TCR and peptides

Abstract

TCR and peptide. T cell receptor (TCR) that binds the Wilms tumor 1 protein (WT1) peptide when presented by the major histocompatibility complex (MHC).

Description

Field of the Invention

[0001] The present invention relates to T cell receptors (TCRs) that bind peptides derived from the Wilms tumor 1 protein (WT1) when presented by the major histocompatibility complex. In this regard, the present invention relates to complementary determining regions (CDRs) that specifically recognize WT1 peptides. The present invention further relates to immunogenic peptides derived from WT1. Background of the Invention

[0003] T cell receptor (TCR) gene therapy is based on the genetic transfer of high-affinity tumor-specific TCR genes into T lymphocytes, thereby being able to specifically target desired tumor-associated antigens and reduce less toxic and more specific and effective therapies. This approach has shown promise in clinical trials. One of the major obstacles limiting the use of TCR gene therapy for clinical cancer treatment is the lack of tumor-specific T cells and corresponding TCRs. Therefore, the low availability of tumor-specific TCRs remains an open problem that limits the widespread application of TCR-based immunotherapy methods.

[0004] Most tumor-associated antigens (TAAs) are self-antigens, so T cells specific for these molecules are destroyed or become unresponsive due to central and peripheral tolerance. Nevertheless, after allogeneic hematopoietic stem cell transplantation (allo-HSCT), naturally occurring tumor-specific T cells have been observed in healthy donors and patients, particularly in patients affected by hematological malignancies, in which the frequency of tumor-specific lymphocytes in the allogeneic hematopoietic stem cell transplantation is linked to disease regression (Kapp, M. et al. Bone Marrow Transplantation 43, 399–410 (2009); and Tyler, EM et al. Blood 121, 308–317 (2013)).

[0005] The selection of tumor antigens to be targeted by immunotherapeutic approaches remains a matter of debate. Ideally, TAAs are highly expressed on tumor cells and minimally expressed in healthy tissues.

[0006] Wilms tumor 1 (WT1) is an intracellular protein that encodes a zinc finger transcription factor that plays an important role in cell growth and differentiation (Yang, L. et al. Leukemia 21, 868-876 (2007)). WT1 is widely expressed in a variety of hematological tumors and solid tumors, while showing limited expression in various healthy tissues (e.g., gonads, uterus, kidneys, mesothelium, progenitor cells in different tissues). Recent evidence suggests a role for WT1 in leukemogenesis and tumorigenesis.

[0007] Several ongoing clinical trials rely on the generation of cytotoxic T lymphocyte (CTL) responses after vaccination with WT1 peptides. However, despite the recognition that WT1 can be used for immunotherapy, a small number of WT1 epitopes (which are restricted to a limited number of HLA alleles) are currently used for vaccination purposes (DiStasi, A. et al. Front. Immunol. (2015)). One such epitope is the WT1 126-134 epitope (RMFPNAPYL; SEQ ID NO: 255), which is presented by the MHC encoded by the HLA-A*0201 allele (i.e., the epitope is restricted by HLA-A*0201).

[0008] Because the major histocompatibility complex (MHC) with the HLA-A*0201 haplotype is expressed in the vast majority (60%) of the Caucasian population, HLA-A*0201-restricted epitopes and corresponding TCRs have attracted attention. Therefore, TCRs targeting HLA-A*0201-restricted WT1 epitopes are particularly advantageous because immunotherapy using such TCRs can be widely applied.

[0009] The WT1 126-134 epitope has been extensively studied in several trials, alone or in combination with other tumor antigens. However, recent reports have highlighted major concerns about the processing of this particular epitope, which may compromise its use for immunotherapy purposes. Notably, the immunoproteasome processes the WT1 126-134 epitope more efficiently than the standard proteasome (Jaigirdar, A. et al. J Immunother. 39(3):105-16 (2016)), which leads to poor recognition of many HLA-A*0201 tumor cell lines or primary leukemia cells that endogenously express WT1.

[0010] Therefore, there remains a need for new WT1 epitopes, particularly WT1 epitopes presented by MHC with prevalent HLA haplotypes (eg, HLA-A*0201).

[0011] One naturally processed HLA-A*0201 restricted epitope that has been identified is WT1 37-45, which has the amino acid sequence VLDFAPPGA (SEQ ID NO: 157, see e.g. Smithgall et al 2001; Blood 98(11 Part 1): 121a). However, few TCR amino acid sequences, particularly CDR sequences, specific for this peptide sequence have been reported (Schmitt, TM et al. (2017) Nat Biotechnol 35: 1188-1195).

[0012] Thus, there remains a need for new WT1 epitopes, particularly those restricted to common HLA alleles, and for new TCRs capable of binding WT1 epitopes. SUMMARY OF THE INVENTION

[0014] We have identified a novel TCR that binds to the WT1 peptide when presented by MHC. Furthermore, we have determined the amino acid sequence of the TCR, including the amino acid sequences of its CDR regions, which are responsible for its binding specificity for WT1. Furthermore, we have demonstrated that T cells expressing the TCR according to the present invention specifically target and kill cells expressing the WT1 protein. Furthermore, we have shown that the TCR of the present invention is restricted to MHC encoded by HLA class 1 and 2 alleles common in the Caucasian population, such as HLA-A*0201 and HLA-B*3501 or HLA-B*3502.

[0015] Thus, in a first aspect, the present invention provides a T cell receptor (TCR) that binds a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR:

[0016] (i) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0017] (ii) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO: 41) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0018] (iii) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0019] (iv) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0020] (v) comprising a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0021] (vi) comprising a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0022] (vii) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO: 57) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0023] (viii) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASTQTPYEQYF (SEQ ID NO: 63) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0024] (ix) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSTVGGEDYGYTF (SEQ ID NO: 69) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0025] (x) comprises a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO: 57) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0026] (xi) comprising a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASTQTPYEQYF (SEQ ID NO: 63) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0027] (xii) comprises a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSTVGGEDYGYTF (SEQ ID NO: 69) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0028] (xiii) comprising a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 75) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO: 80) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0029] (xiv) comprising a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 75) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO: 86) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0030] (xv) comprising a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0031] (xvi) comprising a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0032] (xvii) comprising a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0033] (xviii) comprising a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0034] (xix) comprising a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0035] (xx) comprising a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0036] (xxi) comprises a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0037] (xxii) comprising CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAVTVGNKLVF (SEQ ID NO: 175) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRGWREQFF (SEQ ID NO: 180) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0038] (xxiii) comprising a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CASSWGYQETQYF (SEQ ID NO: 196) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0039] (xxiv) comprising a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0040] (xxv) comprising a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0041] (xxvi) comprises a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CAASSWGYQETQYF (SEQ ID NO: 196) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0042] (xxvii) comprises a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0043] (xxviii) comprises a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0044] (xxix) comprising a CDR3α comprising the amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0045] (xxx) comprises a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0046] (xxxi) comprises a CDR3α comprising the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0047] (xxxii) comprises a CDR3α comprising the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0048] (xxxiii) comprises a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0049] (xxxiv) comprises a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0050] (xxxv) comprises a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0051] (xxxvi) comprises a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0052] (xxxvii) comprises a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0053] (xxxviii) comprises a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288), or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0054] (xxxix) comprises a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0055] (xxxx) comprises a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions, or deletions;

[0056] (xxxxi) comprises a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to 3 amino acid substitutions, additions, or deletions, and a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to 3 amino acid substitutions, additions, or deletions; or

[0057] (xxxxii) comprises CDR3α and CDR3β, wherein CDR3α comprises the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and wherein CDR3β comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0058] In one embodiment, the invention provides a TCR of the invention comprising the following CDR sequences:

[0059] (i) CDR1α-KALYS (SEQ ID NO: 1),

[0060] CDR2α-LLKGGEQ (SEQ ID NO: 2),

[0061] CDR3α-CGTAWINDYKLSF(SEQ ID NO:3),

[0062] CDR1β-SGHDY (SEQ ID NO: 6),

[0063] CDR2β-FNNNVP (SEQ ID NO: 7), and

[0064] CDR3β-CASRKTGGYSNQPQHF(SEQ ID NO:8),

[0065] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0066] (ii)CDR1α-NSASQS (SEQ ID NO:34),

[0067] CDR2α-VYSSGN (SEQ ID NO: 35),

[0068] CDR3α-CVVNLLSNQGGKLIF(SEQ ID NO:36),

[0069] CDR1β-LGHNA (SEQ ID NO: 39),

[0070] CDR2β-YSLEER (SEQ ID NO:40), and

[0071] CDR3β-CASSQDYLVSNEKLFF(SEQ ID NO:41),

[0072] or a variant thereof having up to 3 amino acid substitutions, additions or deletions; (iii) CDR1α-SSVPPY (SEQ ID NO: 12),

[0073] CDR2α-YTSAATLV(SEQ ID NO:13),

[0074] CDR3α-CAVRLSGSARQLTF(SEQ ID NO:14),

[0075] CDR1β-SGHAT (SEQ ID NO: 22),

[0076] CDR2β-FQNNGV (SEQ ID NO: 23), and

[0077] CDR3β-CASSLLGDEQYF(SEQ ID NO:24),

[0078] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0079] (iv)CDR1α-SSVPPY (SEQ ID NO:12),

[0080] CDR2α-YTSAATLV(SEQ ID NO:13),

[0081] CDR3α-CAVRLSGSARQLTF(SEQ ID NO:14),

[0082] CDR1β-SGHTA (SEQ ID NO: 28),

[0083] CDR2β-FQGNSA (SEQ ID NO: 29), and

[0084] CDR3β-CASSLVALQGAGEQYF(SEQ ID NO:30),

[0085] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0086] (v)CDR1α-TSESDYY(SEQ ID NO:17),

[0087] CDR2α-QEAYKQQN(SEQ ID NO:18),

[0088] CDR3α-CAYRSLKYGNKLVF(SEQ ID NO:19),

[0089] CDR1β-SGHAT (SEQ ID NO: 22),

[0090] CDR2β-FQNNGV (SEQ ID NO: 23), and

[0091] CDR3β-CASSLLGDEQYF(SEQ ID NO:24),

[0092] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0093] (vi)CDR1α-TSESDYY (SEQ ID NO:17),

[0094] CDR2α-QEAYKQQN(SEQ ID NO:18),

[0095] CDR3α-CAYRSLKYGNKLVF(SEQ ID NO:19),

[0096] CDR1β-SGHTA (SEQ ID NO: 28),

[0097] CDR2β-FQGNSA (SEQ ID NO: 29), and

[0098] CDR3β-CASSLVALQGAGEQYF(SEQ ID NO:30),

[0099] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0100] (vii)CDR1α–TSINN (SEQ ID NO:45),

[0101] CDR2α-IRSNERE (SEQ ID NO: 46),

[0102] CDR3α–CATDAYSGNTPLVF (SEQ ID NO:47),

[0103] CDR1β-MNHNS (SEQ ID NO: 55),

[0104] CDR2β-SASEGT (SEQ ID NO: 56), and

[0105] CDR3β-CASRAAGLDTEAFF(SEQ ID NO:57),

[0106] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0107] (viii)CDR1α–TSINN (SEQ ID NO:45),

[0108] CDR2α-IRSNERE (SEQ ID NO: 46),

[0109] CDR3α–CATDAYSGNTPLVF (SEQ ID NO:47),

[0110] CDR1β–MNHNY (SEQ ID NO: 61),

[0111] CDR2β-SVGAGI (SEQ ID NO: 62), and

[0112] CDR3β-CASTQTPYEQYF(SEQ ID NO:63),

[0113] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0114] (ix)CDR1α–TSINN (SEQ ID NO:45),

[0115] CDR2α-IRSNERE (SEQ ID NO: 46),

[0116] CDR3α–CATDAYSGNTPLVF (SEQ ID NO:47),

[0117] CDR1β–SGHNS (SEQ ID NO: 67),

[0118] CDR2β-FNNNVP (SEQ ID NO: 68), and

[0119] CDR3β–CASSTVGGEDYGYTF (SEQ ID NO:69),

[0120] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0121] (x)CDR1α-DSAIYN(SEQ ID NO:50),

[0122] CDR2α–IQSSQRE(SEQ ID NO:51),

[0123] CDR3α-CAVRAEYNQGGKLIF(SEQ ID NO:52),

[0124] CDR1β-MNHNS (SEQ ID NO: 55),

[0125] CDR2β-SASEGT (SEQ ID NO: 56), and

[0126] CDR3β-CASRAAGLDTEAFF(SEQ ID NO:57),

[0127] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0128] (xi)CDR1α-DSAIYN (SEQ ID NO:50),

[0129] CDR2α–IQSSQRE(SEQ ID NO:51),

[0130] CDR3α-CAVRAEYNQGGKLIF(SEQ ID NO:52),

[0131] CDR1β–MNHNY (SEQ ID NO: 61),

[0132] CDR2β-SVGAGI (SEQ ID NO: 62), and

[0133] CDR3β-CASTQTPYEQYF(SEQ ID NO:63),

[0134] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0135] (xii)CDR1α-DSAIYN (SEQ ID NO:50),

[0136] CDR2α–IQSSQRE(SEQ ID NO:51),

[0137] CDR3α-CAVRAEYNQGGKLIF(SEQ ID NO:52),

[0138] CDR1β–SGHNS (SEQ ID NO: 67),

[0139] CDR2β-FNNNVP (SEQ ID NO: 68), and

[0140] CDR3β–CASSTVGGEDYGYTF (SEQ ID NO:69),

[0141] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0142] (xiii)CDR1α-DSASNY (SEQ ID NO:73),

[0143] CDR2α–IRSNVGE (SEQ ID NO:74),

[0144] CDR3α-CAASMAGAGSYQLTF(SEQ ID NO:75),

[0145] CDR1β-ENHRY (SEQ ID NO: 78),

[0146] CDR2β–SYGVKD (SEQ ID NO: 79), and

[0147] CDR3β-CAISVGQGALYEQYF (SEQ ID NO:80),

[0148] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0149] (xiv)CDR1α-DSASNY (SEQ ID NO:73),

[0150] CDR2α–IRSNVGE (SEQ ID NO:74),

[0151] CDR3α-CAASMAGAGSYQLTF(SEQ ID NO:75),

[0152] CDR1β–SGDLS (SEQ ID NO:84),

[0153] CDR2β–YYNGEE (SEQ ID NO:85), and

[0154] CDR3β–CASSVARDRRNYGYTF (SEQ ID NO:86),

[0155] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0156] (xv)CDR1α-NSMFDY (SEQ ID NO:90),

[0157] CDR2α–ISSIKDK(SEQ ID NO:91),

[0158] CDR3α-CAANNARLMF(SEQ ID NO:92),

[0159] CDR1β–SGHNS (SEQ ID NO:95),

[0160] CDR2β–FNNNVP (SEQ ID NO: 96), and

[0161] CDR3β–CASSDTRAREQFF(SEQ ID NO:97),

[0162] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0163] (xvi)CDR1α-DSSSTY (SEQ ID NO:101),

[0164] CDR2α–IFSNMDM (SEQ ID NO:102),

[0165] CDR3α-CAERLNTDKLIF(SEQ ID NO:103),

[0166] CDR1β–DFQATT(SEQ ID NO:161),

[0167] CDR2β–SNEGSKA (SEQ ID NO:162), and

[0168] CDR3β–CSARDSVSGNTIYF(SEQ ID NO:163),

[0169] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0170] (xvii)CDR1α-DSSSTY (SEQ ID NO:101),

[0171] CDR2α–IFSNMDM (SEQ ID NO:102),

[0172] CDR3α-CAERLNTDKLIF(SEQ ID NO:103),

[0173] CDR1β–SQVTM (SEQ ID NO:167),

[0174] CDR2β–ANQGSEA (SEQ ID NO:168), and

[0175] CDR3β–CSVGGSGSYNEQFF(SEQ ID NO:169),

[0176] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0177] (xviii)CDR1α-DSVNN (SEQ ID NO:106),

[0178] CDR2α–IPSGT (SEQ ID NO:107),

[0179] CDR3α-CAVEATDSWGKLQF(SEQ ID NO:108),

[0180] CDR1β–DFQATT(SEQ ID NO:161),

[0181] CDR2β–SNEGSKA (SEQ ID NO:162), and

[0182] CDR3β–CSARDSVSGNTIYF(SEQ ID NO:163),

[0183] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0184] (xix)CDR1α-DSVNN (SEQ ID NO:106),

[0185] CDR2α–IPSGT (SEQ ID NO:107),

[0186] CDR3α-CAVEATDSWGKLQF(SEQ ID NO:108),

[0187] CDR1β–SQVTM (SEQ ID NO:167),

[0188] CDR2β–ANQGSEA (SEQ ID NO:168), and

[0189] CDR3β–CSVGGSGSYNEQFF(SEQ ID NO:169),

[0190] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0191] (xx)CDR1α-DSASNY(SEQ ID NO:111),

[0192] CDR2α–IRSNVGE (SEQ ID NO:112),

[0193] CDR3α-CAVRTSYDKVIF(SEQ ID NO:113),

[0194] CDR1β–DFQATT(SEQ ID NO:161),

[0195] CDR2β–SNEGSKA (SEQ ID NO:162), and

[0196] CDR3β–CSARDSVSGNTIYF(SEQ ID NO:163),

[0197] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0198] (xxi)CDR1α-DSASNY(SEQ ID NO:111),

[0199] CDR2α–IRSNVGE (SEQ ID NO:112),

[0200] CDR3α-CAVRTSYDKVIF(SEQ ID NO:113),

[0201] CDR1β–SQVTM (SEQ ID NO:167),

[0202] CDR2β–ANQGSEA (SEQ ID NO:168), and

[0203] CDR3β–CSVGGSGSYNEQFF(SEQ ID NO:169),

[0204] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0205] (xxii)CDR1α-VGISA (SEQ ID NO:173),

[0206] CDR2α–LSSGK(SEQ ID NO:174),

[0207] CDR3α-CAVTVGNKLVF(SEQ ID NO:175),

[0208] CDR1β–MNHNS (SEQ ID NO:178),

[0209] CDR2β–SASEGT (SEQ ID NO: 179), and

[0210] CDR3β–CASRGWREQFF(SEQ ID NO:180),

[0211] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0212] (xxiii)CDR1α-VGISA (SEQ ID NO:184),

[0213] CDR2α–LSSGK(SEQ ID NO:185),

[0214] CDR3α-CAARSYNTDKLIF(SEQ ID NO:186),

[0215] CDR1β–SGHTS (SEQ ID NO:194),

[0216] CDR2β–YDEGEE (SEQ ID NO:195), and

[0217] CDR3β–CASSWGYQETQYF (SEQ ID NO:196),

[0218] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0219] (xxiv)CDR1α-VGISA (SEQ ID NO:184),

[0220] CDR2α–LSSGK(SEQ ID NO:185),

[0221] CDR3α-CAARSYNTDKLIF(SEQ ID NO:186),

[0222] CDR1β–KGHSH (SEQ ID NO:200),

[0223] CDR2β–LQKENI (SEQ ID NO:201), and

[0224] CDR3β–CASSPTGGEYYGYTF (SEQ ID NO:202),

[0225] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0226] (xxv)CDR1α-VGISA (SEQ ID NO:184),

[0227] CDR2α–LSSGK(SEQ ID NO:185),

[0228] CDR3α-CAARSYNTDKLIF(SEQ ID NO:186),

[0229] CDR1β–MNHEY(SEQ ID NO:206),

[0230] CDR2β–SVGAGI (SEQ ID NO: 207), and

[0231] CDR3β–CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO:208),

[0232] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0233] (xxvi)CDR1α-NSMFDY(SEQ ID NO:189),

[0234] CDR2α–ISSIKDK(SEQ ID NO:190),

[0235] CDR3α-CAASYNNARLMF(SEQ ID NO:191),

[0236] CDR1β–SGHTS (SEQ ID NO:194),

[0237] CDR2β–YDEGEE (SEQ ID NO:195), and

[0238] CDR3β–CASSWGYQETQYF (SEQ ID NO:196),

[0239] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0240] (xxvii)CDR1α-NSMFDY (SEQ ID NO:189),

[0241] CDR2α–ISSIKDK(SEQ ID NO:190),

[0242] CDR3α-CAASYNNARLMF(SEQ ID NO:191),

[0243] CDR1β–KGHSH (SEQ ID NO:200),

[0244] CDR2β–LQKENI (SEQ ID NO:201), and

[0245] CDR3β–CASSPTGGEYYGYTF (SEQ ID NO:202),

[0246] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0247] (xxviii)CDR1α-NSMFDY (SEQ ID NO:189),

[0248] CDR2α–ISSIKDK(SEQ ID NO:190),

[0249] CDR3α-CAASYNNARLMF(SEQ ID NO:191),

[0250] CDR1β–MNHEY(SEQ ID NO:206),

[0251] CDR2β–SVGAGI (SEQ ID NO: 207), and

[0252] CDR3β–CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO:208),

[0253] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0254] (xxix)CDR1α-NSMFDY(SEQ ID NO:212),

[0255] CDR2α–ISSIKDK(SEQ ID NO:213),

[0256] CDR3α-CAASGGRDDKIIF (SEQ ID NO:214),

[0257] CDR1β–MNHEY(SEQ ID NO:217),

[0258] CDR2β–SVGAGI (SEQ ID NO: 218), and

[0259] CDR3β–CASSYSRTESTDTQYF(SEQ ID NO:219),

[0260] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0261] (xxx)CDR1α-NSMFDY(SEQ ID NO:90),

[0262] CDR2α–ISSIKDK(SEQ ID NO:91),

[0263] CDR3α-CAANNARLMF(SEQ ID NO:92),

[0264] CDR1β–SGHRS(SEQ ID NO:269),

[0265] CDR2β–YFSETQ (SEQ ID NO:270), and

[0266] CDR3β–CASSPGQHGELFF(SEQ ID NO:271),

[0267] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0268] (xxxi)CDR1α-NSMFDY (SEQ ID NO:264),

[0269] CDR2α–ISSIKDK(SEQ ID NO:265),

[0270] CDR3α-CAASATGNQFYF(SEQ ID NO:266),

[0271] CDR1β–SGHNS (SEQ ID NO:95),

[0272] CDR2β–FNNNVP (SEQ ID NO: 96), and

[0273] CDR3β–CASSDTRAREQFF(SEQ ID NO:97),

[0274] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0275] (xxxii)CDR1α-NSMFDY (SEQ ID NO:264),

[0276] CDR2α–ISSIKDK(SEQ ID NO:265),

[0277] CDR3α-CAASATGNQFYF(SEQ ID NO:266),

[0278] CDR1β–SGHRS(SEQ ID NO:269),

[0279] CDR2β–YFSETQ (SEQ ID NO:270), and

[0280] CDR3β–CASSPGQHGELFF(SEQ ID NO:271),

[0281] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0282] (xxxiii)CDR1α-TSINN (SEQ ID NO:275),

[0283] CDR2α–IRSNERE (SEQ ID NO:276),

[0284] CDR3α-CATDGDSSYKLIF(SEQ ID NO:277),

[0285] CDR1β–DFQATT(SEQ ID NO:161),

[0286] CDR2β–SNEGSKA (SEQ ID NO:162), and

[0287] CDR3β–CSARDSVSGNTIYF(SEQ ID NO:163),

[0288] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0289] (xxxiv)CDR1α-TSINN (SEQ ID NO:275),

[0290] CDR2α–IRSNERE (SEQ ID NO:276),

[0291] CDR3α-CATDGDSSYKLIF(SEQ ID NO:277),

[0292] CDR1β–SQVTM (SEQ ID NO:167),

[0293] CDR2β–ANQGSEA (SEQ ID NO:168), and

[0294] CDR3β–CSVGGSGSYNEQFF(SEQ ID NO:169),

[0295] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0296] (xxxv)CDR1α-TSINN (SEQ ID NO:275),

[0297] CDR2α–IRSNERE (SEQ ID NO:276),

[0298] CDR3α-CATDGDSSYKLIF(SEQ ID NO:277),

[0299] CDR1β–DFQATT(SEQ ID NO:280),

[0300] CDR2β–SNEGSKA (SEQ ID NO:281), and

[0301] CDR3β–CSARDVLTGDYGYTF (SEQ ID NO:282),

[0302] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0303] (xxxvi)CDR1α-TSINN (SEQ ID NO:275),

[0304] CDR2α–IRSNERE (SEQ ID NO:276),

[0305] CDR3α-CATDGDSSYKLIF(SEQ ID NO:277),

[0306] CDR1β–SGHDY(SEQ ID NO:286),

[0307] CDR2β–FNNNVP (SEQ ID NO: 287), and

[0308] CDR3β–CASSLGLSISQETQYF (SEQ ID NO:288),

[0309] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0310] (xxxvii)CDR1α-DSSSTY (SEQ ID NO:101),

[0311] CDR2α–IFSNMDM (SEQ ID NO:102),

[0312] CDR3α-CAERLNTDKLIF(SEQ ID NO:103),

[0313] CDR1β–SGHDY(SEQ ID NO:286),

[0314] CDR2β–FNNNVP (SEQ ID NO: 287), and

[0315] CDR3β–CASSLGLSISQETQYF (SEQ ID NO:288),

[0316] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0317] (xxxviii)CDR1α-DSVNN (SEQ ID NO:106),

[0318] CDR2α–IPSGT (SEQ ID NO:107),

[0319] CDR3α-CAVEATDSWGKLQF(SEQ ID NO:108),

[0320] CDR1β–SGHDY(SEQ ID NO:286),

[0321] CDR2β–FNNNVP (SEQ ID NO: 287), and

[0322] CDR3β–CASSLGLSISQETQYF (SEQ ID NO:288),

[0323] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0324] (xxxix)CDR1α-DSASNY(SEQ ID NO:111),

[0325] CDR2α–IRSNVGE (SEQ ID NO:112),

[0326] CDR3α-CAVRTSYDKVIF(SEQ ID NO:113),

[0327] CDR1β–SGHDY(SEQ ID NO:286),

[0328] CDR2β–FNNNVP (SEQ ID NO: 287), and

[0329] CDR3β–CASSLGLSISQETQYF (SEQ ID NO:288),

[0330] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0331] (xxxx)CDR1α-DSSSTY(SEQ ID NO:101),

[0332] CDR2α–IFSNMDM (SEQ ID NO:102),

[0333] CDR3α-CAERLNTDKLIF(SEQ ID NO:103),

[0334] CDR1β–DFQATT(SEQ ID NO:280),

[0335] CDR2β–SNEGSKA (SEQ ID NO:281), and

[0336] CDR3β–CSARDVLTGDYGYTF (SEQ ID NO:282),

[0337] or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0338] (xxxxi)CDR1α-DSVNN (SEQ ID NO:106),

[0339] CDR2α–IPSGT (SEQ ID NO:107),

[0340] CDR3α-CAVEATDSWGKLQF(SEQ ID NO:108),

[0341] CDR1β–DFQATT(SEQ ID NO:280),

[0342] CDR2β–SNEGSKA (SEQ ID NO:281), and

[0343] CDR3β–CSARDVLTGDYGYTF (SEQ ID NO:282),

[0344] or a variant thereof having up to 3 amino acid substitutions, additions or deletions; or

[0345] (xxxxii)CDR1α-DSASNY(SEQ ID NO:111),

[0346] CDR2α–IRSNVGE (SEQ ID NO:112),

[0347] CDR3α-CAVRTSYDKVIF(SEQ ID NO:113),

[0348] CDR1β–DFQATT(SEQ ID NO:280),

[0349] CDR2β–SNEGSKA (SEQ ID NO:281), and

[0350] CDR3β–CSARDVLTGDYGYTF (SEQ ID NO:282),

[0351] or variants thereof having up to 3 amino acid substitutions, additions or deletions.

[0352] In one embodiment, the invention provides a TCR of the invention comprising:

[0353] (i) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0354] (ii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0355] (iii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0356] (iv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0357] (v) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0358] (vi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0359] (vii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0360] (viii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0361] (ix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0362] (x) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO:53, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO:58, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0363] (xi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO:53, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO:64, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0364] (xii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO:53, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO:70, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0365] (xiii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0366] (xiv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO:76, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO:87, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0367] (xv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0368] (xvi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0369] (xvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0370] (xviii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0371] (xix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0372] (xx) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0373] (xxi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0374] (xxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 176, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 181, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0375] (xxiii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0376] (xxiv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 203, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0377] (xxv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 209, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0378] (xxvi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0379] (xxvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 203, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0380] (xxviii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 209, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0381] (xxix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 215, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 220, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0382] (xxx) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 272, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0383] (xxxi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 267, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0384] (xxxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 267, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 272, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0385] (xxxiii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0386] (xxxiv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0387] (xxxv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0388] (xxxvi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0389] (xxxvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0390] (xxxviii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto;

[0391] (xxxix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0392] (xxxx) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0393] (xxxxi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0394] (xxxxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto. In one embodiment, the invention provides a TCR of the invention comprising

[0395] (i) an alpha chain comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, and variants of SEQ ID NOs:10 and 11 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0396] (ii) an alpha chain comprising the amino acid sequence of SEQ ID NO:38 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, and variants of SEQ ID NOs:43 and 44 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity;

[0397] (iii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, and variants of SEQ ID NOs: 26 and 27 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0398] (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and variants of SEQ ID NOs: 32 and 33 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0399] (v) an alpha chain comprising the amino acid sequence of SEQ ID NO:21 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, and variants of SEQ ID NOs:26 and 27 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0400] (vi) an alpha chain comprising the amino acid sequence of SEQ ID NO:21 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, and variants of SEQ ID NOs:32 and 33 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0401] (vii) an alpha chain comprising the amino acid sequence of SEQ ID NO:49 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:60, and variants of SEQ ID NOs:59 and 60 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0402] (viii) an alpha chain comprising the amino acid sequence of SEQ ID NO:49 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, and variants of SEQ ID NOs:65 and 66 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0403] (ix) an alpha chain comprising the amino acid sequence of SEQ ID NO:49 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, and variants of SEQ ID NOs:71 and 72 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0404] (x) an alpha chain comprising the amino acid sequence of SEQ ID NO:54 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:60, and variants of SEQ ID NOs:59 and 60 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0405] (xi) an alpha chain comprising the amino acid sequence of SEQ ID NO:54 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, and variants of SEQ ID NOs:65 and 66 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0406] (xii) an alpha chain comprising the amino acid sequence of SEQ ID NO:54 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, and variants of SEQ ID NOs:71 and 72 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0407] (xiii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, and variants of SEQ ID NOs: 82 and 83 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0408] (xiv) an alpha chain comprising the amino acid sequence of SEQ ID NO:77 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:88, SEQ ID NO:89, and variants of SEQ ID NOs:88 and 89 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0409] (xv) an alpha chain comprising the amino acid sequence of SEQ ID NO:94 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, and variants of SEQ ID NOs:99 and 100 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0410] (xvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0411] (xvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0412] (xviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0413] (xix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0414] (xx) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0415] (xxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0416] (xxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 177 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 182, SEQ ID NO: 183, and variants of SEQ ID NOs: 182 and 183 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0417] (xxiii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 198, SEQ ID NO: 199, and variants of SEQ ID NOs: 198 and 199 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0418] (xxiv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 204, SEQ ID NO: 205, and variants of SEQ ID NOs: 204 and 205 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0419] (xxv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, and variants of SEQ ID NOs: 210 and 211 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0420] (xxvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 198, SEQ ID NO: 199, and variants of SEQ ID NOs: 198 and 199 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0421] (xxvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 204, SEQ ID NO: 205, and variants of SEQ ID NOs: 204 and 205 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0422] (xxviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, and variants of SEQ ID NOs: 210 and 211 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0423] (xxix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 216 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 221, SEQ ID NO: 222, and variants of SEQ ID NOs: 221 and 222 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0424] (xxx) an alpha chain comprising the amino acid sequence of SEQ ID NO:94 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:273, SEQ ID NO:274, and variants of SEQ ID NOs:273 and 274 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0425] (xxxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 268 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, and variants of SEQ ID NOs: 99 and 100 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0426] (xxxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 268, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 273, SEQ ID NO: 274, and variants of SEQ ID NOs: 273 and 274 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0427] (xxxiii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0428] (xxxiv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0429] (xxxv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0430] (xxxvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0431] (xxxvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0432] (xxxviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0433] (xxxix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0434] (xxxx) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto;

[0435] (xxxxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; or

[0436] (xxxxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285 and variants of SEQ ID NOs: 284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto.

[0437] In one embodiment, the present invention provides a TCR of the present invention, an α chain and a β chain thereof, wherein the α chain comprises the amino acid sequence of SEQ ID NO: 257 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and the β chain comprises the amino acid sequence of SEQ ID NO: 259 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto.

[0438] In one embodiment, the present invention provides a TCR of the present invention, an α chain and a β chain thereof, wherein the α chain comprises the amino acid sequence of SEQ ID NO: 261 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto; and the β chain comprises the amino acid sequence of SEQ ID NO: 263 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity thereto.

[0439] The TCR of the present invention can bind to a WT1 peptide comprising an amino acid sequence selected from the following group: EPASQHTLRSG (SEQ ID NO: 123), YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117) NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254) and variants thereof each having up to 3 amino acid substitutions, additions or deletions.

[0440] In another aspect, the present invention provides a T cell receptor (TCR) that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the WT1 peptide comprises an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123), YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253). NO: 253) PSCQKKFARSDELVR (SEQ ID NO: 254) and variants thereof each having up to 3 amino acid substitutions, additions or deletions.

[0441] In one embodiment, a TCR of the invention binds to an MHC I and / or MHC II peptide complex.

[0442] In one embodiment, the TCR of the invention is restricted to human leukocyte antigen (HLA) alleles. In one embodiment, the TCR of the invention is restricted to HLA-A or HLA-B alleles. In one embodiment, the TCR of the invention is restricted to an HLA-A allele selected from the group consisting of HLA-A*0201, HLA-A*0101, HLA-A*2402, and HLA-A*0301, or an HLA-B allele selected from the group consisting of HLA-B*0702, HLA-B*3501, and HLA-B*3502.

[0443] In one embodiment, the TCR of the invention is restricted to HLA-A*0201.

[0444] In one embodiment, the TCR of the invention is restricted to HLA-B*3502.

[0445] In one embodiment, the TCR of the invention is restricted to HLA-B*3501.

[0446] In one embodiment, the TCR of the invention is restricted to HLA-C alleles. In one embodiment, the TCR of the invention is restricted to HLA-C alleles selected from the group consisting of HLA-C*07:01, HLA-C*03:04, HLA-C*04:01, HLA-C*05:01, HLA-C*06:02, and HLA-C*07:02.

[0447] In one embodiment, a TCR of the invention comprises one or more mutations at the α chain / β chain interface such that the frequency of mispairing between said α chain and said β chain and endogenous TCR α and β chains is reduced when said α chain and said β chain are expressed in a T cell.

[0448] In one embodiment, a TCR of the invention comprises one or more mutations at the α chain / β chain interface such that expression levels of the TCR α and β chains are increased when the α chain and the β chain are expressed in a T cell.

[0449] In one embodiment, one or more mutations introduce a cysteine ​​residue into the constant region of each of said α chain and said β chain, wherein said cysteine ​​residue is capable of forming a disulfide bond between said α chain and said β chain.

[0450] The TCR of the present invention may comprise a murinized constant region.

[0451] In one embodiment, the TCR of the invention is a soluble TCR.

[0452] In another aspect, the present invention provides an isolated polynucleotide encoding the α chain of the T cell receptor (TCR) of the present invention and / or the β chain of the TCR of the present invention.

[0453] In another aspect, the present invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 256 and / or the nucleotide sequence of SEQ ID NO: 258 or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0454] In another aspect, the present invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 260 and / or the nucleotide sequence of SEQ ID NO: 262 or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0455] In one embodiment, the isolated polynucleotide encodes the α chain linked to the β chain.In one embodiment, the isolated polynucleotide encodes one or more short interfering RNAs (siRNAs) or other agents capable of reducing or preventing the expression of one or more endogenous TCR genes.

[0456] In another aspect, the present invention provides a vector comprising a polynucleotide of the present invention. In another embodiment, the vector comprises a polynucleotide encoding one or more CD3 chains, CD8, a suicide gene and / or a selectable marker.

[0457] In another aspect, the present invention provides a cell comprising a TCR of the present invention, a polynucleotide of the present invention or a vector of the present invention,

[0458] In one embodiment, the cell further comprises a vector encoding one or more of a CD3 chain, CD8, a suicide gene and / or a selectable marker.

[0459] In one embodiment, the cell is a T cell, a lymphocyte or a stem cell, such as a hematopoietic stem cell or an induced pluripotent stem cell (iPS). The T cell, the lymphocyte or the stem cell can be selected from the group consisting of CD4 cells, CD8 cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ / δ T cells, natural killer (NK) cells, natural killer T (NKT) cells, double negative T cells, naive T cells, memory stem T cells, central memory T cells, effector memory T cells, effector T cells, hematopoietic stem cells and pluripotent stem cells.

[0460] In one embodiment, the cell is a T cell that has been isolated from a subject.

[0461] In one embodiment, the endogenous gene encoding the TCR alpha chain and / or the endogenous gene encoding the TCR beta chain in the cell is destroyed, preferably so that the endogenous gene encoding the TCR alpha chain and / or the endogenous gene encoding the TCR beta chain is not expressed. In one embodiment, the endogenous gene encoding the TCR alpha chain and / or the endogenous gene encoding the TCR beta chain is destroyed by inserting an expression cassette, and the expression cassette comprises a polynucleotide sequence encoding the TCR of the present invention. In one embodiment, one or more endogenous genes encoding MHC in the cell are destroyed, preferably wherein the cell is a non-autoreactive universal T cell. In one embodiment, the endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / aging / inhibitory signals, homing ability or other T cell functions in the cell is destroyed, preferably wherein the endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / aging / inhibitory signals, homing ability or other T cell functions is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160 and CTLA4. In one embodiment, endogenous genes involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell functions are disrupted by integration of an expression cassette comprising a polynucleotide sequence encoding a TCR of the invention.

[0462] In another aspect, the present invention provides a method for preparing a cell, comprising the steps of introducing the vector of the present invention into the cell in vitro, ex vivo or in vivo, for example, by transfection or transduction.

[0463] In another aspect, the present invention provides a method of preparing a cell, comprising the step of transducing the cell with one or more vectors of the present invention in vitro, ex vivo or in vivo.

[0464] In one embodiment, the cells to be transduced with one or more vectors are selected from the group consisting of T cells, lymphocytes or stem cells, such as hematopoietic stem cells or induced pluripotent stem cells (iPS), optionally, the T cells, the lymphocytes or the stem cells can be selected from the group consisting of CD4 cells, CD8 cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ / δ T cells, natural killer (NK) cells, natural killer T (NKT) cells, double negative T cells, naive T cells, memory stem T cells, central memory T cells, effector memory T cells, effector T cells, hematopoietic stem cells and pluripotent stem cells.

[0465] In one embodiment, the method includes a step of T cell editing, which comprises disrupting an endogenous gene, such as an endogenous gene encoding a TCR α chain and / or an endogenous gene encoding a TCR β chain, using an artificial nuclease, preferably, wherein the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

[0466] In one embodiment, the method includes a step of T cell editing, which comprises disrupting the endogenous gene encoding the TCRα chain and / or the endogenous gene encoding the TCRβ chain with an artificial nuclease, preferably, wherein the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

[0467] In one embodiment, the method includes the step of targeted integration of an expression cassette into the endogenous gene encoding the TCR α chain gene and / or the endogenous gene encoding the TCR β chain destroyed by the artificial nuclease, wherein the expression cassette comprises a polynucleotide encoding the TCR of the present invention or a polynucleotide sequence of the present invention.

[0468] In one embodiment, the method comprises the step of disrupting one or more endogenous genes encoding MHC, preferably wherein the cells produced by said method are non-alloreactive universal T cells.

[0469] In one embodiment, the method comprises the step of disrupting one or more endogenous MHC genes, preferably wherein the cells produced by the method are non-alloreactive universal T cells.

[0470] In one embodiment, the method comprises the steps of disrupting one or more endogenous genes to alter persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability or other T cell functions, preferably, wherein the method comprises the step of targeted integration of an expression cassette into an endogenous gene disrupted by an artificial nuclease, wherein the endogenous gene is involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability or other T cell functions, wherein the expression cassette comprises a polynucleotide sequence encoding a TCR of the present invention, preferably wherein the endogenous gene is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160 and CTLA4.

[0471] In another aspect, the present invention provides cells of the present invention or cells prepared by the method of the present invention for use in adoptive cell transfer, preferably adoptive T cell transfer, optionally the adoptive T cell transfer can be allogeneic adoptive T cell transfer, universal non-autoreactive T cell transfer, or autologous adoptive T cell transfer.

[0472] In another aspect, the invention provides a TCR of the invention, an isolated polynucleotide of the invention, a vector of the invention, a cell of the invention, a cell produced by a method of the invention, or a chimeric molecule of the invention for use in therapy.

[0473] In another aspect, the present invention provides a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell of the present invention, a cell prepared by the method of the present invention, or a chimeric molecule of the present invention for use in treating and / or preventing a disease associated with WT1 expression.

[0474] In another aspect, the present invention provides T cells that have been genetically engineered (genetically edited) to modify persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell functions, wherein the T cells express a TCR alpha chain of the invention and / or a TCR beta chain of the invention.

[0475] In another aspect, the present invention provides T cells genetically engineered (genetically edited) by a protocol comprising the step of targeted integration of an expression cassette into an endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability or other T cell functions that is destroyed by an artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding a TCR alpha chain of the present invention and / or a TCR beta chain of the present invention.

[0476] In another aspect, the present invention provides a method for treating and / or preventing a disease associated with WT1 expression, comprising administering to a subject in need thereof a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell of the present invention, a cell prepared by the method of the present invention, or a chimeric molecule of the present invention.

[0477] Diseases associated with WT1 expression may be proliferative disorders. Preferably, the proliferative disorder may be selected from the group consisting of hematological malignancies such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, myelodisplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma. The proliferative disorder can be selected from the group consisting of solid tumors such as lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0478] In a preferred embodiment, the disease associated with WT1 expression is acute myeloid leukemia (AML).

[0479] In a preferred embodiment, the disease associated with WT1 expression is chronic myeloid leukemia (CML).

[0480] In another aspect, the present invention provides an isolated immunogenic WT1 peptide comprising an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123) YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254). NO: 254) and variants thereof each having up to 3 amino acid substitutions, additions or deletions. BRIEF DESCRIPTION OF THE DRAWINGS

[0482] Figure 1 :The figure shows the results of in vitro expansion of functional WT-1-specific T cells from the peripheral blood of 10 healthy donors

[0483] Peripheral blood mononuclear cells from 10 healthy donors (HD) were stimulated with pooled overlapping WT1 15-mer peptides for 26-30 hours to enrich for CD137 + Cells were cultured and expanded for 9-19 days. The expanded T cells were restimulated for 6 hours with autologous antigen presenting cells (APCs) loaded with irrelevant peptide pools or WT1 peptide pools. In addition, negative controls (unstimulated T cells) and positive controls (T cells cultured in the presence of PMA and ionomycin) were included in the experimental setup (not shown). The dot plots indicate the results of intracellular staining of IFNγ production and CD107a exposure on the cell surface. After several restimulations with autologous APCs loaded with WT1 peptide pools, the specificity of T cells was tested by intracellular staining as described above. The results show the enrichment of WT1-specific T cells in the CD8 T cell compartments of HD1 (a), HD3 (c), HD4 (d), HD5 (e), HD6 (f), HD7 (g) and HD10 (j) and in the CD4 T cell compartments of HD2 (b), HD8 (h) and HD9 (i). WT1, Wilms' tumor 1; PMA, phorbol 12-myristate 13-acetate; IFNγ, interferon-γ; S, stimulated.

[0484] Figure 2: Grid and graph showing identification of WT1 immunogenic peptides by positioning grid strategy

[0485] Intracellular staining was used to identify the epitopes recognized by T cells primed in vitro by repeated stimulation with a pool of overlapping WT1 peptides. Specifically, the percentage of specific T cells that responded to the localized grid of sub-pools of WT1 15 peptides loaded on APCs was assessed. In addition, negative controls (unstimulated T cells and T cells co-cultured with APCs loaded with an irrelevant peptide pool) and positive controls (T cells cultured in the presence of PMA and ionomycin) were included in the experimental setup (unstimulated and PMA / ionomycin-conditioned T cells are not shown).

[0486] (a) Deconvolution grid indicating the percentage of T cells expressing IFNγ and CD107a after co-culture with APCs loaded with different subpools (denoted as SP1-24). IFNγ and CD107a values ​​in bold text indicate subpools containing WT1 epitopes recognized by T cells. Representative dot plots are reported relative to co-culture of T cells with APCs loaded with the responsive subpools, indicating the expression of IFNγ and CD107a. Predominant responses were observed for subpools 4, 5, and 16 in HD1 (b), HD3 (d), HD6 (g), HD7 (h), and HD10 (k); subpools 6, 16, 17, 20, and 23 in HD2 (c); subpools 4, 5, 6, 14, 18, and 21 in HD4 (e); subpools 5, 11, 12, 21, and 22 in HD5 (f); subpools 12 and 14 in HD8 (i); and subpools 5, 13, and 21 in HD9 (j). For HD7, we also observed increased IFNγ secretion and CD107a expression in response to subpools 7, 8, and 20, although at lower percentages compared to the responses observed with subpools 4, 5, and 16. SP, subpools. WT1, Wilms’ tumor 1; APC, antigen-presenting cell; PMA, phorbol 12-myristate 13-acetate; IFNγ, interferon-γ.

[0487] Figure 3 : Epitope specificity of WT1-specific T cells generated by sensitization with the combined peptides.

[0488] To validate the WT1 immunogenic peptides, T cells expanded from each HD were co-cultured for 6 hours in the presence of APCs loaded with the peptides identified after deconvolution of the localization grid and loaded with at least one irrelevant peptide as a negative control. In addition, negative (unstimulated T cells) and positive controls (T cells cultured in the presence of PMA and ionomycin) were included in the experimental setup (not shown). The dot plots show the results of intracellular staining of IFNγ and / or surface CD107a for each HD. For peptides 40 and 41 (for HD1) (a), but not for peptides 42 and 43 (irrelevant peptides); for peptides 54, 77, 90 (for HD2) (b), but not for peptides 42 and 138 (irrelevant peptides); the peptide VLDFAPPGA (SEQ ID NO: 157, VLD, which is a nonamer of the peptide shown in SEQ ID NO: 117 (in Figure 3 c))) for HD3 (c) and with low response to peptides PVLDFAPPG (SEQ ID NO: 158, PVL, which is another nonamer of the peptide set forth in SEQ ID NO: 117) and LDFAPPGAS (SEQ ID NO: 159, LDF, which is a nonamer of the peptide set forth in SEQ ID NO: 116, previously described as an immunogenic peptide (Doubrovina, E. et al. (2012) Blood 120: 1633–1646); peptides 17, 18, 99, 100 for HD4 (d, e) but not to unrelated peptides (15, 16, 63–66, 101, 102, and 132); peptide 101 for HD5 (f) but not to unrelated peptides (63, 107, 108, 113, 119, and 120); peptide VLDFAPPGA (SEQ ID NO: 157, VLD, which is a nonamer of the peptide set forth in SEQ ID NO: Nonamer of the peptide shown in ID NO: 117 (in Figure 3 c)) (referred to as "11-mer" in (c)) (for HD6) (g) and peptide PVLDFAPPG (SEQ ID NO: 158, PVL, which is another nonamer of the peptide shown in SEQ ID NO: 117) but not for peptide LDFAPPGAS (SEQ ID NO: 159, LDF, which is a nonamer of the peptide shown in SEQ ID NO: 116, previously described as an immunogenic peptide (Doubrovina, E. et al. (2012) Blood 120: 1633–1646)); peptides 101, 125, 137 (for HD9) (h); peptide VLDFAPPGA (SEQ ID NO: 157, VLD, which is a nonamer of the peptide shown in SEQ ID NO: 117 (in Figure 3c)) (referred to as “11-mer”) (enrichment of CD107a- and / or IFNγ-positive cells was observed for T cells co-cultured with HD10 (i) but not for irrelevant peptides, respectively. Due to the reduced fitness of the T cells, functional testing was not possible to validate the peptides predicted by deconvolution of the localization grid, namely peptides 40, 41, 91, 92 for HD7 and peptide 24 for HD8.

[0489] To determine the HLA restriction of the WT1 epitope identified by HD4, HD5, and HD10 T cells, donor DNA was sequenced to determine HLA typing. WT1-specific T cells were then co-cultured with different antigen-presenting EBV-BLCL cell lines, each containing a specific HLA allele of interest identified by sequencing HD4, HD5, or HD10 DNA. EBV-BLCL cells were pulsed with HD4 peptide 17, HD5 peptide 101, and VLDFAPPGA (SEQ ID NO: 157) peptides, or with an irrelevant control peptide. After 6 hours of co-culture, we observed substantial responses to WT1 by WT1-specific T cells co-cultured with EBV-BLCL cells expressing the HLA-B*3502 allele for HD4 and pulsed with peptide 17 (j), EBV-BLCL cells expressing the HLA-B*3501 allele for HD5 and pulsed with peptide 101 (k), and EBV-BLCL cells expressing the HLA-A*0201 allele for HD10 and pulsed with the peptide VLDFAPPGA (SEQ ID NO: 157) (l). (m) Table shows peptides recognized by T cells expanded from HD1 to HD10. For HD3, HD6, and HD10, specific nonamers are shown that overlap with peptides 40 and 41 and elicit immune responses. Wilms' tumor 1; APC, antigen-presenting cell; PMA, 2; phorbol 12-myristate 13-acetate; IFNγ, interferon-γ; S, stimulation.

[0490] Figure 4 : Figure and graph showing that expanded T cells of HD1, HD3 and HD4 recognize naturally processed WT1 epitopes

[0491] (a) Graph depicts CD8 T cells expanded from HD1 cells after co-culture with T2 cells pulsed with WT1 pool, K562 cells genetically modified to express the HLA-A*0201 allele and overexpress WT1 protein, or T2 cells pulsed with the nonspecific control MelanA / MART1 pool as a negative control. + CD107 expression on T cells.

[0492] (b) Graph depicts the results of an experiment to determine the ability of HD3-expanded T cells to target cells expressing WT1. Results are expressed as an elimination index, calculated as the total number of target cells remaining after co-culture with WT1-specific T cells divided by the total number of target cells alone. HD3 T cells were co-cultured with T2 cells pulsed with subpool 16 (SP16), containing immunogenic peptides that elicit an immune response; T2 cells pulsed with a MelanA / MART1 pool (Melan A) as a negative control; and wild-type (K562) or K562 cells genetically modified to express the HLA-A*0201 allele and overexpress WT1 protein (K562 A2+WT1+).

[0493] (c) Graph depicts the results of an experiment to determine the ability of WT1-specific T cells from HD4 to eliminate target cells. HD4 T cells were co-cultured with primary CD33+ blasts harvested from HLA-B*3502 patients at a ratio of 10:1, or as a control, with leukemia cells from patients without the HLA-B*3502 allele. After 3 days of co-culture, the results indicate that the ability of WT1-specific T cells (CD3 + When immunized with 100% FcGFR-positive cells (FcGFR-positive cells), CD33+HLA-B*3502 blasts were almost completely eliminated. E, effector; T, target.

[0494] Figure 5 : The figure shows the results of Vβ profile analysis of WT1-specific T cells

[0495] WT1-specific T cells generated from different HDs after several stimulations with WT1 pools were stained with a Vβ immune profiling kit to determine the clonality of the population. In particular, the expression of variable (V) genes of the β chain was determined by FACS analysis. The results indicated the expression of highly dominant Vβ genes in HD1 (TRBV12-3; 12-4), HD2 (TRBV11-2), HD3 (TRBV4-3), HD5 (TRBV20-1), while no obvious enrichment of defined Vβ was detected for HD4, HD6, and HD10. HD4 SP14 indicates T cells stimulated with subpool 14, which contains peptides 17-18 that elicited the highest immune response. HD4 SP18+21 indicates T cells stimulated with subpools 18 and 21, which contain peptides 63-64-65-66 and 99-100-101-102, respectively, which elicited the smallest immune response, as shown in FIG. Figure 3 For HD7, HD8, and HD9, Vβ immune profiling was not possible due to reduced cell fitness.

[0496] Figure 6: The figure shows the TCR sequencing results of enriched WT1-specific T cells over time

[0497] After several stimulations with the WT1 pool, T cells generated from each healthy donor included in the experimental setup were characterized by TCRαβ sequencing. Sequencing results indicate the presence of dominant clonotypes for HD1 (a), HD2 (b), and HD3 (c), HD4 (d), HD5 (e), HD6 (f), HD7 (g), HD8 (h), HD9 (i), and HD10 (j). The bar graph depicts the 10 most dominant CDR3 amino acid sequences identified at each time point (e.g., S9 corresponds to the sequencing result obtained after the 9th round of stimulation). For each bar, starting from the x-axis, the lower segment represents the most dominant CDR sequence. The next 9 most dominant sequences are stacked on top of the lower segment and sorted by decreasing frequency upward. The remaining sequences are grouped together in the top segment. WT1, Wilms' tumor 1; CDR3, complementary determining region 3; S, stimulation.

[0498] Figure 7 : Functional activity of genetically modified T lymphocytes.

[0499] T cells isolated from PBMCs of healthy individuals were transduced with a bidirectional lentiviral vector encoding the α and β chains of TCRs isolated from HD1 and HD3. As a control, T cells were transduced with a previously published TCR that recognizes the WT1126-134 (RMFPNAPYL; SEQ ID NO: 255) peptide when presented by the HLA-A*0201 allele. Transfer (TR) T lymphocytes were co-cultured for 3 days with: (a) T2 cells pulsed or not with WT1 126-134 peptide or VLDFAPPGA (SEQ ID NO: 157) peptide (effector: target ratio = 1: 1); (b) wild type (K562) or K562 cells genetically modified to express the HLA-A*0201 allele (effector: target ratio = 1: 1); (c) 3 different primary AML blasts selected for expression of HLA-A*0201 allele and WT1 antigen (effector: target ratio = 5: 1). For co-culture with T2 and K562 cell lines, we included untransduced T cells as controls. The results indicate the ability of each TCR to recognize target peptides when presented by the HLA-A*0201 allele ( Figure 7a), and that HD1 TCR-transduced T cells had a greater potential to mediate specific and nearly complete elimination of K562 cells containing the HLA*A0201 allele compared to HD3-TR T cells. Notably, no substantial killing of target cells was observed in co-cultures of K562 HLA*A0201 cells with WT1 126-134TR T cells ( Figure 7 b). These results were further confirmed by co-culture experiments using primary AML blasts derived from three different AML patients (pAML1 blasts: WT1- / HLA-A*0201+; pAML2 and pAML3 blasts: WT1+ / HLA-A*0201+) as target cells. In this experimental setup, each individual T cell population was sorted with a specific dextramer prior to co-culture with the target to enrich for effector cell purity. We observed greater depletion of both pAML blasts harboring the HLA-A*0201 allele after co-culture with HD1 TR T cells, whereas HD3 T and WT1126-134 T cells only recognized blasts from pAML3. UT, untransduced; pAML, primary acute myeloid leukemia; TR, metastasis; Dx, dextramer. Detailed Description of the Invention

[0501] As used herein, the term "comprising" is synonymous with "including" or "containing" and is inclusive or open-ended and does not exclude other unrecited members, elements or steps. The term "comprising" also includes the term "consisting of..."

[0502] T cell receptor

[0503] During antigen processing, the antigen is degraded inside the cell and then carried to the cell surface by major histocompatibility complex (MHC) molecules. T cells are able to recognize this peptide:MHC complex on the surface of antigen-presenting cells. There are two different classes of MHC molecules: MHC I and MHC II, each of which delivers peptides from different cellular compartments to the cell surface.

[0504] The T cell receptor (TCR) is a molecule found on the surface of T cells that is responsible for recognizing antigens bound to MHC molecules. Naturally occurring TCR heterodimers are composed of alpha (α) and beta (β) chains in approximately 95% of T cells, while approximately 5% of T cells have TCRs composed of gamma (γ) and delta (δ) chains.

[0505] Binding of the TCR to antigen and MHC results in activation of the T lymphocyte on which the TCR is expressed through a series of biochemical events mediated by associated enzymes, co-receptors and specialized accessory molecules.

[0506] Each chain of a natural TCR is a member of the immunoglobulin superfamily and possesses an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane / transcellular membrane domain, and a short cytoplasmic tail at the C-terminus.

[0507] The variable domains of TCR α chain and β chain have three hypermutations or complementary determining regions (CDRs). TCR α chain or β chain, for example, comprise CDR1, CDR2 and CDR3 in an amino to carboxyl terminal order. Generally, CDR3 is the main CDR responsible for identifying and processing antigens, although it has also been shown that the CDR1 of α chain interacts with the N-terminal portion of antigenic peptides, while the CDR1 of β chain interacts with the C-terminal portion of peptides. It is believed that CDR2 recognizes MHC molecules.

[0508] The constant domain of a TCR may consist of a short linker sequence in which cysteine ​​residues form disulfide bonds, thereby forming a link between the two chains.

[0509] The α chain of the TCR of the present invention may have a constant domain encoded by the TRAC gene. An exemplary amino acid sequence of the α chain constant domain encoded by the TRAC gene is shown below:

[0510] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0511] (SEQ ID NO: 128).

[0512] The TCR of the present invention may comprise an α chain comprising the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto, preferably at least 75% sequence identity thereto.

[0513] The β chain of the TCR of the present invention may have a constant domain encoded by the TRBC1 or TRBC2 gene. An exemplary amino acid sequence of the β chain constant domain encoded by the TRBC1 gene is shown below:

[0514] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0515] (SEQ ID NO: 129).

[0516] An exemplary amino acid sequence of the beta chain constant domain encoded by the TRBC2 gene is shown below:

[0517] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0518] (SEQ ID NO: 130).

[0519] The TCR of the present invention may comprise a β chain comprising the amino acid sequence of SEQ ID NO: 129, SEQ ID NO: 130 or a variant of SEQ ID NO: 129, SEQ ID NO: 130 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto, preferably having at least 75% sequence identity thereto.

[0520] The TCRs of the invention may have one or more additional cysteine ​​residues in each of the α and β chains, such that the TCR may contain two or more disulfide bonds in the constant domain.

[0521] The structure allows the TCR to associate with other molecules such as CD3, which has three unique chains in mammals (gamma, delta, and epsilon) and a zeta chain. These accessory molecules have negatively charged transmembrane regions and are crucial for transmitting signals from the TCR into the cell. The CD3 and zeta chains, together with the TCR, form the so-called T cell receptor complex.

[0522] The signal from the T cell complex is enhanced by simultaneously binding to MHC molecules through specific co-receptors. For helper T cells, this co-receptor is CD4 (specific for class II MHC); and for cytotoxic T cells, this co-receptor is CD8 (specific for class I MHC). Co-receptors allow for prolonged engagement between antigen presenting cells and T cells and recruit essential molecules (e.g., LCK) involved in the signaling of activated T lymphocytes within the cell.

[0523] Thus, as used herein, the term "T cell receptor" (TCR) refers to a molecule capable of recognizing a peptide when presented by an MHC molecule. The molecule can be a heterodimer of two chains α and β (or optionally γ and δ) or it can be a single-chain TCR construct. The TCR of the present invention can be a soluble TCR, for example, omitting or altering one or more constant domains. The TCR of the present invention can comprise constant domains.

[0524] The present invention also provides an α chain or a β chain from such a T cell receptor.

[0525] The TCRs of the present invention may be hybrid TCRs, comprising sequences derived from more than one species. For example, it has been surprisingly found that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, a TCR may comprise murine sequences within human variable and constant regions.

[0526] A disadvantage of this approach is that the murine constant sequences can trigger an immune response, leading to rejection of the transferred T cells. However, the conditioning regimen used to prepare patients for adoptive T cell therapy can result in sufficient immunosuppression to allow engraftment of T cells expressing the murine sequences.

[0527] Complementarity determining regions (CDRs)

[0528] The part that sets up most of the contact with the antigenic peptide that is combined with the major histocompatibility complex (MHC) in TCR is complementary determining region 3 (CDR3), which is unique for each T cell clone. The CDR3 region occurs in the thymus and is related to the somatic rearrangement event of the non-continuous genes belonging to variable (V), diversity (D, for β and δ chains) and connection (J) genes. In addition, the random nucleotides inserted / deleted at the rearrangement locus of each TCR chain gene greatly increase the diversity of highly variable CDR3 sequences. Therefore, the frequency of specific CDR3 sequences in biological samples indicates the abundance of specific T cell populations. The large diversity of the TCR repertoire in healthy humans provides a wide range of protection for the multiple foreign antigens presented by MHC molecules on the surface of antigen presenting cells. In this regard, it is worth noting that theoretically up to 10 15 Different TCRs.

[0529] T cell receptor diversity is concentrated in CDR3, and this region is primarily responsible for antigen recognition.

[0530] The sequence of the CDR3 region of the TCR of the present invention may be selected from those listed in the following Table 1. The TCR may comprise CDRs comprising or consisting of the CDR3α and CDR3β pair described below.

[0531] A CDR may, for example, comprise one, two, or three substitutions, additions, or deletions of a given sequence, provided that the TCR retains the ability to bind to the WT1 peptide when presented by an MHC molecule.

[0532] As used herein, the term "protein" includes single-chain polypeptide molecules as well as multi-polypeptide complexes in which the individual component polypeptides are linked by covalent or non-covalent means. As used herein, the term "polypeptide" refers to a polymer in which the monomers are amino acids and are linked together by peptide or disulfide bonds.

[0533] Variants, derivatives, analogs, homologs and fragments

[0534] In addition to the specific proteins and polynucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs, and fragments thereof.

[0535] In the context of the present invention, a variant of any given sequence is a sequence in which a particular sequence of residues (whether amino acid residues or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. Variant sequences may be obtained by addition, deletion, substitution, modification, substitution and / or variation of at least one residue present in a naturally occurring protein.

[0536] A variant amino acid sequence of the invention referred to as having up to three amino acid substitutions, additions or deletions may have, for example, one, two or three amino acid substitutions, additions or deletions.

[0537] With respect to a protein or polypeptide of the present invention, the term "derivative" as used herein includes any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

[0538] As used herein, the term "analog," with respect to polypeptides or polynucleotides, includes any mimetic, ie, a compound that possesses at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[0539] The proteins used in the present invention may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups of similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0540] Substitutions can involve replacing an amino acid with a similar amino acid (conservative substitutions). Similar amino acids are amino acids that have side chain moieties with related properties that are grouped together, for example, as shown below:

[0541] (i) Basic side chains: lysine (K), arginine (R), histidine (H);

[0542] (ii) acidic side chains: aspartic acid (D) and glutamic acid (E);

[0543] (iii) uncharged polar side chains: asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y); or

[0544] (iv) Non-polar side chains: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W), and cysteine ​​(C).

[0545] Any amino acid changes should maintain the ability of the TCR to bind to the WT1 peptide presented by the MHC molecule.

[0546] Variant sequences may comprise amino acid substitutions, additions, deletions and / or insertions. The variations may be concentrated in one or more regions, such as the constant region, linker or framework region of the α or β chain, or they may be distributed throughout the TCR molecule.

[0547] Conservative substitutions, additions or deletions can be made, for example, according to the following table. Amino acids in the same block in the second column, preferably in the same row in the third column, can be substituted for each other:

[0548]

[0549]

[0550] The present invention also includes homologous substitutions (e.g., substitution and replacement are both used herein to refer to the interchange of an existing amino acid residue with an alternative residue), such as like substitutions, e.g., basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions can also occur, e.g., from one class of residue to another, or alternatively involving the inclusion of unnatural amino acids, such as ornithine.

[0551] As used herein, the term "variant" may refer to an entity having a certain homology with a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" may be equivalent to "identity".

[0552] Variant sequences can include amino acid sequences that are at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, at least 97% or at least 99% identical to the subject sequence. Typically, the variant will contain the same active sites, etc., as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0553] Variant sequences may include nucleotide sequences that are at least 40%, 45%, 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, at least 97% or at least 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0554] Preferably, reference to a sequence having a percentage identity to any SEQ ID NO recited herein refers to the sequence having that percentage identity over the entire length of the referenced SEQ ID NO.

[0555] Identity comparisons can be performed by eye, or more usually with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the homology or identity percentage between two or more sequences.

[0556] Percent homology can be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0557] Although this is a very simple and consistent approach, it fails to take into account that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence may cause a subsequent codon to fall out of alignment, thus potentially leading to a significant reduction in the percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without overly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an effort to maximize local homology.

[0558] In some embodiments, the present invention relates to a method for comparing the sequence of an amino acid sequence with a plurality of gaps. The method comprises the following steps: first, a gap penalty is assigned to each gap that occurs in the alignment, and second, a gap penalty is assigned to each gap that occurs in the alignment. However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible (which reflects a higher correlation between the two compared sequences) will obtain a higher score than an alignment with many gaps. An "affine gap cost" is usually used, which charges a relatively high cost for the existence of a gap and charges a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, a high gap penalty will produce an optimized alignment with fewer gaps. Most alignment programs allow modification of the gap penalty. However, when using this type of software for sequence comparison, it is preferred to use the default value. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0559] Therefore, the calculation of the maximum homology percentage first needs to produce the best comparison, taking into account the gap penalty. The suitable computer program for carrying out such comparison is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can be used for sequence comparison include but are not limited to BLAST packages (see Ausubel et al. (1999) ibid. – Chapter 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and GENEWORKS comparison tool sets. BLAST and FASTA can both be used for offline and online searches (see Ausubel et al. (1999) ibid., 7-58 to 7-60 pages). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool called BLAST 2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0560] In some embodiments, the comparison of the homology percentages is based on the homology of the sequences of interest. The comparison of the sequences of interest ... Although the final homology percentage can be measured according to identity, the comparison process itself is usually not based on all-or-nothing comparisons. Instead, a similarity score matrix of scaling is usually used, which is assigned a score for each paired comparison based on chemical similarity or evolutionary distance. An example of this type of matrix commonly used is the default matrix of the BLOSUM62 matrix-BLAST suite of programs. If provided, the GCG Wisconsin program usually uses public default values ​​or a custom symbol comparison table (for more details, referring to the user manual). For some applications, it is preferred that the GCG software package is used with public default values, or in the case of other software, it is preferred to use a default matrix, such as BLOSUM62.

[0561] Once the software has generated an optimal alignment, it can calculate the percent homology, preferably the percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0562] A "fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0563] Such variants can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. In the case of inserting, the synthetic DNA encoding the insertion and the 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site can be prepared. The flanking regions can contain convenient restriction sites corresponding to the sites in the naturally occurring sequence, thereby allowing the sequence to be cut with an appropriate enzyme and the synthetic DNA to be connected to the nicks. The DNA is then expressed according to the present invention to prepare the encoded protein. These methods only illustrate numerous standard techniques known in the art for manipulating DNA sequences, and other known techniques can also be used.

[0564] Major histocompatibility complex (MHC) molecules

[0565] Typically, a TCR binds a peptide as part of a peptide:MHC complex.

[0566] The MHC molecule may be a class I or class II MHC molecule.The complex may be on the surface of an antigen presenting cell such as a dendritic cell or a B cell, or any other cell, including a cancer cell, or it may be immobilized by, for example, coating on beads or a plate.

[0567] The human leukocyte antigen system (HLA) is the name of the gene complex that encodes the major histocompatibility complex (MHC) in humans and includes HLA class I antigens (A, B, and C) and HLA class II antigens (DP, DQ, and DR). HLA alleles A, B, and C present peptides primarily derived from intracellular proteins, such as proteins expressed within cells. This is particularly relevant because WT1 is an intracellular protein.

[0568] During T cell development in vivo, T cells undergo a positive selection step to ensure recognition of self-MHC, followed by a negative step to remove T cells that bind too strongly to MHC presenting self-antigens. As a result, some T cells and the TCRs they express will only recognize peptides presented by certain types of MHC molecules (i.e., those encoded by specific HLA alleles). This is called HLA restriction.

[0569] One of the HLA alleles of interest is HLA-A*0201, which is expressed in the vast majority (>50%) of the Caucasian population. Therefore, a TCR that binds to a WT1 peptide presented by the MHC encoded by HLA-A*0201 (i.e., restricted by HLA-A*0201) is advantageous because immunotherapy utilizing such a TCR would be suitable for treating a large portion of the Caucasian population.

[0570] Other HLA-A alleles of interest are HLA-A*0101, HLA-A*2402, and HLA-A*0301.

[0571] The widely expressed HLA-B alleles of interest are HLA-B*3501, HLA-B*0702, and HLA-B*3502.

[0572] The TCRs of the present invention may be HLA-A*0201 restricted.

[0573] On the one hand, when the TCR of the present invention comprises a CDR3α comprising the amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0574] On the other hand, when the TCR of the present invention comprises a CDR3α comprising an amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising an amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO: 41) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0575] On the other hand, when the TCR of the present invention comprises a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0576] On the other hand, when the TCR of the invention comprises a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0577] On the other hand, when the TCR of the present invention comprises a CDR3α comprising an amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising an amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0578] On the other hand, when the TCR of the present invention comprises a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0579] On the other hand, when the TCR of the present invention comprises a CDR3α comprising an amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising an amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0580] On the other hand, when the TCR of the present invention comprises a CDR3α comprising an amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising an amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0581] On the other hand, when the TCR of the invention comprises a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0582] On the other hand, when the TCR of the invention comprises a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0583] On the other hand, when the TCR of the invention comprises a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-A*0201 restricted.

[0584] In one embodiment, an HLA-A*0201 restricted TCR of the invention binds to a WT1 peptide comprising the amino acid sequence APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0585] In one aspect, the invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0586] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO:36) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO:41) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO:117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0587] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAANNARLMF (SEQ ID NO:92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSDTRAREQFF (SEQ ID NO:97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO:117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0588] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0589] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0590] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAANNARLMF (SEQ ID NO:92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSPGQHGELFF (SEQ ID NO:271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO:117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0591] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0592] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0593] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO:277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO:163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQID NO:117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0594] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0595] In another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0596] Another broadly expressed HLA allele of interest is HLA-B*3501. The TCRs of the present invention may be HLA-B*3501 restricted.

[0597] In one aspect, when the TCR of the invention comprises a CDR3α comprising an amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising an amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO: 80) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-B*3501 restricted.

[0598] Thus, in another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO:75) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO:80) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-B*3501 restricted, and wherein the WT1 peptide comprises the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO:127) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0599] In one aspect, when the TCR of the invention comprises a CDR3α comprising an amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising an amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO: 86) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-B*3501 restricted.

[0600] Thus, in another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO:75) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO:86) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-B*3501 restricted, and wherein the WT1 peptide comprises the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO:127) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0601] Another broadly expressed HLA allele of interest is HLA-B*3502. The TCRs of the present invention may be HLA-B*3502 restricted.

[0602] In one aspect, where the TCR of the invention comprises a CDR3α comprising the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47) or a variant thereof having up to 3 amino acid substitutions, additions or deletions and a CDR3β comprising the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO: 57) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-B*3502 restricted.

[0603] Thus, in another aspect, the present invention provides a TCR that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises CDR3α and CDR3β, wherein the CDR3α comprises the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO:47) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO:57) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, wherein the TCR is HLA-B*3502 restricted, and wherein the WT1 peptide comprises the amino acid sequence of EPASQHTLRSG (SEQ ID NO:123) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0604] We have demonstrated that T cells expressing a TCR of the invention that binds to a WT1 peptide comprising the amino acid sequence EPASQHTLRSG (SEQ ID NO: 123) are able to selectively eliminate cancer (AML) cells expressing the HLA-B*3502 allele—see Examples 4 and Figure 4 c.

[0605] In one aspect, the TCR of the present invention binds to a WT1 peptide comprising the amino acid sequence of EPASQHTLRSG (SEQ ID NO: 123) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR is HLA-B*3502 restricted.

[0606] In one embodiment, when the TCR of the present invention binds to a WT1 peptide comprising the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-B*0201 restricted.

[0607] In one embodiment, when the TCR of the present invention binds to a WT1 peptide comprising the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, the TCR is HLA-B*3501 restricted.

[0608] Wilms' tumor 1 (WT1) protein

[0609] Wilms' tumor 1 (WT1) is an intracellular protein that encodes a zinc finger transcript that plays an important role in cell growth and differentiation (Yang, L. et al. Leukemia 21, 868-876 (2007)). It is widely expressed in a variety of hematological tumors and solid tumors, while showing limited expression in other tissues (gonads, uterus, kidney, mesothelium, progenitor cells in different tissues). Recent evidence suggests that WT1 plays a role in leukemogenesis and tumorigenesis.

[0610] WT1 has several isoforms, some of which are derived from alternative splicing of the mRNA transcript encoding WT1. The complete amino acid sequence of the WT1 isoform has been previously published (Gessler, M. et al. Nature; 343(6260): 774-778; (1990)). This particular isoform consists of 575 amino acids and includes the first 126 amino acids at the N-terminus, which are absent in the exon 5+ and KTS+ isoforms of WT1.

[0611] An exemplary WT1 protein has the amino acid sequence listed in UniProt entry J3KNN9. Another exemplary WT1 protein has the amino acid sequence listed below:

[0612] SRQRPHPGALRNPTACPLPHFPPSLPPTHSPTHPPRAGTAAQAPGPRRLLAAILDFLLLQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPS LGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYG HTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPG VAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL

[0613] (SEQ ID NO: 131).

[0614] WT1 peptide

[0615] As used herein, the term peptide refers to a plurality of amino acid residues connected by peptide bonds. As defined herein, a peptide can be composed of a length of about 30, less than about 25, less than about 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 amino acid residues. Preferably, the length of the peptide is about 5 to 20 amino acids, more preferably, the length of the peptide is about 8 to 15 amino acid residues.

[0616] The TCR of the present invention binds to the WT1 peptide when presented by MHC. As used herein, the term WT1 peptide should be understood to refer to a peptide comprising an amino acid sequence derived from the WT1 protein.

[0617] For example, the WT1 peptide can comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 25 consecutive amino acid residues of the WT1 protein amino acid sequence.

[0618] The WT1 peptide may comprise or consist of the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to three amino acid substitutions, additions or deletions. Examples of WT1 peptides comprising the amino acid sequence are AAQWAPVLDFAPPGA (SEQ ID NO: 115) and APVLDFAPPGASAYG (SEQ ID NO: 116).

[0619] The WT1 peptide may comprise or consist of an amino acid sequence selected from the group consisting of QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120) or a variant of each of SEQ ID NOs: 118-120 having up to 3 amino acid substitutions, additions or deletions.

[0620] The WT1 peptide may comprise or consist of an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123), YESDNHTTPIL (SEQ ID NO: 126), or a variant of each of SEQ ID NOs: 123 and 126 having up to three amino acid substitutions, additions, or deletions. Exemplary WT1 peptides may have an amino acid sequence selected from the group consisting of TCVPEPASQHTLRSG (SEQ ID NO: 121), EPASQHTLRSGPGCL (SEQ ID NO: 122), HSTGYESDNHTTPIL (SEQ ID NO: 124), and YESDNHTTPILCGAQ (SEQ ID NO: 125).

[0621] The WT1 peptide may comprise or consist of the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0622] The WT1 peptide may comprise or consist of an amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250) or a variant thereof having up to three amino acid substitutions, additions or deletions. Exemplary WT1 peptides may have an amino acid sequence selected from the group consisting of CMTWNQMNLGATLKG (SEQ ID NO: 248) and NQMNLGATLKGVAAG (SEQ ID NO: 249).

[0623] The WT1 peptide may comprise or consist of the amino acid sequence of DPGGIWAKLGAAEAS (SEQ ID NO: 251) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0624] The WT1 peptide may comprise or consist of an amino acid sequence selected from the group consisting of NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254), and variants of SEQ ID NOs: 252, 253, and 254 each having up to 3 amino acid substitutions, additions, or deletions.

[0625] In some embodiments, for WT1 peptides that bind to MHC molecules encoded by the HLA-A*0201 allele, it is preferred that the second amino acid of the peptide (i.e., the second amino acid from the N-terminus) be leucine or methionine, although isoleucine, valine, alanine, and threonine may also be preferred. It is also preferred that the amino acid at position 9 or 10 be valine, leucine, or isoleucine, although alanine, methionine, and threonine may also be preferred. Preferred MHC binding motifs for other HLA alleles are disclosed in Celis et al (Molecular Immunology, Vol. 31, December 8, 1994, pp. 1423-1430).

[0626] The present invention encompasses various uses of the WT1 peptides described herein. For example, the WT1 peptides described herein can be administered to a subject, such as a human subject. Administration of the WT1 peptides of the present invention can elicit an immune response against cells expressing or overexpressing the WT1 protein, i.e., the WT1 peptides are immunogenic WT1 peptides.

[0627] Thus, in one aspect, the present invention provides an isolated immunogenic WT1 peptide comprising an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123), YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254). ID NO: 254) and variants thereof each having up to 3 amino acid substitutions, additions or deletions.

[0628] The WT1 peptides described herein, for example, comprising an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123) and YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254). IDNO: 254) and or variants thereof having up to 3 amino acid substitutions, additions or deletions. For example, T2 cells can be pulsed with the WT1 peptides mentioned herein and incubated with T cell populations isolated from donors. In this method, the expression of cytokines such as CD107a and IFNγ can indicate T cells that recognize the WT1 peptide.

[0629] Thus, in one aspect, the present invention provides a T cell receptor (TCR) that binds a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the WT1 peptide comprises an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123), YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253). NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254), and variants thereof each having up to 3 amino acid substitutions, additions or deletions.

[0630] TCR sequence

[0631] We have determined the amino acid sequences of TCRs that bind to the WT1 peptides described herein. Specifically, we have determined the amino acid sequences of the TCR CDRs that are important for recognition and binding of the WT1 peptides.

[0632] Thus, in one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0633] Thus, in one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO: 41) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0634] Thus, in one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAANNARLMF (SEQ ID NO: 92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0635] Thus, in one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0636] In one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0637] In one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0638] In one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0639] Thus, in one embodiment, the present invention provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0640] The present invention also provides a TCR comprising:

[0641] CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0642] CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0643] CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions; or

[0644] CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions;

[0645] wherein the TCR binds to a WT1 peptide comprising or consisting of an amino acid sequence when presented by MHC, the amino acid sequence being selected from the group consisting of QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), and SQLECMTWNQMNLGA (SEQ ID NO: 120) or variants thereof having up to 3 amino acid substitutions, additions or deletions.

[0646] Thus, in one embodiment, a TCR is provided comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0647] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0648] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0649] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0650] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0651] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0652] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0653] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0654] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0655] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0656] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0657] In one embodiment, a TCR is provided, comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0658] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO: 57) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of EPASQHTLRSG (SEQ ID NO: 123) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0659] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASTQTPYEQYF (SEQ ID NO: 63) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of YESDNHTTPIL (SEQ ID NO: 126) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0660] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSTVGGEDYGYTF (SEQID NO: 69) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of YESDNHTTPIL (SEQ ID NO: 126) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0661] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CAISVGQGALYEQYF (SEQID NO: 80) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0662] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO: 86) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NHTTPILCGAQYRIH (SEQID NO: 127) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0663] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVTVGNKLVF (SEQID NO: 175) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASRGWREQFF (SEQ ID NO: 180) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of DPGGIWAKLGAAEAS (SEQID NO: 251) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0664] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSWGYQETQYF (SEQ ID NO: 196) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NHTTPILCGAQYRIH (SEQID NO: 252) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0665] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of KRHQRRHTGVKPFQC (SEQ ID NO: 253) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0666] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of PSCQKKFARSDELVR (SEQ ID NO: 254) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0667] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAANNARLMF (SEQID NO: 92) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0668] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAASATGNQFYF (SEQID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0669] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAASATGNQFYF (SEQID NO: 266) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0670] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0671] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0672] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0673] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAVRTSYDKVIF (SEQID NO: 113) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0674] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0675] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0676] The present invention further provides a TCR comprising CDR3α and CDR3, wherein the CDR3α comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the CDR3β comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282) or a variant thereof having up to 3 amino acid substitutions, additions or deletions, and the TCR binds to a WT1 peptide when presented by MHC, wherein the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQID NO: 117) or a variant thereof having up to 3 amino acid substitutions, additions or deletions.

[0677] Exemplary TCR amino acid sequences of the invention are provided in Table 1.

[0678] Table 1

[0679] Donor: HD1

[0680]

[0681]

[0682] Donor: HD2

[0683]

[0684]

[0685]

[0686]

[0687] Donor: HD3

[0688]

[0689]

[0690] Donor: HD4

[0691]

[0692]

[0693]

[0694]

[0695]

[0696] Donor: HD5

[0697]

[0698]

[0699]

[0700] Donor: HD6

[0701]

[0702]

[0703]

[0704]

[0705] Donor: HD7

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712] Donor: HD8

[0713]

[0714]

[0715]

[0716] Donor: HD9

[0717]

[0718]

[0719]

[0720]

[0721] Donor: HD10

[0722]

[0723]

[0724] Thus, the present invention provides isolated polypeptides comprising one or more amino acid sequences selected from SEQ ID NOs: 1-114 and 160-222, fragments, variants and homologues thereof.

[0725] In one aspect, the present invention provides a TCR comprising a TCR alpha chain sequence selected from the HD1-HD10 alpha chain sequences of Table 1 and a TCR beta chain sequence independently selected from the HD1-HD10 beta chain sequences of Table 1. The chain sequences of Table 1 reduce mispairing and improve TCR expression

[0726] The TCRs of the present invention can be expressed in T cells to alter the antigen specificity of the T cells. TCR-transduced T cells can express at least two TCRα and two TCRβ chains. Endogenous TCRα / β chains form self-tolerant receptors, while the introduced TCRα / β chains form receptors with a defined specificity for a given target antigen.

[0727] However, TCR gene therapy requires sufficient expression of the transferred TCR. The presence of endogenous TCRs may dilute the transferred TCRs, resulting in suboptimal expression of tumor-specific TCRs. In addition, mispairing between endogenous and introduced chains may occur to form new receptors, which may display unexpected specificity for self-antigens and cause autoimmune damage when transferred into the patient.

[0728] Therefore, several strategies have been explored to reduce the risk of mispairing between endogenous and introduced TCR chains. Mutation of the TCR alpha / beta interface is one strategy currently being used to reduce unwanted mispairing. For example, the introduction of cysteines in the constant domains of the alpha and beta chains allows for disulfide bond formation and enhances pairing of the introduced chain while reducing mispairing with the wild-type chain.

[0729] Thus, the TCRs of the invention may comprise one or more mutations at the α chain / β chain interface such that when the α chain and β chain are expressed in a T cell, the mispairing frequency between the chains is reduced compared to endogenous TCR α and β chains. In one embodiment, the one or more mutations introduce a cysteine ​​residue into the constant region of each of the α chain and the β chain, wherein the cysteine ​​residue is capable of forming a disulfide bond between the α chain and the β chain.

[0730] Another strategy to reduce mismatch relies on the introduction of polynucleotide sequences encoding siRNAs, which are added to the genes encoding tumor-specific TCR α and / or β chains and are designed to limit the expression of endogenous TCR genes (Okamoto S. Cancer Res 69, 9003-9011, 2009).

[0731] Thus, a vector or polynucleotide encoding a TCR of the invention may contain one or more siRNAs or other agents designed to limit or eliminate endogenous TCR gene expression.

[0732] Artificial nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR / Cas systems can also be combined to target the constant regions of endogenous genes, such as TCR genes (TRAC and / or TRBC), to achieve permanent disruption of endogenous TCR α and / or β chain genes, thereby allowing for full expression of tumor-specific TCRs and thus reducing or eliminating the risk of TCR mispairing. This process is known as TCR gene editing and has been shown to be superior to TCR gene transfer both in vitro and in vivo (Provasi E., Genovese P., Nature Medicine May; 18(5): 807-15; 2012).

[0733] Thus, the TCRs of the invention can be used to edit T cell specificity through TCR disruption and genetic addition of tumor-specific TCRs.

[0734] In addition, genome editing technology allows the targeted integration of an expression cassette comprising a polynucleotide encoding a TCR of the present invention and optionally one or more promoter regions and / or other expression control sequences into an endogenous gene that is destroyed by an artificial nuclease (Lombardo A., Nature biotechnology 25, 1298-1306; 2007).

[0735] Therefore, the TCR of the present invention can be used to edit T cell specificity by targeted integration of a polynucleotide encoding the TCR of the present invention in a genomic region. The integration can be targeted by an artificial nuclease.

[0736] Another strategy to increase the expression of transferred TCRs and reduce TCR mispairing has been developed, including "murinization," which replaces the human TCRα and TCRβ constant regions (e.g., TRAC, TRBC1, and TRBC2 regions) with their murine counterparts. Murizination of TCR constant regions is described, for example, in Sommermeyer and Uckert, J. Immunol; J. Immunol. Chem. Soc., 1992, 6, 1897. 2010 (184: 6223-6231). Thus, the TCRs of the present invention can be humanized.

[0737] Isolated polynucleotides

[0738] The present invention relates to an isolated polynucleotide encoding a TCR receptor of the invention or a portion thereof, such as an α chain and / or a β chain, a variable domain or a portion thereof.

[0739] An isolated polynucleotide may be double-stranded or single-stranded, and may be RNA or DNA.

[0740] Those skilled in the art will appreciate that due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. In addition, it should be understood that the skilled artisan can use conventional techniques to make nucleotide substitutions, additions or deletions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention to reflect the codon usage of any particular host organism. The polypeptides of the present invention will be expressed.

[0741] The polynucleotides described herein can be modified by any method available in the art. Such modifications can be made in order to enhance the in vivo activity or lifespan of the polynucleotides of the present invention.

[0742] Polynucleotides such as DNA polynucleotides can be produced recombinantly, synthetically or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0743] Typically, recombinant methods, such as polymerase chain reaction (PCR) cloning techniques, will be used to generate longer polynucleotides. This will involve preparing a pair of primers (e.g., about 15 to 30 nucleotides) flanking the target sequence of the desired clone, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that cause amplification of the desired region, isolating the amplified fragments (e.g., by purifying the reaction mixture with agarose gel) and recovering the amplified DNA. The primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0744] Table 2 provides examples of nucleotide sequences encoding TCRs according to the present invention.

[0745] Table 2

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776] Accordingly, the present invention provides an isolated polynucleotide comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 132-156, 223-247, and 292-299, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0777] The present invention also provides a TCR comprising an alpha chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 132, 135, 138, 141, 144, 147, 152, 223, 226, 227, 228, 233, 236, 237, 238, 245, 292, 295 and variants thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0778] The present invention also provides a TCR comprising a beta chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID No: 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 150, 151, 153, 154, 155, 156, 224, 225, 229, 230, 231, 232, 234, 235, 239, 240, 241, 242, 243, 244, 246, 247, 293, 294, 296-299, and variants thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0779] The present invention further provides isolated polynucleotide sequences derived from the sequences present in Table 2. For example, the present invention provides isolated polynucleotides encoding the variable region of a TCR according to the present invention, wherein the isolated polynucleotide comprises a segment of nucleotides of any one of SEQ ID Nos: 132-156, 223-247 and 292-299.

[0780] The variant sequence may have one or more base additions, deletions or substitutions. If the variation involves one or more additions or deletions, they may occur in groups of three or be balanced (i.e., one addition per deletion) such that the variation does not cause a frameshift in the translation of the remainder of the sequence.

[0781] Some or all variations may be "silent" in the sense that they do not affect the protein-encoded sequence due to the degeneracy of the genetic code.

[0782] As explained above, some or all variants may produce conservative amino acid substitutions, additions or deletions. The variations may be concentrated in one or more regions, such as the regions encoding the constant regions of the α or β chains, the linker or the framework regions, or they may be distributed throughout the molecule.

[0783] The variant sequence should retain the ability to encode all or part of the TCR amino acid sequence that binds to the WT1 peptide.

[0784] Codon optimization

[0785] The polynucleotides used in the present invention can be codon optimized. Codon optimization has previously been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in the selection of their specific codons. This codon bias corresponds to the bias of the relative abundance of a specific tRNA in the cell type. By changing the codons in the sequence so that they are suitable for matching with the relative abundance of the corresponding tRNA, expression can be increased. Similarly, expression can be reduced by intentionally selecting codons of the corresponding tRNA that are known to be rare in a specific cell type. Therefore, an additional degree of translational control can be obtained.

[0786] Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by changing these to correspond to commonly used mammalian codons, increased expression of packaging components in mammalian production cells can be achieved. Codon usage tables are known in the art for mammalian cells and a variety of other organisms.

[0787] Codon optimization may also involve the removal of mRNA destabilizing motifs and cryptic splice sites.

[0788] carrier

[0789] The present invention provides vectors comprising the polynucleotides described herein.

[0790] A vector is a tool that allows or promotes the transfer of an entity from one environment to another. According to the present invention, and for example, some vectors used in recombinant nucleic acid technology allow entities, such as nucleic acid segments (e.g., heterologous DNA segments, such as heterologous cDNA segments) to be transferred into target cells. Vectors can be used to maintain heterologous nucleic acids (DNA or RNA) in cells, promote the replication of vectors containing nucleic acid segments, or promote the expression of proteins encoded by nucleic acid segments. Vectors can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, chromosomes, artificial chromosomes, and viruses. Vectors can be single-stranded or double-stranded. It can be linear, and optionally, the vector comprises one or more homology arms. The vector can also be, for example, naked nucleic acid (e.g., DNA). In its simplest form, the vector itself can be the nucleotide of interest.

[0791] The vector used in the present invention may be, for example, a plasmid or a viral vector, and may comprise a promoter for expressing the polynucleotide and optionally a regulator of the promoter.

[0792] A vector comprising a polynucleotide for use in the present invention can be introduced into cells using a variety of techniques known in the art, such as transformation, transfection, and transduction. Several techniques are known in the art, such as transduction with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral, and herpes simplex viral vectors, Sleeping Beauty vectors; direct injection of nucleic acids and biolistic transformation.

[0793] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes the process of delivering genes to target cells using non-viral vectors. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofection, cationic agent-mediated transfection, cationic facial amphiphiles (CFA) (Nature Biotechnology 1996 14; 556) and combinations thereof.

[0794] The term "transfection" should be understood to include delivery of polynucleotides to cells by both viral and non-viral delivery.

[0795] Additionally, the present invention may employ gene targeting approaches, such as the delivery of DNA modifying agents.

[0796] The term "vector" includes expression vectors, i.e., constructs capable of in vivo or in vitro / ex vivo expression, expression being controlled by vector sequences or, for example, by target sequences in the case of insertion into a target site. The vector may be integrated or tethered to the DNA of the cell.

[0797] Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.

[0798] Retroviruses are RNA viruses with a life cycle that differs from that of lytic viruses. In this respect, retroviruses are infectious entities that replicate via a DNA intermediate. When a retrovirus infects a cell, its genome is converted to DNA form by the enzyme reverse transcriptase. The DNA copy serves as a template for the production of a new RNA genome and the virally encoded proteins necessary for the assembly of infectious viral particles.

[0799] There are many retroviruses, such as murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelomatosis virus type 29 (MC29) and avian erythroblastosis virus (AEV) and all other retroviruses, including lentiviruses.

[0800] A detailed list of retroviruses can be found in Coffin et al ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp758-763).

[0801] Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non-dividing cells (Lewis et al (1992) EMBO J. 3053-3058).

[0802] The vector can be capable of transferring the nucleotide sequence encoding the WT1-specific TCR described herein to cells, such as T cells, so that the cells express the WT1-specific TCR. Preferably, the vector will be capable of sustained high-level expression in T cells, so that the introduced TCR can successfully compete with the endogenous TCR for the limited CD3 molecule pool.

[0803] Increasing the supply of CD3 molecules can increase the expression of TCR, for example, in cells that have been modified in a manner to express the TCR of the present invention. Therefore, the vector of the present invention may further include one or more genes encoding CD3-gamma, CD3-delta, CD3-epsilon and / or CD3-zeta. In one embodiment, the vector of the present invention includes a gene encoding CD3-zeta. The vector may include a gene encoding CD8. The vector may encode a selection marker or a suicide gene to increase the safety profile of genetically engineered cells, such as cells of the present invention or cells modified to express the TCR of the present invention (Bonini, Science 1997, Ciceri, Bonini Lancet Oncol. 2009, Oliveira et al., STM2015). The genes included in the vector of the present invention can be connected by a self-cleavage sequence, such as a 2A self-cleavage sequence.

[0804] Alternatively, one or more separate vectors encoding CD3 genes may be provided for co-transfer to cells simultaneously, sequentially or separately with one or more vectors of the invention, such as one or more vectors encoding a TCR of the invention.

[0805] cell

[0806] The present invention relates to a cell comprising a polynucleotide or a vector according to the invention.

[0807] The cell can be a T cell, a lymphocyte or a stem cell. The T cell, the lymphocyte or the stem cell can be selected from the group consisting of CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells and pluripotent stem cells.

[0808] The type of cells can be selected to provide desired and advantageous persistence in vivo, and to provide desired and advantageous functions and properties for the cells of the invention.

[0809] The cells may have been isolated from a subject.

[0810] The cells of the present invention can be provided for adoptive cell transfer. As used herein, the term "adoptive cell transfer" refers to administering a cell population to a patient. Typically, the cells are T cells that are isolated from a subject and then genetically modified and cultured in vitro to express the TCR of the present invention prior to administration to the patient.

[0811] Adoptive cell transfer can be allogeneic or autologous.

[0812] By "autologous cell transfer" it is understood that the starting cell population (which is then transduced according to the methods of the invention, or transduced with a vector according to the invention) is obtained from the same subject as the subject to whom the transduced T cell population is administered. Autologous transfer is advantageous because it avoids problems associated with immunological incompatibility and can be used for subjects regardless of the availability of a genetically matched donor.

[0813] "Allogeneic cell transfer" is understood to mean that the starting cell population (which is then transduced according to the methods of the invention or transduced with the vectors of the invention) is obtained from a subject different from the subject to which the transduced cell population is administered. Preferably, the donor is genetically matched to the subject to whom the cells are administered to minimize the risk of immunological incompatibility. Alternatively, the donor may be a mismatch and unrelated to the patient.

[0814] An appropriate dosage of the transduced cell population is, for example, effective therapeutically and / or prophylactically. The dosage to be administered may depend on the subject and the condition to be treated, and may be readily determined by a skilled artisan.

[0815] The cells can be derived from T cells isolated from a subject. The T cells can be part of a mixed cell population isolated from a subject, such as a peripheral blood lymphocyte (PBL) population. T cells within a PBL population can be activated by methods known in the art, such as using anti-CD3 and / or anti-CD28 antibodies or cell-sized beads conjugated to anti-CD3 and / or anti-CD28 antibodies.

[0816] T cells can be CD4 + Helper T cells or CD8 + Cytotoxic T cells. Cells can be found in CD4 + Helper T cells / CD8 + Polyclonal activation (e.g., using anti-CD3 antibodies, optionally in combination with anti-CD28 antibodies) will trigger the CD4 + and CD8 + Proliferation of T cells.

[0817] Cells can be isolated from subjects to whom genetically modified cells are to be adopted for transfer. In this regard, cells can be prepared by isolating T cells from subjects, optionally activating T cells, and transferring TCR genes to cells ex vivo. Subsequent immunotherapy of the subject can then be performed by adoptive transfer of TCR-transduced cells. As used herein, this process refers to autologous T cell transfer, i.e., administering TCR-transduced cells to the same subject from which the T cells were initially derived.

[0818] Alternatively, T cells can be isolated from different subjects so that they are allogeneic. T cells can be isolated from donor subjects. For example, if the subject is undergoing allogeneic hematopoietic stem cell transplantation (Allo-HSCT) or solid organ transplantation or cell transplantation or stem cell therapy, the cell can be derived from the donor of the derived organ, tissue or cell. The donor and the subject undergoing treatment can be brothers and sisters.

[0819] Alternatively, the cell may be or may be derived from a stem cell, such as a hematopoietic stem cell (HSC). Since stem cells do not express CD3 molecules, gene transfer to HSC does not result in TCR expression on the cell surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, activation of CD3 expression results in surface expression of the TCR introduced in thymocytes.

[0820] The advantage of this approach is that, once generated, mature T cells express only the introduced TCR and very few or no endogenous TCR chains, because expression of the introduced TCR chains inhibits the rearrangement of endogenous TCR gene segments to form functional TCR α and β genes. Another advantage is that the genetically modified stem cells are a continuous source of mature T cells with the desired antigen specificity. Thus, the cells can be genetically modified stem cells, preferably genetically modified hematopoietic stem cells, which, upon differentiation, give rise to T cells expressing the TCR of the present invention.

[0821] Other methods known in the art can be used to reduce, limit, prevent, silence or eliminate the expression of endogenous genes in the cells of the invention or cells produced by the methods of the invention.

[0822] As used herein, the term "disruption" refers to reducing, limiting, preventing, silencing or eliminating the expression of a gene. Those skilled in the art can use any method known in the art to disrupt endogenous genes, for example, any suitable method for genome editing, gene silencing, gene knockdown or gene knockout.

[0823] For example, endogenous genes can be destroyed using artificial nucleases. Artificial nucleases are artificial restriction enzymes that are engineered to selectively target specific polynucleotide sequences (e.g., encoding a gene of interest) and induce double-strand breaks in the polynucleotide sequence. Typically, double-strand breaks (DSBs) are repaired by error-prone non-homologous end joining (NHEJ), resulting in the formation of non-functional polynucleotide sequences that are unable to express the endogenous gene.

[0824] In some embodiments, the artificial nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), and CRISPR / Cas (eg, CRISPR / Cas9).

[0825] The method for preparing cells (e.g., T cells) of the present invention can include a step of targeting an expression cassette for integration into an endogenous gene (e.g., endogenous TCR α chain gene and / or endogenous TCR β chain gene). As used herein, the term expression cassette refers to a polynucleotide sequence (e.g., a DNA polynucleotide sequence) comprising one or more polynucleotide sequences encoding one or more genes of interest, such that the gene of interest can be expressed. The endogenous sequence can promote expression from the expression cassette and / or the transcription control sequence within the expression cassette can promote expression. For example, the expression cassette can include a polynucleotide sequence of the present invention or a polynucleotide sequence encoding the TCR of the present invention that is operably connected to an expression control sequence, such as a promoter or enhancer sequence. One or more genes of interest can be located between one or more groups of restriction sites. Suitably, restriction sites can promote the integration of the expression cassette into, for example, a vector, a plasmid, or genomic DNA (e.g., host cell genomic DNA).

[0826] For example, an expression cassette of the invention can be transferred from a first polynucleotide sequence (e.g., on a vector) to another by "cutting," e.g., excising, the expression cassette using one or more appropriate restriction enzymes, and "pasting," e.g., integrating, the expression cassette into a second polynucleotide sequence.

[0827] The expression cassette may comprise a polynucleotide of the present invention. The expression cassette may comprise a polynucleotide encoding one or more TCRs of the present invention. The expression cassette may further comprise an antibiotic resistance gene or other selection marker gene that allows identification of cells that have successfully integrated the expression cassette into their DNA. The polynucleotide sequence contained in the expression cassette may be operably linked to an expression control sequence, such as a suitable promoter or enhancer sequence. Those skilled in the art will be able to select a suitable expression control sequence.

[0828] The present invention also contemplates cells expressing the TCR of the present invention, which have been engineered to destroy one or more endogenous MHC genes. The destruction of endogenous MHC genes can reduce or prevent the expression of MHC on the engineered cell surface. Therefore, such engineered cells with reduced MHC expression or without MHC expression will have a limited ability to present antigens on their cell surface or no such ability. Such cells are particularly advantageous for adoptive cell transfer because the cells will be non-allogeneic, for example, the cells will not present antigens that can be recognized by the immune system of the subject receiving the adoptively transferred cells. Therefore, the transferred cells will not be identified as "non-self" and can avoid adverse immune responses to the cells. Such cells are referred to as "universal cells" because they are suitable for adoptive transfer to a variety of different hosts, regardless of HLA type.

[0829] Therefore, the present invention provides a method for preparing non-autoreactive universal T cells expressing the TCR of the present invention. The present invention further provides non-autoreactive universal T cells expressing the TCR of the present invention.

[0830] The present invention further contemplates cells that have been engineered to destroy one or more endogenous genes to modify cells, thereby enhancing favorable properties, characteristics, or functions of the cells and / or reducing undesirable properties, characteristics, or functions. For example, by destroying endogenous cells, persistence, expansion, activity, resistance to exhaustion / aging / inhibitory signals, homing ability, or other cellular functions can be modified. As used in this context, the term "modification" refers to a change in one or more characteristics relative to an equivalent unmodified cell, e.g., a cell in which an endogenous gene has not yet been destroyed. For example, a change can be an increase, enhancement, or introduction of a cellular characteristic or function relative to an equivalent unmodified cell. Alternatively, a change can be a reduction, inhibition, or elimination of a cellular characteristic or function relative to an equivalent unmodified cell.

[0831] The polynucleotides and vectors of the present invention can be transferred into specific T cell subsets, including CD4 and or CD8, naive, memory stem T cells, central memory, effector memory or effector cells, or into other cell subsets, for example to promote different in vivo persistence lengths and functions in the cells of the present invention.

[0832] The polynucleotides and vectors of the present invention can also be transferred into T cell subsets, such as naive and memory stem T cells, central memory cells, effector memory cells, and effector cells.

[0833] Depending on the cytokine background most suitable for targeting a specific tumor type, the polynucleotides and vectors of the present invention can also be transferred into T cell subsets with different polarizations, such as Th0 / Tc0, Th1 / Tc1, Th2 / Tc2, Th17, Th22, etc.

[0834] In addition, the polynucleotides and vectors of the present invention encoding the antigen-specific regions of the TCR of the present invention can be transferred in other cell subsets, including γ / δ T cells, NK cells, NKT cells, hematopoietic stem cells or other cells to obtain therapeutic effects.

[0835] The present invention further provides a method for preparing cells, comprising the step of transducing cells in vitro or ex vivo with the vector of the present invention. Various methods for transducing cells with vectors are known in the art (see, for example, Sambrook et al.).

[0836] The present invention also provides a method for producing T cells expressing the TCR of the present invention by inducing differentiation of stem cells comprising the polynucleotide or vector of the present invention.

[0837] Cell colonies can be selectively purified for cells that exhibit a specific phenotype or characteristic and for other cells that never exhibit this phenotype or characteristic or that exhibit this phenotype or characteristic to a lesser extent. For example, cell colonies expressing specific markers (e.g., CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) can be purified from a starting cell population. Alternatively or in addition, cell colonies that do not express another marker can be purified.

[0838] By "enriching" a cell population for a certain type of cell, it is understood that the concentration of that type of cell within the population is increased. The concentrations of other cell types may concomitantly decrease.

[0839] Purification or enrichment can result in a population of cells that is substantially pure of other cell types.

[0840] Purification or enrichment of cell populations expressing a specific marker (e.g., CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) can be achieved by using a reagent that binds to the marker, preferably a reagent that actually specifically binds to the marker. The reagent that specifically binds to a cell marker can be an antibody, such as an antibody that binds to CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4.

[0841] The term "antibody" refers to intact antibodies or antibody fragments capable of binding to a selected target, including Fv, ScFv, F(ab') and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies, including chimeric antibodies, CDR-implanted antibodies and humanized antibodies, as well as artificially selected antibodies produced using phage display or alternative technologies.

[0842] Additionally, alternatives to classical antibodies may also find use in the present invention, such as "avibodies," "avimers," "anticalins," "nanobodies," and "DARPins."

[0843] The reagent that can be combined with specific marker can be marked so that any one of multiple techniques known in the art can be used for identification. Reagent can be intrinsically labeled, or can be modified by being conjugated to a marker. By "conjugating", it should be understood that reagent and marker are operably connected. This means that reagent and marker are connected together so that both can play their function (such as binding marker, allowing fluorescence identification or allowing separation when placed in a magnetic field) without hindrance. Suitable conjugation methods are well known in the art, and those skilled in the art will easily identify.

[0844] For example, the label can allow purification from its environment (e.g., the reagent can be labeled with magnetic beads or affinity tags such as avidin) and / or detection of the labeled reagent and any cells bound to it. Detectable labels suitable for use as labels include fluorophores (e.g., green, cherry, cyan, and orange fluorescent proteins) and peptide tags (e.g., His-tag, Myc-tag, FLAG-tag, and HA-tag).

[0845] Many techniques for isolating cell populations expressing specific markers are known in the art. These include magnetic bead-based separation techniques (e.g., separation based on closed-loop magnetic beads), flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g., using affinity columns or beads, such as biotin columns to separate avidin-labeled reagents), and microscopy-based techniques.

[0846] It is also possible to use a combination of different techniques for separation, such as a magnetic bead-based separation step followed by flow cytometry sorting of the resulting cell population for one or more additional (positive or negative) markers.

[0847] For example, you can use Clinical grade separations are performed using the Miltenyi system. This is an example of a closed-loop magnetic bead-based separation technology.

[0848] It is also envisioned that dye exclusion properties (eg, side population or rhodamine labeling) or enzymatic activity (eg, ALDH activity) can be used to enrich for HSCs.

[0849] chimeric molecules

[0850] In another aspect, the invention provides a chimeric molecule comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, or a portion thereof, conjugated to a non-cellular substrate. The conjugation may be covalent or non-covalent.

[0851] The acellular substrate can be a nanoparticle, an exosome, or any acellular substrate known in the art.

[0852] The chimeric molecules of the present invention may be soluble.

[0853] In another aspect, the invention provides a chimeric molecule comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, or a portion thereof, conjugated to a toxin or an antibody.

[0854] The toxin or antibody can be cytotoxic. The toxin can be a cytotoxic molecule or compound, such as a radioactive molecule or compound. The TCR portion of the chimeric molecule can confer the ability to recognize cells expressing a WT1 protein or peptide. Thus, the chimeric molecule can specifically recognize and / or bind to tumor cells expressing WT1. Thus, the chimeric molecules of the present invention can provide WT1-targeted delivery of cytotoxic toxins, antibodies, and / or compounds.

[0855] WT1-related diseases

[0856] WT1 is widely expressed in a variety of hematological malignancies and solid tumors, while showing limited expression in various healthy tissues (e.g., gonads, uterus, kidneys, mesothelium, and progenitor cells in various tissues). The present inventors have identified and determined the amino acid sequence of a TCR that recognizes the WT1 peptide. Furthermore, they have demonstrated that T cells expressing the TCR according to the present invention target and kill cells that present the WT1 peptide or overexpress the WT1 protein.

[0857] Therefore, the present invention provides a method for treating and / or preventing a disease associated with WT1 expression, comprising administering a TCR, isolated polynucleotide, vector, or cell of the present invention to a subject in need thereof. The present invention also provides a method for treating and / or preventing a disease associated with WT1 expression, comprising administering a cell prepared by the method of the present invention to a subject in need thereof.

[0858] The present invention also provides the TCR of the present invention, the isolated polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, or the cell prepared by the method of the present invention for use in treating and / or preventing diseases associated with WT1 expression.

[0859] The term "prevent" is intended to mean avoiding, delaying, preventing or hindering the development of a disease. Treatment can, for example, prevent or reduce the likelihood of developing or developing a disease associated with WT1 expression.

[0860] As used herein, "treat" or "treatment" refers to the care of a subject with a disease in order to ameliorate, cure, or alleviate the symptoms of the disease, or to lessen, arrest, or delay the progression of the disease.

[0861] The subject can be a human subject. The human subject can be a child. For example, the child can be less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, or less than 2 years old. The human subject can be an infant.

[0862] Based on the expression of WT1, it may have been previously determined that the subject needs the TCR, isolated polynucleotides, vectors or cells of the present invention, or the cells prepared by the methods of the present invention. For example, the subject may have a cell population that exhibits increased WT1 expression relative to a healthy control cell population. The expression of WT1 can be determined using a variety of techniques known in the art, for example, quantitative RT-PCR can be used to determine the amount of WT1 RNA transcripts, which indicates WT1 protein expression. It will also be understood by those skilled in the art that WT1 protein expression can be determined by western blotting using commercial antibodies specific for WT1.

[0863] The subject may also have been previously identified as having a change (e.g., mutation or deletion) in the WT1 gene. Such changes may be hereditary. Therefore, the disease associated with WT1 expression may be a hereditary disease. Examples of hereditary diseases associated with WT1 expression include, but are not limited to, WAGR (Wilms tumor-aniridia-urogenital malformation-delay) syndrome, Denys-Drash syndrome (DDS), Frasier syndrome (FS), urogenital anomalies (reproductive and urinary system anomalies) syndrome.

[0864] A subject with a genetic disease associated with WT1 expression may have a higher risk of developing a proliferative disorder, such as cancer.

[0865] The disease associated with WT1 expression may be a proliferative disorder.

[0866] The proliferative disorder may be a hematological malignancy or a solid tumor. The hematological malignancy may be selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

[0867] The solid tumor may be selected from the group consisting of lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial carcinoma, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0868] Diseases associated with WT1 expression can be selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-Hodgkin's lymphoma and Hodgkin's lymphoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial carcinoma, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0869] Pharmaceutical composition

[0870] The TCRs of the present invention, the polynucleotides of the present invention, the vectors of the present invention, the cells of the present invention, the cells prepared by the methods of the present invention, the chimeric molecules of the present invention, or the mixed cell populations of the present invention can be formulated with a pharmaceutically acceptable carrier, diluent, or excipient for administration to a subject. Suitable carriers and diluents include isotonic saline solutions, such as phosphate-buffered saline, and potentially containing human serum albumin.

[0871] Preferably, the processing of cell therapy products is carried out according to the FACT-JACIE international cell therapy standards.

[0872] Treatment

[0873] In another aspect, the present invention provides a method for treating and / or preventing a disease associated with WT1 expression, comprising the step of administering to a subject in need thereof a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell of the present invention, a cell prepared by the method of the present invention, a chimeric molecule of the present invention, or a mixed cell population of the present invention.

[0874] The subject can be a human subject. The subject can be a non-human animal subject.

[0875] The subject may have a disease associated with WT1 expression. The subject may be at risk for developing a disease associated with WT1 expression. The subject may have previously been determined to be at risk for developing a disease associated with WT1 expression. The subject may have an increased risk for developing a disease associated with WT1.

[0876] The increased risk may have been determined by genetic screening and / or by reviewing the subject's family history.The subject may express genetic markers that indicate an increased risk of developing a disease associated with WT1 expression.

[0877] Suitably, those skilled in the art will be aware of genetic risk factors (e.g., genetic markers) associated with an increased risk of developing a disease associated with WT1. A skilled person may be able to use any suitable method or technique known in the art to determine whether a subject has an increased risk of developing a disease associated with WT1 expression.

[0878] The subject may have previously been treated for a disease associated with WT1 expression. The subject may be in remission. The subject may be resistant to chemotherapy. The subject may be resistant to anti-WT1 therapy.

[0879] In one embodiment, the method for treating and / or preventing a disease associated with WT1 expression comprises administering chemotherapy to a subject. The chemotherapy can be administered to the subject simultaneously, sequentially, or separately from the TCR of the present invention, the isolated polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, the cell prepared by the method of the present invention, or the chimeric molecule of the present invention.

[0880] In another aspect, the present invention provides a method for treating and / or preventing a disease associated with WT1 expression, comprising the step of administering a mixed cell population, wherein the mixed cell population comprises a plurality of cell populations each expressing a different TCR of the present invention.

[0881] In another aspect, the present invention provides a mixed cell population comprising a plurality of cell populations each expressing a different TCR of the present invention.

[0882] In another aspect, the present invention provides a method for preparing a mixed cell population comprising a plurality of cell populations each expressing a different TCR of the present invention, wherein the method comprises the step of transducing cells in vitro or ex vivo with the vector of the present invention.

[0883] In another aspect, the present invention provides a mixed cell population for treating and / or preventing a disease associated with WT1 expression, wherein the mixed cell population comprises a plurality of cell populations each expressing a different TCR of the present invention.

[0884] For example, a mixed cell population can include a first cell population expressing a first TCR of the present invention and a second cell population expressing a second TCR of the present invention. For example, a mixed cell population can include a first cell population expressing a first TCR of the present invention, a second cell population expressing a second TCR of the present invention, and a third cell population expressing a third TCR of the present invention, etc.

[0885] Each cell population in a mixed cell population may, for example, express only a single TCR of the present invention. The endogenous TCR gene of the cell population in the mixed cell population may be destroyed or deleted. The expression of the endogenous TCR gene of the cells in the mixed cell population can be destroyed, for example, by gene editing with an artificial nuclease.

[0886] In another aspect, the present invention provides a use of the TCR of the present invention, the isolated polynucleotide of the present invention, the vector of the present invention, the cell of the present invention, the cell prepared by the method of the present invention, the chimeric molecule of the present invention, or the mixed cell population of the present invention for preparing a medicament for treating a disease associated with WT1 expression.

[0887] Both human and veterinary treatments are within the scope of the present invention.

[0888] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology, histology, immunology, and oncology, which are within the capabilities of a person of ordinary skill in the art and are explained in the literature.

[0889] See, for example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Chapters 9, 13 and 16, John Wiley & Sons; Roetree, B., Crab, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; and Lilley, DMand Dahlberg,JE(1992)Methods in Enzymology:DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general textbooks is incorporated herein by reference.

[0890] Various preferred features and embodiments of the invention will now be described by way of non-limiting examples. Example

[0891] Example 1: Generation of functional WT1-specific cytotoxic T lymphocytes (CTLs) from healthy donors (HD)

[0892] To identify novel TCRs specific for WT1 epitopes restricted to different HLA alleles, we stimulated peripheral blood mononuclear cells (PBMCs) from 10 different HD patients with a pool of overlapping 11 amino acids (15-mers) spanning the complete sequence of the WT1 protein (see Materials and Methods). This peptide design ensures that it is specific for CD4 + and CD8 + Optimal stimulation of T cells.

[0893] After 26-30 hours of stimulation, we enriched T cells expressing CD137. CD137 is a molecule that is upregulated upon T cell receptor engagement and has previously been shown to be a reliable marker for the rapid identification, isolation, and in vitro expansion of antigen-specific memory and naive CD4 T cells. + and CD8 + The CD137 negative fraction was further depleted of CD3 fraction, then irradiated with 30 Gy and used as CD137 + fraction of antigen presenting cells (APC). Sorted CD137 + Cells were expanded in vitro for approximately 9 days and restimulated every 7-14 days with autologous APCs presented by CD3-depleted cells or immortalized autologous B cells loaded with the peptide pool. Figure 1 As shown in the cytofluorescence results presented in (aj), this procedure resulted in the enrichment of WT1-specific T lymphocytes. Functional characterization of T cells was performed at different time points. In more detail, T cells were co-cultured with autologous APCs loaded with the peptide pool, and after 6 hours of co-culture, the expression of CD107a and IFNγ in the T cell population was identified by intracellular staining. The expression of CD107a after antigen encounter indicates the potential for antigen-induced degranulation and lysis.

[0894] Cells were stimulated with an irrelevant peptide pool as a negative control. Control stimulation resulted in minimal secretion of IFNγ and CD107a by T cells derived from each healthy donor.

[0895] Example 2: Localization of WT1 epitopes that trigger T cell responses

[0896] To identify WT1 epitopes recognized by T cells, IFNγ secretion was quantified after WT1-stimulated / enriched T cells were co-cultured with autologous APCs for 6 hours in vitro. The autologous APCs were loaded with peptide subpools, each containing up to 12 peptides according to a positioning grid. The positioning grid consisted of 24 subpools, each of which had each peptide uniquely contained within two intersecting subpools (Doubrovina, E. et al. Blood 120, 1633–1646 (2012)). Figure 2 The results are summarized in a.

[0897] FACS analysis showed substantial expression of IFNγ and CD107a by HD1, HD3, HD6, HD7, and HD10-derived T cells after stimulation with subpools 4, 5, and 16 ( Figure 2 b, d, g, h, k). Substantial expression of IFNγ was observed for HD2-derived T cells stimulated with subpools 6, 16, 17, and 20 ( Figure 2c), while subcombinations 4, 5, 6, 14, 18, and 21 stimulated the expression of IFNγ and CD107a in HD4-derived T cells ( Figure 2 e), and subcombinations 5, 11, 12, 21, 22 stimulated IFNγ expression in HD5 ( Figure 2 f). For HD7, we additionally observed increased expression of IFNγ and CD107a following stimulation with subcombinations 7, 8, and 20, albeit at lower levels compared to those observed with subcombinations 4, 5, and 16 ( Figure 2 h). In addition, we observed that HD8-derived T cells stimulated with subcombinations 12 and 14 ( Figure 2 i) and HD9-derived T cells stimulated with subcombinations 5, 13, 21 ( Figure 2 j) After 4 h, the expression of IFNγ and CD107a increased.

[0898] Thereafter, HD-derived T cells were stimulated for 6 hours with APCs pulsed with a single pentadecapeptide shared by the subpools eliciting the highest immune response and with at least one irrelevant 15mer. FACS analysis indicated that for peptides 40 and 41 in HD1 T cells ( Figure 3 a), peptides 54, 77, and 90 for HD2 T cells ( Figure 3 b) The peptide VLDFAPPGA (SEQ ID NO: 157; which is a nonamer of the peptide shown in SEQ ID NO: 117) is effective for HD3 T cells ( Figure 3 c), peptides 17, 18, 99, and 100 for HD4 ( Figure 3 d,e), peptide 101 for HD5 ( Figure 3 f), peptide VLDFAPPGA (SEQ ID NO: 157; which is a nonamer of the peptide shown in SEQ ID NO: 117) for HD6 T cells ( Figure 3 g), peptides 101, 125 and 137 for HD9 T cells ( Figure 3 h), the peptide VLDFAPPGA (SEQ ID NO: 157; which is a nonamer of the peptide shown in SEQ ID NO: 117) for HD10 T cells ( Figure 3i) increased expression of CD107a and / or IFNγ. In this way, dominant immunogenic sequences were identified. No relevant immune response (i.e., increased expression of CD107a and / or IFNγ) was observed after co-culture with irrelevant control peptides. For HD7 and HD8, due to reduced cell fitness, it was not possible to perform incubation experiments to identify immunogenic peptides. However, we were able to predict the recognized peptides by deconvolution of the localization grid. We identified overlapping sequences of peptides 41 and 42 (derived from SP4, 5, 16) of HD7, as well as overlapping sequences of peptides 91 and 92 (derived from SP7, 8, 20) and peptide 24 of HD8.

[0899] To determine the HLA restriction of WT1 immunogenic peptides recognized by T cells expanded from HD4, HD5, and HD10, T cells from these were co-cultured for 6 hours with a panel of different target EBV-BLCL cells expressing different HLA or HLA-B alleles, which had been pulsed with relevant peptides (peptide 17 from HD4; peptide 101 from HD5; peptide VLDFAPPGA (SEQ ID NO: 157) from HD10) or an irrelevant control peptide. The results showed that peptide 17 was presented by the HLA-B*3502 allele and recognized by T cells derived from HD4 ( Figure 3 j), peptide 101 is presented by HLA-B*3501 and recognized by HD5-derived T cells ( Figure 3 k); the peptide VLDFAPPGA (SEQ ID NO: 157) is presented by HLA-A*0201 and recognized by T cells derived from HD10 ( Figure 3 l).

[0900] The sequences of WT1 peptides recognized by WT1-specific T cells expanded from HD1-HD10 are shown in Table 3 below.

[0901] Table 3

[0902]

[0903]

[0904] Example 3: WT1-specific T cells selectively eliminate cells expressing WT1

[0905] To determine the HLA restriction of WT1-specific T cells and their ability to eliminate WT1-expressing cells, T cells were co-cultured with different target cells.

[0906] HD1-derived T cells

[0907] Recognizing that HD1 contains the HLA-A*0201 allele, we co-cultured enriched WT1-specific T cells with different target cells: T2 cells pulsed with an overlapping peptide pool containing peptides 40 and 41 (see Table 3); T2 cells pulsed with a MelanA / MART1 pool as a negative control (T2 MelanA / MART1 pool); or K562 cells genetically modified to express both the HLA-A*0201 allele and overexpress WT1 protein (K562 HLA-A*0201WT1).

[0908] After 6 hours of co-culture, the expression of CD107a was established by FACS. The results of HD1 indicated that >60% of CD8 + CD107a expression in T cells ( Figure 4 a). Similarly, co-culture of WT1-specific T cells with genetically modified K562 cells (expressing the HLA-A*0201 allele and WT1 protein) resulted in CD8 + T cells expressed more than 60% CD107a ( Figure 4 a).

[0909] In contrast, CD8 + CD107a expression on T cells was minimal ( Figure 4 a).

[0910] These results demonstrate that isolated HD1-derived T cells specifically recognize a WT1 peptide comprising the sequence APVLDFAPPGA (SEQ ID NO: 117) when presented by an MHC molecule encoded by the HLA-A*0201 allele. Furthermore, the results show that HD1-derived T cells are able to specifically target cells overexpressing the WT1 protein. Thus, these experimental data demonstrate that TCRs expressed by HD1-derived T cells specifically bind to a peptide comprising the amino acid sequence APVLDFAPPGA (SEQ ID NO: 117) and that such TCRs are HLA-A*0201-restricted.

[0911] HD3-derived T cells

[0912] Recognizing that HD3 contains the HLA-A*0201 allele, we co-cultured the enriched WT1-specific T cells with different target cells: T2 cells pulsed with subpool 16, which was previously determined to contain immunogenic peptides that elicit an immune response (T2-SP16); T2 cells pulsed with the MelanA / MART1 pool as a negative control (T2-Melan A); wild-type K562 cells (K562) as a negative control; or K562 cells genetically modified to express both the HLA-A*0201 allele and overexpress WT1 protein (K562 A2+WT1+).

[0913] After 4 days of coculture, the ability of HD3-derived T cells to kill target cells was expressed as the elimination index—calculated as the total number of target cells remaining after coculture with WT1-specific T cells divided by the total number of target cells alone.

[0914] The results demonstrated the ability of WT1-specific T cells to eliminate target cells expressing the specific WT1 epitopes identified ( Figure 4 b). In particular, HD3-derived T cells eliminated approximately 95% of T2 cells pulsed with a WT1 peptide comprising the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) (subpool 16; SP16). In addition, HD3-derived T cells eliminated approximately 78% of K562 cells expressing MHC molecules encoded by the HLA-A*0201 allele and overexpressing WT1 protein. In contrast, none of the T2 cells pulsed with the negative control MelanA / MART1 pool were eliminated by HD3-derived T cells. Similarly, there was minimal depletion of control wild-type K562 cells ( Figure 4 b).

[0915] These results demonstrate that isolated HD3-derived T cells specifically recognize a WT1 peptide comprising the amino acid sequence APVLDFAPPGA (SEQ ID NO: 117). Furthermore, the results show that HD3-derived T cells are able to specifically target and kill cells overexpressing the WT1 protein through peptide presentation via MHC encoded by HLA-A*0201. Therefore, these experimental data demonstrate the specificity of the WT1 peptide for the TCR expressed by HD3-derived T cells and that the HD3-derived TCR is HLA-A*0201-restricted.

[0916] HD4-derived T cells

[0917] T4 cells were isolated from primary leukemic blasts (CD33 +The ability of HD4-derived T cells to eliminate target cells was assessed by co-culture with leukemic blasts from patients with AML who were selected based on high WT1 antigen expression and HLA typing (HLA-B*3502). As a negative control, HD4-derived T cells were co-cultured with leukemic blasts from patients with AML who do not express the HLA-B*3502 allele.

[0918] After three days of co-culture at an effector to target ratio of 10:1, FACS analysis showed that the HD4WT1-specific T cells (CD3 + Leukemic blasts (CD33 + ) of the almost complete elimination ( Figure 4 c, upper panel) In fact, only 0.54% of the remaining total cell population was positive for CD33 expression.

[0919] In contrast, no expression of CD33 was observed after co-culture of WT1-specific T cells with irrelevant control AML blasts. + Cell clearance ( Figure 4 c, lower panel). Indeed, in control samples, 7.9% of the total cell population was positive for CD33 expression after co-culture with HD4-derived WT1-specific T cells.

[0920] Importantly, these results demonstrated the ability of HD4-derived WT1-specific T cells to specifically target and kill leukemic blasts (AML cancer cells) that overexpress WT1 and the MHC encoded by HLA-B*3502. Thus, HD4-derived TCRs are able to specifically target and kill cancer cells in an HLA-B*3502-restricted manner.

[0921] Example 4: Immune profiling of Vβ sequences in WT1-specific T cells

[0922] To better characterize the TCRs involved in antigen recognition of WT1-specific T cells from HD1-6 and 10 (for HD7, HD8 and HD9, Vβ immune profiling was not possible due to reduced cell fitness), we first performed a multiparameter FACS analysis to quantitatively determine the TCR Vβ repertoire. Therefore, to determine the clonality of the expanded WT1-specific T cells, we used the IOTest Beta Mark TCR V beta repertoire kit according to the manufacturer's recommendations. This kit allows the detection of 24 different V beta gene expression in 8 individual tubes. In particular, using this method, a coverage of 75% of the entire V beta repertoire was ensured. The results of the FACS staining indicated a greater prevalence of specific Vβ for HD1, HD2, HD3, HD5 - see Figure 5For HD4, HD6, and HD10, a thorough determination of the predominant Vβ was not possible, likely due to inherent limitations of the kit, which contains antibodies covering 75% of the existing Vβ proteins.

[0923] Example 5: High-throughput sequencing of TCR α and β chains isolated from WT1-specific T cells

[0924] WT1-specific T cells were collected at different time points within the co-culture time frame and their RNA was extracted using the Arcturus Pico Pure RNA extraction kit. The CDR3 sequences of WT1-specific T cells were amplified using a modified RACE method, which included magnetic capture after cDNA synthesis to improve the specificity of the reaction and eliminate unwanted templates (Ruggiero et al. Nat. Commun. 6, 8081 (2015)). The samples were sequenced using an Illumina MiSeq sequencer and CDR3 clonotypes were identified using MiXTCR software (Bolotin, DA et al. Nature Methods 12, 380–381 (2015)). In addition, CDR1, CDR2 and CDR3 were further defined using the IMGT V-quest tool (Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008). PMID: 18503082; Giudicelli, V., Brochet, X., Lefranc, M.-P., Cold Spring Harb Protoc. 2011 Jun 1; 2011(6). pii:pdb.prot5633.doi:10.1101 / pdb.prot5633. PMID: 21632778 abstract, also in the IMGT booklet kindly provided by Cold Spring Harbor (CSH) Project).

[0925] Sequencing results demonstrated that for both TCR chains in HD1-10, the prevalence of specific CDR3 clonotypes in the WT1-specific T cell population increased over time. Figure 6 The predominant α and β chain genotypes and CDR3 sequences are provided.

[0926] In addition, the full-length α and β chain amino acid sequences of the TCR derived from HD1-10 were determined - see Table 1. The corresponding nucleotide sequences were also determined - see Table 2.

[0927] Example 6: Functional validation of newly identified TCRs

[0928] TCR alpha and beta sequences that were isolated from HD1 and HD3 and recognize the WT1 VLDFAPPGA (SEQ ID NO: 157) peptide when presented by the HLA-A*0201 allele were cloned into a lentiviral vector under the control of a bidirectional promoter to enhance robust and coordinated expression of both TCR chains in transduced lymphocytes. T cells from healthy individuals were transduced with viral vectors encoding either the HD1 TCR or the HD3 TCR. As a control, we also transduced cells with the WT1 126-134 TCR. The transduced T cells were functionally validated by co-culturing with different target cells as follows: T2 cells (P<0.05) pulsed with one of the two recognized peptides (VLDFAPPGA (SEQ ID NO: 157) for HD1 and HD3 TCRs; RMFPNAPYL (SEQ ID NO: 255) for WT1 126-134 TCR). Figure 7 a), K562 cells either wild-type or engineered to express the HLA-A*0201 allele ( Figure 7 b), Primary AML blasts derived from three AML patients and selected based on HLA-A*0201 allele expression and WT1 expression ( Figure 7 c). After 3 days of co-culture, we observed the ability of each transduced T cell population to specifically recognize the target peptide when presented by the HLA-A*0201 allele ( Figure 7 a) and the greater potency of HD1 T cells in mediating the near-complete elimination of engineered K562 cells. Results from co-culture with pAML blasts further confirmed the higher potency of the HD1 TCR in recognizing target antigens. Here, we observed greater elimination of pAML blasts harboring the HLA-A*0201 allele when co-cultured with HD1 TR T cells compared to conditions using HD3 TR and WT1 126-134 TR T lymphocytes as effector cells.

[0929] Materials and methods

[0930] Peptides for stimulating and isolating WT1-specific T cells were designed using the WT1 protein sequence previously published by Gessler et al. (Gessler, M. et al. (1990) Nature 343:774-778). This sequence contains 575 amino acids and lacks the first 126 amino acids at the N-terminus of the WT1 (exon 5+, KTS+) isoform. We designed 141 pentadecapeptides spanning the entire sequence of the WT1 protein, each overlapping the next by 11 amino acids.

[0931] Peptides were synthesized by PRIMM to verify the specifications of the sequence, 70% purity, sterility and lack of endotoxin. These peptides were mixed in equal amounts in the WT1 pool at a concentration of 1 μg / ml for each peptide. In addition, 24 sub-pools were generated, each containing up to 12 peptides (4.17 μg / ml / each peptide) according to a specific positioning matrix, so that each peptide was included in only two overlapping sub-pools, as shown in Table 4 (see the positioning grid strategy in Doubrovina, E. et al. Blood 120, 1633–1646 (2012)).

[0932] Table 4

[0933]

[0934] Isolation of peripheral blood mononuclear cells

[0935] Peripheral blood was obtained from 10 healthy donors at San Raffaele Hospital after informed consent. Peripheral blood mononuclear cells were isolated using Ficoll-Hypaque density gradient centrifugation.

[0936] Immortalized B cells

[0937] Autologous B cells were isolated from PBMCs of healthy donors using CD19 microbeads (Miltenyi Biotech). Cells were then transduced with a lentiviral vector containing the BCL-6 / BCL-XL transgene (Kwakkenbos, MJ et al. Nat. Med. 16, 123 (2009)) and H / F alpha (Lévy, C. et al. Molecular Therapy 20 9, 1699–1712, (2012)) and cultured in IMDM supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and 10 ng / ml of IL-21 (Miltenyi Biotech). B cells were restimulated every 5 days by co-culturing with irradiated (50 Gy) CD40L-expressing mouse L cell fibroblasts (3T3-CD40L) at a B cell:3T3-CD40L ratio of 10:1.

[0938] cell lines

[0939] We cultured T2 and K562 cell lines in RPMI 1640 (GIBCO-BRL) supplemented with penicillin, streptomycin, glutamine and 10% FBS (BioWhittaker).

[0940] Leukemia cells

[0941] Primary AML cells were obtained from the San Raffaele Hospital (OSR) Leukemia Biobank and selected based on WT1 expression and HLA typing by quantitative PCR. All EBV-BLCL and primary leukemia cells were typed for HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ alleles at high resolution in the OSR HLA laboratory.

[0942] Flow cytometry

[0943] We used antibodies against human CD3, CD4, CD8, CD107a, IFNγ, TNFα, CD33, CD117, CD34, CD14, Vβ21.3, Vβ8, Vβ7.2, and HLA-A2 conjugated with FITC, PE, PerCP, APC, PE-cytochrome 7–, APCCychrome 7–, Pacific Blue, and Brillant Violet. APC-fluorescently labeled WT1 VLDFAPPGA (SEQ ID NO: 157) and PE-fluorescently labeled WT1 RMFPNAPYL (SEQ ID NO: 255) dextramers were used according to the manufacturer's instructions. Cells were incubated with antibodies at 4°C for 15 minutes and washed with phosphate-buffered saline (PBS) containing 1% FBS. Samples were run on a fluorescence-activated cell sorter (FACS) Canto II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Treestar Inc). For intracellular assessment of cytokine secretion and expression of degranulation markers, Fix / Perm buffer set (Biolegend) was used according to the manufacturer's instructions.

[0944] Stimulation, isolation, and expansion of WT1-specific T cells

[0945] Freshly isolated PBMCs were resuspended in X-VIVO medium supplemented with 5% human AB serum, 2 mM glutamine and 1 μg / ml CD28 monoclonal antibody. 7 Cells were plated at a density of 10 cells / ml and stimulated with a pool of WT1 overlapping peptides, each peptide present at a concentration of 1 μg / ml.

[0946] Antigen-specific T cells were isolated 26-30 hours later by CD137 expression. More specifically, cells were stained with PE-conjugated CD137 antibody and sorted using anti-PE microbeads (Miltenyi Biotech). CD137 was expressed using CD3-microbeads (Miltenyi Biotech). -The fraction was depleted of CD3 cells, irradiated with 30 Gy, and in the presence of CD137 + The fractions were mixed in a ratio of 100:1 (when possible) or at least 20:1 and 5x10 6 Peptide-loaded APCs were co-cultured at a final density of 10 cells / ml. X-VIVO medium supplemented with 5% human AB serum, 5 ng / ml IL7, 5 ng / ml IL15, and 10 ng / ml IL21 was used as the culture medium. The cytokine-containing culture medium was replaced every 2-3 days.

[0947] Re-stimulation of expanded antigen-specific T cells

[0948] Cells were restimulated every 7-14 days with autologous APCs (PBMC CD3-depleted cells; immortalized B cells) pulsed with WT1. In the initial restimulation, cells were washed 2 days before and plated in cytokine-free medium. APCs were irradiated with 30 Gy, pulsed overnight with peptide pools, and then co-cultured with effector cells in X-VIVO supplemented with 5% human AB serum, 1 μg / ml CD28 monoclonal antibody, and IL7 (5 ng / ml), IL15 (5 ng / ml), and IL21 (10 ng / ml).

[0949] Assessment of T cell responses

[0950] The percentage of T cells that responded to the WT1 peptide pool was measured by co-culturing the effector cells with autologous APCs (at least a 1:1 ratio) pulsed with the desired antigen (WT1 peptide pool, WT1 sub-pool, WT1 individual peptide, irrelevant peptide pool as a control) for 6 hours. The co-cultures were plated in X-VIVO supplemented with 5% human AB serum and supplemented with CD28 monoclonal antibody (1 μg / ml), Golgi Stop (BD) and CD107a-FITC antibody to assess degranulation. The cells were then fixed, permeabilized and intracellularly stained to determine the expression of IFNγ and CD107a in CD3 + CD8 + or CD3 + CD4 + The percentage of cells.

[0951] Localization of immunogenic peptides

[0952] T cells stimulated with the WT1 pool were seeded into separate wells and co-cultured with autologous APCs loaded with one of each WT1 subpool at a ratio of at least 1:1. T cell responses to each subpool were measured as previously described by FACS analysis. Deconvolution of the localization grid was crucial for determining which shared peptides elicited a T cell response. Once immunogenic peptides were identified, T cells were further stimulated with APCs loaded with the individual peptides to confirm their immunogenicity.

[0953] Assessing the ability of T cells to recognize WT1-expressing cells

[0954] Different experimental procedures were used to measure the WT1-specific and HLA-restricted ability of T cells to recognize target cells. For T cells derived from HD1, the secretion of CD107a was determined by FACS analysis after 6 hours of coculture with target cells. For T cells derived from HD3, the elimination index was calculated as the total number of target cells still present after 4 days of coculture with WT1-specific T cells divided by the total number of target cells alone. For T cells derived from HD4, the elimination index of CD3 was assessed by cytofluorimetry. + The percentage of CD33+ target cells (primary AML cells containing the HLA allele of interest and, as a control, primary AML cells without the specific HLA allele) that remain after 3 days of co-culture with WT1-specific T cells.

[0955] Assessment of T cell clonality

[0956] To determine the clonality of expanded WT1-specific T cells, IOTest BetaMark TCR V beta panel was used according to the manufacturer's recommendations.

[0957] TCR repertoire sequencing

[0958] WT1-specific T cells were collected at different time points within the co-culture time frame and RNA was extracted using the Arcturus Pico Pure RNA extraction kit. The complementary determining region (CDR) 3 sequences of WT1-specific T cells were amplified using a modified RACE method (Ruggiero, E. et al. Nat. Commun. 6, 8081 (2015)). The samples were sequenced using an Illumina MiSeq sequencer and CDR3 clonotypes were identified using MiXCR software (Bolotin, DA et al. Nature Methods 12, 380–381 (2015)).

[0959] Lentiviral vectors

[0960] The TCR α and β chain genes isolated from HD1 and HD3 were codon-optimized, cysteine-modified, and cloned in a lentiviral vector (LV) under a bidirectional promoter. The amino acid (aa) and nucleotide (nt) sequences are:

[0961]

[0962]

[0963]

[0964]

[0965] LVs were packaged with an integrase-competent third generation construct and pseudotyped with the vesicular stomatitis virus (VSV) envelope. As a control, we included LVs encoding the WT1 126-134 TCR that recognizes the RMFPNAPYL (SEQ ID NO: 255) peptide.

[0966] Vector transduction

[0967] For transduction with the WT1-TCR lentiviral vector, T lymphocytes isolated from healthy individuals were activated and sorted using magnetic beads conjugated with CD3 and CD28 antibodies (ClinExVivo CD3 / CD28; Invitrogen) according to the manufacturer's instructions and cultured in a 5% PBS-supplemented medium supplemented with penicillin, streptomycin, 10% FBS, and 5 ng / ml of each. -1 T lymphocytes were cultured in Iscove's modified Dulbecco's medium (IMDM) (GIBCO-BRL) supplemented with IL-7 and IL-15 (PeproTech). 6 cells ml -1 After 24 hours of infection, T cells were divided into 10 6 cells ml -1 Culture and expand. Transduction efficiency was determined by measuring the percentage of CD3 T cells expressing specific dextramers. Cells were sorted using anti-APC or anti-PE microbeads (Miltenyi Biotec) according to the manufacturer's instructions, using APC or PE fluorescently labeled HLA-A*0201 dextramer specific for VLDFAPPGA (SEQ ID NO: 157) or RMFPNAPYL (SEQ ID NO: 255) peptides (Immudex).

[0968] Functional assays

[0969] The ability of HD1, HD3, and WT1 126-134 TCR-transferred T cells to recognize WT1-expressing target cells was measured after co-culture with: (a) T2 cells pulsed with WT1 126-134 peptide or VLDFAPPGA (SEQ ID NO: 157) peptide (effector: target ratio = 1:1); (b) wild-type (K562) or K562 cells genetically modified to express the HLA-A*0201 allele (effector: target ratio = 1:1); and (c) three different primary AML blasts selected for HLA-A*0201 allele expression and WT1 antigen expression (effector: target ratio = 5:1). For co-culture with T2 and K562 cell lines, we included untransduced T cells as a control. After 3 days of culture, the percentage of target cells was assessed by cytofluorimetry. The elimination index was calculated as follows: 1-(total number of target cells remaining after 3 days of co-culture with WT1-specific T cells / total number of target cells alone).

[0970] All publications mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the described present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. In fact, various modifications of the described modes that are obvious to those skilled in the art of cell biology, immunology, immunotherapy, molecular biology, oncology or related fields are intended to be within the scope of the appended claims. Sequence Listing <110> Ospedale San Raffaele Srl San Raffaele Central Foundation <120> TCR and peptides <130> P111125PCT <150> US 62 / 489,226 <151> 2017-04-24 <160> 299 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> TCR (T-cell receptor) amino acid sequence, donor HD1 <400> 1 Lys Ala Leu Tyr Ser 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 2 Leu Leu Lys Gly Gly Glu Gln 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 3 Cys Gly Thr Ala Trp Ile Asn Asp Tyr Lys Leu Ser Phe 1 5 10 <210> 4 <211> 132 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 4 Met Glu Thr Leu Leu Lys Val Leu Ser Gly Thr Leu Leu Trp Gln Leu 1 5 10 15 Thr Trp Val Arg Ser Gln Gln Pro Val Gln Ser Pro Gln Ala Val Ile 20 25 30 Leu Arg Glu Gly Glu Asp Ala Val Ile Asn Cys Ser Ser Ser Lys Ala 35 40 45 Leu Tyr Ser Val His Trp Tyr Arg Gln Lys His Gly Glu Ala Pro Val 50 55 60 Phe Leu Met Ile Leu Leu Lys Gly Gly Glu Gln Lys Gly His Glu Lys 65 70 75 80 Ile Ser Ala Ser Phe Asn Glu Lys Lys Gln Gln Ser Ser Leu Tyr Leu 85 90 95 Thr Ala Ser Gln Leu Ser Tyr Ser Gly Thr Tyr Phe Cys Gly Thr Ala 100 105 110 Trp Ile Asn Asp Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val Thr 115 120 125 Val Arg Ala Asn​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Leu Tyr Ser Val His Trp Tyr Arg Gln Lys His Gly Glu Ala Pro Val 50 55 60 Phe Leu Met Ile Leu Leu Lys Gly Gly Glu Gln Lys Gly His Glu Lys 65 70 75 80 Ile Ser Ala Ser Phe Asn Glu Lys Lys Gln Gln Ser Ser Leu Tyr Leu 85 90 95 Thr Ala Ser Gln Leu Ser Tyr Ser Gly Thr Tyr Phe Cys Gly Thr Ala 100 105 110 Trp Ile Asn Asp Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val Thr 115 120 125 Val Arg Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg 130 135 140 Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser 180 185 190 Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe 195 200 205 Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser 210 215 220 Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn 225 230 235 240 Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 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> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 6 Ser Gly His Asp Tyr 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 7 Phe Asn Asn Asn Val Pro 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 8 Cys Ala Ser Arg Lys Thr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe 1 5 10 15 <210> 9 <211> 136 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 9 Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Arg Lys Thr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe Gly Asp 115 120 125 Gly Thr Arg Leu Ser Ile Leu Glu 130 135 <210> 10 <211> 312 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD1 <400> 10 Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Arg Lys Thr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe Gly Asp 115 120 125 Gly Thr Arg Leu Ser Ile 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> 11 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> TCR Amino Acid Sequence, Donor HD1 <400> 11 Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Arg Lys Thr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe Gly Asp 115 120 125 Gly Thr Arg Leu Ser Ile Leu Glu Asp Leu Lys Asn 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 Tyr 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 Glu 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 Ser Arg Gly 305 310 <210> 12 <211> 6 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 12 Ser Ser Val Pro Pro Tyr 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 13 Tyr Thr Ser Ala Ala Thr Leu Val 1 5 <210> 14 <211> 14 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 14 Cys Ala Val Arg Leu Ser Gly Ser Ala Arg Gln Leu Thr Phe 1 5 10 <210> 15 <211> 134 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 15 Met Leu Leu Leu Leu Val Pro Val Leu Glu Val Ile Phe Thr Leu Gly 1 5 10 15 Gly Thr Arg Ala Gln Ser Val Thr Gln Leu Gly Ser His Val Ser Val 20 25 30 Ser Glu Gly Ala Leu Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Val 35 40 45 Pro Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Asn Gln Gly Leu Gln 50 55 60 Leu Leu Leu Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn 65 70 75 80 Gly Phe Glu Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr 85 90 95 Lys Pro Ser Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val 100 105 110 Arg Leu Ser Gly Ser Ala Arg Gln Leu Thr Phe Gly Ser Gly Thr Gln 115 120 125 Leu Thr Val Leu Pro Asp 130 <210> 16 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> TCR Amino Acid Sequence, Donor HD2 <400> 16 Met Leu Leu Leu Leu Val Pro Val Leu Glu Val Ile Phe Thr Leu Gly 1 5 10 15 Gly Thr Arg Ala Gln Ser Val Thr Gln Leu Gly Ser His Val Ser Val 20 25 30 Ser Glu Gly Ala Leu Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Val 35 40 45 Pro Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Asn Gln Gly Leu Gln 50 55 60 Leu Leu Leu Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn 65 70 75 80 Gly Phe Glu Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr 85 90 95 Lys Pro Ser Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val<B 100 105 110 Arg Leu Ser Gly Ser Ala Arg Gln Leu Thr Phe Gly Ser Gly Thr Gln 115 120 125 Leu Thr Val Leu Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <210> 17 <211> 7 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 17 Thr Ser Glu Ser Asp Tyr Tyr 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 18 Gln Glu Ala Tyr Lys Gln Gln Asn 1 5 <210> 19 <211> 14 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 19 Cys Ala Tyr Arg Ser Leu Lys Tyr Gly Asn Lys Leu Val Phe 1 5 10 <210> 20 <211> 136 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 20 Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys 100 105 110 Ala Tyr Arg Ser Leu Lys Tyr Gly Asn Lys Leu Val Phe Gly Ala Gly '115 120 125 Thr Ile Leu Arg Val Lys Ser Tyr 130 135 <210> 21 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> TCR Amino Acid Sequence, Donor HD2 <400> 21 Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys 100 105 110 Ala Tyr Arg Ser Leu Lys Tyr Gly Asn Lys Leu Val Phe Gly Ala Gly 115 120 125 Thr Ile Leu Arg Val Lys Ser Tyr Ile Gln Asn Pro Asp Pro Ala Val 130 135 140 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 165 170 175 Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe 180 185 190 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220 Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 225 230 235 240 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 245 250 255 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 260 265 270 Leu Trp Ser Ser 275 <210> twenty two <211> 5 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> twenty two Ser Gly His Ala Thr 1 5 <210> twenty three <211> 6 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> twenty three Phe Gln Asn Asn Gly Val 1 5 <210> twenty four <211> 12 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> twenty four Cys Ala Ser Ser Leu Leu Gly Asp Glu Gln Tyr Phe 1 5 10 <210> 25 <211> 132 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 25 Met Gly Thr Arg Leu Leu Cys Trp Ala Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Glu Leu Thr Glu Ala Gly Val Ala Gln Ser Pro Arg Tyr Lys Ile Ile 20 25 30 Glu Lys Arg Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His 35 40 45 Ala Thr Leu Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu 50 55 60 Leu Ile Gln Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Leu Gly Asp Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr Glu 130 <210> 26 <211> 308 <212> PRT <213> Artificial Sequence <220> [[ID=2,6]]<223> TCR Amino Acid Sequence, Donor HD2 <400> 26 Met Gly Thr Arg Leu Leu Cys Trp Ala Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Glu Leu Thr Glu Ala Gly Val Ala Gln Ser Pro Arg Tyr Lys Ile Ile 20 25 30 Glu Lys Arg Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His 35 40 45 Ala Thr Leu Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu 50 55 60 Leu Ile Gln Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Leu Gly Asp Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr Glu Asp Leu Asn Lys 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 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 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 苏氨酸 谷氨酰胺 天冬氨酸 精氨酸 丙氨酸 赖氨酸 脯氨酸 缬氨酸 苏氨酸 谷氨酰胺 异亮氨酸 缬氨酸 丝氨酸 丙氨酸 谷氨酸 丙氨酸 245 250 255 色氨酸 甘氨酸 精氨酸 丙氨酸 天冬氨酸 半胱氨酸 甘氨酸 苯丙氨酸 苏氨酸 丝氨酸 缬氨酸 丝氨酸 酪氨酸 谷氨酰胺 谷氨酰胺 甘氨酸 260 265 270 缬氨酸 亮氨酸 丝氨酸 丙氨酸 苏氨酸 异亮氨酸 亮氨酸 酪氨酸 谷氨酸 异亮氨酸 亮氨酸 亮氨酸 甘氨酸 赖氨酸 丙氨酸 苏氨酸 275 280 285 亮氨酸 酪氨酸 丙氨酸 缬氨酸 亮氨酸 缬氨酸 丝氨酸 丙氨酸 亮氨酸 缬氨酸 亮氨酸 甲硫氨酸 丙氨酸 甲硫氨酸 缬氨酸 赖氨酸 290 295 300<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Leu Ile Gln Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Leu Gly Asp Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr 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> 28 <211> 5 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 28 Ser Gly His Thr Ala 1 5 <210> 29 <211> 6 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 29 Phe Gln Gly Asn Ser Ala 1 5 <210> 30 <211> 16 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 30 Cys Ala Ser Ser Leu Val Ala Leu Gln Gly Ala Gly Glu Gln Tyr Phe 1 5 10 15 <210> 31 <211> 136 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD2 <400> 31 Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr 20 25 30 Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His 35 40 45 Thr Ala Leu Tyr Trp Tyr Arg Gln Ser Leu Gly Gln Gly Leu Glu Phe 50 55 60 Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro 65 70 75 80 Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Gly Ser Val Ser Thr Leu 85 90 95 Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Val Ala Leu Gln Gly Ala Gly Glu Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Thr Glu 130 135 <210> 32 <211> 312 <212> PRT <213> Artificial Sequence <220> ​​​​​​​​​​​​​​​​​​​​​​​​​​​ Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Val Ala Leu Gln Gly Ala Gly Glu Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Thr 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> 33 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> TCR Amino Acid Sequence, Donor HD2 <400> 33 Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr 二十 二十五 三十 Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His 35 40 45 Thr Ala Leu Tyr Trp Tyr Arg Gln Ser Leu Gly Gln Gly Leu Glu Phe 50 55 60 Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro 65 70 75 80 Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Gly Ser Val Ser Thr Leu 85 90 95 Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Val Ala Leu Gln Gly Ala Gly Glu Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn 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 Tyr 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 Glu 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 Ser Arg Gly 305 310 <210> 34 <211> 6 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 34 Asn Ser Ala Ser Gln Ser 1 5 <210> 35 <211> 6 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 35 Val Tyr Ser Ser Gly Asn 1 5 <210> 36 <211> 15 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 36 Cys Val Val Asn Leu Leu Ser Asn Gln Gly Gly Lys Leu Ile Phe 1 5 10 15 <210> 37 <211> 134 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 37 Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe 20 25 30 Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn 35 40 45 Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu 50 55 60 Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg 65 70 75 80 Phe Thr Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile 85 90 95 Arg Asp Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Asn 100 105 110 Leu Leu Ser Asn Gln Gly Gly Lys Leu Ile Phe Gly Gln Gly Thr Glu 115 120 125 Leu Ser Val Lys Pro Asn 130 <210> 38 <211> 274 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 38 Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe 20 25 30 Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn 35 40 45 Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu 50 55 60 Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg 65 70 75 80 Phe Thr Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile 85 90 95 Arg Asp Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Asn 100 105 110 Leu Leu Ser Asn Gln Gly Gly Lys Leu Ile Phe Gly Gln Gly Thr Glu 115 120 125 Leu Ser Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <210> 39 <211> 5 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 39 Leu Gly His Asn Ala 1 5 <210> 40 <211> 6 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 40 Tyr Ser Leu Glu Glu Arg 1 5 <210> 41 <211> 16 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 41 Cys Ala Ser Ser Gln Asp Tyr Leu Val Ser Asn Glu Lys Leu Phe Phe 1 5 10 15 <210> 42 <211> 138 <212> PRT <213> Artificial sequence <220> <223> TCR amino acid sequence, donor HD3 <400> 42 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Glu Leu Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His 20 25 30 Leu Val Met Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His 35 40 45 Leu Gly His Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro 50 55 60 Leu Glu Leu Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn 65 70 75 80 Ser Val Pro Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu 85 90 95 Phe Leu His Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu 100 105 110 Cys Ala Ser Ser Gln Asp Tyr Leu Val Ser Asn Glu Lys Leu Phe Phe 115 120 125 Gly Ser Gly Thr Gln Leu Ser Val Leu Glu 130 135 <210> 43 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> TCR Amino Acid Sequence, Donor HD3 <400> 43 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Glu Leu Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His 20 25 30 Leu Val Met Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His 35 40 45 Leu Gly His Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro 50 55 60 Leu Glu Leu Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn 65 70 75 80 Ser Val Pro Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu 85 90 95 Phe Leu His Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu 100 105 110 Cys Ala Ser Ser Gln Asp Tyr Leu Val Ser Asn Glu Lys Leu Phe Phe 115 120 125 Gly Ser Gly Thr Gln Leu Ser Val Leu Glu Asp Leu Asn Lys Val Phe 130 135 140 Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His 145 150 155 160 Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp 165 170 175 His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 180 185 190 Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp 195 200 205 Ser Arg Tyr Cys Leu Se...

Claims

1. A T cell receptor (TCR) that binds a Wilms tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the TCR comprises the following CDR sequences: CDR1α-KALYS (SEQ ID NO: 1), CDR2α-LLKGGEQ (SEQ ID NO:2), CDR3α-CGTAWINDYKLSF (SEQ ID NO:3), CDR1β-SGHDY (SEQ ID NO: 6), CDR2β-FNNNVP (SEQ ID NO: 7), and CDR3β-CASRKTGGYSNQPQHF (SEQ ID NO:8).

2. A TCR according to claim 1, comprising an α chain variable domain comprising the amino acid sequence of SEQ ID NO:4 or a variant thereof having at least 75% sequence identity; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO:9 or a variant thereof having at least 75% sequence identity.

3. The TCR of claim 1, comprising an α chain comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, and variants thereof having at least 75% sequence identity thereto.

4. A TCR according to claim 1, wherein the TCR is restricted to HLA-A*0201.

5. A TCR according to claim 1, comprising one or more mutations at the α chain / β chain interface such that when the α chain and the β chain are expressed in a T cell, the mispairing frequency between the chains and endogenous TCR α and β chains is reduced.

6. A TCR according to claim 5, wherein the one or more mutations introduce a cysteine ​​residue into the constant region of each of the α chain and the β chain, wherein the cysteine ​​residue is capable of forming a disulfide bond between the α chain and the β chain.

7. The TCR according to claim 1, comprising a murinized constant region.

8. The TCR according to claim 1, wherein the TCR is a soluble TCR.

9. An isolated polynucleotide encoding the alpha chain of the T cell receptor (TCR) according to claim 1, and the beta chain of the TCR according to claim 1.

10. The isolated polynucleotide according to claim 9, wherein said polynucleotide encodes said alpha chain linked to said beta chain.

11. An isolated polynucleotide according to claim 9, encoding one or more short interfering RNAs (siRNAs) or other agents capable of reducing or preventing the expression of one or more endogenous TCR genes.

12. A vector comprising the polynucleotide according to claim 9.

13. A vector according to claim 12, comprising a polynucleotide encoding one or more of a CD3 chain, CD8, a suicide gene and / or a selectable marker.

14. A cell comprising the TCR according to claim 1 or polynucleotides encoding the α chain and β chain thereof.

15. The cell according to claim 14, wherein the cell is a lymphocyte or a stem cell.

16. The cell according to claim 15, wherein the cell is a T cell.

17. The cell according to claim 15, wherein the cell is a T cell that has been isolated from a subject.

18. The cell according to claim 14, wherein the endogenous gene encoding the TCR alpha chain and / or the endogenous gene encoding the TCR beta chain is disrupted.

19. A method for preparing a cell, comprising the steps of: The vector according to claim 12 is introduced into cells in vitro or ex vivo.

20. The method for preparing cells according to claim 19, comprising a step of T cell editing, said step comprising destroying the endogenous gene encoding the TCR α chain and / or the endogenous gene encoding the TCR β chain using an artificial nuclease.

21. A method for preparing cells according to claim 20, comprising the step of targeted integration of an expression cassette into the endogenous gene encoding the TCR α chain and / or the endogenous gene encoding the TCR β chain destroyed by the artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding the TCR of claim 1.

22. A method for preparing a cell according to claim 19, comprising the step of disrupting one or more endogenous genes encoding MHC.

23. The method for preparing cells according to claim 19, comprising the following steps: Disruption of one or more endogenous genes to alter persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell functions.

24. A cell according to claim 14 or a cell prepared by the method of claim 19 for use in adoptive cell transfer.

25. A chimeric molecule comprising the TCR of claim 1 conjugated to a non-cellular substrate, a toxin and / or an antibody.

26. Use of a TCR according to claim 1, an isolated polynucleotide according to claim 9, a vector according to claim 12, a cell according to claim 14, a cell prepared by the method of claim 19, or a chimeric molecule according to claim 25 in the preparation of a composition for treating and / or preventing acute myeloid leukemia.

27. Use of a TCR according to claim 1, an isolated polynucleotide according to claim 9, a vector according to claim 12, a cell according to claim 14, a cell prepared by the method of claim 19, or a chimeric molecule according to claim 25 in the preparation of a composition for treating and / or preventing chronic myeloid leukemia.

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