), and MAGE-A4-negative HLA-A2-negative tumor cells (A549) were used as target cells. In the cytotoxicity assay, the co-culture consisted of 40,000 TCR transgenic T cells and 5,000 (NCI-H1755, NCI-H1703, A549) and 2,500 (Saos-2) tumor cells transduced with fluorescent marker genes, with an effector-to-target ratio of approximately 8-16:1 (dependent on target cell size). Saturated concentration of MAGE-A4 was used. GVY Peptides (10 -5 Tumor cells loaded with M) were used as an internal positive control. The decrease in fluorescent target cells (cell count per well) was measured every 3 hours over a total period of 172 hours using live cell monitoring (IncuCyte® ZOOM, Essen Bioscience). To analyze cytokine release, the co-culture supernatant was harvested after 24 hours, and each IFN-γ concentration was analyzed using a standard sandwich ELISA (BD Human IFN-γ ELISA set).

[0190] result: Two endogenous MAGE-A4-positive HLA-A2-positive tumor cell lines (NCI-H1703, NCI-H1755) were recognized and lysed by all MAGE-A4-TCR transgenic T cells. A MAGE-A4-negative but HLA-A2-positive tumor cell line (Saos-2) was recognized and lysed by saturated MAGE-A4 GVY The peptide was recognized and lysed only when it was externally loaded into the cells. The negative control cell line (A549) was neither recognized nor lysed by any MAGE-A4-TCR. These results demonstrate that MAGE-A4-TCR transgenic T cells can remarkably selectively and efficiently lyse endogenously MAGE-A4-positive tumor cells (Figures 4a, 4b, and 4c).

[0191] Example 5 MAGE-A4-TCR transgenic T cells do not recognize normal human cells. Using a panel of normal human cells, we analyzed the potential on-target / off-tumor toxicity and off-target toxicity that can be caused by MAGE-A4-TCR transgenic T cells.

[0192] Primary cells and induced pluripotent stem cell (iPS)-derived cells representing basic tissues or organs were tested for recognition by MAGE-A4-TCR transduced T cells. HLA-A2 transiently expressed in HLA-A2-ivtRNA in HLA-A2-negative NHBE cells by electroporation. iCell neurons were treated with IFN-γ for 72 hours prior to the initiation of co-culture to induce cell surface HLA-A2 expression. HLA-A2 expression in all cell types was confirmed by flow cytometry. For cytotoxicity assays, co-cultures were set up with 20,000 TCR transgenic T cells and cell type-specific amounts of target cells. As an internal positive control, all normal human cells were treated with a final concentration of 10 -5M's MAGE-A4 GVY Peptides were loaded. To analyze cytokine release, the co-culture supernatant was harvested after 24 hours, and IFN-γ or IL-2 concentrations were analyzed using a standard sandwich ELISA (BD Human IFN-γ or IL-2 ELISA set). For co-cultures with iCell neurons pre-treated with IFN-γ to induce HLA-A2 surface expression, IL-2 release was determined.

[0193] result: At the start of co-culture with MAGE-A4-TCR transgenic T cells, both primary cells and induced pluripotent stem cell (iPS)-derived cells were HLA-A2 positive. Furthermore, MAGE-A4-TCR transgenic T cells also produced MAGE-A4 in individual target cells. GVY When peptides were loaded, all normal cells were efficiently recognized. Unloaded normal cells were not recognized by any MAGE-A4 transgenic T cells. MAGE-A4 transgenic T cells showed no signs of on-target / off-tumor toxicity or off-target toxicity (Figures 5a, 5b, and 5c).

[0194] Example 6 A lentiviral vector encoding a fully human MAGE-A4 TCR. The TCR polynucleotide sequences identified in Example 1 were optimized for expression. TCRs were expressed using lentiviral vectors encoding a polycistronic TCR construct. The polycistronic TCR construct contains a TCRα or TCRβ chain, an optional furin cleavage site, a P2A ribosome skip sequence, and the corresponding TCRα or TCRβ chain. The lentiviral vectors were produced according to known methods. See, for example, Kutner et al., BMC Biotechnol. 2009;9:10.doi:10.1186 / 1472-6750-9-10 and Kutner et al. Nat. Protoc. 2009;4(4):495-505.doi:10.1038 / nprot. 2009.22.

[0195] The polycistronic polynucleotide (SEQ ID NO:93) encoding the MAGE-A4 TCR-4 polyprotein (SEQ ID NO:94) contains a β-chain encoded by SEQ ID NO:70, a polynucleotide encoding a ribosome skip sequence, and an α-chain encoded by SEQ ID NO:69.

[0196] The polycistronic polynucleotide (SEQ ID NO:95) encoding the MAGE-A4 TCR-5 polyprotein (SEQ ID NO:96) contains a β-chain encoded by SEQ ID NO:78, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosome skip sequence, and an α-chain encoded by SEQ ID NO:77.

[0197] The polycistronic polynucleotide (SEQ ID NO:97) encoding the MAGE-A4 TCR-6 polyprotein (SEQ ID NO:98) contains a β-chain encoded by SEQ ID NO:86, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosome skip sequence, and an α-chain encoded by SEQ ID NO:85.

[0198] Example 7 T cells expressing the MAGE-A4 fully human TCR are MAGE-A4 GVY -Binds to MHC polymers CD3 + T cells were isolated from PBMCs of healthy donors and transduced with lentiviral vectors encoding three different fully human MAGE-A4 TCRs and a control TCR that does not recognize MAGE-A4. After expression, the transduced T cells were subjected to MAGE-A4 GVY -MHC polymer (MAGE-A4 230-239 The cells were stained with GVYDGREHTV (immuneAware) and an antibody against CD3. The population included viable CD3 cells. + Cellular and multimer staining were used for gate detection. All MAGE-A4-TCR transgenic T cell populations were MAGE-A4 GVY - Binds to MHC polymers very efficiently (>70%). MAGE-A4 GVY -MHC multimer staining was not observed in the control TCR.

[0199] result: These results demonstrate that TCRs isolated from MAGE-A4-responsive T cell clones can be transgenically expressed in T cells from healthy donors (Figure 6).

[0200] Example 8 T cells expressing the MAGE-A4 fully human TCR are MAGE-A4 GVY Recognizes peptides We confirmed the MAGE-A4 specificity of TCR transgenic T cells using antigen-dependent cytokine expression. T cells transduced with a lentiviral vector encoding the fully human MAGE-A4 TCR described in Example 6 were subjected to 10 ng / mL of MAGE-A4 GVYT2 cells (HLA-A*02 positive) pulsed with peptides or unrelated control peptides, and untransduced A549 / HLA-A2 cells or A549 / HLA-A2 cells transduced with the MAGE-A4 gene, were co-cultured in a 2:1 effector-to-target cell ratio. After 20–24 hours, IFN-γ concentrations in the co-culture supernatant were analyzed using the Luminex assay.

[0201] result: MAGE-A4-TCR transgenic T cells are MAGE-A4 GVY T2 and MAGE-A4-loaded cells recognized transduced A549 cells, but control target cells were not. These results suggest that healthy human donor T cells expressing the fully human MAGE-A4 TCR recognize MAGE-A4 GVY This demonstrates that the peptide specifically reacts with target cells that display it (Figure 7).

[0202] Example 9 T cells expressing the MAGE-A4 fully human TCR exhibit high functional avidity. MAGE-A4 GVY We analyzed the differences in functional avidity of T cells expressing fully human MAGE-A4-TCR using tumor cell lines that display peptides. T cells transduced with the lentiviral vector encoding the fully human MAGE-A4 TCR described in Example 6 were co-cultured with MAGE-A4-positive HLA-A2-positive tumor cell lines (A375, NCI-H1703, NCI-H1755), MAGE-A4-positive HLA-A2-negative tumor cell line (NCI-H520), and MAGE-A4-negative HLA-A2-positive tumor cell line (A549) at an effector-to-target cell ratio of 5:1. After 20-24 hours, the IFN-γ concentration in the co-culture supernatant was analyzed using the Luminex assay.

[0203] result: When multiple donors were combined, MAGE-A4 TCR5 showed the highest functional avidity against MAGE-A4-positive HLA-A2-positive tumor cell lines. MAGE-A4 TCR1 and MAGE-A4 TCR-6 showed reduced functional avidity compared to MAGE-A4 TCR-5 (Figure 8).

[0204] Example 10 T cells expressing the MAGE-A4 full human TCR lyse MAGE-A4-positive HLA-A2-positive tumor cell lines T cells transduced with the lentiviral vector encoding the full human MAGE-A4 TCR described in Example 6 were co-cultured with MAGE-A4-positive HLA-A2-positive tumor cell lines (A375, NCI-H1703, A549-HLA-A2-MAGE-A4) and MAGE-A4-negative HLA-A2-positive tumor cell line (A549-HLA-A2) at an effector-to-target cell ratio of 5:1. After 6 hours of co-culture, cytotoxicity against the tumor cell lines was measured by impedance assay.

[0205] result: MAGE-A4-positive HLA-A2-positive tumor cell lines (A375, NCI-H1703, A549-HLA-A2-MAGE-A4) were recognized and lysed by all MAGE-A4-TCR transgenic T cells. Lysis of the MAGE-A4-negative HLA-A2-positive tumor cell line (A549-HLA-A2) by MAGE-A4-TCR transgenic T cells was not significantly different from the lysis observed using non-transduced control T cells (Figure 9).

[0206] Example 11 T cells expressing the MAGE-A4 full human TCR suppress MAGE-A4-positive tumors in vivo <  5×10 6 Individual MAGE-A4-positive A375 tumor cells were injected into the flanks of each of 10 NSG mice. Ten days after tumor transplantation, 3.5×10 7 Individual MAGE-A4-TCR transgenic T cells, 3.5×107 Individual control non-transduced T cells or vehicle PBS were administered to mice. After treatment, all mice were measured twice weekly with calipers for their respective tumor volumes.

[0207] result: Mice treated with non-transduced T cells and vehicle PBS were unable to suppress tumor growth and were sacrificed once tumors reached the maximum size tolerated by the protocol. Mice treated with MAGE-A4-TCR transgenic T cells suppressed tumor growth until 35 days after T cell injection (Figure 10).

[0208] Example 12 Enhanced human MAGE-A4 TCR To enhance expression and functional avidity, the TCR polynucleotide sequences identified in Example 1 were modified. The TCR α and β chain constant regions were minimally murineized and hydrophobic amino acid substitutions were introduced into the transmembrane domain of the TCR α chain constant region. Exemplary polynucleotide sequences of the enhanced MAGE-A4 TCR are shown in SEQ ID NOs: 99-101, 105-107, and 111-113. Exemplary polypeptide sequences of the enhanced MAGE-A4 TCR are shown in SEQ ID NOs: 102-104, 108-110, and 114-116.

[0209] Enhanced MAGE-A4 TCRs (TCR-7, TCR-8, and TCR-9) were expressed using lentiviral vectors encoding polycistronic TCR constructs. The polycistronic TCR constructs contain a TCRα or TCRβ chain, an optional furin cleavage site, a P2A ribosome skip sequence, and the corresponding TCRα or TCRβ chain. The lentiviral vectors were produced according to known methods. See, for example, Kutner et al., BMC Biotechnol. 2009;9:10.doi:10.1186 / 1472-6750-9-10 and Kutner et al. Nat. Protoc. 2009;4(4):495-505.doi:10.1038 / nprot.2009.22.

[0210] The polycistronic polynucleotide (SEQ ID NO:101) encoding the MAGE-A4 TCR-7 polyprotein (SEQ ID NO:104) contains a β-chain encoded by SEQ ID NO:100, a polynucleotide encoding a ribosome skip sequence, and an α-chain encoded by SEQ ID NO:99.

[0211] The polycistronic polynucleotide (SEQ ID NO:107) encoding the MAGE-A4 TCR-8 polyprotein (SEQ ID NO:110) contains a β-chain encoded by SEQ ID NO:106, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosome skip sequence, and an α-chain encoded by SEQ ID NO:105.

[0212] The polycistronic polynucleotide (SEQ ID NO:113) encoding the MAGE-A4 TCR-9 polyprotein (SEQ ID NO:116) contains a β-chain encoded by SEQ ID NO:112, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosome skip sequence, and an α-chain encoded by SEQ ID NO:111.

[0213] Example 13 T cells expressing MAGE-A4 fully human TCR or enhanced MAGE-A4 TCR can be efficiently expressed on human T cells. Peripheral blood mononuclear cells (PBMCs) were isolated from three unrelated healthy donors, activated, and transduced with lentiviral vectors encoding fully human MAGE-A4 TCR (TCR-5) or enhanced MAGE-A4 TCR (TCR-8), or kept as negative controls without transduction. Cells were cultured for in vitro proliferation, and vector integration was analyzed by measuring the vector copy number (VCN), as well as by MAGE-A4. GVY -MHC polymer (MAGE-A4 230-239 Expression was analyzed using flow cytometry on cells stained with antibodies against GVYDGREHTV (immuneAware) and CD3.

[0214] result: While the VCNs of TCR-5 and TCR-8 were comparable, TCR surface expression and density were higher in cells transduced with TCR-8 and cells transduced with TCR-5, respectively. Figures 11A–11C.

[0215] Example 14 T cells expressing fully human MAGE-A4 TCR or enhanced MAGE-A4 TCR specifically recognize and kill MAGE-A4+ cell lines in vitro. Peripheral blood mononuclear cells (PBMCs) were isolated from three unrelated healthy donors, activated, and transduced with lentiviral vectors encoding fully human MAGE-A4 TCR (TCR-5) or enhanced MAGE-A4 TCR (TCR-8), or used as untransduced (UTD) cells as negative controls. T cells expressing the TCR were evaluated for specific responsiveness to the following MAGEA4-positive (+) and MAGEA4-negative (-) tumor cell lines: A549.A2 (A2+, MAGE-A4(-)); NCI-H2023 (A2+, MAGE-A4(+)); A375 (A2+, MAGE-A4(+)); A549.A2.MAGEA4 (A2+, MAGE-A4(+)); and U2OS (A2+, MAGE-A4(low)).

[0216] result: TCR-5 T cells and TCR-8 T cells released IFNγ when co-cultured with HLA-A2+ / MAGEA4(+) tumor cell lines, but did not release IFNγ when co-cultured with HLA-A2+ / MAGEA4(-) cells or when cultured in the absence of target cells. UTD T cells did not release IFNγ under any culture conditions. Figure 12A.

[0217] TCR-5 T cells and TCR-8 T cells effectively killed HLA-A2+ / MAGEA4(+) tumor cell lines at E:T ratios of 10:1, 5:1, and 2.5:1. UTD T cells did not kill HLA-A2+ / MAGEA4(+) tumor cell lines at any E:T ratio. Figure 12B.

[0218] Example 15 T cells expressing fully human MAGE-A4 TCR or enhanced MAGE-A4 TCR mediate regression of MAGE-A4-expressing tumors in vivo. MAGE-A4-positive A375 tumor cells were injected into each flank of five NSG mice. 50 mm 3 Mice with A375 tumors were given PBS (medium), untransduced T cells (UTDs), and 5 × 10⁻¹⁴ cells. 6Individual TCR-5 or TCR-8 T cells (left abdomen), or 1.5 × 10 6 Individual TCR-5 or TCR-8 T cells (right abdomen) were administered. To mice with 100 mm 3 A375 tumors, PBS (vehicle), untransduced (UTD) T cells, or 10 × 10 6 Individual TCR-5 or TCR-8 T cells were administered. Tumor growth was measured twice a week, and the antitumor activity of the TCR T cells was evaluated by comparison with mice given UTD controls and vehicle controls.

[0219] result: TCR-5 T cells and TCR-8 T cells mediated equivalent tumor regression of 50 mm 6 A375 tumors with a dose of 5 × 10 3 Individual TCR+ T cells. TCR-8 T cells mediated enhanced tumor regression of 50 mm 6 A375 tumors with a dose of 1.5 × 10 3 Individual TCR+ T cells, or 100 mm 6 A375 tumors with a dose of 10 × 10 3 Individual TCR+ T cells. The vehicle and UTD T cells did not mediate regression of A375 tumors under any conditions.

[0220] The present invention further features the following items.

[0221] Item 1: An isolated T cell receptor (TCR) specific for MAGE-A4.

[0222] Item 2: a) A variable TCRα region comprising a CDR1 having the amino acid sequence of SEQ ID NO:2, a CDR2 having the amino acid sequence of SEQ ID NO:3, and a CDR3 having the amino acid sequence of SEQ ID NO:4, A variable TCRβ region comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6, and a CDR3 having the amino acid sequence of SEQ ID NO:7; or b) A variable TCRα region comprising CDR1 having the amino acid sequence of SEQ ID NO:12, CDR2 having the amino acid sequence of SEQ ID NO:13, and CDR3 having the amino acid sequence of SEQ ID NO:14, A variable TCRβ region comprising CDR1 having the amino acid sequence of SEQ ID NO:15, CDR2 having the amino acid sequence of SEQ ID NO:16, and CDR3 having the amino acid sequence of SEQ ID NO:17; or c) A variable TCRα region comprising CDR1 having the amino acid sequence of SEQ ID NO:22, CDR2 having the amino acid sequence of SEQ ID NO:23, and CDR3 having the amino acid sequence of SEQ ID NO:24. A variable TCRβ region containing CDR1 having the amino acid sequence of SEQ ID NO:25, CDR2 having the amino acid sequence of SEQ ID NO:26, and CDR3 having the amino acid sequence of SEQ ID NO:27. A MAGE-A4-specific isolated T cell receptor (TCR), including [specific component].

[0223] Section 3: An isolated TCR according to any one of the above items, which specifically recognizes the amino acid sequence or fragment of SEQ ID NO:1.

[0224] Section 4: An isolated TCR according to any one of the above sections, which specifically recognizes the amino acid sequence of SEQ ID NO:1 in a form bound to HLA-A2.

[0225] Item 5: An isolated TCR according to any one of the above items, which specifically recognizes the amino acid of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01.

[0226] Item 6: An isolated TCR according to any one of the above items, comprising a TCRα chain containing a complementarity-determining region 3 (CDR3) having a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:14, and SEQ ID NO:24.

[0227] Item 7: An isolated TCR according to any one of the above items, comprising a TCRβ chain containing a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, and SEQ ID NO: 27.

[0228] Item 8: a) A variable TCRα region having an amino acid sequence at least 80% identical to SEQ ID NO:8 and a variable TCRβ region having an amino acid sequence at least 80% identical to SEQ ID NO:9; or b) A variable TCRα region having an amino acid sequence at least 80% identical to SEQ ID NO:18 and a variable TCRβ region having an amino acid sequence at least 80% identical to SEQ ID NO:19; or c) Variable TCRα region having an amino acid sequence at least 80% identical to SEQ ID NO:28 and variable TCRβ region having an amino acid sequence at least 80% identical to SEQ ID NO:29 An isolated TCR according to any one of the above items, including:

[0229] Item 9: a) A variable TCRα region having the amino acid sequence of SEQ ID NO:8 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:9; or b) A variable TCRα region having the amino acid sequence of SEQ ID NO:18 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:19; or c) Variable TCRα region having amino acid sequence SEQ ID NO:28 and variable TCRβ region having amino acid sequence SEQ ID NO:29 An isolated TCR according to any one of the above items, including:

[0230] Item 10: a) TCRα chain having an amino acid sequence at least 80% identical to SEQ ID NO:10 and TCRβ chain having an amino acid sequence at least 80% identical to SEQ ID NO:11; or b) A TCRα chain having an amino acid sequence at least 80% identical to SEQ ID NO:20 and a TCRβ chain having an amino acid sequence at least 80% identical to SEQ ID NO:21; or c) TCRα chain having an amino acid sequence at least 80% identical to SEQ ID NO:30 and TCRβ chain having an amino acid sequence at least 80% identical to SEQ ID NO:31 An isolated TCR according to any one of the above items, including:

[0231] Item 11: a) TCRα chain having the amino acid sequence of SEQ ID NO:10 and TCRβ chain having the amino acid sequence of SEQ ID NO:11; or b) A TCRα chain having the amino acid sequence of SEQ ID NO:20 and a TCRβ chain having the amino acid sequence of SEQ ID NO:21; or c) TCRα chain having amino acid sequence SEQ ID NO:30 and TCRβ chain having amino acid sequence SEQ ID NO:31 An isolated TCR according to any one of the above items, including:

[0232] Section 12: A TCR comprises a TCRα chain and a TCRβ chain. a) The variable TCRα region has an amino acid sequence that is at least 80% identical to SEQ ID NO:8 and includes CDR3 encoded by the amino acid sequence described in SEQ ID NO:4. - The variable TCRβ region has an amino acid sequence that is at least 80% identical to SEQ ID NO:9 and contains CDR3 encoded by the amino acid sequence described in SEQ ID NO:7; or b) The variable TCRα region has an amino acid sequence that is at least 80% identical to SEQ ID NO:18 and includes CDR3 encoded by the amino acid sequence described in SEQ ID NO:14; or - The variable TCRβ region has an amino acid sequence that is at least 80% identical to SEQ ID NO:19 and contains CDR3 encoded by the amino acid sequence described in SEQ ID NO:17; or c) The variable TCRα region has an amino acid sequence that is at least 80% identical to SEQ ID NO:28 and includes CDR3 encoded by the amino acid sequence described in SEQ ID NO:24; or - The variable TCRβ region has an amino acid sequence that is at least 80% identical to SEQ ID NO:29 and contains CDR3 encoded by the amino acid sequence described in SEQ ID NO:27. The isolated TCR described in any one of the above items.

[0233] Item 13: a) TCRα chain having the amino acid sequence of SEQ ID NO:10 and TCRβ chain having the amino acid sequence of SEQ ID NO:11; b) A TCRα chain having the amino acid sequence of SEQ ID NO:20 and a TCRβ chain having the amino acid sequence of SEQ ID NO:21; or c) TCRα chain having amino acid sequence SEQ ID NO:30 and TCRβ chain having amino acid sequence SEQ ID NO:31 An isolated TCR as described in any one of paragraphs 1 to 5, including the above.

[0234] Item 14: a) TCRα chain having the amino acid sequence of SEQ ID NO: 87 and TCRβ chain having the amino acid sequence of SEQ ID NO: 88; b) A TCRα chain having the amino acid sequence of SEQ ID NO:89 and a TCRβ chain having the amino acid sequence of SEQ ID NO:90; or c) TCRα chain having amino acid sequence SEQ ID NO:91 and TCRβ chain having amino acid sequence SEQ ID NO:92 An isolated TCR as described in any one of paragraphs 1 to 5, including the above.

[0235] Item 15: a) TCRα chain having the amino acid sequence of SEQ ID NO: 102 and TCRβ chain having the amino acid sequence of SEQ ID NO: 103; b) A TCRα chain having the amino acid sequence of SEQ ID NO:108 and a TCRβ chain having the amino acid sequence of SEQ ID NO:109; or c) TCRα chain having amino acid sequence SEQ ID NO:114 and TCRβ chain having amino acid sequence SEQ ID NO:115 An isolated TCR as described in any one of paragraphs 1 to 5, including the above.

[0236] Paragraph 16: A purified isolated TCR as described in any one of the preceding paragraphs.

[0237] Item 17: An isolated TCR according to any one of the preceding items, wherein the amino acid sequence comprises one or more phenotypically silent substitutions.

[0238] paragraph 18: An isolated TCR according to any one of the preceding paragraphs, wherein its amino acid sequence has been modified to include a detectable label, therapeutic agent or pharmacokinetic modifier.

[0239] Section 19: An isolated TCR as described in Section 18, wherein the therapeutic agent is selected from the group consisting of immunoeffector molecules, cytotoxic agents, and radionuclides.

[0240] Section 20: An isolated TCR as described in Section 19, wherein the immune effector molecule is a cytokine.

[0241] Item 21: An isolated TCR according to any one of the preceding items, which is soluble or membrane-bound.

[0242] Claim 22: The isolated TCR according to Claim 18, wherein the pharmacokinetic modification portion is at least one polyethylene glycol repeating unit, at least one glycol group, at least one sialyl group, or a combination thereof.

[0243] Item 23: An isolated TCR according to any one of the preceding items, wherein the TCRα chain and the TCRβ chain are linked by a linker sequence.

[0244] Section 24: An isolated TCR according to any one of sections 1 to 23, wherein the TCRα chain or TCRβ chain has been modified to include an epitope tag.

[0245] Paragraph 25: An isolated polypeptide comprising a functional portion of the TCR described in any one of paragraphs 1 to 24, wherein the functional portion comprises at least one of the amino acid sequences of SEQ ID NO: 4, 7, 14, 17, 24, and 27.

[0246] Section 26: The isolated polypeptide described in Section 25, wherein the functional portion comprises a TCRα variable chain and / or a TCRβ variable chain.

[0247] Item 27: A fusion protein comprising a TCRα chain and a TCRβ chain, comprising an amino acid sequence represented by any one of SEQ ID NO: 94, 96, 98, 104, 110, and 116.

[0248] Paragraph 28: A polyvalent TCR complex comprising at least two TCRs described in any one of paragraphs 1 to 24.

[0249] Paragraph 29: A polyvalent TCR complex as described in paragraph 28, wherein the therapeutic agent is bound to at least one of the TCRs.

[0250] Section 30: An isolated TCR according to any one of sections 1 to 24, a polypeptide according to section 25 or 26, a fusion protein according to section 27, or a polyvalent TCR complex according to section 28 or 29, wherein IFN-γ secretion is induced by binding to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01.

[0251] Paragraph 31: A nucleic acid encoding a TCR as described in any one of paragraphs 1 to 24, a polypeptide as described in paragraph 25 or 26, or a fusion protein as described in paragraph 27.

[0252] Section 32: The nucleic acid described in Section 31, wherein the nucleic acid sequence encoding the TCRα chain is represented by one of the following SEQ ID NOs: 69, 77, 85, 99, 105, and 111.

[0253] Section 33: The nucleic acid described in Section 31 or 32, wherein the nucleic acid sequence encoding the TCRβ chain is indicated by one of SEQ ID NO: 70, 78, 86, 100, 106, and 112.

[0254] Subparagraph 34: The nucleic acid described in subparagraph 31, wherein the TCR comprises an α chain encoded by SEQ ID NO:69 and a β chain encoded by SEQ ID NO:70; an α chain encoded by SEQ ID NO:77 and a β chain encoded by SEQ ID NO:78; an α chain encoded by SEQ ID NO:85 and a β chain encoded by SEQ ID NO:86; an α chain encoded by SEQ ID NO:99 and a β chain encoded by SEQ ID NO:100; an α chain encoded by SEQ ID NO:105 and a β chain encoded by SEQ ID NO:106; or an α chain encoded by SEQ ID NO:111 and a β chain encoded by SEQ ID NO:112.

[0255] Section 35: The nucleic acid described in Section 31, wherein the fusion protein is encoded by a nucleic acid sequence represented by one of SEQ ID NO: 93, 95, 97, 101, 107, and 113.

[0256] Paragraph 36: A vector containing the nucleic acid described in any one of paragraphs 31 to 35.

[0257] Section 37: A vector comprising nucleic acids encoding (a) polypeptide sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88; (b) polypeptide sequences shown in SEQ ID NO: 89 and SEQ ID NO: 90; (c) polypeptide sequences shown in SEQ ID NO: 91 and SEQ ID NO: 92; (d) polypeptide sequences shown in SEQ ID NO: 102 and SEQ ID NO: 103; (e) polypeptide sequences shown in SEQ ID NO: 108 and SEQ ID NO: 109; or (f) polypeptide sequences shown in SEQ ID NO: 114 and SEQ ID NO: 115.

[0258] Section 38: An expression vector, as described in Section 36 or 37.

[0259] Paragraph 39: A retroviral vector, as described in any one of paragraphs 36 to 38.

[0260] Paragraph 40: A lentiviral vector, as described in any one of paragraphs 36 to 39.

[0261] Item 41: Cells expressing any one of the TCRs described in items 1 through 24.

[0262] Item 42: Cells containing the vector described in any one of items 36 to 40.

[0263] Section 43: Cells described in Section 41 or 3942, which are isolated or non-natural.

[0264] Item 44: A cell containing a nucleic acid as described in any one of items 31 to 35 or a vector as described in any one of items 36 to 40.

[0265] Paragraph 45: a) An expression vector comprising at least one nucleic acid as described in any one of paragraphs 28 to 32, b) A first expression vector comprising a nucleic acid encoding the alpha chain of the TCR described in any one of paragraphs 1 to 21, and a second expression vector comprising a nucleic acid encoding the beta chain of the TCR described in any one of paragraphs 1 to 21. Cells as described in any one of paragraphs 41 to 44, including the above.

[0266] Item 46: A cell that is a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC), as described in any one of items 41 to 45.

[0267] Item 49: A T cell, as described in any one of items 41 to 48.

[0268] Item 50: A T cell, as described in any one of items 41 to 48.

[0269] Item 51: An antibody or antigen-binding fragment that specifically binds to the portion of the TCR described in any one of items 1 to 24 that mediates specificity to MAGE-A4.

[0270] Section 52: The portion of the TCR that mediates MAGE-A4 specificity is a) CDR3 of the alpha strand of SEQ ID NO:4 and / or CDR3 of the beta strand of SEQ ID NO:7, b) CDR3 of the alpha strand of SEQ ID NO:14 and / or CDR3 of the beta strand of SEQ ID NO:17, c) CDR3 of the alpha strand of SEQ ID NO:24 and / or CDR3 of the beta strand of SEQ ID NO:27 The antibody described in paragraph 51, including the following.

[0271] Item 53: A composition comprising a TCR as described in any one of items 1 to 24, a polypeptide as described in item 25 or 26, a fusion protein as described in item 27, a polyvalent TCR complex as described in item 28 or 29, a nucleic acid as described in any one of items 31 to 35, a vector as described in any one of items 36 to 40, a cell as described in any one of items 41 to 50, or an antibody as described in item 51 or 52.

[0272] Item 54: A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as described in any one of items 1 to 24, a polypeptide as described in item 25 or 26, a fusion protein as described in item 27, a polyvalent TCR complex as described in item 28 or 29, a nucleic acid as described in any one of items 31 to 35, a vector as described in any one of items 36 to 40, a cell as described in any one of items 41 to 50, or an antibody as described in item 51 or 52.

[0273] 55: A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a cell as described in any one of the paragraphs 41 to 50.

[0274] Item 56: A TCR as described in any one of items 1 to 24, a polypeptide as described in item 25 or 26, a fusion protein as described in item 27, a polyvalent TCR complex as described in item 28 or 29, a nucleic acid as described in any one of items 31 to 35, a vector as described in any one of items 36 to 40, a cell as described in any one of items 41 to 50, an antibody as described in item 51 or 52, a composition as described in item 53, or a pharmaceutical composition as described in item 54 or 55, for use as a pharmaceutical.

[0275] paragraph 57: A TCR as described in any one of paragraphs 1 to 24, a polypeptide as described in paragraph 25 or 26, a fusion protein as described in paragraph 27, a polyvalent TCR complex as described in paragraph 28 or 29, a nucleic acid as described in any one of paragraphs 31 to 35, a vector as described in any one of paragraphs 36 to 40, a cell as described in any one of paragraphs 41 to 50, an antibody as described in paragraph 51 or 52, a composition as described in paragraph 53, or a pharmaceutical composition as described in paragraph 54 or 55, for use in the treatment of cancer.

[0276] Section 58: A TCR, polypeptide, fusion protein, polyvalent TCR complex, nucleic acid, cell, antibody, composition, or pharmaceutical composition as described in Section 57, wherein the cancer is a hematological malignancy or a solid tumor.

[0277] paragraph 59: A TCR, polypeptide, fusion protein, polyvalent TCR complex, nucleic acid, cell, antibody, composition, or pharmaceutical composition as described in paragraph 57 or 58, wherein the cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia.

[0278] Item 60: The TCR, polypeptide, fusion protein, polyvalent TCR complex, nucleic acid, cell, antibody, composition or pharmaceutical composition described in Item 50 or 51, wherein the cancer is preferably selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer or colorectal cancer, sarcoma or osteosarcoma.

[0279] References TIFF0007877420000005.tif209150TIFF0007877420000006.tif202149TIFF0007877420000007.tif160150

[0280] In general, the terms used in the attached claims should not be interpreted to limit the claims to the specific embodiments disclosed herein and in the claims, but rather to encompass all conceivable embodiments relating to the entire scope of equivalents recognized in those claims. Thus, the claims are not limited by this disclosure.

[0281] Sequence information SEQUENCE LISTING <110> MEDIGENE IMMUNOTHERAPIES GMBH 2SEVENTY BIO, INC. <120> MAGE A4 T CELL RECEPTORS <150> EP 19165387.2 <151> 2019-03-27 <160> 116 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Gly Val Tyr Asp Gly Arg Glu His Thr Val 1 5 10 <210> 2 <211> 7 <212> PRT <213> Homo sapiens <400> 2 Thr Ser Asp Gln Ser Tyr Gly 1 5 <210> 3 <211> 8 <212> PRT <213> Homo sapiens <400> 3 Gln Gly Ser Tyr Asp Glu Gln Asn 1 5 <210> 4 <211> 14 <212> PRT <213> Homo sapiens <400> 4 Cys Ala Met Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe 1 5 10 <210> 5 <211> 5 <212> PRT <213> Homo sapiens <400> 5 Lys Gly His Asp Arg 1 5 <210> 6 <211> 6 <212> PRT <213> Homo sapiens <400> 6 Ser Phe Asp Val Lys Asp 1 5 <210> 7 <211> 17 <212> PRT <213> Homo sapiens <400> 7 Cys Ala Thr Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr 1 5 10 15 Phe <210> 8 <211> 136 <212> PRT <213> Homo sapiens <400> 8 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe Gly Gly Gly 115 120 125 Thr Arg Leu Met Val Lys Pro His 130 135 <210> 9 <211> 135 <212> PRT <213> Homo sapiens <400> 9 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu 130 135 <210> 10 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> with mininmal murinized constant region <400> 10 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe Gly Gly Gly 115 120 125 Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val 130 135 140 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 165 170 175 Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe 180 185 190 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220 Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 225 230 235 240 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 245 250 255 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 260 265 270 Leu Trp Ser Ser 275 <210> 11 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> with minimal murinized constant region <400> 11 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser 260 265 270 Tyr His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Phe 305 310 <210> 12 <211> 7 <212> PRT <213> Homo sapiens <400> 12 Thr Ser Asp Pro Ser Tyr Gly 1 5 <210> 13 <211> 8 <212> PRT <213> Homo sapiens <400> 13 Gln Gly Ser Tyr Asp Gln Gln Asn 1 5 <210> 14 <211> 15 <212> PRT <213> Homo sapiens <400> 14 Cys Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe 1 5 10 15 <210> 15 <211> 5 <212> PRT <213> Homo sapiens <400> 15 Ser Gly Asp Leu Ser 1 5 <210> 16 <211> 6 <212> PRT <213> Homo sapiens <400> 16 Tyr Tyr Asn Gly Glu Glu 1 5 <210> 17 <211> 13 <212> PRT <213> Homo sapiens <400> 17 Cys Ala Ser Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe 1 5 10 <210> 18 <211> 137 <212> PRT <213> Homo sapiens <400> 18 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 115 120 125 Gly Thr Arg Leu Phe Val Lys Ala Asn 130 135 <210> 19 <211> 131 <212> PRT <213> Homo sapiens <400> 19 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu 130 <210> 20 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> with minimal murinized constant region <400> 20 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 115 120 125 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <210> 21 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> with minimal murinized constant region <400> 21 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Arg Ser Tyr His Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <210> 22 <211> 6 <212> PRT <213> Homo sapiens <400> 22 Asp Ser Ala Ser Asn Tyr 1 5 <210> 23 <211> 7 <212> PRT <213> Homo sapiens <400> 23 Ile Arg Ser Asn Val Gly Glu 1 5 <210> 24 <211> 14 <212> PRT <213> Homo sapiens <400> 24 Cys Ala Ala Ser Arg Gly Thr Gly Phe Gln Lys Leu Val Phe 1 5 10 <210> 25 <211> 5 <212> PRT <213> Homo sapiens <400> 25 Leu Gly His Asp Thr 1 5 <210> 26 <211> 6 <212> PRT <213> Homo sapiens <400> 26 Tyr Asn Asn Lys Glu Leu 1 5 <210> 27 <211> 16 <212> PRT <213> Homo sapiens <400> 27 Cys Ala Ser Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe 1 5 10 15 <210> 28 <211> 133 <212> PRT <213> Homo sapiens <400> 28 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Arg Gly Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu 115 120 125 Leu Val Ser Pro Asn 130 <210> 29 <211> 134 <212> PRT <213> Homo sapiens <400> 29 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr 130 <210> 30 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> with minimal murinized constant region <400> 30 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Arg Gly Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu 115 120 125 Leu Val Ser Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Ser Asp 210 215 220 Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Dear <210> 31 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> with minimal murinized constant region <400> 31 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr 260 265 270 His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Phe 305 310 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized <400> 32 accagcgatc agagctacgg c 21 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 33 cagggcagct acgacgagca gaat 24 <210> 34 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 34 tgtgccatga gcggcgatag cgccggcaac atgcttacat tt 42 <210> 35 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 35 aagggccacg accgg 15 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 36 agcttcgacg tgaaggac 18 <210> 37 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 37 tgcgccacca gcgactggga tagaagcggc gacaaagaga cacagtactt c 51 <210> 38 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> with minimal murinized constant region; codon optimized <400> 38 atgagcctga gcagcctgct gaaggtcgtg acagcctctc tgtggctcgg acctggaatc 60 gcccagaaga tcacccagac acagcccggc atgttcgtgc aagagaaaga agccgtgaca 120 ctggactgca cctacgacac cagcgatcag agctacggcc tgttctggta caagcagcct 180 agcagcggcg agatgatctt cctgatctac cagggcagct acgacgagca gaatgccacc 240 gagggcagat acagcctgaa cttccagaag gcccggaagt ccgccaacct ggtcatttct 300 gcttctcagc tgggcgacag cgccatgtac ttttgtgcca tgagcggcga tagcgccggc 360 aacatgctta catttggcgg cggaacccgg ctgatggtca agccccatat tcagaacccc 420 gatcctgccg tgtaccagct gagagacagc aagagcagcg acaagagcgt gtgtctgttc 480 accgacttcg acagccagac caacgtgtcc cagagcaagg acagcgacgt gtacatcacc 540 gacaagaccg tgctggacat gcggagcatg gacttcaaga gcaacagcgc cgtggcctgg 600 tccaacaaga gcgatttcgc ctgcgccaac gccttcaaca atagcattat ccccgaggac 660 acattcttcc ccagctccga tgtgccctgc gacgtgaagc tggtggaaaa gagcttcgag 720 acagacacca acctgaactt ccagaacctg agcgtgatcg gcttcagaat cctgctgctg 780 aaggtggccg gcttcaatct gctgatgacc ctgagactgt ggtccagctg a 831 <210> 39 <211> 939 <212> DNA <213> Artificial Sequence <220> <223> with minimal murinized constant region; codon optimized <400> 39 atggccagcc tgctgttctt ctgcggcgcc ttttatctgc tcggcaccgg ctctatggac 60 gccgacgtta cacagacccc tcggaacaga atcaccaaga ccggcaagcg gatcatgctg 120 gaatgcagcc agaccaaggg ccacgaccgg atgtactggt acagacagga ccctggcctg 180 ggcctgagac tgatctacta cagcttcgac gtgaaggaca tcaacaaggg cgagatcagc 240 gacggctaca gcgtgtcaag acaggctcag gccaagttca gcctgtctct ggaaagcgct 300 atccccaacc agacagccct gtacttctgc gccaccagcg actgggatag aagcggcgac 360 aaagagacac agtacttcgg ccctggcacc agactgctgg tgctggaaga tctgaacaag 420 gtgttccctc cagaggtggc cgtgttcgag ccttctaagg ccgagattgc ccacacacag 480 aaagccacac tcgtgtgcct ggctaccggc ttctttcctg accacgtgga actgtcttgg 540 tgggtcaacg gcaaagaggt gcacagcggc gtcagcacag atccccagcc tctgaaagaa 600 cagcccgctc tgaacgacag ccggtactgt ctgagcagca gactgagagt gtccgccaca 660 ttctggcaga accccagaaa ccacttcaga tgccaggtgc agttctacgg cctgagcgag 720 aacgatgagt ggacccagga tagagccaag cctgtgacac agatcgtgtc tgccgaagcc 780 tggggcagag ccgattgtgg aattaccagc gccagctacc atcagggcgt gctgtctgcc 840 acaatcctgt acgagatcct gctgggcaaa gccactctgt acgccgtgct ggtgtctgcc 900 ctggtgctga tggccatggt caagagaaag gacttttga 939 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 40 accagcgatc ctagctacgg c 21 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 41 cagggcagct acgaccagca gaat 24 <210> 42 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 42 tgtgccatga gcggcggcta caccggcggc ttcaagacaa tcttt 45 <210> 43 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 43 agcggcgacc tgagc 15 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 44 tactacaacg gcgaggaa 18 <210> 45 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 45 tgtgccagct ctggcggaga tggcgacgag cagtttttt 39 <210> 46 <211> 834 <212> DNA <213> Artificial Sequence <220> <223> with minimal murinized constant region; codon optimized <400> 46 atgagcctga gcagcctgct gaaggtcgtg acagcctctc tgtggctcgg acctggaatc 60 gcccaagaga tcacccagac acagcccggc atgttcgtgc aagagaaaga agccgtgaca 120 ctggactgca cctacgacac cagcgatcct agctacggcc tgttctggta caagcagcct 180 agcagcggcg agatgatctt cctgatctac cagggcagct acgaccagca gaatgccacc 240 gagggcagat acagcctgaa cttccagaag gcccggaagt ccgccaacct ggtcatttct 300 gctagccagc tgggcgacag cgccatgtac ttttgtgcca tgagcggcgg ctacaccggc 360 ggcttcaaga caatctttgg cgccggaacc agactgttcg tgaaggccaa tattcagaac 420 cccgatcctg ccgtgtacca gctgagagac agcaagagca gcgacaagag cgtgtgtctg 480 ttcaccgact tcgacagcca gaccaacgtg tcccagagca aggacagcga cgtgtacatc 540 accgacaaga ccgtgctgga catgcggagc atggacttca agagcaacag cgccgtggcc 600 tggtccaaca agagcgattt cgcctgcgcc aacgccttca acaatagcat tatccccgag 660 gacacattct tccccagctc cgatgtgccc tgcgacgtga agctggtgga aaagagcttc 720 gagacagaca ccaacctgaa cttccagaac ctgagcgtga tcggcttcag aatcctgctg 780 ctgaaggtgg ccggcttcaa tctgctgatg accctgagac tgtggtccag ctga 834 <210> 47 <211> 933 <212> DNA <213> Artificial Sequence <220> <223> with minimal murinized constant region; codon optimized <400> 47 atgggcttca gactgctgtg ctgcgtggcc ttttgtctgc ttggagccgg acctgtggat 60 agcggcgtta cccagacacc taagcacctg atcacagcca caggccagcg cgtgaccctg 120 agatgttctc ctagaagcgg cgacctgagc gtgtactggt atcagcagtc tctggaccag 180 ggcctgcagt tcctgatcca gtactacaac ggcgaggaaa gagccaaggg caacatcctg 240 gaacggttca gcgcccagca gttcccagat ctgcacagcg agctgaacct gagcagcctg 300 gaactgggag atagcgccct gtacttctgt gccagctctg gcggagatgg cgacgagcag 360 ttttttggcc ctggcaccag actgaccgtg ctggaggacc tgaagaacgt gttccctccg 420 gaggtggccg tgttcgagcc cagcaaagcc gagatcgcgc acacccagaa ggccaccctg 480 gtgtgcctgg ccaccggctt ctaccccgac cacgtggagc tgagctggtg ggtgaacggc 540 aaggaggtgc acagcggcgt gagcaccgac ccccagcccc tgaaggagca gcccgccctg 600 aacgacagcc gctactgcct gagcagccgc ctgcgcgtga gcgccacctt ctggcagaac 660 ccccgcaacc acttccgctg ccaggtgcag ttctacggcc tgagcgagaa cgacgagtgg 720 acccaggacc gcgccaagcc cgtgacccag atcgtgagcg ccgaggcctg gggccgcgcc 780 gactgcggca ttaccagccg cagctaccat cagggcgtgc tgagcgccac catcctgtac 840 gagatcctgc tgggcaaggc caccctgtac gccgtgctgg tgagcgccct ggtgctgatg 900 gcgatggtga agcgcaagga cagccgcggc tga 933 <210> 48 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 48 gacagcgcca gcaactac 18 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 49 atccggtcca acgtgggcga g 21 <210> 50 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 50 tgcgctgcca gcagaggcac cggcttccag aaactggtgt tt 42 <210> 51 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 51 ctgggccacg acacc 15 <210> 52 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 52 tacaacaaca aagagctg 18 <210> 53 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> codon optimized <400> 53 tgtgccagca gccagttctg ggatggcgct ggcgacgagc agtatttt 48 <210> 54 <211> 822 <212> DNA <213> Artificial Sequence <220> <223> with minimal murinized constant region; codon optimized <400> 54 atgaccagca tccgggccgt gttcatcttc ctgtggctgc agctggacct ggtcaacggc 60 gagaatgtgg aacagcaccc cagcacactg agcgtgcaag agggcgattc tgccgtgatc 120 aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca agagctgggc 180 aaaagacccc agctgatcat cgacatccgg tccaacgtgg gcgagaagaa ggaccagaga 240 atcgccgtga cactgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacacag 300 cctgaggata gcgccgtgta cttttgcgct gccagcagag gcaccggctt ccagaaactg 360 gtgtttggca ccggcaccag actgctggtg tccccaaata ttcagaaccc cgatcctgcc 420 gtgtaccagc tgagagacag caagagcagc gacaagagcg tgtgtctgtt caccgacttc 480 gagagccaga ccaacgtgtc ccagagcaag ggagcgacg tgtacatcac cgacagacc 540 gtgctggaca tgcggagcat ggactcaag agcacagcg ccgtggcctg gtccaacaag 600 agcgatttcg cctgcgccaa cgccttcaac atagcatta tccccgagga cacattctc 660 cccagctccg atgtgccctg cgacgtgaag ctggtggaaa agagcttcga gatacacacc 720 aacctgaact tccagaacct gagcgtgatc ggcttcagaa tcctgctgct gaaggtggcc 780 ggcttcaatc tgctgatgac cctgagactg tggtccagct ga 822 <210> 55 <211> 936 <212> DNA <213> Artificial Sequence <220> <223> with minimal murinized constant region; codon optimized <400> 55 atgggctgca gactgctgtg ctgtgtggtg tctgctgc tgcaagccgg acctctggat 60 acagccgtgt ctcagacccc taagtacctg gtcacccaga tggcacga cagagcatc 120 aagtgcgagc agaacctggg ccacgacacc atgtactggt acaagcagga cagcaagaaa 180 ttcctgaaga tcatgttcag ctacaacaac aaagagctga tcatcaacga gacagtgccc 240 aacagattca gccctaagag ccccgataag gcccacctga acctgcacat caacagcctg 300 gaactgggcg acagcgccgt gtacttttgt gccagcagcc agttctggga tggcgctggc 360 gacgagcagt attttggccc tggcaccaga ctgaccgtga ccgaggacct gaacaaggtg 420 ttccctccgg aggtggccgt gttcgagccc agcaaagccg agatcgcgca cacccagaag 480 gccaccctgg tgtgcctggc caccggcttc ttccccgacc acgtggagct gagctggtgg 540 gtgaacggca aggaggtgca cagcggcgtg agcaccgacc cccagcccct gaaggagcag 600 cccgccctga acgacagccg ctactgcctg agcagccgcc tgcgcgtgag cgccaccttc 660 tggcagaacc cccgcaacca cttccgctgc caggtgcagt tctacggcct gagcgagaac 720 gacgagtgga cccaggaccg cgccaagccc gtgacccaga tcgtgagcgc cgaggcctgg 780 ggccgcgccg actgcggcat taccagcgcg agctaccatc agggcgtgct gagcgccacc 840 atcctgtacg agatcctgct gggcaaggcc accctgtacg ccgtgctggt gagcgccctg 900 gtgctgatgg cgatggtgaa gcgcaaggac ttctga 936 <210> 56 <211> 10 <212> PRT <213> Homo sapiens <400> 56 Lys Leu Tyr Gly Leu Asp Trp Ala Glu Leu 1 5 10 <210> 57 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> murinized <400> 57 With Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe Gly Gly Gly 115 120 125 Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Glu Pro Ala Val 130 135 140 Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly 165 170 175 Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser 180 185 190 Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys 195 200 205 Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val 210 215 220 Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn 225 230 235 240 Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu 245 250 255 Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <210> 58 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> murinized <400> 58 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro 130 135 140 Lys Val Thr Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp 225 230 235 240 Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys 290 295 300 Lys Lys Asn Ser 305 <210> 59 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> murinized <400> 59 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 115 120 125 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Glu Pro Ala 130 135 140 Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 165 170 175 Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp 180 185 190 Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 195 200 205 Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 210 215 220 Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Star <210> 60 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> murinized <400> 60 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr Leu 130 135 140 Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg 195 200 205 Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln 210 215 220 Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser 225 230 235 240 Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala 245 250 255 Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala 260 265 270 Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val 275 280 285 Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser 290 295 300 <210> 61 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> murinized <400> 61 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Arg Gly Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu 115 120 125 Leu Val Ser Pro Asn Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140 Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190 Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205 Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220 Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe 225 230 235 240 Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala 245 250 255 Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 62 <211> 307 <212> PRT <213> Artificial Sequence <220> <223> murinized <400> 62 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr Glu Asp Leu Arg Asn Val Thr Pro Pro Lys 130 135 140 Val Thr Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser 195 200 205 Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe 210 215 220 Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro 225 230 235 240 Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp 245 250 255 Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val 260 265 270 Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu 275 280 285 Tyr Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys 290 295 300 Lys Asn Ser 305 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 63 acttcagacc agtcgtacgg t 21 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 64 cagggatcgt acgacgagca gaac 24 <210> 65 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 65 tgcgcaatgt ccggcgatag cgcaggaaac atgctgactt tc 42 <210> 66 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 66 aagggccacg accgc 15 <210> 67 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 67 tccttcgacg tgaaggac 18 <210> 68 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 68 tgtgccacct cggattggga ccgatccggc gacaaggaaa ctcagtactt c 51 <210> 69 <211> 831 <212> DNA <213> human optimized <400> 69 atgagcctct cttccctgct caaagtggtc actgcctccc tgtggctggg accgggaatc 60 gcccagaaga tcactcagac ccagcctgga atgttcgtgc aagagaaaga ggccgtgacc 120 ctggattgta cttatgacac ttcagaccag tcgtacggtt tgttctggta caagcagccg 180 tcctccggag aaatgatctt cctgatctac cagggatcgt acgacgagca gaacgctacc 240 gagggcagat attccctcaa cttccaaaag gcccggaaat ccgcgaacct cgtgatctcg 300 gcctcacaac ttggggactc cgctatgtat ttctgcgcaa tgtccggcga tagcgcagga 360 aacatgctga ctttcggcgg tggactagg ctgatggtca agccccacat tcaaaaccct 420 gacccagcag tctaccagtt gcgggattcc aagtcttccg ataaatccgt gtgtctcttt 480 agagcttcg atagccagac caacgtgtcc cagagcaaag acagcgacgt gtacattact 540 gacaagactg tgctgggacat gcggtccatg gacttcaaga gcaactccgc cgtcgcttgg 600 tccaacaagt ctgactttgc gtgcgcgaac gctttcaaca acagcattat cccggaggac 660 acctttttcc cttcccccga gtcaagctgc gatgtcaagc ttgtggaaaa gtcgttcgaa 720 accgacacca acctgaactt ccagaacctg tccgtcatcg ggttccgcat tctgctgctg 780 aaggtcgccg gcttcaatct cctgatgact ctccgcttgt ggtcctcata a 831 <210> 70 <211> 936 <212> DNA <213> human optimized <400> 70 atggcgtccc tgctgttctt ctgcggtgcc ttctaccttc tgggaaccgg ctcgatggac 60 gccgacgtga cccaaacccc tcgcaaccgc atcaccaaga ctggaaagcg gatcatgctg 120 gaatgctccc agaccaaggg ccacgaccgc atgtactggt acagacagga cccgggtctg 180 ggattgcgcc tgatctacta ctccttcgac gtgaaggaca tcaacaaggg ggagatctcc 240 gatggatact cagtctcgag acaagcccag gctaagtttt ccctgtccct cgaatccgcc 300 attcccaatc agaccgcgct gtacttctgt gccacctcgg attgggaccg atccggcgac 360 aaggaaactc agtacttcgg accaggaacc aggctcctgg tgctggagga tctgaacaag 420 gtgttcccgc cggaagtggc agtgttcgag ccatccgaag ccgagatctc gcatacgcag 480 aaggccaccc tcgtgtgcct ggccactggg tttttccctg accacgtgga gctctcgtgg 540 tgggtcaacg gaaaggaagt gcacagcggt gtctcaaccg acccgcaacc tctcaaggaa 600 cagcccgcgc tcaatgattc gcggtactgc ctgagcagcc ggctcagagt gtccgccact 660 ttctggcaaa acccgcggaa ccatttccgg tgccaagtgc aattctacgg gctgtcggaa 720 aacgacgaat ggacccagga cagggccaag cccgtgaccc agattgtgtc cgctgaagcc 780 tgggggagag ctgattgcgg tttcaccagc gtgtcgtatc agcagggcgt gctgtcagcc 840 accattctgt acgaaatcct cctggggaag gccacactgt acgccgtgct cgtgtccgcc 900 ctggtgctga tggccatggt caagcggaag gacttc 936 <210> 71 <211> 21 <212> DNA <213> human optimized <400> 71 acctccgacc cgtcctacgg g 21 <210> 72 <211> 24 <212> DNA <213> human optimized <400> 72 caaggctcct atgatcagca gaac 24 <210> 73 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 73 tgtgccatgt cgggcggata caccggggga ttcaagacca ttttc 45 <210> 74 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 74 tctggcgacc tctcc 15 <210> 75 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 75 tactacaacg gagaggag 18 <210> 76 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 76 tgcgcaagca gcggtggtga cggggatgaa cagttcttt 39 <210> 77 <211> 834 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 77 atgtccctta gctcactgct gaaagtggtc actgcgagcc tgtggctggg accaggcatt 60 gctcaaaaga tcacccagac tcagcctggg atgttcgtgc aagagaagga agccgtgacc 120 ctcgactgca cttacgacac ctccgacccg tcctacgggc tgttctggta caagcagccg 180 tcctccggag agatgatctt cctcatctac caaggctcct atgatcagca gaacgccacc 240 gaaggacgct acagcctgaa cttccagaag gctcggaagt cggcgaacct cgtgatcagc 300 gcatcccaac tgggggacag cgccatgtac ttctgtgcca tgtcgggcgg atacaccggg 360 ggattcaaga ccattttcgg ggccggcact agactgttcg tgaaggccaa catccagaat 420 cctgatccgg cggtgtatca gctgcgcgac tccaagtctt ccgataaatc cgtgtgtctc 480 tttacagact tcgactccca aaccaacgtg tcacagtcca aggacagcga tgtgtacatc 540 accgacaaaa ccgtgctgga catgcggtcc atggacttca agtcaaacag cgcagtcgcc 600 tggtccaaca agtccgactt cgcctgtgcg aacgccttca acaactccat cattccggaa 660 gatacctttt tcccttcacc tgagtcgagc tgtgatgtga agctcgtgga aaagtcgttt 720 gaaacggaca ccaacctgaa ctttcagaac ctgtccgtga ttggtttccg catcctgctg 780 ctgaaggtcg ccggcttcaa cttgctgatg acgctccggc tgtggtcctc gtaa 834 <210> 78 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 78 atgggattcc gccttctgtg ctgcgtggcc ttctgcttgc ttggagctgg tcccgtcgac 60 tcgggagtga cccagacgcc gaagcacttg attaccgcta ctgggcagcg cgtgactctg 120 cgatgctcac cacggtctgg cgacctctcc gtgtactggt atcagcagag cctggaccag 180 ggactgcagt tcctgatcca gtactacaac ggagaggagc gggctaaggg aaacatactg 240 gagcggttct cggcgcaaca attccccgat ctgcactccg aactgaacct gtcctccctg 300 gaattgggag actccgccct gtacttctgc gcaagcagcg gtggtgacgg ggatgaacag 360 ttctttggcc ctggaaccag actcaccgtg ctcgaggacc tcaagaacgt gttcccaccc 420 gaagtcgcgg tgttcgagcc ctccgaagcg gaaatcagcc atactcagaa agccactctc 480 gtgtgcctgg ccaccggatt ctacccggac cacgtcgagc tctcttggtg ggtgaacggg 540 aaagaggtcc acagcggcgt gagcactgat ccgcagccgc tgaaggaaca acccgccttg 600 aacgactcgc ggtactgtct gtcctcccgg ctgagagtgt cggccacctt ctggcaaaac 660 cccaggaacc actttaggtg ccaagtccag ttctacggcc tgagcgaaaa cgatgagtgg 720 acccaggaca gagccaagcc tgtgacccag attgtgtcag ccgaggcttg gggtagagca 780 gactgcggat tcacctccga gtcctaccaa caaggcgtcc tctcggcgac cattctgtac 840 gaaatcctgc ttgggaaggc cactctgtac gccgtgctgg tgtccgccct ggtgctgatg 900 gccatggtca agcggaagga ctcccggggg 930 <210> 79 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 79 gactcagcat caaactat 18 <210> 80 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 80 atccgctcca acgtcggaga g 21 <210> 81 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 81 tgtgcggcct cgaggggcac tgggtttcag aagctcgtgt tc 42 <210> 82 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 82 ctcggacacg acacc 15 <210> 83 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 83 tacaacaaca aggaactg 18 <210> 84 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 84 tgcgcgtcca gccagttctg ggacggagcg ggcgacgaac agtacttc 48 <210> 85 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 85 atgacgtcca ttagagccgt gttcattttc ctgtggctgc aactggacct cgtgaatgga 60 gaaaacgtcg agcagcaccc atccaccctg agcgtgcagg agggagactc cgcagtcatc 120 aagtgcactt actccgactc agcatcaaac tatttcccgt ggtataagca ggaactcgga 180 aagcggcctc agctcatcat tgacatccgc tccaacgtcg gagagaagaa ggaccagaga 240 attgccgtga cactcaacaa gaccgccaag catttctccc ttcacatcac cgaaacccag 300 cccgaggaca gcgccgtcta cttttgtgcg gcctcgaggg gcactgggtt tcagaagctc 360 gtgttcggca ctgggacccg gctgctggtg tcgccaaaca tccagaatcc agaccccgcg 420 gtgtaccagc tgagagactc gaagtcttcc gataaatccg tgtgtctctt tacagacttc 480 gatagccaga ctaacgtgtc ccagtccaag gactccgatg tgtacatcac cgacaagact 540 gtgctggata tgcggagcat ggactttaag tccaattcag cggtcgcgtg gagcaacaag 600 tccgacttcg cctgcgctaa cgctttcaac aactccatta tcccggagga taccttcttc 660 ccgtcaccgg aatcctcgtg cgacgtgaag ctggtcgaga agtccttcga aaccgatacc 720 aacctgaact tccaaaacct ctccgtgatc ggcttcagaa tcctgctgct gaaagtggct 780 ggcttcaatt tgctgatgac cctgcggctc tggagcagc 819 <210> 86 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 86 atgggttgtc ggttgctgtg ttgcgtcgtg ttctgccttc ttcaagctgg tcctctcgat 60 actgccgtga gccaaacccc taagtacctt gtcacccaaa tgggcaacga caagtccatc 120 aaatgcgaac agaacctcgg acacgacacc atgtactggt acaaacagga ttccaagaag 180 ttcctgaaga ttatgttctc atacaacaac aaggaactga ttatcaacga aactgtgccg 240 aaccggttct caccgaagtc gcctgacaag gctcatctca acttgcatat caactcgctg 300 gagctcggcg actccgccgt gtacttctgc gcgtccagcc agttctggga cggagcgggc 360 gacgaacagt acttcggccc gggcactcgg ctgaccgtga ccgaagatct gaacaaagtg 420 ttcccccccg aagtggccgt gttcgaacct tccgaggccg agatcagcca cacccaaaag 480 gccactctgg tctgcctggc caccggtttc ttccccgatc acgtggaact gtcttggtgg 540 gtgaacggaa aagaagtgca ctcgggggtg tccacggacc cccagcctct gaaggaacag 600 ccggcactga atgactcacg ctactgtctg tcgtcacggc tgcgcgtgtc ggccaccttc 660 tggcaaaacc cgcgaaacca ctttcgctgc caagtgcagt tttacgggct ttccgagaac 720 gacgagtgga ctcaggacag agcgaagccc gtgacccaaa tcgtgtccgc cgaggcctgg 780 ggacgcgccg actgcggttt cacctccgtg agctaccaac agggcgtgct gtcagctacc 840 atcctttacg agatcctcct gggaaaggcc accctctacg ccgtcctggt gtccgcactg 900 gtgctgatgg ctatggtcaa gcggaaggat 930 <210> 87 <211> 276 <212> PRT <213> Homo sapiens <400> 87 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe Gly Gly Gly 115 120 125 Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val 130 135 140 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 165 170 175 Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe 180 185 190 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220 Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 225 230 235 240 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 245 250 255 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 260 265 270 Leu Trp Ser Ser 275 <210> 88 <211> 312 <212> PRT <213> Homo sapiens <400> 88 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Phe 305 310 <210> 89 <211> 277 <212> PRT <213> Homo sapiens <400> 89 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 115 120 125 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <210> 90 <211> 310 <212> PRT <213> Homo sapiens <400> 90 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <210> 91 <211> 273 <212> PRT <213> Homo sapiens <400> 91 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Arg Gly Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu 115 120 125 Leu Val Ser Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 To be <210> 92 <211> 310 <212> PRT <213> Homo sapiens <400> 92 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr 260 265 270 Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp 305 310 <210> 93 <211> 1833 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 93 atggcgtccc tgctgttctt ctgcggtgcc ttctaccttc tgggaaccgg ctcgatggac 60 gccgacgtga cccaaacccc tcgcaaccgc atcaccaaga ctggaaagcg gatcatgctg 120 gaatgctccc agaccaaggg ccacgaccgc atgtactggt acagacagga cccgggtctg 180 ggattgcgcc tgatctacta ctccttcgac gtgaaggaca tcaacaaggg ggagatctcc 240 gatggatact cagtctcgag acaagcccag gctaagtttt ccctgtccct cgaatccgcc 300 attcccaatc agaccgcgct gtacttctgt gccacctcgg attgggaccg atccggcgac 360 aaggaaactc agtacttcgg accaggaacc aggctcctgg tgctggagga tctgaacaag 420 gtgttcccgc cggaagtggc agtgttcgag ccatccgaag ccgagatctc gcatacgcag 480 aaggccaccc tcgtgtgcct ggccactggg tttttccctg accacgtgga gctctcgtgg 540 tgggtcaacg gaaaggaagt gcacagcggt gtctcaaccg acccgcaacc tctcaaggaa 600 cagcccgcgc tcaatgattc gcggtactgc ctgagcagcc ggctcagagt gtccgccact 660 ttctggcaaa acccgcggaa ccatttccgg tgccaagtgc aattctacgg gctgtcggaa 720 aacgacgaat ggacccagga cagggccaag cccgtgaccc agattgtgtc cgctgaagcc 780 tgggggagag ctgattgcgg tttcaccagc gtgtcgtatc agcagggcgt gctgtcagcc 840 accattctgt acgaaatcct cctggggaag gccacactgt acgccgtgct cgtgtccgcc 900 ctggtgctga tggccatggt caagcggaag gacttcggca gcggagctac caacttctcc 960 ctgctgaagc aggccggcga tgtggaagaa aatcccggac ctatgagcct ctcttccctg 1020 ctcaaagtgg tcactgcctc cctgtggctg ggaccgggaa tcgcccagaa gatcactcag 1080 acccagcctg gaatgttcgt gcaagagaaa gaggccgtga ccctggattg tacttatgac 1140 acttcagacc agtcgtacgg tttgttctgg tacaagcagc cgtcctccgg agaaatgatc 1200 ttcctgatct accagggatc gtacgacgag cagaacgcta ccgagggcag atattccctc 1260 aacttccaaa aggcccggaa atccgcgaac ctcgtgatct cggcctcaca acttggggac 1320 tccgctatgt atttctgcgc aatgtccggc gatagcgcag gaaacatgct gactttcggc 1380 ggtggaacta ggctgatggt caagccccac attcaaaacc ctgacccagc agtctaccag 1440 ttgcgggatt ccaagtcttc cgataaatcc gtgtgtctct ttacagactt cgatagccag 1500 accaacgtgt cccagagcaa agacagcgac gtgtacatta ctgacaagac tgtgctggac 1560 atgcggtcca tggacttcaa gagcaactcc gccgtcgctt ggtccaacaa gtctgacttt 1620 gcgtgcgcga acgctttcaa caacagcatt atcccggagg acaccttttt cccttccccc 1680 gagtcaagct gcgatgtcaa gcttgtggaa aagtcgttcg aaaccgacac caacctgaac 1740 ttccagaacc tgtccgtcat cgggttccgc attctgctgc tgaaggtcgc cggcttcaat 1800 ctcctgatga ctctccgctt gtggtcctca taa 1833 <210> 94 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> human optimized <400> 94 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Phe Gly Ser Gly Ala Thr Asn Phe Ser 305 310 315 320 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser 325 330 335 Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu Gly Pro 340 345 350 Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe Val Gln 355 360 365 Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser Asp Gln 370 375 380 Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu Met Ile 385 390 395 400 Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr Glu Gly 405 410 415 Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn Leu Val 420 425 430 Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys Ala Met 435 440 445 Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg 450 455 460 Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 465 470 475 480 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 485 490 495 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 500 505 510 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 515 520 525 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 530 535 540 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 545 550 555 560 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 565 570 575 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 580 585 590 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 595 600 605 Ser Ser 610 <210> 95 <211> 1842 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 95 atgggattcc gccttctgtg ctgcgtggcc ttctgcttgc ttggagctgg tcccgtcgac 60 tcgggagtga cccagacgcc gaagcacttg attaccgcta ctgggcagcg cgtgactctg 120 cgatgctcac cacggtctgg cgacctctcc gtgtactggt atcagcagag cctggaccag 180 ggactgcagt tcctgatcca gtactacaac ggagaggagc gggctaaggg aaacatactg 240 gagcggttct cggcgcaaca attccccgat ctgcactccg aactgaacct gtcctccctg 300 gaattgggag actccgccct gtacttctgc gcaagcagcg gtggtgacgg ggatgaacag 360 ttctttggcc ctggaaccag actcaccgtg ctcgaggacc tcaagaacgt gttcccaccc 420 gaagtcgcgg tgttcgagcc ctccgaagcg gaaatcagcc atactcagaa agccactctc 480 gtgtgcctgg ccaccggatt ctacccggac cacgtcgagc tctcttggtg ggtgaacggg 540 aaagaggtcc acagcggcgt gagcactgat ccgcagccgc tgaaggaaca acccgccttg 600 aacgactcgc ggtactgtct gtcctcccgg ctgagagtgt cggccacctt ctggcaaaac 660 cccaggaacc actttaggtg ccaagtccag ttctacggcc tgagcgaaaa cgatgagtgg 720 acccaggaca gagccaagcc tgtgacccag attgtgtcag ccgaggcttg gggtagagca 780 gactgcggat tcacctccga gtcctaccaa caaggcgtcc tctcggcgac cattctgtac 840 gaaatcctgc ttgggaaggc cactctgtac gccgtgctgg tgtccgccct ggtgctgatg 900 gccatggtca agcggaaagga ctcccggggg agagcaaaga ggggatcggg agccaccaat 960 tttagcctgc tgaagcaggc cggcgatgtg gaaaaatc ctggccccat gtcccttagc 1020 tcactgctga aagtggtcac tgcgagcctg tgggctggac caggcattgc tcaaaagatc 1080 acccagactc agcctgggat gttcgtgcaa gagagaag ccgtgaccct cgactgcact 1140 tacgacacct ccgacccgtc ctacgggctg ttctggtaca agcagccgtc ctccggagag 1200 atgatcttcc tcatctacca aggctcctat gatcagcaga acgccaccga aggacgctac 1260 agcctgaact tccagaaggc tcggaagtcg gcgaacctcg tgatcagcgc atcccaactg 1320 ggggacagcg ccatgtactt ctgtgccatg tcgggcggat acaccgggg attcaagacc 1380 attttcgggg ccggcactag actgttcgtg aaggccaaca tccagaatcc tgatccggcg 1440 gtgtatcagc tgcgcgactc caagtcttcc gataaatccg tgtgtctctt tacagacttc 1500 gactcccaaa ccaacgtgtc acagtccaag gacagcgatg tgtacatcac cgacaaaacc 1560 gtgctggaca tgcggtccat ggacttcaag tcaaacagcg cagtcgcctg gtccaacaag 1620 tccgacttcg cctgtgcgaa cgccttcaac aactccatca ttccggaaga tacctttttc 1680 ccttcacctg agtcgagctg tgatgtgaag ctcgtggaaa agtcgtttga aacggacacc 1740 aacctgaact ttcagaacct gtccgtgatt ggtttccgca tcctgctgct gaaggtcgcc 1800 ggcttcaact tgctgatgac gctccggctg tggtcctcgt aa 1842 <210> 96 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> human optimized <400> 96 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn 305 310 315 320 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 325 330 335 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 340 345 350 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 355 360 365 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 370 375 380 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 385 390 395 400 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 405 410 415 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 420 425 430 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 435 440 445 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 450 455 460 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 465 470 475 480 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 485 490 495 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 500 505 510 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 515 520 525 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 530 535 540 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 545,550,555,560 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 565,570,575 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 580,585,590 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 595,600,605 Arg Leu Trp Ser Ser 610 <210> 97 <211> 1830 <212> DNA <213> Artificial Sequence <220> <223> human optimized <400> 97 atgggttgtc ggttgctgtg ttgcgtcgtg ttctgccttc ttcaagctgg tcctctcgat 60 actgccgtga gccaaacccc taagtacctt gtcacccaaa tgggcaacga caagtccatc 120 aaatgcgaac agaacctcgg acacgacacc atgtactggt acaaacagga ttccaagaag 180 ttcctgaaga tttgttctc atacaacaac aaggaactga tttcaacga aactgtgccg 240 aaccggttct caccgaagtc gcctgacaag gctcatctca acttgcatat caactcgctg 300 gagctcggcg actccgccgt gtacttctgc gcgtccagcc agttctggga cggagcggc 360 gacgaacagt acttcggccc gggcactcgg ctgaccgtga ccgaagatct gaacaaagtg 420 ttccccccg aagtggccgt gttcgaacct tccgaggccg agatcagcca cacccaaaag 480 gccactctgg tctgcctggc caccggttc ttccccgatc acgtggaact gtcttggtgg 540 gtgaacggaa aagaagtgca ctcgggggtg tccacggacc cccagcctct gaaggaacag 600 ccggcactga atgactcacg ctactgtctg tcgtcacggc tgcgcgtgtc ggccaccttc 660 tggcaaaacc cgcgaaacca cttcgctgc caagtgcagt tttacgggct ttccgagaac 720 gacgagtgga ctcaggacag agcgaagccc gtgacccaaa tcgtgtccgc cgaggcctgg 780 ggacgcgccg actgcggttt cacctccgtg agctaccaac agggcgtgct gtcagctacc 840 atcctttacg agatcctcct gggaaaggcc accctctacg ccgtcctggt gtccgcactg 900 gtgctgatgg ctatggtcaa gcggaaggat tttagggcca aacgcgggtc cggagcgacc 960 aacttctcgc tgttgaagca ggccggcgat gtggaagaga accctggacc gatgacgtcc 1020 attagagccg tgttcatttt cctgtggctg caactggacc tcgtgaatgg agaaaacgtc 1080 gagcagcacc catccaccct gagcgtgcag gagggagact ccgcagtcat caagtgcact 1140 tactccgact cagcatcaaa ctatttcccg tggtataagc aggaactcgg aaagcggcct 1200 cagctcatca ttgacatccg ctccaacgtc ggagagaaga aggaccagag aattgccgtg 1260 acactcaaca agaccgccaa gcatttctcc cttcacatca ccgaaaccca gcccgaggac 1320 agcgccgtct acttttgtgc ggcctcgagg ggcactgggt ttcagaagct cgtgttcggc 1380 actgggaccc ggctgctggt gtcgccaaac atccagaatc cagaccccgc ggtgtaccag 1440 ctgagagact cgaagtcttc cgataaatcc gtgtgtctct ttacagactt cgatagccag 1500 actaacgtgt cccagtccaa ggactccgat gtgtacatca ccgacaagac tgtgctggat 1560 atgcggagca tggactttaa gtccaattca gcggtcgcgt ggagcaacaa gtccgacttc 1620 gcctgcgcta acgctttcaa caactccatt atcccggagg ataccttctt cccgtcaccg 1680 gaatcctcgt gcgacgtgaa gctggtcgag aagtccttcg aaccgatac caacctgaac 1740 ttccaaaacc tctccgtgat cggcttcaga atcctgctgc tgaaagtggc tggcttcaat 1800 ttgctgatga ccctgcggct ctggagcagc 1830 <210> 98 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> human optimized <400> 98 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr 260 265 270 Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Phe Arg Ala Lys Arg Gly Ser Gly Ala Thr 305 310 315 320 Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly 325 330 335 Pro Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu 340 345 350 Asp Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser 355 360 365 Val Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser 370 375 380 Ala Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro 385 390 395 400 Gln Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln 405 410 415 Arg Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His 420 425 430 Ile Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala 435 440 445 Ser Arg Gly Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg 450 455 460 Leu Leu Val Ser Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 465 470 475 480 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 485 490 495 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 500 505 510 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 515,520,525 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 530 535 540 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 545 550 555 560 Glu Ser Ser Cys Asp Val Leu Val Glu Lys Ser Phe Glu Thr Asp 565,570,575 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 580,585,590 Lys Val Ala Gly Phe Asn and Met Thr and Arg and Trp 595,600,605 Ser Ser 610 <210> 99 <211> 828 <212> DNA <213> Artificial Sequence <220> <223> Full TCR-7 <400> 99 atgagcctct cttccctgct caagtgtc actgcctccc tgtggctggg accgggaatc 60 gcccagaaga tcactcagac ccagcctgga atgttcgtgc aagagaaaga ggccgtgacc 120 ctggattgta cttatgacac ttcagaccag tcgtacggtt tgttctgta caagcagccg 180 tcctccggag aaatgatctt cctgatctac cagggatcgt acgacgagca gaacgctacc 240 gagggcagat attccctcaa cttccaaaag gcccggaaat ccgcgaacct cgtgatctcg 300 gcctcacaac ttggggactc cgctatgtat ttctgcgcaa tgtccggcga tagcgcagga 360 aacatgctga ctttcggcgg tggaactagg ctgatggtca agccccacat tcaaaaccct 420 gacccagcag tctaccagtt gcgggattcc aagtcttccg ataaatccgt gtgtctcttt 480 acagacttcg atagccagac caacgtgtcc cagagcaaag acagcgacgt gtacattact 540 gacaagactg tgctggacat gcggtccatg gacttcaaga gcaactccgc cgtcgcttgg 600 tccaacaagt ctgactttgc gtgcgcgaac gctttcaaca acagcattat cccggaggac 660 acctttttcc cttccagcga cgtcccctgc gatgtcaagc ttgtggaaaa gtcgttcgaa 720 accgacacca acctgaactt ccagaacctg ctagtgattg tgctccgcat tctgctgctg 780 aaggtcgccg gcttcaatct cctgatgact ctccgcttgt ggtcctca 828 <210> 100 <211> 936 <212> DNA <213> Artificial Sequence <220> <223> TCR-7 b complete <400> 100 atggcgtccc tgctgttctt ctgcggtgcc ttctaccttc tgggaaccgg ctcgatggac 60 gccgacgtga cccaaacccc tcgcaaccgc atcaccaaga ctggaaagcg gatcatgctg 120 gaatgctccc agaccaaggg ccacgaccgc atgtactggt agacagga cccgggtctg 180 ggattgcgcc tgatctacta ctccttgac gtgaaggaca tcaacaaggg ggagatctcc 240 gatggatact cagtctcgag acaagcccag gctaagttt ccctgtccct cgaatccgcc 300 attcccaatc agaccgcgct gtacttctgt gccacctcgg attgggaccg atccggcgac 360 aaggaactc agtacttcgg accaggaacc aggctcctgg tgctggagga tctgaacaag 420 480 aaggccaccc tcgtgtgcct ggccactggg tttttccctg accacgtgga gctctcgtgg 540 tgggtcaacg gaaggaagt gcacagcggt gtctcaaccg acccgcaacc tctcaaggaa 600 cagcccgcgc tcaatgattc gcggtactgc ctgagcagcc ggctcagagt gtccgccact 660 ttctggcaaa acccgcggaa ccatttccgg tgccaagtgc aattctacgg gctgtcggaa 720 aacgacgaat ggacccagga cagggccaag cccgtgaccc agattgtgtc cgctgaagcc 780 tgggggagag ctgattgcgg tatcaccagc gccagctacc accagggcgt gctgtcagcc 840 accattctgt acgaaatcct cctggggaag gccacactgt acgccgtgct cgtgtccgcc 900 ctggtgctga tggccatggt caagcggaag gacttc 936 <210> 101 <211> 1833 <212> DNA <213> Artificial Sequence <220> <223> TCR-7 polycistronic construct <400> 101 atggcgtccc tgctgttctt ctgcggtgcc ttctaccttc tgggaaccgg ctcgatggac 60 gccgacgtga cccaaacccc tcgcaaccgc atcaccaaga ctggaaagcg gatcatgctg 120 gaatgctccc agaccaaggg ccacgaccgc atgtactggt acagacagga cccgggtctg 180 ggattgcgcc tgatctacta ctccttcgac gtgaaggaca tcaacaaggg ggagatctcc 240 gatggatact cagtctcgag acaagcccag gctaagtttt ccctgtccct cgaatccgcc 300 attcccaatc agaccgcgct gtacttctgt gccacctcgg attgggaccg atccggcgac 360 aaggaactc agtacttcgg accaggaacc aggctcctgg tgctggagga tctgaacaag 420 480 aaggccaccc tcgtgtgcct ggccactggg tttttccctg accacgtgga gctctcgtgg 540 tgggtcaacg gaaggaagt gcacagcggt gtctcaaccg acccgcaacc tctcaaggaa 600 cagcccgcgc tcaatgattc gcggtactgc ctgagcagcc ggctcagagt gtccgccact 660 ttctggcaaa acccgcggaa ccatttccgg tgccaagtgc aattctacgg gctgtcggaa 720 aacgacgaat ggacccag cagggccaag cccgtgaccc agattgtgtc cgctgaagcc 780 tggggagag ctgattgcgg tatcaccagc gccagctacc accagggcgt gctgtcagcc 840 accattctgt acgaaatcct cctgggggaag gccacactgt acgccgtgct cgtgtccgcc 900 ctggtgctga tggccatggt caagggaag gacttcggca gcggagctac caacttctcc 960 ctgctgaagc aggccggcga tgtggagaa aatcccggac ctatgagcct ctcttccctg 1020 ctcaaagtgg tcactgcctc cctgtggctg ggaccgggaa tcgcccagaa gatcactcag 1080 acccagcctg gaatgttcgt gcaagagaaa gaggccgtga ccctggattg tacttatgac 1140 acttcagacc agtcgtacgg tttgttctgg tacaagcagc cgtcctccgg agaaatgatc 1200 ttcctgatct accagggatc gtacgacgag cagaacgcta ccgagggcag atattccctc 1260 aacttccaaa aggcccggaa atccgcgaac ctcgtgatct cggcctcaca acttggggac 1320 tccgctatgt atttctgcgc aatgtccggc gatagcgcag gaaacatgct gactttcggc 1380 ggtggaacta ggctgatggt caagccccac attcaaaacc ctgacccagc agtctaccag 1440 ttgcgggatt ccaagtcttc cgataaatcc gtgtgtctct ttacagactt cgatagccag 1500 accaacgtgt cccagagcaa agacagcgac gtgtacatta ctgacaagac tgtgctggac 1560 atgcggtcca tggacttcaa gagcaactcc gccgtcgctt ggtccaacaa gtctgacttt 1620 gcgtgcgcga acgctttcaa caacagcatt atcccggagg acaccttttt cccttccagc 1680 gacgtcccct gcgatgtcaa gcttgtggaa aagtcgttcg aaaccgacac caacctgaac 1740 ttccagaacc tgctagtgat tgtgctccgc attctgctgc tgaaggtcgc cggcttcaat 1800 ctcctgatga ctctccgctt gtggtcctca taa 1833 <210> 102 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> TCR-7 a complete <400> 102 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe Gly Gly Gly 115 120 125 Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val 130 135 140 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 165 170 175 Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe 180 185 190 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220 Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 225 230 235 240 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val Leu Arg 245 250 255 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 260 265 270 Leu Trp Ser Ser 275 <210> 103 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> TCR-7 b complete <400> 103 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser 260 265 270 Tyr His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Phe 305 310 <210> 104 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> TCR-7 polyprotein <400> 104 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Asp Trp Asp Arg Ser Gly Asp Lys Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser 260 265 270 Tyr His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Phe Gly Ser Gly Ala Thr Asn Phe Ser 305 310 315 320 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser 325 330 335 Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu Gly Pro 340 345 350 Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe Val Gln 355 360 365 Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser Asp Gln 370 375 380 Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu Met Ile 385 390 395 400 Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr Glu Gly 405 410 415 Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn Leu Val 420 425 430 Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys Ala Met 435 440 445 Ser Gly Asp Ser Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg 450 455 460 Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 465 470 475 480 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 485 490 495 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 500 505 510 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 515 520 525 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 530 535 540 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Ser 545 550 555 560 Asp Val Pro Cys Asp Val Leu Val Glu Lys Ser Phe Glu Thr Asp 565,570,575 Thr Asn Leo Asn Phe Gln Asn Leo Leo Val Is Val Leo Arg Is Leo 580,585,590 Lys Val Ala Gly Phe Asn and Met Thr and Arg and Trp 595,600,605 Ser Ser 610 <210> 105 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> Full TCR-8 <400> 105 atgtccctta gctcactgct gaaagtgtc actgcgagcc tgtggctgg accaggcatt 60 gctcaaaga tcaccagac tcagcctggg atgttcgtgc aagagaagga agccgtgacc 120 ctcgactgca cttacgacac ctccgaccg tcctacgggc tgttctgta caagcagccg 180 tcctccggag agatctt cctcatctac caggctcct atgatcagca gaacgccacc 240 gaaggacgct acagcctgaa cttccagaag gctcggaagt cggcgaacct cgtgatcagc 300 gcatcccaac tgggggacag cgccatgtac ttctgtgcca tgtcgggcgg atacaccggg 360 ggattcaaga ccattttcgg ggccggcact agactgttcg tgaaggccaa catccagaat 420 cctgatccgg cggtgtatca gctgcgcgac tccaagtctt ccgataaatc cgtgtgtctc 480 tttacagact tcgactccca aaccaacgtg tcacagtcca aggacagcga tgtgtacatc 540 accgacaaaa ccgtgctgga catgcggtcc atggacttca agtcaaacag cgcagtcgcc 600 tggtccaaca agtccgactt cgcctgtgcg aacgccttca acaactccat cattccggaa 660 gatacctttt tcccttcaag cgacgtcccc tgtgatgtga agctcgtgga aaagtcgttt 720 gaaacggaca ccaacctgaa ctttcagaac ctgctagtga ttgtgctccg catcctgctg 780 ctgaaggtcg ccggcttcaa cttgctgatg acgctccggc tgtggtcctc g 831 <210> 106 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> TCR-8 b complete <400> 106 atgggattcc gccttctgtg ctgcgtggcc ttctgcttgc ttggagctgg tcccgtcgac 60 tcgggagtga cccagacgcc gaagcacttg attaccgcta ctgggcagcg cgtgactctg 120 cgatgctcac cacggtctgg cgacctctcc gtgtactggt atcagcagag cctggaccag 180 ggactgcagt tcctgatcca gtactacaac ggagaggagc gggctaaggg aaacatactg 240 gagcggttct cggcgcaaca attccccgat ctgcactccg aactgaacct gtcctccctg 300 gaattgggag actccgccct gtacttctgc gcaagcagcg gtggtgacgg ggatgaacag 360 ttctttggcc ctggaaccag actcaccgtg ctcgaggacc tcaagaacgt gttcccaccc 420 gaagtcgcgg tgttcgagcc ctccaaggcg gagatcgccc atactcagaa agccactctc 480 gtgtgcctgg ccaccggatt ctacccggac cacgtcgagc tctcttggtg ggtgaacggg 540 aaagaggtcc acagcggcgt gagcactgat ccgcagccgc tgaaggaaca acccgccttg 600 aacgactcgc ggtactgtct gtcctcccgg ctgagagtgt cggccacctt ctggcaaaac 660 cccaggaacc actttaggtg ccaagtccag ttctacggcc tgagcgaaaa cgatgagtgg 720 acccaggaca gagccaagcc tgtgacccag attgtgtcag ccgaggcttg gggtagagca 780 gactgcggaa tcaccagcgc cagctaccac caaggcgtcc tctcggcgac cattctgtac 840 gaaatcctgc ttgggaaggc cactctgtac gccgtgctgg tgtccgccct ggtgctgatg 900 gccatggtca agcggaagga ctcccggggg 930 <210> 107 <211> 1842 <212> DNA <213> Artificial Sequence <220> <223> TCR-8 polycistronic construct <400> 107 atgggattcc gccttctgtg ctgcgtggcc ttctgcttgc ttggagctgg tcccgtcgac 60 tcgggagtga cccagacgcc gaagcacttg attaccgcta ctgggcagcg cgtgactctg 120 cgatgctcac cacggtctgg cgacctctcc gtgtactggt atcagcagag cctggaccag 180 ggactgcagt tcctgatcca gtactacaac ggagaggagc gggctaaggg aaacatactg 240 gagcggttct cggcgcaaca attccccgat ctgcactccg aactgaacct gtcctccctg 300 gaattgggag actccgccct gtacttctgc gcaagcagcg gtggtgacgg ggatgaacag 360 ttctttggcc ctggaaccag actcaccgtg ctcgaggacc tcaagaacgt gttcccaccc 420 gaagtcgcgg tgttcgagcc ctccaaggcg gagatcgccc atactcagaa agccactctc 480 gtgtgcctgg ccaccggatt ctacccggac cacgtcgagc tctcttggtg ggtgaacggg 540 aaagaggtcc acagcggcgt gagcactgat ccgcagccgc tgaaggaaca acccgccttg 600 aacgactcgc ggtactgtct gtcctcccgg ctgagagtgt cggccacctt ctggcaaaac 660 cccaggaacc actttaggtg ccaagtccag ttctacggcc tgagcgaaaa cgatgagtgg 720 acccaggaca gagccaagcc tgtgacccag attgtgtcag ccgaggcttg gggtagagca 780 gactgcggaa tcaccagcgc cagctaccac caaggcgtcc tctcggcgac cattctgtac 840 gaaatcctgc ttgggaaggc cactctgtac gccgtgctgg tgtccgccct ggtgctgatg 900 gccatggtca agcggaaagga ctcccggggg agagcaaaga ggggatcggg agccaccaat 960 tttagcctgc tgaagcaggc cggcgatgtg gaaaaatc ctggccccat gtcccttagc 1020 tcactgctga aagtggtcac tgcgagcctg tgggctggac caggcattgc tcaaaagatc 1080 acccagactc agcctgggat gttcgtgcaa gagagaag ccgtgaccct cgactgcact 1140 tacgacacct ccgacccgtc ctacgggctg ttctggtaca agcagccgtc ctccggagag 1200 atgatcttcc tcatctacca aggctcctat gatcagcaga acgccaccga aggacgctac 1260 agcctgaact tccagaaggc tcggaagtcg gcgaacctcg tgatcagcgc atcccaactg 1320 ggggacagcg ccatgtactt ctgtgccatg tcgggcggat acaccggggg attcaagacc 1380 attttcgggg ccggcactag actgttcgtg aaggccaaca tccagaatcc tgatccggcg 1440 gtgtatcagc tgcgcgactc caagtcttcc gataaatccg tgtgtctctt tacagacttc 1500 gactcccaaa ccaacgtgtc acagtccaag gacagcgatg tgtacatcac cgacaaaacc 1560 gtgctggaca tgcggtccat ggacttcaag tcaaacagcg cagtcgcctg gtccaacaag 1620 tccgacttcg cctgtgcgaa cgccttcaac aactccatca ttccggaaga tacctttttc 1680 ccttcaagcg acgtcccctg tgatgtgaag ctcgtggaaa agtcgtttga aacggacacc 1740 aacctgaact ttcagaacct gctagtgatt gtgctccgca tcctgctgct gaaggtcgcc 1800 ggcttcaact tgctgatgac gctccggctg tggtcctcgt aa 1842 <210> 108 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> TCR-8 a complete <400> 108 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 115 120 125 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val Leu 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <210> 109 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> TCR-8 b complete <400> 109 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <210> 110 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> TCR-8 polyprotein <400> 110 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn 305 310 315 320 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 325 330 335 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 340 345 350 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 355 360 365 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 370 375 380 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 385 390 395 400 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 405 410 415 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 420 425 430 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 435 440 445 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 450 455 460 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 465 470 475 480 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 485 490 495 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 500 505 510 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 515 520 525 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 530 535 540 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 545 550 555 560 Pro Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe 565,570,575 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val Leu 580 585 590 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 595,600,605 Arg Leu Trp Ser Ser 610 <210> 111 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> TCR-9 is complete <400> 111 atgacgtcca ttagagccgt gttcatttc ctgtggctgc aactggacct cgtgaatgga 60 gaaaacgtcg agcagcaccc atccaccctg agcgtgcagg agggagactc cgcagtcatc 120 aagtgcactt actccgactc agcatcaaac tatttcccgt ggtataagca ggaactcgga 180 aagcggcctc agctcatcat tgacatccgc tccaacgtcg gagagaagaa ggaccagaga 240 attgccgtga cactcaacaa gaccgccaag cattctccc ttcacatcac cgaaacccag 300 cccgaggaca gcgccgtcta cttttgtgcg gcctcgaggg gcactgggtt tcagaagctc 360 gtgttcggca ctgggacccg gctgctggtg tcgccaaaca tccagaatcc agaccccgcg 420 gtgtaccagc tgagagactc gaagtcttcc gataaatccg tgtgtctctt tacagacttc 480 gatagccaga ctaacgtgtc ccagtccaag gactccgatg tgtacatcac cgacaagact 540 gtgctggata tgcggagcat ggactttaag tccaattcag cggtcgcgtg gagcaacaag 600 tccgacttcg cctgcgctaa cgctttcaac aactccatta tcccggagga taccttcttc 660 ccgtcaagcg acgtcccctg cgacgtgaag ctggtcgaga agtccttcga aaccgatacc 720 aacctgaact tccaaaacct cctagtgatt gtgctcagaa tcctgctgct gaaagtggct 780 ggcttcaatt tgctgatgac cctgcggctc tggagcagc 819 <210> 112 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> TCR-9 b complete <400> 112 atgggttgtc ggttgctgtg ttgcgtcgtg ttctgccttc ttcaagctgg tcctctcgat 60 actgccgtga gccaaacccc taagtacctt gtcacccaaa tgggcaacga caagtccatc 120 aaatgcgaac agaacctcgg acacgacacc atgtactggt acaaacagga ttccaagaag 180 ttcctgaaga tttgttctc atacaacaac aaggaactga tttcaacga aactgtgccg 240 aaccggttct caccgaagtc gcctgacaag gctcatctca acttgcatat caactcgctg 300 gagctcggcg actccgccgt gtacttctgc gcgtccagcc agttctggga cggagcggc 360 gacgaacagt acttcggccc gggcactcgg ctgaccgtga ccgaagatct gaacaaagtg 420 ttccccccg aagtggccgt gttcgaacct tccaaggcgg agatcgccca cacccaaaag 480 gccactctgg tctgcctggc caccggttc ttccccgatc acgtggaact gtcttggtgg 540 gtgaacggaa aagaagtgca ctcgggggtg tccacggacc cccagcctct gaaggaacag 600 ccggcactga atgactcacg ctactgtctg tcgtcacggc tgcgcgtgtc ggccaccttc 660 tggcaaaacc cgcgaaacca cttcgctgc caagtgcagt tttacgggct ttccgagaac 720 gacgagtgga ctcaggacag agcgaagccc gtgacccaaa tcgtgtccgc cgaggcctgg 780 ggacgcgccg actgcggtat caccagcgcc agctaccacc agggcgtgct gtcagctacc 840 atcctttacg agatcctcct gggaaaggcc accctctacg ccgtcctggt gtccgcactg 900 gtgctgatgg ctatggtcaa gcggaaggat 930 <210> 113 <211> 1833 <212> DNA <213> Artificial Sequence <220> <223> TCR-9 polycistronic construct <400> 113 atgggttgtc ggttgctgtg ttgcgtcgtg ttctgccttc ttcaagctgg tcctctcgat 60 actgccgtga gccaaacccc taagtacctt gtcacccaaa tgggcaacga caagtccatc 120 aaatgcgaac agaacctcgg acacgacacc atgtactggt acaaacagga ttccaagaag 180 ttcctgaaga ttatgttctc atacaacaac aaggaactga ttatcaacga aactgtgccg 240 aaccggttct caccgaagtc gcctgacaag gctcatctca acttgcatat caactcgctg 300 gagctcggcg actccgccgt gtacttctgc gcgtccagcc agttctggga cggagcgggc 360 gacgaacagt acttcggccc gggcactcgg ctgaccgtga ccgaagatct gaacaaagtg 420 ttcccccccg aagtggccgt gttcgaacct tccaaggcgg agatcgccca cacccaaaag 480 gccactctgg tctgcctggc caccggtttc ttccccgatc acgtggaact gtcttggtgg 540 gtgaacggaa aagaagtgca ctcgggggtg tccacggacc cccagcctct gaaggaacag 600 ccggcactga atgactcacg ctactgtctg tcgtcacggc tgcgcgtgtc ggccaccttc 660 tggcaaaacc cgcgaaacca ctttcgctgc caagtgcagt tttacgggct ttccgagaac 720 gacgagtgga ctcaggacag agcgaagccc gtgacccaaa tcgtgtccgc cgaggcctgg 780 ggacgcgccg actgcggtat caccagcgcc agctaccacc agggcgtgct gtcagctacc 840 atcctttacg agatcctcct gggaaaggcc accctctacg ccgtcctggt gtccgcactg 900 gtgctgatgg ctatggtcaa gcggaaggat tttagggcca aacgcgggtc cggagcgacc 960 aacttctcgc tgttgaagca ggccggcgat gtggaagaga accctggacc gatgacgtcc 1020 attagagccg tgttcatttt cctgtggctg caactggacc tcgtgaatgg agaaaacgtc 1080 gagcagcacc catccaccct gagcgtgcag gagggagact ccgcagtcat caagtgcact 1140 tactccgact cagcatcaaa ctatttcccg tggtataagc aggaactcgg aaagcggcct 1200 cagctcatca ttgacatccg ctccaacgtc ggagagaaga aggaccagag aattgccgtg 1260 acactcaaca agaccgccaa gcatttctcc cttcacatca ccgaaaccca gcccgaggac 1320 agcgccgtct acttttgtgc ggcctcgagg ggcactgggt ttcagaagct cgtgttcggc 1380 actgggaccc ggctgctggt gtcgccaaac atccagaatc cagaccccgc ggtgtaccag 1440 ctgagagact cgaagtcttc cgataaatcc gtgtgtctct ttacagactt cgatagccag 1500 actaacgtgt cccagtccaa ggactccgat gtgtacatca ccgacaagac tgtgctggat 1560 atgcggagca tggactttaa gtccaattca gcggtcgcgt ggagcaacaa gtccgacttc 1620 gcctgcgcta acgctttcaa caactccatt atcccggagg ataccttctt cccgtcaagc 1680 gacgtcccct gcgacgtgaa gctggtcgag aagtccttcg aaaccgatac caacctgaac 1740 ttccaaaacc tcctagtgat tgtgctcaga atcctgctgc tgaaagtggc tggcttcaat 1800 ttgctgatga ccctgcggct ctggagcagc taa 1833 <210> 114 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> TCR-9 a complete <400> 114 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Arg Gly Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu 115 120 125 Leu Val Ser Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Ser Asp 210 215 220 Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val Leu Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 To be <210> 115 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> TCR-9 b complete <400> 115 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr 260 265 270 His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp 305 310 <210> 116 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> TCR-9 polyprotein <400> 116 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Phe Trp Asp Gly Ala Gly Asp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr 260 265 270 His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Phe Arg Ala Lys Arg Gly Ser Gly Ala Thr 305 310 315 320 Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly 325 330 335 Pro Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu 340 345 350 Asp Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser 355 360 365 Val Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser 370 375 380 Ala Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro 385 390 395 400 Gln Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln 405 410 415 Arg Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His 420 425 430 Ile Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala 435 440 445 Ser Arg Gly Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg 450 455 460 Leu Leu Val Ser Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 465 470 475 480 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 485 490 495 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 500 505 510 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 515 520 525 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 530 535 540 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Ser 545 550 555 560 Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 565 570 575 Thr Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val Leu Arg Ile Leu 580 585 590 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 595 600 605 Cheese Cheese 610

Claims

1. A TCRα chain containing a variable TCRα region including CDR1 having the amino acid sequence of SEQ ID NO:12, CDR2 having the amino acid sequence of SEQ ID NO:13, and CDR3 having the amino acid sequence of SEQ ID NO:14, and A TCRβ chain containing a variable TCRβ region including CDR1 having the amino acid sequence of SEQ ID NO:15, CDR2 having the amino acid sequence of SEQ ID NO:16, and CDR3 having the amino acid sequence of SEQ ID NO:

17. A T cell receptor (TCR) specific to MAGE-A4, wherein the sequence of the TCR is modified to include minimally mouse-like Cα and Cβ regions.

2. The TCR according to claim 1, which specifically recognizes the amino acid sequence or fragment thereof of SEQ ID NO:1 in a form bound to HLA-A2.

3. The TCR according to claim 1, which specifically recognizes the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:

01.

4. The TCR according to any one of claims 1 to 3, wherein the variable TCRα region has the amino acid sequence of SEQ ID NO: 18, and the variable TCRβ region has the amino acid sequence of SEQ ID NO:

19.

5. The TCR according to any one of claims 1 to 3, wherein the TCRα chain has the amino acid sequence of SEQ ID NO: 20 and the TCRβ chain has the amino acid sequence of SEQ ID NO:

21.

6. The TCR according to any one of claims 1 to 3, wherein the TCRα chain has the amino acid sequence of SEQ ID NO: 89 and the TCRβ chain has the amino acid sequence of SEQ ID NO:

90.

7. The TCR according to any one of claims 1 to 3, wherein the TCRα chain has the amino acid sequence of SEQ ID NO: 108 and the TCRβ chain has the amino acid sequence of SEQ ID NO:

109.

8. A polyvalent TCR complex comprising at least two TCRs according to any one of claims 1 to 7.

9. A fusion protein specific to MAGE-A4, containing a TCRα chain and a TCRβ chain, and containing the amino acid sequence shown in SEQ ID NO:

110.

10. A fusion protein specific to MAGE-A4, containing a TCRα chain and a TCRβ chain, and containing the amino acid sequence shown in SEQ ID NO:

96.

11. A nucleic acid encoding a TCR according to any one of claims 1 to 7 or a fusion protein according to any one of claims 9 to 10.

12. The nucleic acid according to claim 11, comprising SEQ ID NO:105 and SEQ ID NO:

106.

13. The nucleic acid according to claim 11, comprising SEQ ID NO:77 and SEQ ID NO:

78.

14. The nucleic acid according to claim 11, comprising SEQ ID NO:46 and SEQ ID NO:

47.

15. The nucleic acid according to claim 11, wherein the fusion protein is encoded by the nucleic acid sequence shown in SEQ ID NO:

95.

16. The nucleic acid according to claim 11, wherein the fusion protein is encoded by the nucleic acid sequence shown in SEQ ID NO:

107.

17. A vector comprising the nucleic acid according to any one of claims 11 to 16.

18. A cell expressing the TCR described in any one of claims 1 to 7.

19. A cell comprising the nucleic acid or vector according to any one of claims 11 to 17.

20. The cells according to claim 18 or 19, which are immune effector cells.

21. The cell according to claim 20, wherein the immune effector cell is a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.

22. A composition comprising a TCR according to any one of claims 1 to 7, a polyvalent TCR complex according to claim 8, a fusion protein according to any one of claims 9 to 10, a nucleic acid according to any one of claims 11 to 16, a vector according to claim 17, or a cell according to any one of claims 18 to 21.

23. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, a TCR according to any one of claims 1 to 7, a polyvalent TCR complex according to claim 8, a fusion protein according to any one of claims 9 to 10, a nucleic acid according to any one of claims 11 to 16, a vector according to claim 17, or a cell according to any one of claims 18 to 21.

24. A composition according to claim 22, or a pharmaceutical composition according to claim 23, for use in the treatment of blood cancer or solid tumors.

25. (a) The cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia; or (b) The cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer or colorectal cancer, sarcoma, or osteosarcoma. The composition or pharmaceutical composition according to claim 24.

26. The composition or pharmaceutical composition according to claim 24 or 25, wherein the cancer is synovial sarcoma.

Images