MAGE a4 t cell receptors

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 March 27, 2019, which is incorporated herein by reference in its entirety.

[0002] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text format in lieu of hard copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is MED16702PCT_ST25. This text file is 146KB, was created on March 24, 2020, and was submitted electronically via EFS-Web simultaneously with the filing of this application.

[0003] The present invention relates to isolated T cell receptors (TCRs) specific for MAGE-A4, polypeptides comprising functional parts of the TCRs, multivalent TCR complexes, nucleic acids encoding the TCRs, cells expressing the TCRs, and compositions and pharmaceutical compositions comprising them. The present invention also relates to methods of using the same in methods of medical treatment or for formulation, and / or as medicaments, in particular for use in the treatment of cancer. [Background technology]

[0004] 2. Description of Related Art 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 allow recognition of peptides presented on major histocompatibility complex (MHC) molecules expressed on nucleated cells (known as antigen presentation). Antigens derived from pathogens, i.e. foreign antigens presented by MHC molecules, elicit a strong T cell response, whereas self-antigens do not usually 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 is thus able to distinguish between nucleated cells presenting foreign antigens and those presenting self-antigens, and can specifically target and eradicate infected cells by the strong cytokine release and cellular cytotoxicity mechanisms of T cells.

[0005] The capabilities of the immune system have been recognized as a promising tool for future cancer treatment. In the past decades, research has begun to exploit the unique properties of T cells by using adoptive cell therapy (ACT), which involves the administration of ex vivo expanded lymphocytes from the patient. ACT is an attractive idea for cancer treatment because it does not require the patient's immune competence and it can direct the specificity of the transferred lymphocytes towards tumor antigens that are non-mutated and therefore poorly immunogenic and generally cannot effectively trigger autologous T cell responses. Although ACT has been shown to be a promising treatment for various types of cancer, its widespread application as a clinical treatment has been hindered by the need to custom isolate and characterize tumor-specific T cells from each patient, a difficult and time-consuming process, as well as the frequent lack of availability of 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 allows for the rapid generation of tumor-reactive T lymphocytes with a given antigen specificity, even in immunocompromised patients, thus overcoming some of the limitations of current ACT. However, the identification of suitable T cell clones carrying TCRs that specifically recognize tumor antigens and exhibit the desired antitumor effect in vivo is still the subject of ongoing research. Given that there were approximately 14.1 million new cases of cancer worldwide in 2012, and that cancer is currently responsible for approximately 14.6% of all human deaths worldwide, there is an urgent need for new and efficient treatment options. The object of the present invention is to meet the above-mentioned needs.

[0007] MAGE-A4 belongs to the melanoma antigen (MAGE) family, which is expressed in a variety of malignant tumor types, including melanoma, colon, lung, breast and other tumors. Specifically, the MAGE family is classified into two groups based on its tissue expression pattern, of which the MAGE-A subfamily is expressed in germ cells of the testis and ectopically re-expressed in malignant tumors, which is also the case 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 cancer 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). Summary of the Invention

[0009] overview It is an object of the present invention to provide an isolated T cell receptor (TCR) specific for MAGE-A4.

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

[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 comprises a variable TCR alpha 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 TCR beta 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 TCR beta 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 TCR beta 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 a variable TCR beta 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. and a variable TCR β region, which comprises a CDR1 having the amino acid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQ ID NO:26 and a CDR3 having the amino acid sequence of SEQ ID NO:27. These TCRs are described in further detail below.

[0013] In particular embodiments, the TCR comprises a variable TCR alpha region having the amino acid sequence of SEQ ID NO:8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:9; or a variable TCR alpha region having the amino acid sequence of SEQ ID NO:18 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:19; or a variable TCR alpha region having the amino acid sequence of SEQ ID NO:28 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:29.

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

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

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

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

[0018] In some embodiments, the invention relates to an isolated TCR as described herein, wherein binding of the TCR expressed on an effector cell to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01 can result in greater than 100-fold, preferably greater than 500-fold, more preferably greater than 2000-fold greater IFN-γ secretion when bound to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01 compared to binding to an unrelated peptide presented by a 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 routinely coupled to the TCR or TCR variant, optionally via linkers of various lengths. The term "label" or "label group" refers to any detectable label. Additionally or alternatively, the amino acid sequence may be modified to include a therapeutic agent or a pharmacokinetic-modifying moiety. The therapeutic agent may be selected from the group consisting of an immune effector molecule, a cytotoxic agent, and a radionuclide. The immune effector molecule may be, for example, a cytokine. The pharmacokinetic-modifying moiety may be at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialyl group, or a combination thereof.

[0020] TCRs, particularly soluble forms of TCRs contemplated herein, can be modified by attaching functional moieties, for example, to reduce immunogenicity, increase hydrodynamic size (size in solution), solubility and / or stability (e.g., by increasing protection from proteolysis), and / or increase serum half-life. Other useful functional moieties and modifications include "suicide switches" or "safety switches" that can be used to shut down or start effector host cells carrying the TCRs of the invention in the patient. TCRs with altered glycosylation patterns are also contemplated herein.

[0021] It is also contemplated that the TCRs, particularly soluble forms of the TCRs of the invention, may be loaded with drugs or therapeutic entities, such as small molecule compounds. The TCRs, particularly soluble forms of the TCRs of the invention, may also be further modified to introduce additional domains (tags) that aid in the identification, tracking, purification and / or isolation of the respective molecules.

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

[0023] Another aspect relates to a polypeptide comprising a functional portion of a TCR described herein, wherein the functional portion comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs:4, 7, 14, 17, 24 and 27.

[0024] In a specific embodiment, the functional portion comprises a TCR alpha variable region and / or a TCR beta variable region.

[0025] A specific embodiment relates to a multivalent TCR complex comprising at least two TCRs as described herein, in a further specific embodiment at least one of the TCRs is linked to a therapeutic agent.

[0026] Particular embodiments relate to fusion proteins comprising a TCR alpha chain and a TCR beta chain and comprising the amino acid sequence set forth in any one of SEQ ID NOs:94, 96, 98, 104, 110 and 116.

[0027] In specific aspects, the fusion protein further comprises a furin cleavage site and / or a ribosomal skipping sequence.

[0028] Another aspect relates to a nucleic acid encoding a TCR as described herein or a nucleic acid encoding a polypeptide or fusion protein as described above.

[0029] In one aspect, the nucleic acid sequence encoding the TCR alpha chain is set forth in any one of SEQ ID NOs:69, 77, 85, 99, 105 and 111. In another aspect, the nucleic acid sequence encoding the TCR beta chain is set forth in any one of SEQ ID NOs:70, 78, 86, 100, 106 and 112. In another aspect, the TCR comprises an alpha chain encoded by SEQ ID NO:69 and a beta chain encoded by SEQ ID NO:70; an alpha chain encoded by SEQ ID NO:77 and a beta chain encoded by SEQ ID NO:78; an alpha chain encoded by SEQ ID NO:85 and a beta chain encoded by SEQ ID NO:86; an alpha chain encoded by SEQ ID NO:99 and a beta chain encoded by SEQ ID NO:100; an alpha chain encoded by SEQ ID NO:105 and a beta chain encoded by SEQ ID NO:106; or an alpha chain encoded by SEQ ID NO:111 and a beta chain encoded by SEQ ID NO:112.

[0030] A further aspect relates to a fusion protein encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs:93, 95, 97, 101, 107 and 113.

[0031] A further aspect relates to a plasmid or vector comprising the nucleic acid of the present application as described above.A further aspect relates to a plasmid or vector comprising the nucleic acid encoding the polypeptide sequence shown in SEQ ID NO:87 and SEQ ID NO:88; the polypeptide sequence shown in SEQ ID NO:89 and SEQ ID NO:90; the polypeptide sequence shown in SEQ ID NO:91 and SEQ ID NO:92; the polypeptide sequence shown in SEQ ID NO:102 and SEQ ID NO:103; the polypeptide sequence shown in SEQ ID NO:108 and SEQ ID NO:109; or the polypeptide sequence shown in SEQ ID NO:114 and SEQ ID NO:115.Preferably, the vector is an expression vector or a vector suitable for transducing or transfecting cells, particularly eukaryotic cells.The vector can be, for example, a retroviral vector, for example a gamma retroviral vector or a lentiviral vector.

[0032] Another aspect pertains to cells expressing the TCRs described herein. The cells may be isolated or non-naturally occurring.

[0033] Another aspect relates to a cell comprising the above-mentioned nucleic acid or the above-mentioned plasmid or vector. More specifically, the cell may comprise an expression vector comprising one or more of the above-mentioned nucleic acids, or may comprise a first expression vector comprising a nucleic acid encoding the alpha chain of the TCR as described herein and a second expression vector comprising a nucleic acid encoding the beta chain of the TCR as described herein.

[0034] The cells can be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). Typically, the 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.

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

[0036] Another aspect pertains to a composition comprising a TCR as described herein, a polypeptide as described herein, a fusion protein as described herein, a multivalent 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.

[0037] Another aspect pertains to a pharmaceutical composition comprising a TCR as described herein, a polypeptide as described herein, a fusion protein as described herein, a multivalent 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, a pharmaceutical composition includes at least one pharma- ceutically acceptable carrier.

[0039] Another aspect relates to the TCR described herein, the polypeptide described herein, the multivalent TCR complex described herein, the nucleic acid described herein, the vector described herein, the cell described herein, the antibody described herein, the composition described herein, or the pharmaceutical composition for use as a medicament, in particular for use in the treatment of cancer. The cancer may be a hematological cancer or a solid tumor. The cancer may be selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin's 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. Preferably, the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer, or colorectal cancer, sarcoma, and osteosarcoma. [The present invention 1001] a) a variable TCR alpha 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 TCR beta 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 TCR alpha region, comprising a CDR1 having the amino acid sequence of SEQ ID NO:12, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:14; A variable TCR beta region comprising a CDR1 having the amino acid sequence of SEQ ID NO:15, a CDR2 having the amino acid sequence of SEQ ID NO:16, and a CDR3 having the amino acid sequence of SEQ ID NO:17; or c) a variable TCR alpha region, comprising a CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having the amino acid sequence of SEQ ID NO:23, and a CDR3 having the amino acid sequence of SEQ ID NO:24; A variable TCR beta region comprising a CDR1 having the amino acid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQ ID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:27. An isolated T cell receptor (TCR) specific for MAGE-A4. [The present invention 1002] An isolated TCR of the present invention 1001, which specifically recognizes the amino acid sequence of SEQ ID NO:1 or a fragment thereof, preferably specifically recognizes the amino acid sequence of SEQ ID NO:1 in the form bound to HLA-A2, more preferably specifically recognizes the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01. [The present invention 1003] a) a variable TCR α region having the amino acid sequence of SEQ ID NO:8 and a variable TCR β region having the amino acid sequence of SEQ ID NO:9; or b) a variable TCR α region having the amino acid sequence of SEQ ID NO:18 and a variable TCR β region having the amino acid sequence of SEQ ID NO:19; or c) a variable TCR α region having the amino acid sequence of SEQ ID NO:28 and a variable TCR β region having the amino acid sequence of SEQ ID NO:29 Any of the isolated TCRs of the present invention. [The present invention 1004] Any of the isolated TCRs of the present invention, comprising a functional portion comprising at least one of the amino acid sequences of SEQ ID NOs:4, 7, 14, 17, 24 and 27. [The present invention 1005] a) a TCR α chain having the amino acid sequence of SEQ ID NO:10 and a TCR β chain having the amino acid sequence of SEQ ID NO:11; b) a TCR α chain having the amino acid sequence of SEQ ID NO:20 and a TCR β chain having the amino acid sequence of SEQ ID NO:21; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:30 and a TCR β chain having the amino acid sequence of SEQ ID NO:31 The isolated TCR of any of claims 1001 to 1004. [The present invention 1006] a) a TCR α chain having the amino acid sequence of SEQ ID NO:87 and a TCR β chain having the amino acid sequence of SEQ ID NO:88; b) a TCR α chain having the amino acid sequence of SEQ ID NO:89 and a TCR β chain having the amino acid sequence of SEQ ID NO:90; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:91 and a TCR β chain having the amino acid sequence of SEQ ID NO:92 The isolated TCR of any of claims 1001 to 1004. [The present invention 1007] a) a TCR α chain having the amino acid sequence of SEQ ID NO:102 and a TCR β chain having the amino acid sequence of SEQ ID NO:103; b) a TCR α chain having the amino acid sequence of SEQ ID NO:108 and a TCR β chain having the amino acid sequence of SEQ ID NO:109; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:114 and a TCR β chain having the amino acid sequence of SEQ ID NO:115 The isolated TCR of any of claims 1001 to 1004. [The present invention 1008] An isolated TCR of any of claims 1001 to 1007 or a multivalent TCR complex of claim 1008, which induces IFN-γ secretion by binding to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01. [The present invention 1009] An isolated TCR of any of the present inventions 1001 to 1007, wherein IFN-γ secretion induced by binding of a TCR expressed on an effector cell to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01 can be more than 100-fold, preferably more than 500-fold, and more preferably more than 2000-fold higher when bound to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01, compared to binding to an unrelated peptide presented by a molecule encoded by HLA-A*02:01. [The present invention 1010] A multivalent TCR complex comprising at least two TCRs according to any one of claims 1001 to 1007 of the present invention. [The present invention 1011] A fusion protein comprising a TCR alpha chain and a TCR beta chain and comprising an amino acid sequence as set forth in any one of SEQ ID NOs:94, 96, 98, 104, 110 and 116. [The present invention 1012] A nucleic acid encoding any one of the TCRs of the present inventions 1001 to 1007 or the fusion protein of the present invention 1011. [The present invention 1013] 1012. A nucleic acid of the present invention, wherein the nucleic acid sequence encoding the TCR alpha chain is set forth in any one of SEQ ID NOs:69, 77, 85, 99, 105 and 111. [The present invention 1014] 1012. A nucleic acid of the present invention, wherein the nucleic acid sequence encoding the TCR β chain is set forth in any one of SEQ ID NOs:70, 78, 86, 100, 106 and 112. [The present invention 1015] TCR, a) a TCR α chain encoded by SEQ ID NO:69 and a TCR β chain encoded by SEQ ID NO:70; b) a TCR α chain encoded by SEQ ID NO:77 and a TCR β chain encoded by SEQ ID NO:78; c) a TCR α chain encoded by SEQ ID NO:85 and a TCR β chain encoded by SEQ ID NO:86; d) a TCR α chain encoded by SEQ ID NO:99 and a TCR β chain encoded by SEQ ID NO:100; e) a TCR α chain encoded by SEQ ID NO:105 and a TCR β chain encoded by SEQ ID NO:106; or f) The TCR α chain encoded by SEQ ID NO:111 and the TCR β chain encoded by SEQ ID NO:112 The nucleic acid of the present invention 1010, comprising [The present invention 1016] The nucleic acid of the present invention 1012, wherein the fusion protein is encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs:93, 95, 97, 101, 107 and 113. [The present invention 1017] A vector comprising any one of the nucleic acids of the present inventions 1012 to 1016, which is preferably an expression vector, more preferably a retroviral vector, and further preferably a lentiviral vector. [The present invention 1018] a) the polypeptide sequences shown in SEQ ID NO:87 and SEQ ID NO:88; b) the polypeptide sequences shown in SEQ ID NO:89 and SEQ ID NO:90; c) the polypeptide sequences shown in SEQ ID NO:91 and SEQ ID NO:92; d) the polypeptide sequences shown in SEQ ID NO:102 and SEQ ID NO:103; e) the polypeptide sequences shown in SEQ ID NO:108 and SEQ ID NO:109; or f) the polypeptide sequences shown in SEQ ID NO:114 and SEQ ID NO:115 A vector comprising a nucleic acid encoding the above-mentioned compound, said vector being preferably an expression vector, more preferably a retroviral vector, and even more preferably a lentiviral vector. [The present invention 1019] A cell expressing any one of the TCRs of the present invention Nos. 1001 to 1047. [The present invention 1020] A cell comprising the vector of the present invention 1017 or 1018. [The present invention 1021] The cell of the present invention 1019 or 1020, which is an immune effector cell. [The present invention 1022] The cell of any one of claims 1019 to 1021, wherein the immune effector cell is a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. [The present invention 1023] An antibody or antigen-binding fragment thereof that specifically binds to a portion of the TCR that mediates specificity for MAGE-A4 according to any one of claims 1001 to 1007 of the present invention, preferably wherein the portion of the TCR that mediates MAGE-A4 specificity is a) the CDR3 of the alpha chain of SEQ ID NO:4 and / or the CDR3 of the beta chain of SEQ ID NO:7, or b) the CDR3 of the alpha chain of SEQ ID NO:14 and / or the CDR3 of the beta chain of SEQ ID NO:17, or c) the CDR3 of the alpha chain of SEQ ID NO:24 and / or the CDR3 of the beta chain of SEQ ID NO:27 The antibody or antigen-binding fragment thereof. [The present invention 1024] A composition comprising a TCR of any one of 1001 to 1007 of the present invention, a multivalent TCR complex of 1008 of the present invention, a fusion protein of 1011 of the present invention, a nucleic acid of any one of 1012 to 1016 of the present invention, a vector of 1017 or 1018 of the present invention, a cell of any one of 1019 to 1022 of the present invention, or an antibody of 1023 of the present invention. [The present invention 1025] A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a TCR according to any one of claims 1001 to 1007 of the present invention, a multivalent TCR complex according to claim 1008 of the present invention, a fusion protein according to claim 1011 of the present invention, a nucleic acid according to any one of claims 1012 to 1016 of the present invention, a vector according to claim 1017 or 1018 of the present invention, a cell according to any one of claims 1019 to 1022 of the present invention, or an antibody according to claim 1023 of the present invention. [The present invention 1026] A TCR of any one of 1001 to 1007, a multivalent TCR complex of the present invention 1008, a fusion protein of the present invention 1011, a nucleic acid of any one of 1012 to 1016, a vector of the present invention 1017 or 1018, a cell of any one of 1019 to 1022, an antibody of the present invention 1023, a composition of the present invention 1024, or a pharmaceutical composition of the present invention 1025, for use as a pharmaceutical. [The present invention 1027] Cancer, preferably a cancer that is a blood cancer or a solid tumor, more preferably a cancer selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin's 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, most preferably NSCLC, SCLC, breast cancer, ovarian cancer, A TCR according to any one of claims 1001 to 1007, a multivalent TCR complex according to any one of claims 1008, a fusion protein according to any one of claims 1011, a nucleic acid according to any one of claims 1012 to 1016, a vector according to any one of claims 1017 or 1018, a cell according to any one of claims 1019 to 1022, an antibody according to any one of claims 1023, a composition according to any one of claims 1024, or a pharmaceutical composition according to any one of claims 1025, for use in treating a cancer selected from the group consisting of human cancer, colorectal cancer, sarcoma, or osteosarcoma. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows MAGE-A4GVY-MHC multimer binding of CD8+ T cells transduced with different MAGE-A4-reactive TCRs. CD8+ 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. Transduced CD8+ T cells were enriched by FACS using mouse constant beta region as a marker for transduction. After expansion of these cells, they were stained with MAGE-A4GVY-MHC multimers and antibodies against CD8 and mouse constant beta region (mmCb) and analyzed by flow cytometry. The population was gated on viable CD8+ / mCb+ cells. Multimer / CD8 staining is shown. [Diagram 2]Figure 2 shows that MAGE-A4-TCR transgenic T cells recognize MAGE-A4GVY peptide presented on HLA-A2. Transgenic T cells were co-cultured with T2 cells exogenously loaded with MAGE-A4GVY peptide or with K562 / HLA-A2 cells transfected with the MAGE-A4 gene. As negative controls, T2 cells loaded with control peptide and non-transduced K562 / HLA-A2 cells were used, respectively. Target cell recognition was analyzed by measuring IFN-γ concentrations in the co-culture supernatants by standard ELISA. [Diagram 3] Figure 3 shows the functional avidity of MAGE-A4-TCR transgenic T cells. Transgenic T cells were co-cultured with T2 cells exogenously loaded with graded concentrations of MAGE-A4GVY peptide (10-12M to 10-4M). IFN-γ concentrations in the co-culture supernatants were measured by standard ELISA. [Figure 4A] Figure 4A-C shows 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 MAGE-A4GVY peptide are used as positive control. Cytotoxicity against tumor cell lines stably transduced with fluorescent markers was measured by taking pictures every 2 hours with an IncuCyte® ZOOM device (Essen Bioscience). To analyze cytokine release, co-culture supernatants were harvested after 24 h and IFN-γ concentrations were analyzed by standard sandwich ELISA (BD human IFN-γ ELISA set). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A]Figure 5A-C shows 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 MAGE-A4GVY peptide are used as positive control. HLA-A2 expression was induced in neurons 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, co-culture supernatants were harvested after 24 h and IFN-γ and IL-2 concentrations were analyzed by standard sandwich ELISA (BD human IFN-γ or IL-2 ELISA set). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6] Figure 6 shows MAGE-A4GVY-MHC multimer binding of CD3+ T cells transduced with different MAGE-A4-reactive 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 expansion of these cells, they were stained with antibodies against MAGE-A4GVY-MHC multimers and CD3 and analyzed by flow cytometry. Populations were gated on viable CD3+ cells and multimer staining. [Figure 7]Figure 7 shows that MAGE-A4 fully human TCR transgenic T cells recognize MAGE-A4GVY peptide presented on HLA-A2. Transgenic T cells were co-cultured with T2 cells exogenously loaded with MAGE-A4GVY peptide or A549 / HLA-A2 cells transfected with MAGE-A4 gene. As negative controls, T2 cells loaded with control peptide and non-transduced A549 / HLA-A2 cells were used, respectively. Target cell recognition was analyzed by measuring IFN-γ concentration in the co-culture supernatant by Luminex assay. [Figure 8] Figure 8 shows the ability of MAGE-A4 fully human TCR transgenic T cells to specifically react 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, co-culture supernatants were 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 starting 6 hours after the start of co-culture. [Figure 10] FIG. 10 shows the ability of MAGE-A4 fully human TCR transgenic T cells to suppress MAGE-A4 positive tumors implanted in NSG mice. [Figure 11]Figure 11A-C shows vector copy number (VCN) and expression of MAGE-A4 TCR. Peripheral blood mononuclear cells (PBMCs) were transduced with lentiviral vectors encoding the fully human MAGE-A4 TCR (TCR-5) or enhanced variant (TCR-8). A) VCN measurements in TCR-5 and TCR-8 T cells were comparable. B) TCR expression on the T cell surface was assessed using GVY-specific tetramer detection by flow cytometry and shown as a percentage of total CD3+ T cells. TCR-8 expression is increased relative to TCR-5 expression. C) Density of TCR molecules on the T cell surface was assessed using GVY-specific tetramer detection by flow cytometry and shown as geometric mean fluorescence intensity (gMFI) of total tetramer+ TCR T cells. TCR-8 expression density is increased relative to TCR-5 expression density. [Figure 12]Figure 12A-B shows that T cells expressing fully human MAGE-A4 TCR (TCR-5) or 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(+)) at a 1:1 E:T ratio. After 24 hours, IFNγ release was assessed as a biomarker for T cell activity. TCR-5 and TCR-8 T cells secreted INFγ when co-cultured with target cells expressing MAGE-A4, but not in the presence of MAGE-A4-negative cells. B) TCR-5, TCR-8 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 after 6 hours using impedance as a normalized percentage relative to tumor cells alone. TCR-5 and TCR-8 T cells mediated comparable cytotoxicity against the three MAGE-A4 expressing cell lines. [Figure 13]Figure 13A-C shows that T cells expressing fully human MAGE-A4 TCR (TCR-5) or enhanced variant (TCR-8) mediate regression in mice bearing MAGE-A4 expressing tumors. Five NSG mice (for each condition) were subcutaneously injected with MAGEA4(+) A375 tumor cells and treated with vehicle, UTD T cells, or T cells expressing fully human MAGE-A4 TCR (TCR-5) or enhanced variant (TCR-8). Tumor growth was measured twice weekly and antitumor activity of TCR T cells was assessed compared to mice receiving UTD control and vehicle control. A-B) Mice bearing 50 mm3 A375 tumors were given 5x106 (A) or 1.5x106 (B) UTD T cells, TCR-5 T cells, or TCR-8 T cells. Both TCR-5 and TCR-8 T cells suppressed tumors at a T cell dose of 5x106, while TCR-8 T cells showed increased tumor suppression at a lower dose of 1.5x106 T cells. C) Mice bearing 100mm3 A375 tumors received 10x106 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description A. Overview The expression pattern of MAGE-A4 makes it 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) that is presented by HLA-A2 molecules (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 MAGE-A antigens for cancer immunotherapy. The present disclosure aims to address this unmet medical need with respect to T cell receptors that efficiently target MAGE-A4 antigens on cancer cells.

[0042] B. Definition Before describing aspects of the present invention in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0043] The invention(s) illustratively described below may suitably be practiced in the absence of one or more elements or limitation not specifically disclosed herein.

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

[0045] When the term "comprising" is used in the present specification and claims, it does not exclude other elements. In the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising of". Hereinafter, when a group is defined as comprising at least a certain number of embodiments, it should be understood to also disclose a group consisting of preferably only those embodiments.

[0046] In the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". If, hereinafter, for example, it is specified that an antibody is obtainable from a specific source, it should be understood to also disclose the antibody obtained from that source.

[0047] When an indefinite or definite article is used to refer to a singular noun, such as "a", "an" or "the", it includes a plural of that noun unless some specific statement is made otherwise. The term "about" or "approximately" refers in the present invention to an interval of accuracy that a person skilled in the art would understand to still ensure 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 indicated numerical value.

[0048] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to all of the elements in the series. The term "at least one" refers to one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Numerous equivalents to the specific embodiments of the invention described herein will be known to those skilled in the art, or will be ascertainable using no more than routine experimentation. Such equivalents are intended to be encompassed by the present invention.

[0049] The term "and / or" whenever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by that term".

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

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

[0052] Throughout the specification and claims, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating the plural as well as the singular, unless the context otherwise requires.

[0053] Technical terms are used with their usual meaning or meaning to those of ordinary skill in the art. Where special meanings are ascribed to certain terms, definitions of the terms are given below in the context in which those terms are used.

[0054] Additional definitions are set forth throughout this disclosure.

[0055] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manuals, etc.), both supra and infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In the event that any material incorporated by reference conflicts or is incompatible with this specification, the present specification will take precedence over any such material.

[0056] The contents of all publications and patent documents cited herein are hereby incorporated by reference in their entirety.

[0057] C. TCR Basics TCRs are composed of two distinct and separate 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. The rearranged V(D)J regions of both the TCR α and TCR β chains contain hypervariable regions (CDRs, complementarity determining regions), especially the CDR3 region, which determines the specific epitope recognition. Both the TCR α and TCR β chains contain a hydrophobic transmembrane domain in the C-terminal region and terminate in a short cytoplasmic tail.

[0058] Typically, TCRs are heterodimers of one α-chain and one β-chain that can bind to peptide-presenting MHC molecules.

[0059] In this context, the term "variable TCR alpha region" or "TCR alpha variable chain" or "variable domain" refers to the variable region of the TCR alpha chain. In this context, the term "variable TCR beta region" or "TCR beta variable chain" refers to the variable region of the TCR beta chain.

[0060] TCR loci and 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 A first aspect relates to an isolated T cell receptor (TCR) specific for MAGE-A4.

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

[0063] In particular, the TCR contemplated herein is a TCR that binds amino acids 230 to 239 of MAGE-A4, i.e., the amino acid sequence SEQ ID NO:1 (GVYDGREHTV, herein MAGE-A4 GVY It specifically recognizes an epitope comprising the nucleotide sequence of the nucleotide sequence of the nucleotide a, nucleotide b, or a fragment thereof.

[0064] Typically, TCRs recognize peptide fragments of antigens when presented by major histocompatibility complex (MHC) molecules.

[0065] The human leukocyte antigen (HLA) system or HLA complex is a complex of genes that encodes major histocompatibility complex (MHC) proteins in humans. HLA-A*02 (HLA-A2) is a specific set 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] Thus, 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 an HLA-A*02:01 encoded molecule.

[0067] The TCR is highly specific for MAGE-A4 and does not exhibit cross-reactivity to other peptides, meaning that the TCR does not recognize normal human cell lines, including cardiomyocytes, endothelial cells, lung 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] The term "specific for" in this context means that the TCR specifically binds to a target. In a particular embodiment, the TCR is specific for MAGE-A4 and specifically binds to the amino acid sequence shown in SEQ ID NO:1 presented by HLA-A*02:01 encoded molecules.

[0069] E. TCR-specific sequences The CDR3 of the TCR alpha 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 TCR β 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 TCR alpha chain and a TCR beta chain, wherein a) the TCR alpha chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:4, and the TCR beta chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:7; or b) the TCR alpha chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:14, and the TCR beta chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:17; or c) the TCR alpha chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:24, and the TCR beta chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:27.

[0072] More specific embodiments include: a) a TCR alpha chain 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, and a TCR beta chain 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 TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:12, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:14, and a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:15, a CDR2 having the amino acid sequence of SEQ ID NO:16, and a CDR3 having the amino acid sequence of SEQ ID NO:17; or c) a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having the amino acid sequence of SEQ ID NO:23, and a CDR3 having the amino acid sequence of SEQ ID NO:24. The present invention relates to an isolated TCR comprising a TCR alpha chain comprising a CDR3 having the amino acid sequence of SEQ ID NO:24, a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQ ID NO:26 and a CDR3 having the amino acid sequence of SEQ ID NO:27.

[0073] A preferred embodiment relates to an isolated TCR defined by the CDRs of the TCR alpha and TCR beta chains described above, in particular by CDR3, wherein the recombinant TCR sequence has been modified to contain murinized Calpha and Cbeta regions, preferably minimal murinized Calpha and Cbeta regions.

[0074] In a particularly preferred embodiment, the isolated TCR is defined by the CDRs of the TCR alpha and beta chains described above, in particular by CDR3, and the recombinant TCR sequence is modified to contain minimally murinized Calpha and Cbeta regions and hydrophobic amino acid mutations in the Calpha transmembrane domain. In a particular embodiment, these TCRs have increased expression and functional avidity compared to TCRs that are not minimally murinized and do not contain hydrophobic mutations in the Calpha transmembrane domain.

[0075] In a further preferred embodiment, the isolated TCR is defined by the CDRs of the TCR alpha and beta chains described above, in particular by CDR3, and the recombinant TCR sequence has not been modified to contain murinized or minimally murinized Calpha and Cbeta regions.

[0076] Some embodiments relate to an isolated TCR comprising: a) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:8 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:9; or b) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:18 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:19; or c) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:28 and a variable TCR beta 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 an amino acid sequence at least 80% identical," includes amino acid sequences that are 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 disclosed amino acid sequence.

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

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

[0080] The following table provides a summary of exemplary TCRs: TIFF2025024103000001.tif31165

[0081] As can be seen from the examples, the TCRs contemplated herein are specific for MAGE-A4.

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

[0083] The TCR contemplated herein is isolated or purified. "Isolated" means that the TCR is not present in the context in which it originally occurred in nature. "Purified" means, for example, that the TCR is free or substantially free of other proteins and non-proteinaceous parts of the cell from which it was originally derived.

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

[0085] A "phenotypically silent substitution" is also called a "conservative amino acid substitution". The concept of "conservative amino acid substitution" is understood by those skilled in the art and preferably means that codons encoding positively charged residues (H, K and R) are replaced with codons encoding positively charged residues, codons encoding negatively charged residues (D and E) are replaced with codons encoding negatively charged residues, codons encoding polar neutral residues (C, G, N, Q, S, T and Y) are replaced with codons encoding polar neutral residues, and codons encoding non-polar neutral residues (A, F, I, L, M, P, V and W) are replaced with codons encoding non-polar neutral residues. These mutations may occur spontaneously, may be introduced by random mutagenesis, or may be introduced by directed mutagenesis. These changes can be made without destroying the essential characteristics of these polypeptides. One of skill in the art can readily routinely screen the mutant amino acids and / or the nucleic acids encoding them to determine whether the mutations substantially reduce or abolish ligand binding ability, using methods known in the art.

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

[0087] Non-limiting examples of detectable labels are radioactive labels, fluorescent labels, nucleic acid probes, enzymes and contrast agents. Therapeutic agents that can be bound to the TCR 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 will avoid damage during transport in the body and ensure that the therapeutic agent, e.g., toxins, exerts its maximum effect after the TCR binds to the relevant antigen-presenting cells. Further examples of therapeutic agents include: peptide cytotoxins, i.e., proteins or peptides capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase. Small molecule cytotoxic agents, i.e., compounds capable of killing mammalian cells and having a molecular weight of less than 700 daltons. Such compounds may contain toxic metals that can have a cytotoxic effect. It should also be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release a cytotoxic agent. Such agents include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, racheromycin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, mitoxantrone, auristatin E, vincristine and doxorubicin; radionuclides, for example iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225 and astatine-213. Binding of radionuclides to the TCR or derivatives thereof can be effected, for example, by chelators; immunostimulants, also known as immunopotentiators, i.e. immune effector molecules that stimulate the immune response.Exemplary immune stimulants 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 characteristics; superantigens, i.e., antigens that cause non-specific activation of T cells resulting in polyclonal T cell activation and massive cytokine release, and variants thereof; chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating activity protein, complement activators; heterologous protein domains, allologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0088] Antigen receptor molecules (T cell receptor molecules) on human T lymphocytes are non-covalently associated with the CD3 (T3) molecular complex on the cell surface. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Thus, some embodiments relate to TCRs as described herein to which anti-CD3 antibodies or functional fragments or variants of said CD3 antibodies are attached (usually by fusion to the N- or C-terminus of the alpha or beta chain). Antibody fragments and variants / analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab') 2 These include molecules comprising synthetic single immunoglobulin variable heavy domains derived from camelids (camel or llama or alpaca) antibodies), as well as domain antibodies (comprising affinity matured single immunoglobulin variable heavy or light domains (Domantis (Belgium)), or alternative protein scaffolds exhibiting antibody-like binding characteristics, such as Affibodies (comprising an engineered Protein A scaffold, Affibody (Sweden)) or Anticalins (comprising engineered Anticalins, Pieris (Germany)).

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

[0090] The pharmacokinetic modifying moiety may be, for example, at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialyl group, or a combination thereof. The linkage of at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialyl group may be brought about by several methods known to those skilled in the art. In a preferred embodiment, said unit is covalently linked to the TCR. The TCR contemplated herein may be modified with one or several pharmacokinetic modifying moieties. In particular, the soluble form of the TCR is modified with one or several pharmacokinetic modifying moieties. The pharmacokinetic modifying moiety may bring about beneficial changes in the pharmacokinetic profile of the therapeutic agent, such as improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility.

[0091] The TCR contemplated herein may be soluble or membrane-bound. The term "soluble" refers to the TCR being soluble (i.e., having no transmembrane or cytoplasmic domains) for use, for example, as a targeting agent for delivering therapeutic agents to antigen-presenting cells. For stability, the soluble αβ heterodimeric TCR preferably has disulfide bonds introduced between the residues of each constant domain, for example, as described in WO 03 / 020763. One or both of the constant domains present in the αβ heterodimer may be truncated at one or more C-termini, for example, by up to 15, or up to 10, or up to 8 or less amino acids. For use in adoptive therapy, the αβ heterodimeric TCR may be transfected as a full-length chain, for example, having both cytoplasmic and transmembrane domains. The TCR may contain disulfide bonds corresponding to those found naturally between each alpha and beta constant domain, and in addition, or alternatively, non-native disulfide bonds may be present.

[0092] Thus, TCRs, particularly soluble forms of TCRs contemplated herein, can be modified by attaching additional functional moieties, for example, to reduce immunogenicity, increase hydrodynamic size (size in solution), solubility and / or stability (e.g., by enhanced protection from proteolysis), and / or increase serum half-life. Other useful functional moieties and modifications include "suicide switches" or "safety switches" that can be used to shut down effector host cells bearing the TCRs of the invention in the patient. 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 well-known methods to express a caspase 9 domain, the dimerization of which is dependent on a small molecule dimerizing drug such as AP1903 / CIP, which dimerization results in rapid induction of apoptosis in the modified effector cells. This system is described, for example, in EP 2173869(A2). Other examples of "suicide switches" or "safety switches" are known in the art, such as herpes simplex virus thymidine kinase (HSV-TK), expression of CD20 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] Also contemplated herein are TCRs with altered glycosylation patterns. As known in the art, glycosylation patterns may depend on the amino acid sequence (e.g., the presence or absence of specific glycosylated amino acid residues, as described below) and / or the host cell or host organism in which the protein is produced. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Addition of N-linked glycosylation sites to the binding molecule is conveniently accomplished by altering the amino acid sequence such 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 may be introduced by adding or substituting one or more serine or threonine residues to the starting sequence.

[0094] Another means of glycosylating the TCR is by chemical or enzymatic coupling of glycosides to the protein. Depending on the coupling method used, sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. Similarly, deglycosylation (i.e., removal of carbohydrate moieties present on the binding molecule) may be accomplished chemically, for example by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically, using endo- and exoglycosidases.

[0095] It is also conceivable to add drugs such as small molecule compounds to TCR, particularly to the soluble form of the TCR of the present invention. Linkage can be achieved by covalent bond or non-covalent interaction, such as electrostatic force. Various linkers known in the art can be used to form drug conjugates.

[0096] The TCRs, particularly soluble forms of the TCRs of the invention, may also 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 TCR α or TCR β chains may 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 the binding of specific antibodies, allowing the identification and tracking of the binding and translocation of soluble TCRs or host cells within a patient or cultured (host) cells. Detection of epitope tags, and thus tagged TCRs, can be achieved using several different techniques. Tags can also be used to stimulate and grow host cells bearing the TCRs of the invention by culturing the cells in the presence of a binding molecule (antibody) specific for the tag.

[0098] Generally, in some instances, the TCR can be modified with various mutations that modify the affinity and off-rate of the TCR for the target antigen. In particular, these mutations can increase affinity and / or decrease off-rate. Thus, the TCR can be mutated in at least one of its CDRs and variable domain framework regions.

[0099] However, in a preferred embodiment, the CDR regions of the TCR are not modified and are not subjected to in vitro affinity maturation, as in the case of the TCR receptors of the examples. This means that the CDR regions have natural sequences. This can be advantageous, since in vitro affinity maturation can lead to immunogenicity of the TCR molecules, which can lead to the production of anti-drug antibodies that reduce or inactivate the therapeutic effect and treatment, and / or induce adverse effects.

[0100] The mutations can be 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, ligation-independent cloning methods, etc., as described in Sambrook, Cold Spring Harbor Laboratory Press 2012.

[0101] Theoretically, mispairing between endogenous and exogenous TCR chains may result in unpredictable TCR specificity with the risk of cross-reactivity. To avoid mispairing of TCR sequences, recombinant TCR sequences may be modified to contain minimal murine Cα and Cβ regions, a technique that has been shown to efficiently enhance correct pairing of several different transduced TCR chains. Mouserization of TCRs (i.e., replacing the human constant regions in the alpha and beta chains with their respective murine counterparts) is a commonly applied technique to improve the cell surface expression of TCRs in host cells. Without wishing to be bound by a particular theory, it is believed that murine TCRs bind more effectively to CD3 coreceptors and / or pair preferentially with each other, and are less likely to form mixed TCRs on human T cells genetically modified ex vivo to express TCRs with the desired antigen specificity, while still retaining and expressing each "original" TCR.

[0102] Nine amino acids responsible for improved expression of murinized TCRs have been identified (Sommermeyer and Uckert, J Immunol. 2010;184(11):6223-6231), and it is envisaged to substitute one or all of these amino acid residues in the TCR alpha and / or beta chain constant regions for their respective murine counterparts. This technique, also called "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, thus reducing the risk of immunogenicity.

[0103] In a preferred embodiment, the TCR containing the minimal murine Cα and Cβ regions is TCR-1 comprising an α chain of SEQ ID NO:10 and a β chain of SEQ ID NO:11, TCR-2 comprising an α chain of SEQ ID NO:20 and a β chain of SEQ ID NO:21, and TCR-3 comprising an α chain of SEQ ID NO:30 and a β chain of SEQ ID NO:31.

[0104] In a preferred embodiment, the TCR contains minimally murine Cα and Cβ regions and further comprises 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 stability of the entire chain, thereby affecting the formation and surface expression of the entire TCR-CD3 complex.Replacing three amino acids in the TCRα transmembrane domain with hydrophobic amino acids leucine or valine increases the expression and functional avidity of the TCR.Haga-Friedman et al.J.Immunology 2012;188:5538-5546.

[0105] In a preferred embodiment, the TCR containing minimally murine Cα and Cβ regions and hydrophobic amino acid substitutions in the TCR α chain is TCR-7 comprising an α chain of SEQ ID NO:102 and a β chain of SEQ ID NO:103; TCR-8 comprising an α chain of SEQ ID NO:108 and a β chain of SEQ ID NO:109; and TCR-9 comprising an α chain of SEQ ID NO:114 and a β chain of SEQ ID NO:115.

[0106] Some embodiments relate to isolated TCRs as described herein that are of the single chain type, in which the TCR alpha and beta chains are linked by a linker sequence, which is optionally cleavable.

[0107] A suitable single chain TCR format comprises a first segment encoded by an amino acid sequence corresponding to a variable TCR alpha region, a second segment composed of an amino acid sequence corresponding to a variable TCR beta region fused to the N-terminus of an amino acid sequence corresponding to a TCR beta chain constant region extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may be composed of an amino acid sequence corresponding to a TCR beta chain variable region and the second segment may be composed of an amino acid sequence corresponding to a TCR alpha chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR alpha chain constant region extracellular sequence. The single chain TCR may further comprise a disulfide bond between the first and second chain, the length of the linker sequence and the position of the disulfide bond being such that the variable domain sequences of the first and second segments are oriented with respect to each other essentially in the same way as they are in the native T cell receptor. More specifically, the first segment may be constituted by an amino acid sequence corresponding to a TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR α chain constant region extracellular sequence, the second segment may be constituted by an amino acid sequence corresponding to a TCR β chain variable region fused to the N-terminus of an amino acid sequence corresponding to a TCR β chain constant region extracellular sequence, and a disulfide bond may be provided between the first and second chains. The linker sequence may be any sequence that does not impair the function of the TCR.

[0108] By "functional" TCR α and / or β chain fusion protein is meant a TCR or TCR variant that has been modified, e.g. by addition, deletion or substitution of amino acids, which retains at least substantial biological activity. In the case of a TCR α and / or β chain, this is meant that both chains (together with the unmodified α and / or β chain, or with the α and / or β chain of another fusion protein of the invention) are still capable of forming a T cell receptor that exerts its biological function, in particular binding of the TCR to a specific peptide-MHC complex and / or functional signaling upon specific peptide:MHC interaction.

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

[0110] The functional T cell receptor (TCR) alpha and / or beta chain fusion protein of the present invention is preferred, in which the epitope tag is 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. Myc tags, T7 tags, GST tags, and GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, for example, U.S. Pat. Nos. 4,703,004 and 4,851,341). The myc tag is preferably used, since 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 the recognition by the antibody. 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 TCR alpha and beta chains are separated by one or more polypeptide cleavage signals. In a particular embodiment, the fusion protein may comprise, from 5' to 3', the TCR alpha chain, one or more polypeptide cleavage signals, and the TCR beta chain. In a particular embodiment, the fusion protein may comprise, from 5' to 3', the TCR beta chain, one or more polypeptide cleavage signals, and the TCR alpha chain.

[0112] Polypeptide cleavage signals contemplated herein include, but are not limited to, protease cleavage sites and ribosomal skipping sequences. A polypeptide cleavage signal may be placed between each of the polypeptide domains described herein, for example, between the TCR alpha and TCR beta chains. In addition, a polypeptide cleavage signal may be placed in 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 ribosomal skipping sequences (see deFelipe and Ryan, 2004.Traffic,5(8);616-26).

[0113] Illustrative examples of protease cleavage sites suitable for use in particular embodiments include furin (e.g., Arg-XX-Arg, e.g., Arg-X-Lys / Arg-Arg or Arg-Gln / Tyr-Lys / Arg-Arg. Furin may also cleave the sequence 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 / PC7IPC8 / SPC7 and SKI-I); enterokinase (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*); caspase-3 (e.g., Asp-Glu-Val-Asp*).

[0114] Specific examples of self-cleaving viral peptides or ribosomal 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 particular embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide or a cardiovirus 2A peptide. In preferred embodiments, 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 teschovirus-1 2A peptide (P2A), theirovirus 2A peptide and encephalomyocarditis virus 2A peptide.

[0115] In certain embodiments, the fusion protein comprises a TCR alpha chain, a proteolytic cleavage site and / or a ribosomal skipping sequence, and a TCR beta chain. In a preferred embodiment, the fusion protein comprises a TCR alpha chain, a furin cleavage site and / or a P2A ribosomal skipping sequence, and a TCR beta chain. In another preferred embodiment, the fusion protein comprises a TCR alpha chain, a P2A ribosomal skipping sequence, and a TCR beta chain.

[0116] In a particular embodiment, the fusion protein comprises a TCR beta chain, a proteolytic cleavage site and / or a ribosomal skipping sequence, and a TCR alpha chain. In a preferred embodiment, the fusion protein comprises a TCR beta chain, a furin cleavage site and / or a P2A ribosomal skipping sequence, and a TCR alpha chain. In another preferred embodiment, the fusion protein comprises a TCR beta chain, a P2A ribosomal skipping sequence, and a TCR alpha chain.

[0117] In a preferred embodiment, the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs:94, 96, 98, 104, 110 and 116.

[0118] F. TCR Fragments and Variants Another aspect pertains to a polypeptide comprising a functional portion of a TCR as described herein. The functional portion may comprise at least one of the amino acid sequences 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, SEQ ID NO:27.

[0119] In a specific embodiment, the polypeptide may comprise only a functional portion of the TCR, e.g., a soluble form. Alternatively, the polypeptide may be combined with other domains.

[0120] The functional portion may mediate binding of the TCR to an antigen, in particular to an antigen-MHC complex. In one embodiment, the functional portion comprises a TCR alpha and / or beta variable chain as described herein.

[0121] TCR variant molecules, i.e. molecules that combine a polypeptide containing a functional portion of a TCR with other domains, may have the binding properties of a TCR, but may also be combined with a signaling domain of an effector cell (other than a T cell), in particular a signaling domain of a NK cell. Thus, some embodiments relate to proteins that include a functional portion of a TCR as described herein combined with a signaling domain of an immune effector cell, such as a NK cell.

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

[0123] Another aspect relates to a multivalent TCR complex comprising at least two TCRs as described herein. In one embodiment of this aspect, at least two TCR molecules are linked by a linker moiety to form a multivalent complex. Preferably, the complex is water-soluble, so the linker moiety should be selected accordingly. Preferably, the linker moiety is capable of attachment to a predefined location on the TCR molecule, so that the structural diversity of the complex formed is minimized. One embodiment of this aspect is provided by a TCR complex in which a polymer chain or peptide linker sequence 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 of medical use, the linker moiety should be selected with due consideration given to its pharmaceutical suitability, 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 are hydrophilic polymers and peptide linkers. An example of a hydrophilic polymer is polyalkylene glycol. The most commonly used of this type are based on polyethylene glycol, i.e. PEG. However, there are other suitable, optionally substituted, polyalkylene glycol-based linkers, including, for example, polypropylene glycol and copolymers of ethylene glycol and propylene glycol. Peptide linkers are composed of chains of amino acids and serve to generate simple linkers or multimerization domains to which TCR molecules can be attached.

[0125] Certain embodiments relate to multivalent TCR complexes in which at least one of the TCRs is bound by a therapeutic agent.

[0126] G. Cytokine and Chemokine Release Some embodiments relate to an isolated TCR as described herein, a polypeptide as described herein, or a multivalent TCR complex as described herein, in which IFN-γ secretion is induced upon binding of the TCR of the invention expressed on an effector cell to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01.

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

[0128] Cytokine and chemokine release, e.g., IFN-γ secretion, was induced by transfecting K562 cells (Greiner et al. 2006, Blood. 2006 Dec 15;108(13):4109-17) with ivtRNA or transducing them to express the amino acid sequence of SEQ ID NO:1 or an unrelated peptide, respectively, and incubating them with CD8+ cells expressing the TCR of interest. + Enriched PBMC and / or non-CD8 + This can be measured using an in vitro assay in which T2 cells are incubated with enriched PBMCs or exogenously loaded with SEQ ID NO:1 or an irrelevant peptide and then incubated with CD8 + Enriched PBMC and / or non-CD8 + This can be measured in an in vitro assay by co-incubation with enriched PBMCs.

[0129] Some embodiments relate to an isolated TCR as described herein, a polypeptide as described herein or a multivalent TCR complex as described herein, in which binding of a TCR of the invention expressed on an effector cell 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, induces IFN-γ secretion below a pre-determined threshold. The threshold may be determined by using a particular effector to target ratio of at least 2:1.

[0130] "Effector cells" can be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). 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] When the TCR of the present invention expressed on an effector cell binds to the amino acid sequence of SEQ ID NO:1 presented by the molecule encoded by HLA-A*02:01, IFN-γ secretion is at least 10 -7 [M], preferably at least 10 -8 [M], more preferably 10 -9 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 an effector cell 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 It can be induced at a MAGE-A4 peptide concentration of [M].

[0132] The present invention also 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, 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 the target and / or mediates an immune response.

[0133] Whether a candidate TCR or a fragment thereof mediates an immune response can be determined by measuring cytokine secretion, such as, for example, IFN-γ secretion. As described above, cytokine secretion can be measured by, for example, measuring K562 cells (or other APCs) transfected with ivtRNA encoding the amino acid sequence of SEQ ID NO:1, in vitro, or in vivo, in vivo, in vivo, in vivo, in vivo, or in vivo, in vivo, in vivo, in vivo, in vivo, in vivo, in vivo, or in vivo, in vivo. + This can be measured by an in vitro assay involving incubation with enriched PBMCs.

[0134] H. Nucleic Acids, Vectors Another aspect pertains to nucleic acids encoding the TCRs described herein, or polynucleotides encoding the TCRs described herein.

[0135] The table below shows the nucleotide sequence encoding each peptide sequence. TIFF2025024103000002.tif88128TIFF2025024103000003.tif212126TIFF2025024103000004.tif21128

[0136] "Nucleic acid molecule" and "nucleotide sequence" generally refer to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or derived from a natural source (e.g., isolated and / or purified), and may contain natural nucleotides, non-natural or modified nucleotides, and may contain natural internucleotide linkages or non-natural or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiesters found between nucleotides of unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant nucleic acids. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining natural or synthetic nucleic acid segments into a nucleic acid molecule capable of replicating in a living cell, or (ii) a molecule resulting from the replication of a molecule described in (i) above. Replication herein can be in vitro or in vivo replication. Nucleic acids can be constructed using techniques known in the art or commercially available (e.g., from companies such as Genscript, Thermo Fisher, etc.), based on chemical synthesis and / or enzymatic ligation reactions. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or using variously modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (see Sambrook et al.). Nucleic acids can include any nucleotide sequence that encodes a recombinant TCR, recombinant polypeptide or recombinant protein, or any functional portion or variant thereof.

[0137] The disclosure also provides variants of isolated or purified nucleic acids 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 encoding functional TCRs that specifically recognize MAGE-A4.

[0138] The disclosure also provides isolated or purified nucleic acids comprising a nucleotide sequence complementary to any of the nucleotide sequences of the nucleic acids described herein or that hybridize under stringent conditions to any of the nucleotide sequences of the nucleic acids described herein.

[0139] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence hybridizes to a target sequence (any nucleotide sequence of the nucleic acids described herein) in an amount detectably stronger than non-specific hybridization. High stringency conditions include conditions that allow for the discrimination of polynucleotides with exact complementary sequences or those containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more likely to melt than full-length complementary strands of 14-17 bases or more, allowing them to be easily distinguished in high stringency hybridization. Relatively high stringency conditions may include, for example, those provided by low salt and / or high temperature conditions, e.g., about 0.02-0.1 M NaCl or equivalent, and a temperature of about 50-70°C. Such high stringency conditions tolerate little mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the TCRs described herein. It is generally understood that the stringency of the conditions can be increased by adding increasing amounts of formamide.

[0140] In certain embodiments, the nucleic acid is codon optimized. As used herein, the term "codon optimized" refers to the substitution of codons in a polynucleotide encoding a polypeptide to increase expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or between synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene or set of genes; (iii) systematic variation of codons with context; (iv) variation of codons according to their respective decoding tRNAs; (v) variation of codons according to GC% across a triplet or at one position in a triplet; (vi) variation in similarity to a reference sequence, e.g., a natural sequence; (vii) variation of codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based; (x) systematic variation of the codon set for each amino acid; and / or (xi) isolated removal of spurious translation initiation sites.

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

[0142] A "vector" is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell where 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 comprise DNA or RNA and / or may comprise a liposome. A vector may be a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, a lentiviral vector, an adenoviral vector, or a particle and / or vector used in gene therapy. A vector may comprise a nucleic acid sequence, such as an origin of replication, that allows it to replicate in a host cell. A vector may also comprise one or more selectable marker genes, and other genetic elements known to those skilled in the art. A vector is preferably an expression vector that comprises a nucleic acid of the invention operably linked to a sequence that allows expression of the nucleic acid of the invention.

[0143] In a preferred embodiment, the vector comprises a nucleic acid encoding a TCR β chain having the amino acid sequence shown in SEQ ID NO:88 and a TCR α chain having the amino acid sequence shown in SEQ ID NO:87; a TCR β chain having the amino acid sequence shown in SEQ ID NO:90 and a TCR α chain having the amino acid sequence shown in SEQ ID NO:89; a TCR β chain having the amino acid sequence shown in SEQ ID NO:92 and a TCR α chain having the amino acid sequence shown in SEQ ID NO:91; a TCR β chain having the amino acid sequence shown in SEQ ID NO:103 and a TCR α chain having the amino acid sequence shown in SEQ ID NO:102; a TCR β chain having the amino acid sequence shown in SEQ ID NO:109 and a TCR α chain having the amino acid sequence shown in SEQ ID NO:108; or a TCR β chain having the amino acid sequence shown in SEQ ID NO:115 and a TCR α 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 particularly a gamma retroviral vector or a lentiviral vector.

[0145] I. Cells, cell lines Another aspect relates to a cell expressing a TCR as described herein. In some embodiments, the cell is isolated or non-naturally occurring. In a specific embodiment, the cell may contain a nucleic acid encoding a TCR as described herein, or a vector containing the nucleic acid.

[0146] The cell can be introduced with the vector described above that contains the nucleic acid sequence encoding the TCR described above, or with ivtRNA encoding the TCR. The cell can be a peripheral blood lymphocyte, such as a T cell. Methods for cloning and exogenous expression of TCR are described, for example, in Engels et al. Cancer Cell, 2013; 23(4): 516-526. Transduction of primary human T cells with 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, for example, a eukaryotic host cell. It should be noted that the introduction or transfer of a nucleic acid sequence is not limited to the method mentioned, and can be achieved by many means, such as electroporation, microinjection, gene gun delivery, lipofection or superfection.

[0148] The term "transduction" refers to the introduction of an exogenous nucleic acid sequence into a host cell using a viral vector, such as an adenovirus, adeno-associated virus (AAV), vaccinia virus, herpes virus, retrovirus, or lentivirus.

[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 alpha chain of a TCR described herein and a second expression vector comprising a nucleic acid encoding the beta chain of a TCR described herein.

[0150] In some embodiments, the cells are peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). The cells can 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 + The cells can be T cells or double negative T cells, i.e., T cells that do not express 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 a co-receptor, i.e., the TCRs, e.g., TCR-5 and TCR-8, function independently of CD8 + CD4 in cells + It also works in cells.

[0152] Stem cell-like memory T cells (TSCM) are CD8 +They are a less differentiated subpopulation of T cells, characterized by their ability to self-renew and persist for long periods of time. When these cells encounter their respective antigens in vivo, they further differentiate into central memory T cells (TCM), effector memory T cells (TEM) and terminally differentiated effector memory T cells (TEMRA), while some TSCM remain quiescent (Flynn et al., Clinical & Translational Immunology 2014;3(7):e20). These residual TSCM cells show the ability to establish durable immune 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 restrict the T cell pool to stem cell memory T cell subtypes (see Riddell et al. Cancer Journal 2014;20(2):141-144).

[0153] J. Antibodies Targeting the TCR Another aspect relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of the TCR described herein that mediates specificity for MAGE-A4. In one embodiment, the portion of the TCR that mediates MAGE-A4 specificity comprises a CDR3 of a 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 a beta chain selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17 or SEQ ID NO:27.

[0154] The antibody or antigen-binding fragment thereof may modulate the activity of the TCR, which may or may not block the binding of the TCR to MAGE-A4, and may be used to modulate the therapeutic activity of the TCR or for diagnostic purposes.

[0155] K. Pharmaceutical Compositions, Medical Treatments and Kits Another aspect relates to a composition comprising a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding a TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody which specifically binds to a portion of a TCR as described in the invention.

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

[0157] These active ingredients of the present invention are preferably mixed with acceptable carriers or carrier materials in a dose capable of treating or at least alleviating the disease, and used in the above-mentioned pharmaceutical compositions. Such compositions can contain (in addition to the active ingredients and carriers) fillers, salts, buffers, stabilizers, solubilizers and other materials that are known in the art.

[0158] The term "pharmacologically acceptable" refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. The choice of carrier depends on the application.

[0159] The pharmaceutical composition may contain additional components that enhance the activity of the active component or supplement the treatment. Such additional components and / or factors can be part of the pharmaceutical composition to obtain synergistic effects or to minimize adverse or unwanted effects.

[0160] The techniques for formulation or preparation and application / medication of the active ingredients 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, PA, Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.Suitable applications are parenteral applications, such as intramuscular injection, subcutaneous injection, intramedullary injection, as well as intrathecal injection, direct intravenous injection, intranodal injection, intraperitoneal injection or intratumoral injection.Intravenous injection or intravenous infusion is the preferred patient treatment.

[0161] According to a preferred embodiment, the pharmaceutical composition is administered by infusion or injection. The injectable composition is a pharma- ceutical acceptable fluid composition that contains at least one active ingredient, such as an expanded T cell population expressing 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 minor amounts of one or more non-toxic auxiliary substances, such as emulsifiers, preservatives, and pH buffering agents. The injectable compositions useful for use with the fusion proteins of the present disclosure are conventional, and suitable formulations are well known to those skilled in the art.

[0162] Typically, a pharmaceutical composition includes at least one pharma- ceutically acceptable carrier.

[0163] Thus, another aspect relates to a TCR as described herein, a polypeptide comprising a functional part of the TCR, a multivalent 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 part of the TCR, a composition comprising one or more cells expressing a TCR as described herein, or a pharmaceutical composition, for use as a medicament.

[0164] Some embodiments relate to a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or a composition or pharmaceutical composition comprising same, for use in the treatment of cancer.

[0165] In one embodiment, the cancer is a blood cancer or solid tumor. Blood cancer is also called blood cancer, and does not form solid tumors, and therefore spreads throughout the body. Examples of blood cancer are leukemia, lymphoma, or multiple myeloma. Solid tumors are broadly divided into two types: sarcoma and carcinoma. Sarcoma is, for example, a tumor of blood vessels, bone, adipose tissue, ligament, lymphatic vessel, muscle, or tendon.

[0166] In one embodiment, the cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), non-Hodgkin's 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, the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer or colorectal cancer, or sarcoma.More preferably, the 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] Also contemplated herein are pharmaceutical compositions and kits that contain one or more of: (i) an isolated TCR as described herein; (ii) a viral particle that comprises a nucleic acid encoding a recombinant TCR; (iii) an immune cell, such as a T cell or a NK cell, that has been modified to express a recombinant TCR as described herein; (iv) a nucleic acid encoding a recombinant TCR as described herein. In some embodiments, the present disclosure provides a composition that comprises a lentiviral vector particle that comprises a nucleotide sequence encoding a recombinant TCR as described herein (or a T cell that has been modified using a vector particle as described herein to express a recombinant TCR). Such compositions can be administered to a subject in the methods of the present disclosure, as further described herein.

[0169] Compositions comprising the modified T cells described herein can be utilized in methods and compositions for adoptive immunotherapy using known techniques or variations thereof that will be apparent to those of skill in the art based on this disclosure.

[0170] In some embodiments, the cells are formulated in a therapeutically effective amount by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutical acceptable" carrier). Suitable infusion media can include any isotonic medium formulation, typically saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's can also be utilized. Infusion media can be supplemented with human serum albumin.

[0171] The number of cells in the composition for effective treatment is typically greater than 10 cells, up to 10 6 cells, up to 10 8 or 10 9 cells or less, 10 10The number of cells will depend on the end use of the composition and the type of cells contained therein. For example, if cells specific for a particular antigen are desired, the population will contain more than 70%, typically 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, which can be 500 ml or less, or even 250 ml or less or 100 ml or less. Thus, the desired cell density is typically 10 6 cells / ml, typically 10 7 >10 cells / ml, typically 8 A clinically relevant number of immune cells is greater than 10 9 More than 10 cells 10 More than 10 cells 11 The cells can be divided into multiple injections.

[0172] The pharmaceutical compositions provided herein may take various forms, such as solid, liquid, powder, aqueous solution, or lyophilized form. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and / or additives can be formulated in a conventional manner and administered to subjects in appropriate doses. Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can protect the composition from degradation in the body. Compositions intended for administration by injection can include one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonicity agents.

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

[0174] Also contemplated herein is a kit comprising a polynucleotide encoding a gene of interest (e.g., a recombinant TCR). Also contemplated herein is a kit comprising a viral vector encoding a sequence of interest (e.g., a recombinant TCR) and, optionally, a polynucleotide sequence encoding an immune checkpoint inhibitor.

[0175] The kits contemplated herein also include kits for carrying out methods for detecting the presence of polynucleotides encoding any one or more of the TCRs disclosed herein. In particular, such diagnostic kits may include a set of suitable amplification and detection primers and other related reagents for performing deep sequencing to detect polynucleotides encoding the TCRs disclosed herein. In further embodiments, the kits herein may include reagents for detecting the TCRs disclosed herein, such as antibodies or other binding molecules. The 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. The kits may also include instructions. The instructions typically include concrete expressions that describe the components included in the kit and the methods for administration, including methods for determining the proper condition of the subject, the proper dosage, and the appropriate method of administration. The instructions may also include guidance for monitoring the subject during the duration of the treatment period.

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

[0177] The kit provided herein can also include a device for administering a compound, such as a T cell activator or T cell stimulator, or a TLR agonist, such as a TLR4 agonist, to a subject.Any of the various devices known in the art for administering pharmaceuticals to a subject can be included in the kit provided herein.Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injections, hypodermic needles, intravenous needles, catheters, needleless injection devices, liquid dispensers such as inhalers and eyedroppers.Typically, the device for administering the compound of the kit will be compatible with the desired method of administration of the compound.

[0178] In certain embodiments, the formulation of pharma- ceutically acceptable carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for use in various treatment regimens of certain compositions described herein, including, for example, enteral and parenteral administration, such as intravascular, intravenous, intraarterial, intraosseous, intravenous, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulations.It will be understood by those skilled in the art that certain embodiments contemplated herein may include other formulations, such as those well known in the pharmaceutical field and described, for example, in Remington: The Science and Practice of Pharmacy, Vol. I and Vol. II, 22nd Edition, Edited by Loyd V. Allen Jr., Philadelphia, PA, Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0179] All publications, patent applications, and issued patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0180] Although the above embodiments have been described in some detail by way of illustration and examples for clarity of understanding, it will be apparent to one of ordinary skill in the art in light of the teachings contemplated herein that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims. The following examples are offered by way of illustration only, and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to provide essentially similar results. EXAMPLES

[0181] Example 1 MAGE-A4-TCR transgenic T cells express MAGE-A4 GVY - Binds to MHC multimers To isolate MAGE-A4-reactive T cell clones, we used an in vitro sensitization approach, in which 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 responder cells. In vitro transcribed RNA (ivtRNA) encoding the human MAGEA4 gene served as the source of specific antigen. After electroporation into mDCs, ivtRNA encoding MAGE-A4 was transcribed into protein, which was subsequently processed and presented as peptides on mDCs by molecules encoded by HLA-A*02:01. For 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 de novo induction of antigen-specific T cells, which served as the source of the corresponding TCR. Antigen-specific T cells could be enriched in different ways and cloned by limiting dilution or FACS-based single-cell sorting. The sequences of the TCR alpha and beta chains of MAGE-A4-reactive T cell clones were identified by next-generation sequencing, and the constant TCR regions were exchanged with their murine counterparts before cloning into the retroviral vector pES.12-6. PBMCs from healthy donors were isolated by Ficoll gradient centrifugation. CD8 + T cells were enriched and stimulated in non-tissue culture 24-well plates precoated 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 HEK293T cells with each TCR-encoding retroviral plasmid and two expression plasmids. Two days after stimulation, CD8 + T cells were transduced and on day 12, transduced CD8 T cells were identified by FACS using the mouse constant beta region as a marker for transduction. +The cells were concentrated and then expanded by a rapid expansion protocol (Riddell SR, Science, 1992 Jul 10;257(5067):238-41).

[0182] The experiments described in Examples 2-5 tested TCRs containing murine Cα and Cβ regions (i.e., TCR-1 comprising an α chain of SEQ ID NO:57 and a β chain of SEQ ID NO:58, TCR-2 comprising an α chain of SEQ ID NO:59 and a β chain of SEQ ID NO:60, and TCR-3 comprising an α chain of SEQ ID NO:61 and a β chain of SEQ ID NO:62). The same types of experiments described in Examples 2-5 may also be performed with TCRs containing the minimal murine Cα and Cβ regions described above.

[0183] result: CD8 + T cells were transduced with three different TCRs isolated from MAGE-A4-reactive T cell clones and one control TCR that does not recognize MAGE-A4. GVY -MHC multimers (MAGE-A4 230-239 , GVYDGREHTV; immuneAware) and antibodies against CD8 and mouse constant beta region. GVY -Bound extremely efficiently to MHC multimers (>70%). MAGE-A4 GVY No -MHC multimer staining was observed with control TCRs. These results demonstrate that TCRs isolated from MAGE-A4-reactive T cell clones can be transgenically expressed in T cells from healthy donors (Figure 1).

[0184] Example 2 MAGE-A4-TCR transgenic T cells express 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) were injected with a saturating dose (10 -5 M)'s MAGE-A4 GVY The target cells were loaded with either the IFN-γ peptide (SEQ ID NO:1) or an irrelevant control peptide. In addition, K562 cells were transduced with HLA-A*02:01 and MAGE-A4 genes (K562 / A2 / MAGE-A4). K562 cells transduced with HLA-A*02:01 alone were used as a control (K562 / A2). Each target cell line was incubated with TCR transgenic T cells in a 2:1 ratio, with 20,000 T cells and 10,000 target cells. After 20-24 hours, IFN-γ concentrations in the co-culture supernatants were analyzed by standard sandwich ELISA (BD human IFN-γ ELISA set).

[0185] result: MAGE-A4-TCR transgenic T cells express MAGE-A4 GVY MAGE-A4-reactive T cell clones recognized MAGE-A4-loaded T2 cells and MAGE-A4-transduced K562 cells, but not control target cells. TCR-3-transduced T cells showed recognition of the K562 / A2 control. These results indicate that TCRs isolated from MAGE-A4-reactive 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 used peptide-loaded T2 cells to analyze differences in the functional avidity of MAGE-A4-TCR transgenic T cells.

[0187] MAGE-A4 in T2 cells GVY The peptides were diluted in graded concentrations (10 -11 M~10 -5M) and co-cultured TCR transgenic T cells with 10,000 T2 cells and 20,000 T cells at a ratio of 1:2. After 20–24 h, IFN-γ concentrations in the co-culture supernatants were analyzed by standard sandwich ELISA (BD human IFN-γ ELISA set).

[0188] result: Collectively, across multiple donors, MAGE-A4 was loaded on HLA-A*02. GVY The highest functional avidity for the peptide was exhibited by TCR-2, with TCR-1 and TCR-3 showing 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 lines (Saos-2) and MAGE-A4 negative HLA-A2 negative tumor cells (A549) were used as target cells. For cytotoxicity assays, co-cultures were set up with 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, respectively, at an effector-to-target ratio of approximately 8–16:1 (depending on target cell size). Saturating concentrations of MAGE-A4 GVY Peptides (10 -5 Tumor cells loaded with IFN-γ (IgA, M) were used as an internal positive control. The reduction of fluorescent target cells (cell counts 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, co-culture supernatants were harvested after 24 hours and the respective IFN-γ concentrations were analyzed by 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 saturating concentrations of MAGE-A4. GVY Only when the peptide was exogenously loaded into the cells were they recognized and lysed. A negative control cell line (A549) was not recognized or lysed by any of the MAGE-A4-TCRs. These results indicate that MAGE-A4-TCR transgenic T cells can lyse endogenously MAGE-A4 positive tumor cells with remarkable selectivity and efficiency (Figure 4a, b, and c).

[0191] Example 5 MAGE-A4-TCR transgenic T cells do not recognize normal human cells A panel of normal human cells was used to analyze potential on-target / off-tumor and off-target toxicities that may be caused by MAGE-A4-TCR transgenic T cells.

[0192] Primary 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-A*02:01-negative NHBE cells were transiently expressed with HLA-A2 by transfecting with HLA-A2-ivtRNA by electroporation. iCell Neuron were treated with IFN-γ for 72 h to induce cell surface HLA-A2 expression prior to the initiation of coculture. HLA-A2 expression in all cell types was confirmed by flow cytometry. For cytotoxicity assays, cocultures 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 IFN-γ at a final concentration of 10 -5M's MAGE-A4 GVY To analyze cytokine release, co-culture supernatants were harvested after 24 h and IFN-γ or IL-2 concentrations were analyzed by standard sandwich ELISA (BD human IFN-γ or IL-2 ELISA set). For co-cultures with iCell neurons that had been 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 (iPSC)-derived cells were HLA-A2 positive. Furthermore, MAGE-A4-TCR transgenic T cells were able to express MAGE-A4 on individual target cells. GVY All normal cells could be efficiently recognized when loaded with peptides. Unloaded normal cells were not recognized by any of the MAGE-A4 transgenic T cells. The MAGE-A4 transgenic T cells showed no signs of on-target / off-tumor and off-target toxicity (Figure 5a, Figure 5b, and Figure 5c).

[0194] Example 6 Lentiviral vector encoding the fully human MAGE-A4 TCR The TCR polynucleotide sequence identified in Example 1 was optimized for expression. The TCR was expressed using a lentiviral vector encoding a polycistronic TCR construct. The polycistronic TCR construct contains a TCR alpha or beta chain, an optional furin cleavage site, a P2A ribosomal skip sequence, and the corresponding TCR alpha or beta chain. The lentiviral vector was produced according to known methods. See, for example, Kutner et al., BMC Biotechnol. 2009; 9:10. doi:10.1186 / 1472-6750-9-10, 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 beta chain encoded by SEQ ID NO:70, a polynucleotide encoding a ribosomal skip sequence, and an alpha 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 beta chain encoded by SEQ ID NO:78, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosomal skip sequence, and an alpha 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 beta chain encoded by SEQ ID NO:86, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosomal skip sequence, and an alpha chain encoded by SEQ ID NO:85.

[0198] Example 7 T cells expressing the MAGE-A4 fully human TCR GVY - Binds to MHC multimers 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 GVY -MHC multimers (MAGE-A4 230-239 The population was stained with IgG4-specific IgG4 antibodies (GVYDGREHTV; immuneAware) and antibodies against CD3. + Gating was performed on cells and multimer staining. Both MAGE-A4-TCR transgenic T cell populations expressed MAGE-A4 GVY -Bound extremely efficiently to MHC multimers (>70%). MAGE-A4 GVY -MHC multimer staining was not observed with control TCR.

[0199] result: These results demonstrate that TCRs isolated from MAGE-A4-reactive 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 GVY Recognizes peptides Antigen-dependent cytokine expression was used to confirm MAGE-A4 specificity of TCR transgenic T cells. T cells transduced with lentiviral vectors encoding the fully human MAGE-A4 TCR as described in Example 6 were incubated with 10 ng / mL of MAGE-A4 GVYT2 cells (HLA-A*02 positive) pulsed with peptides or an irrelevant control peptide were cocultured with untransduced A549 / HLA-A2 cells or A549 / HLA-A2 cells transduced with the MAGE-A4 gene at an effector-to-target cell ratio of 2:1. After 20-24 hours, IFN-γ concentrations in the coculture supernatants were analyzed using a Luminex assay.

[0201] result: MAGE-A4-TCR transgenic T cells express MAGE-A4 GVY These results suggest that healthy human donor T cells expressing the fully human MAGE-A4 TCR recognize MAGE-A4-loaded T2 and MAGE-A4-transduced A549 cells, but not control target cells. GVY It has been shown that it specifically reacts with target cells displaying the peptide (Figure 7).

[0202] Example 9 T cells expressing the MAGE-A4 fully human TCR exhibit high functional avidity MAGE-A4 GVY The peptide-displaying tumor cell lines were used to analyze the difference in functional avidity of T cells expressing the fully human MAGE-A4-TCR. T cells transduced with lentiviral vectors 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, IFN-γ concentrations in the co-culture supernatants were analyzed using a Luminex assay.

[0203] result: Across multiple donors, MAGE-A4 TCR5 showed the highest functional avidity for MAGE-A4-positive HLA-A2-positive tumor cell lines, whereas 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 fully human TCR lyse MAGE-A4-positive, HLA-A2-positive tumor cell lines T cells transduced with lentiviral vectors 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, A549-HLA-A2-MAGE-A4) and MAGE-A4 negative HLA-A2 positive tumor cell lines (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 untransduced control T cells (Figure 9).

[0206] Example 11 T cells expressing the MAGE-A4 fully human TCR suppress MAGE-A4-positive tumors in vivo 5×10 6 MAGE-A4 positive A375 tumor cells were injected into each flank of 10 NSG mice. Ten days after tumor implantation, 3.5 × 10 7 MAGE-A4-TCR transgenic T cells, 3.5 x 107 Mice were administered control non-transduced T cells or vehicle PBS. 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 the vehicle PBS were unable to suppress tumor growth and were sacrificed when tumors reached the maximum size allowed by the protocol. Mice treated with MAGE-A4-TCR transgenic T cells suppressed tumor growth up to 35 days after T cell injection (Figure 10).

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

[0209] Enhanced MAGE-A4 TCR (TCR-7, TCR-8 and TCR-9) was 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 ribosomal 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, 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 beta chain encoded by SEQ ID NO:100, a polynucleotide encoding a ribosomal skip sequence, and an alpha 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 beta chain encoded by SEQ ID NO:106, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosomal skip sequence, and an alpha 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 beta chain encoded by SEQ ID NO:112, a polynucleotide encoding a furin cleavage site, a polynucleotide encoding a ribosomal skip sequence, and an alpha chain encoded by SEQ ID NO:111.

[0213] Example 13 T cells expressing the 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 the fully human MAGE-A4 TCR (TCR-5) or enhanced MAGE-A4 TCR (TCR-8), or left untransduced as a negative control. Cells were cultured for in vitro expansion and analyzed for vector integration by measuring vector copy number (VCN) and MAGE-A4 TCR expression. GVY -MHC multimers (MAGE-A4 230-239 Expression was analyzed using flow cytometry on cells stained with antibodies against CD3 (immuneAware) and CD4.

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

[0215] Example 14 T cells expressing the MAGE-A4 fully human 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 the fully human MAGE-A4 TCR (TCR-5) or enhanced MAGE-A4 TCR (TCR-8), or untransduced (UTD) cells as a negative control. T cells expressing the TCR were assessed for specific reactivity against 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 and TCR-8 T cells released IFNγ when co-cultured with HLA-A2+ / MAGEA4(+) tumor cell lines, but not when co-cultured with HLA-A2+ / MAGEA4(-) cells or in the absence of target cells. UTD T cells did not release IFNγ under any culture condition. Figure 12A.

[0217] TCR-5 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 the MAGE-A4 fully human 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. 3 Mice bearing A375 tumors were treated with PBS (vehicle), untransduced T cells (UTD), or 5 × 10 6TCR-5 or TCR-8 T cells (left flank), or 1.5 × 10 6 TCR-5 or TCR-8 T cells (right flank) were administered. 3 Mice bearing A375 tumors were treated with PBS (vehicle), untransduced (UTD) T cells, or 10 × 10 6 Mice were administered either TCR-5 or TCR-8 T cells. Tumor growth was measured twice weekly and the anti-tumor activity of the TCR T cells was assessed in comparison to mice receiving a UTD control and a vehicle control.

[0219] result: TCR-5 and TCR-8 T cells were administered at a dose of 5 × 10 6 TCR+ T cells, 50mm 3 TCR-8 T cells mediated comparable tumor regression of A375 tumors at a dose of 1.5 × 10 compared with TCR-5 T cells. 6 50mm for TCR+ T cells 3 of A375 tumors, or a dose of 10 x 10 6 100mm for TCR+ T cells 3 Vehicle and UTD T cells did not mediate regression of A375 tumors under any condition.

[0220] The present invention is further characterized by the following items.

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

[0222] Clause 2: a) a variable TCR alpha 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 TCR beta 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 TCR alpha region, comprising a CDR1 having the amino acid sequence of SEQ ID NO:12, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:14; A variable TCR beta region comprising a CDR1 having the amino acid sequence of SEQ ID NO:15, a CDR2 having the amino acid sequence of SEQ ID NO:16, and a CDR3 having the amino acid sequence of SEQ ID NO:17; or c) a variable TCR alpha region, comprising a CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having the amino acid sequence of SEQ ID NO:23, and a CDR3 having the amino acid sequence of SEQ ID NO:24; A variable TCR beta region comprising a CDR1 having the amino acid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQ ID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:27. An isolated T cell receptor (TCR) specific for MAGE-A4.

[0223] Item 3: An isolated TCR according to any one of the preceding items, which specifically recognises the amino acid sequence of SEQ ID NO:1 or a fragment thereof.

[0224] Item 4: An isolated TCR according to any one of the preceding items, which specifically recognises the amino acid sequence of SEQ ID NO:1 in its HLA-A2-bound form.

[0225] Item 5: An isolated TCR according to any one of the preceding items, which specifically recognises 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 preceding items, comprising a TCR alpha chain comprising 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 preceding items, comprising a TCR beta chain comprising 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] Clause 8: a) a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:8 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:9; or b) a variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID NO:18 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID NO:19; or c) a variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID NO:28 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID NO:29 The isolated TCR of any one of the preceding items.

[0229] Item 9: a) a variable TCR alpha region having the amino acid sequence of SEQ ID NO:8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:9; or b) a variable TCR α region having the amino acid sequence of SEQ ID NO:18 and a variable TCR β region having the amino acid sequence of SEQ ID NO:19; or c) a variable TCR α region having the amino acid sequence of SEQ ID NO:28 and a variable TCR β region having the amino acid sequence of SEQ ID NO:29 The isolated TCR of any one of the preceding items.

[0230] Clause 10: a) a TCR alpha chain having an amino acid sequence at least 80% identical to SEQ ID NO:10 and a TCR beta chain having an amino acid sequence at least 80% identical to SEQ ID NO:11; or b) a TCR α chain having an amino acid sequence at least 80% identical to SEQ ID NO:20 and a TCR β chain having an amino acid sequence at least 80% identical to SEQ ID NO:21; or c) a TCR α chain having an amino acid sequence at least 80% identical to SEQ ID NO:30 and a TCR β chain having an amino acid sequence at least 80% identical to SEQ ID NO:31. The isolated TCR of any one of the preceding items.

[0231] Item 11: a) a TCR alpha chain having the amino acid sequence of SEQ ID NO:10 and a TCR beta chain having the amino acid sequence of SEQ ID NO:11; or b) a TCR α chain having the amino acid sequence of SEQ ID NO:20 and a TCR β chain having the amino acid sequence of SEQ ID NO:21; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:30 and a TCR β chain having the amino acid sequence of SEQ ID NO:31 The isolated TCR of any one of the preceding items.

[0232] Clause 12: The TCR comprises a TCR alpha chain and a TCR beta chain; a)-the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:8 and comprises a CDR3 encoded by the amino acid sequence set forth in SEQ ID NO:4; - the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID NO:9 and comprises a CDR3 encoded by the amino acid sequence set forth in SEQ ID NO:7; or b)-the variable TCR alpha region has an amino acid sequence at least 80% identical to SEQ ID NO:18 and comprises a CDR3 encoded by the amino acid sequence set forth in SEQ ID NO:14; or - the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID NO:19 and comprises a CDR3 encoded by the amino acid sequence set forth in SEQ ID NO:17; or c)-the variable TCR alpha region has an amino acid sequence at least 80% identical to SEQ ID NO:28 and comprises a CDR3 encoded by the amino acid sequence set forth in SEQ ID NO:24; or - the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID NO:29 and includes a CDR3 encoded by the amino acid sequence set forth in SEQ ID NO:27; An isolated TCR according to any one of the preceding items.

[0233] Item 13: a) a TCR alpha chain having the amino acid sequence of SEQ ID NO:10 and a TCR beta chain having the amino acid sequence of SEQ ID NO:11; b) a TCR α chain having the amino acid sequence of SEQ ID NO:20 and a TCR β chain having the amino acid sequence of SEQ ID NO:21; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:30 and a TCR β chain having the amino acid sequence of SEQ ID NO:31 6. An isolated TCR according to any one of paragraphs 1 to 5, comprising:

[0234] Item 14: a) a TCR alpha chain having the amino acid sequence of SEQ ID NO:87 and a TCR beta chain having the amino acid sequence of SEQ ID NO:88; b) a TCR α chain having the amino acid sequence of SEQ ID NO:89 and a TCR β chain having the amino acid sequence of SEQ ID NO:90; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:91 and a TCR β chain having the amino acid sequence of SEQ ID NO:92 6. An isolated TCR according to any one of paragraphs 1 to 5, comprising:

[0235] Item 15: a) a TCR alpha chain having the amino acid sequence of SEQ ID NO:102 and a TCR beta chain having the amino acid sequence of SEQ ID NO:103; b) a TCR α chain having the amino acid sequence of SEQ ID NO:108 and a TCR β chain having the amino acid sequence of SEQ ID NO:109; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:114 and a TCR β chain having the amino acid sequence of SEQ ID NO:115 6. An isolated TCR according to any one of paragraphs 1 to 5, comprising:

[0236] Item 16: The isolated TCR of any one of the preceding items, which is purified.

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

[0238] Clause 18: An isolated TCR according to any one of the preceding clauses, the amino acid sequence of which has been modified to include a detectable label, a therapeutic agent or a pharmacokinetic-modifying moiety.

[0239] Clause 19: The isolated TCR of clause 18, wherein the therapeutic agent is selected from the group consisting of an immune effector molecule, a cytotoxic agent and a radionuclide.

[0240] Item 20: The isolated TCR described in item 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] Item 22: The isolated TCR of item 18, wherein the pharmacokinetic modifying moiety is at least one polyethylene glycol repeat 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, which is a single-chain type in which the TCR alpha chain and the TCR beta chain are linked by a linker sequence.

[0244] Clause 24: An isolated TCR described in any one of clauses 1 to 23, wherein the TCR alpha chain or the TCR beta chain has been modified to include an epitope tag.

[0245] Paragraph 25: An isolated polypeptide comprising a functional portion of a 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] Clause 26: The isolated polypeptide of clause 25, wherein the functional moiety comprises a TCR alpha variable chain and / or a TCR beta variable chain.

[0247] Item 27: A fusion protein comprising a TCR alpha chain and a TCR beta chain, the fusion protein comprising an amino acid sequence as set forth in any one of SEQ ID NOs:94, 96, 98, 104, 110 and 116.

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

[0249] Clause 29: A multivalent TCR complex according to clause 28, wherein at least one of the TCRs is linked to a therapeutic agent.

[0250] Clause 30: An isolated TCR described in any one of clauses 1 to 24, a polypeptide described in clause 25 or clause 26, a fusion protein described in clause 27, or a multivalent TCR complex described in clause 28 or clause 29, which induces IFN-γ secretion by binding to the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-A*02:01.

[0251] Clause 31: A nucleic acid encoding a TCR according to any one of clauses 1 to 24, encoding a polypeptide according to clause 25 or clause 26, or encoding a fusion protein according to clause 27.

[0252] Item 32: The nucleic acid of item 31, wherein the nucleic acid sequence encoding the TCR alpha chain is set forth in any one of SEQ ID NOs:69, 77, 85, 99, 105 and 111.

[0253] Item 33: The nucleic acid of item 31 or 32, wherein the nucleic acid sequence encoding the TCR β chain is set forth in any one of SEQ ID NOs:70, 78, 86, 100, 106 and 112.

[0254] Clause 34: The nucleic acid of clause 31, wherein the TCR comprises an alpha chain encoded by SEQ ID NO:69 and a beta chain encoded by SEQ ID NO:70; an alpha chain encoded by SEQ ID NO:77 and a beta chain encoded by SEQ ID NO:78; an alpha chain encoded by SEQ ID NO:85 and a beta chain encoded by SEQ ID NO:86; an alpha chain encoded by SEQ ID NO:99 and a beta chain encoded by SEQ ID NO:100; an alpha chain encoded by SEQ ID NO:105 and a beta chain encoded by SEQ ID NO:106; or an alpha chain encoded by SEQ ID NO:111 and a beta chain encoded by SEQ ID NO:112.

[0255] Clause 35: The nucleic acid of clause 31, wherein the fusion protein is encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs:93, 95, 97, 101, 107 and 113.

[0256] Item 36: A vector comprising the nucleic acid according to any one of items 31 to 35.

[0257] Item 37: A vector comprising a nucleic acid encoding (a) the polypeptide sequence set forth in SEQ ID NO:87 and SEQ ID NO:88; (b) the polypeptide sequence set forth in SEQ ID NO:89 and SEQ ID NO:90; (c) the polypeptide sequence set forth in SEQ ID NO:91 and SEQ ID NO:92; (d) the polypeptide sequence set forth in SEQ ID NO:102 and SEQ ID NO:103; (e) the polypeptide sequence set forth in SEQ ID NO:108 and SEQ ID NO:109; or (f) the polypeptide sequence set forth in SEQ ID NO:114 and SEQ ID NO:115.

[0258] Clause 38: A vector according to clause 36 or clause 37 which is an expression vector.

[0259] Clause 39: The vector according to any one of clauses 36 to 38, which is a retroviral vector.

[0260] Clause 40: The vector according to any one of clauses 36 to 39, which is a lentiviral vector.

[0261] Paragraph 41: A cell expressing the TCR described in any one of paragraphs 1 to 24.

[0262] Clause 42: A cell comprising the vector described in any one of clauses 36 to 40.

[0263] Clause 43: The cell of clause 41 or clause 3942, which is isolated or non-naturally occurring.

[0264] Clause 44: A cell comprising a nucleic acid according to any one of clauses 31 to 35 or a vector according to any one of clauses 36 to 40.

[0265] Item 45: a) an expression vector comprising at least one nucleic acid according to any one of items 28 to 32; b) a first expression vector comprising a nucleic acid encoding the alpha chain of the TCR according to any one of paragraphs 1 to 21 and a second expression vector comprising a nucleic acid encoding the beta chain of the TCR according to any one of paragraphs 1 to 21. The cell according to any one of items 41 to 44, comprising:

[0266] Clause 46: The cell of any one of clauses 41 to 45, which is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).

[0267] Clause 49: The cell of any one of clauses 41 to 48, which is a T cell.

[0268] Clause 50: The cell described in any one of clauses 41 to 48, which is a T cell.

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

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

[0271] Clause 53: A composition comprising a TCR according to any one of clauses 1 to 24, a polypeptide according to clause 25 or clause 26, a fusion protein according to clause 27, a multivalent TCR complex according to clause 28 or clause 29, a nucleic acid according to any one of clauses 31 to 35, a vector according to any one of clauses 36 to 40, a cell according to any one of clauses 41 to 50, or an antibody according to clause 51 or clause 52.

[0272] Clause 54: A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a TCR according to any one of clauses 1 to 24, a polypeptide according to clause 25 or clause 26, a fusion protein according to clause 27, a multivalent TCR complex according to clause 28 or clause 29, a nucleic acid according to any one of clauses 31 to 35, a vector according to any one of clauses 36 to 40, a cell according to any one of clauses 41 to 50, or an antibody according to clause 51 or clause 52.

[0273] Clause 55: A pharmaceutical composition comprising at least one pharma- ceutically acceptable carrier and a cell according to any one of clauses 41 to 50.

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

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

[0276] Clause 58: The TCR, polypeptide, fusion protein, multivalent TCR complex, nucleic acid, cell, antibody, composition or pharmaceutical composition according to clause 57, wherein the cancer is a blood cancer or a solid tumor.

[0277] Clause 59: The TCR, polypeptide, fusion protein, multivalent TCR complex, nucleic acid, cell, antibody, composition or pharmaceutical composition according to clause 57 or 58, wherein the cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin's 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.

[0278] Clause 60: The TCR, polypeptide, fusion protein, multivalent TCR complex, nucleic acid, cell, antibody, composition or pharmaceutical composition according to clause 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 TIFF2025024103000005.tif209150TIFF2025024103000006.tif202149TIFF2025024103000007.tif160150

[0280] In general, in the appended claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Thus, the claims are not limited by the present 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 Read 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 Read 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 and Gly Phe Asn and Met and 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 actgcctcc 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 and Gly Phe Asn and Met and 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) a variable TCR alpha 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 TCR beta 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 TCR alpha region, comprising a CDR1 having the amino acid sequence of SEQ ID NO:12, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:14; A variable TCR beta region comprising a CDR1 having the amino acid sequence of SEQ ID NO:15, a CDR2 having the amino acid sequence of SEQ ID NO:16, and a CDR3 having the amino acid sequence of SEQ ID NO:17; or c) a variable TCR alpha region, comprising a CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having the amino acid sequence of SEQ ID NO:23, and a CDR3 having the amino acid sequence of SEQ ID NO:24; A variable TCR beta region comprising a CDR1 having the amino acid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQ ID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:

27.

1. An isolated T cell receptor (TCR) specific for MAGE-A4, comprising: The isolated T cell receptor (TCR) which specifically recognizes the amino acid sequence of SEQ ID NO:1 or a fragment thereof in a form bound to HLA-A2.

2. The amino acid sequence of SEQ ID NO:1 presented by the HLA-A*02:01-encoded molecule The isolated TCR of claim 1, which specifically recognizes

3. a) a variable TCR α region having the amino acid sequence of SEQ ID NO:8 and a variable TCR β region having the amino acid sequence of SEQ ID NO:9; or b) a variable TCR α region having the amino acid sequence of SEQ ID NO:18 and a variable TCR β region having the amino acid sequence of SEQ ID NO:19; or c) a variable TCR alpha region having the amino acid sequence of SEQ ID NO:28 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:29 3. The isolated TCR of claim 1 or 2, comprising:

4. a) a TCR α chain having the amino acid sequence of SEQ ID NO:10 and a TCR β chain having the amino acid sequence of SEQ ID NO:11; b) a TCR α chain having the amino acid sequence of SEQ ID NO:20 and a TCR β chain having the amino acid sequence of SEQ ID NO:21; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:30 and a TCR β chain having the amino acid sequence of SEQ ID NO:31 The isolated TCR of any one of claims 1 to 3, comprising:

5. a) a TCR α chain having the amino acid sequence of SEQ ID NO:87 and a TCR β chain having the amino acid sequence of SEQ ID NO:88; b) a TCR α chain having the amino acid sequence of SEQ ID NO:89 and a TCR β chain having the amino acid sequence of SEQ ID NO:90; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:91 and a TCR β chain having the amino acid sequence of SEQ ID NO:92 The isolated TCR of any one of claims 1 to 3, comprising:

6. a) a TCR α chain having the amino acid sequence of SEQ ID NO:102 and a TCR β chain having the amino acid sequence of SEQ ID NO:103; b) a TCR α chain having the amino acid sequence of SEQ ID NO:108 and a TCR β chain having the amino acid sequence of SEQ ID NO:109; or c) a TCR α chain having the amino acid sequence of SEQ ID NO:114 and a TCR β chain having the amino acid sequence of SEQ ID NO:115 The isolated TCR of any one of claims 1 to 3, comprising:

7. A multivalent TCR complex comprising at least two TCRs according to any one of claims 1 to 6.

8. A fusion protein comprising a TCR alpha chain and a TCR beta chain and comprising an amino acid sequence as set forth in any one of SEQ ID NOs:94, 96, 98, 104, 110 and 116.

9. A nucleic acid encoding a TCR according to any one of claims 1 to 6 or a fusion protein according to claim 8.

10. TCR, a) a TCR α chain encoded by SEQ ID NO:69 and a TCR β chain encoded by SEQ ID NO:70; b) a TCR α chain encoded by SEQ ID NO:77 and a TCR β chain encoded by SEQ ID NO:78; c) a TCR α chain encoded by SEQ ID NO:85 and a TCR β chain encoded by SEQ ID NO:86; d) a TCR α chain encoded by SEQ ID NO:99 and a TCR β chain encoded by SEQ ID NO:100; e) a TCR α chain encoded by SEQ ID NO:105 and a TCR β chain encoded by SEQ ID NO:106; or f) The TCR α chain encoded by SEQ ID NO:111 and the TCR β chain encoded by SEQ ID NO:112 The nucleic acid of claim 9, comprising:

11. The nucleic acid of claim 9, wherein the fusion protein is encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs:93, 95, 97, 101, 107 and 113.

12. A vector comprising the nucleic acid according to any one of claims 9 to 11.

13. A cell expressing the TCR according to any one of claims 1 to 6.

14. A cell comprising the nucleic acid or vector of any one of claims 9 to 12.

15. 15. The cell of claim 13 or 14, which is an immune effector cell.

16. The cell of claim 15, wherein the immune effector cell is a T cell, a natural killer (NK) cell or a natural killer T (NKT) cell.

17. A composition comprising a TCR according to any one of claims 1 to 6, a multivalent TCR complex according to claim 7, a fusion protein according to claim 8, a nucleic acid according to any one of claims 9 to 11, a vector according to claim 12, or a cell according to any one of claims 13 to 16.

18. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a TCR according to any one of claims 1 to 6, a multivalent TCR complex according to claim 7, a fusion protein according to claim 8, a nucleic acid according to any one of claims 9 to 11, a vector according to claim 12, or a cell according to any one of claims 13 to 16.

19. 19. The composition of claim 17, or the pharmaceutical composition of claim 18, for use in the treatment of a blood cancer or a solid tumor.

20. (a) the cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin's 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, ovarian or colorectal cancer, sarcoma, or osteosarcoma; 20. The composition or pharmaceutical composition of claim 19.

21. 21. The composition or pharmaceutical composition of claim 19 or 20, wherein the cancer is synovial sarcoma.

Images