MAGE-B2-specific T cell receptor

Abstract

When expressed recombinantly on the T cell surface, HLA-A * Provided herein are T cell receptors (TCRs) capable of recognizing the MAGE-B2 derived peptide GVYDGEEHSV (SEQ ID NO: 1) when presented by 02:01 sufficiently to activate recombinant T cells. Certain TCRs provided herein are also capable of recognizing the MAGE-A4 derived peptide GVYDGREHTV (SEQ ID NO: 2) sufficiently to activate recombinant T cells. Importantly, the exemplary TCRs provided herein have been thoroughly screened for lack of cross-reactivity with similar peptides that may be presented by normal cells or tissues, and for alloreactivity.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 129,447, filed December 22, 2020, the entirety of which is incorporated by reference herein for all purposes as if fully set forth herein.

[0002] The present invention relates to T cell receptors that, when recombinantly expressed on the surface of a T cell, are capable of recognizing a peptide sufficiently to activate the recombinant T cell.

[0003] Sequence Listing This application contains, as a separate part of the disclosure, a sequence listing in computer readable format (filename: A-2668-WO-PCT_ST25.txt, created on Nov. 2, 2021, and having a size of 113 KB), which is incorporated by reference in its entirety. [Background technology]

[0004] Adoptive T cell therapy offers tremendous opportunities to treat cancer. Chimeric antigen receptor (CAR)-T cell therapy is an approved adoptive T cell therapy for hematological malignancies, but it has a limited targeting range due to its recognition only against cell surface antigens that make up about 25% of the genome. Unlike CAR-T cells, TCR-T cells engineered to express T cell receptors (TCRs) specific for tumor antigens can recognize peptide-MHC complexes (pMHC) derived from intracellular proteins that make up about 75% of the genome, thus enabling a broader range of targets for multiple cancer indications. Intracellular proteins are processed and presented by the major histocompatibility complex (MHC) as pMHC complexes.

[0005] Cancer-testis antigens (CTAs) are attractive targets for cancer immunotherapy, such as TCR-T cell therapy, due to their restricted expression in germ cells and aberrant reactivation in various cancers, as well as their immunogenicity. Germ cells, such as the testis (an immune privileged site), do not normally express HLA class I / II molecules, allowing them to avoid attack from the immune system. MAGE-B2 and MAGE-A4 are members of the melanoma antigen (MAGE) gene family, most of which are classified as intracellular cancer-testis antigens, such as MAGE-B2 and MAGE-A4. Recent studies suggest that MAGE assembly with E3 RING ubiquitin ligases acts as a regulator of ubiquitin binding, plays a role in cell proliferation and oncogenic activity, and regulates cellular stress responses. However, the functions of most MAGE genes, such as MAGE-B2 and MAGE-A4, are not fully understood.

[0006] TCR-T cells have been found to be highly potent and sensitive modalities for tumor-specific peptide-MHC targets, while TCRs can recognize multiple peptides. DNA rearrangements required for TCR formation generate a certain number of T cells that recognize self-antigens. During early T cell development, autoreactive T cells are negatively selected and eliminated in the medulla of the thymus through indiscriminate expression of a wide range of self-antigens in thymic medullary epithelial cells. This negative selection in the thymus serves as a major mechanism of central immune tolerance, shaping the T cell repertoire and avoiding autoimmunity. TCRs engineered to increase their affinity for a specific pMHC or to introduce cross-reactivity to multiple pMHCs do not have the advantage of the negative selection that occurs in the thymus. It is noteworthy that affinity-enhanced MAGE-A3 TCR-T cells cause lethal toxicity due to cross-reactivity to titin expressed in the myocardium (Cameron et al., Sci Transl Med. 2013 5(197)). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Cameron et al.,Sci Transl Med.2013 5(197) Summary of the Invention [Means for solving the problem]

[0008] Identification of TCR sequences that recognize tumor-specific antigens has proven to be very challenging in the art, especially due to the rarity of tumor-specific T cells in the blood of patients, the difficulty of expanding the very few tumor-specific T cell clones ex vivo, and the potential exhaustion or suppression of tumor-specific T cells in tumor-infiltrating lymphocytes (TILs). Despite these challenges, MAGE-B2 peptide-MHC (GVYDGEEHSV / HLA-A) was identified using healthy donor blood and ex vivo stimulation methods. * 02:01) and MAGE-A4 peptide-MHC (GVYDGREHTV / HLA-A * As demonstrated in the Examples herein, exemplary TCR-T cells that recognize tumor-specific MAGE-B2 pMHC, and in some embodiments, MAGE-A4, pMHC, exert cytotoxicity and inhibit MAGE-B2+ / HLA-A by producing cytokines. * 02:01+ and / or MAGE-A4+ / HLA-A * 02:01+ could be highly potent therapeutics for the treatment of tumors. These TCR-T cell therapies will be important treatment options for a wide variety of cancer indications.

[0009] TCR-T cells are the most potent and sensitive modality in vitro for pMHC targets. The TCR-T cells provided herein exhibit high potency even against very low target expressing cells. This high potency of TCR-T cells arises from the complex of the transduced TCR and endogenous CD3 subunits. To further enhance in vivo efficacy, the exemplary TCR-T cells contain an activation-dependent IL12 payload incorporated into the TCR-T construct, where IL12 expression is controlled by TCR activation under a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter. Thus, IL12 is produced when the TCR-T-IL12 cells encounter tumor antigens. As shown in the mouse studies provided in the examples, the IL12 payload enhances the efficacy of adoptive T cell therapy in vivo, thus reducing the potential clinical dose (10-100-fold).

[0010] In a first aspect, the present invention is an expression vector comprising a nucleic acid sequence encoding a T cell receptor (TCR) alpha chain and a TCR beta chain, the TCR alpha chain and the TCR beta chain being a. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 13, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 24; b. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 14, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 25; c. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 15, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 26; d. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 16, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 27; e. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 17, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 28; f. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 29; g. A TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 30; h. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:20, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:31; i. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:21, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:32; j. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO:22, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO:33; and k. A TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO:23, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO:34. is selected from the group consisting of:

[0011] Any expression vector of the first aspect may further comprise a nucleic acid encoding interleukin-12 (IL-12) or a functional variant thereof and may be a viral vector, such as a retroviral or lentiviral vector.

[0012] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 13 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 24. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 35 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 46. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 68.

[0013] In a particular embodiment of the first aspect, the expression vector encodes a TCR α chain comprising the CDR3 region amino acid sequence set forth in SEQ ID NO: 14 and a TCR β chain comprising the CDR3 region amino acid sequence set forth in SEQ ID NO: 25. In a preferred embodiment, the mature TCR α chain comprises the amino acid sequence set forth in SEQ ID NO: 36 and the mature TCR β chain comprises the amino acid sequence set forth in SEQ ID NO: 47. The expression vector may encode a full-length TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 58 and a full-length TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 69.

[0014] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 15 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 26. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 37 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 48. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 59 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0015] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 16 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 27. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 38 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 49. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 60 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 71.

[0016] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 17 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 28. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 39 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 50. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 61 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 72.

[0017] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 18 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 29. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 40 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 51. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 62 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 73.

[0018] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 19 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 30. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 41 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 52. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 63 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 74.

[0019] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 20 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 31. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 42 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 53. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 64 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 75.

[0020] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 21 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 32. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 43 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 54. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 65 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 76.

[0021] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 22 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 33. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 44 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 55. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 66 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 77.

[0022] In certain embodiments of the first aspect, the expression vector encodes a TCR alpha chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 23 and a TCR beta chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 34. In a preferred embodiment, the mature TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 45 and the mature TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 56. The expression vector may encode a full-length TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 67 and a full-length TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 78.

[0023] A second embodiment is a cell expressing a recombinant T cell receptor (TCR), said TCR comprising: a. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 13, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 24; b. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 14, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 25; c. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 15, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 26; d. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 16, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 27; e. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 17, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 28; f. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 18, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 29; g. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 19, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 30; h. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 20, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 31; i. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 21, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 32; j. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 22, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 33; or k. a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 23, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 34; Includes.

[0024] In a preferred embodiment of the second aspect, the cell comprises a. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 35, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 46; b. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:36, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:47; c. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:37, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:48; d. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:38, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:49; e. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 39, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 50; f. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 40, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 51; g. A TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:41, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:52; h. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 42, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 53; i. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 43, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 54; j. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 44, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 55; or k. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:45, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:56; The present invention relates to a recombinant TCR comprising:

[0025] The cells of the second aspect may further express recombinant IL-12 or a functional variant thereof.

[0026] In certain embodiments of the second aspect, the cell comprises one or more expression vectors of the first aspect.

[0027] The cell can be a T cell and the TCR is HLA-A * It binds to the peptide of SEQ ID NO:1 or SEQ ID NO:2 in the context of 02:01, which binding leads to the activation of IFNγ, TNFα, IL-12, or the production of granzyme B by the cell.

[0028] In a third aspect of the invention, a pharmaceutical composition comprises a therapeutically effective amount of a cell of the second aspect or an expression vector of the first aspect.

[0029] In a fourth aspect, the present invention provides a method for producing a cell of the second aspect or a pharmaceutical composition of the third aspect, comprising introducing into a cell an expression vector comprising a nucleic acid sequence encoding a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain are: a. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 13, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 24; b. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 14, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 25; c. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 15, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 26; d. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 16, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 27; e. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 17, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 28; f. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 18, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 29; g. A TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 19, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 30; h. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 20, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 31; i. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 21, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 32; j. a TCR alpha chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 22, and a TCR beta chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 33; or k. a TCR α chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO:23, and a TCR β chain comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO:34; The method is selected from the group consisting of:

[0030] In a preferred embodiment of the fourth aspect, the TCR alpha and beta chains comprise a. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 35, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 46; b. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:36, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:47; c. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:37, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:48; d. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:38, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:49; e. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 39, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 50; f. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 40, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 51; g. A TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:41, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:52; h. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 42, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 53; i. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 43, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 54; j. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO:44, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO:55; and k. a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO:45, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO:56; is selected from the group consisting of:

[0031] In certain embodiments of the fourth aspect, a nucleic acid sequence encoding IL-12 or a functional variant thereof is also introduced into the cell and may be on an expression vector encoding the alpha and / or beta chain or may be encoded on a separate vector.

[0032] The cells produced by the method of the fourth aspect may be primary T cells isolated from a cancer patient.

[0033] In a fifth aspect, the invention provides a method of treating a MAGE-B2 or MAGE-A4 expressing cancer, said method comprising administering to a cancer patient a therapeutically effective amount of a cell of the second aspect, a pharmaceutical composition of the third aspect, or a cell produced by the method of the fourth aspect. In a particular embodiment of the fifth aspect, the patient is tested prior to administration to determine the presence of a MAGE-B2 or MAGE-A4 expressing cancer. The testing may detect MAGE-B2- or MAGE-A4-encoding nucleic acid, MAGE-B2 or MAGE-A4 protein, or MAGE-B2- or MAGE-A4-derived peptides. In a preferred embodiment, the patient is HLA-A * Identified as carrying the 02:01 allele. [Brief description of the drawings]

[0034] [Figure 1A-1] (A) MAGE-A4 and MAGE-B2 mRNA expression in various cancers (TCGA and internal RNA-seq data). (B) MAGE-A4 and MAGE-B2 mRNA expression in human normal tissues (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that MAGE-A4 protein is expressed in the majority of tumor cells within squamous non-small cell lung cancer tumors. A representative IHC line of squamous non-small cell lung cancer tumor shows 100% MAGE-A4 positive tumor cells and staining intensity 3+. [Figure 1A-2] (A) MAGE-A4 and MAGE-B2 mRNA expression in various cancers (TCGA and internal RNA-seq data). (B) MAGE-A4 and MAGE-B2 mRNA expression in human normal tissues (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that MAGE-A4 protein is expressed in the majority of tumor cells within squamous non-small cell lung cancer tumors. A representative IHC line of squamous non-small cell lung cancer tumor shows 100% MAGE-A4 positive tumor cells and staining intensity 3+. [Figure 1B] (A) MAGE-A4 and MAGE-B2 mRNA expression in various cancers (TCGA and internal RNA-seq data). (B) MAGE-A4 and MAGE-B2 mRNA expression in human normal tissues (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that MAGE-A4 protein is expressed in the majority of tumor cells within squamous non-small cell lung cancer tumors. A representative IHC line of squamous non-small cell lung cancer tumor shows 100% MAGE-A4 positive tumor cells and staining intensity 3+. [Figure 1C] (A) MAGE-A4 and MAGE-B2 mRNA expression in various cancers (TCGA and internal RNA-seq data). (B) MAGE-A4 and MAGE-B2 mRNA expression in human normal tissues (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that MAGE-A4 protein is expressed in the majority of tumor cells within squamous non-small cell lung cancer tumors. A representative IHC line of squamous non-small cell lung cancer tumor shows 100% MAGE-A4 positive tumor cells and staining intensity 3+. [Figure 2A] (A) Mass spectrometry (MS) data (Immatics) demonstrate that MAGE-B2 peptide-HLA-A*02:01 is expressed in tumors and not in normal tissues. (B) MAGE-B2 pMHC frequency in representative tumors as measured by MS is shown in the table. [Figure 2B] (A) Mass spectrometry (MS) data (Immatics) demonstrate that MAGE-B2 peptide-HLA-A*02:01 is expressed in tumors and not in normal tissues. (B) MAGE-B2 pMHC frequency in representative tumors as measured by MS is shown in the table. [Diagram 3]The patient population in a given cancer indication was estimated based on pMHC target frequency multiplied by new cases (new patients) per year in the US population. pMHC target frequency in each cancer indication was calculated by MAGE-B2 and / or MAGE-A4 mRNA expression frequency (TCGA) multiplied by HLA-A*02:01 carrier frequency in the US population (0.41). MAGE-B2 / A4 indicates MAGE-B2 and / or MAGE-A4 positive cancer patients. [Figure 4A] Identification of MAGE-B2 pMHC-specific TCRs from healthy human PBMCs. (A) Schematic illustrating the procedure to identify MAGE-B2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometric identification of MAGE-B2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorochromes (PE and APC) following multiple rounds of enrichment by stimulation with MAGE-B2 peptide-loaded autologous antigen presenting cells. Representative screen results show that positive donor A showed enriched MAGE-B2 pMHC-specific T cells after multiple ex vivo stimulations, whereas negative donor B had no Dex+ T cells. (C) IFNγ ELISPOT analysis of selected CD8+Dex+ T cells stimulated with T2 cells pulsed with MAGE-B2 peptide or an irrelevant AFP peptide as a negative control. [Figure 4B]Identification of MAGE-B2 pMHC-specific TCRs from healthy human PBMCs. (A) Schematic illustrating the procedure to identify MAGE-B2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometric identification of MAGE-B2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorochromes (PE and APC) following multiple rounds of enrichment by stimulation with MAGE-B2 peptide-loaded autologous antigen presenting cells. Representative screen results show that positive donor A showed enriched MAGE-B2 pMHC-specific T cells after multiple ex vivo stimulations, whereas negative donor B had no Dex+ T cells. (C) IFNγ ELISPOT analysis of selected CD8+Dex+ T cells stimulated with T2 cells pulsed with MAGE-B2 peptide or an irrelevant AFP peptide as a negative control. [Figure 4C] Identification of MAGE-B2 pMHC-specific TCRs from healthy human PBMCs. (A) Schematic illustrating the procedure to identify MAGE-B2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometric identification of MAGE-B2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorochromes (PE and APC) following multiple rounds of enrichment by stimulation with MAGE-B2 peptide-loaded autologous antigen presenting cells. Representative screen results show that positive donor A showed enriched MAGE-B2 pMHC-specific T cells after multiple ex vivo stimulations, whereas negative donor B had no Dex+ T cells. (C) IFNγ ELISPOT analysis of selected CD8+Dex+ T cells stimulated with T2 cells pulsed with MAGE-B2 peptide or an irrelevant AFP peptide as a negative control. [Diagram 5]MAGE-B2 TCR screen using Jurkat-luciferase activation assay. Activity of individual TCRs was expressed as the mean fold change in luciferase activity (light emission) in the presence of T2 cells loaded with MAGE-B2 peptides compared to T2 cells with vehicle alone. Error bars represent standard error. [Figure 6A] Selection of the top four MAGE-B2 TCR-T by different functional assays. (A) Cytotoxicity summary of MAGE-B2 TCR-T in T2 / MAGE-B2 peptide cytotoxicity assays including peptide titration and E:T titration studies (EC90 average of peptide concentrations (M) from three donors or E:T). (B) Cytotoxicity study using T2 / peptide assay with MAGE-B2 peptide concentration at E:T=1:1. (C) Cytotoxicity study using T2 / peptide assay with E:T titration was performed at MAGE-B2 peptide concentration of 10-8M. (For B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T against SK-Mel-5 cancer cell line with MAGE-B2 expression of 27.5 FPKM. TCR1●, TCR2■, TCR8☆. (E) Representative data from a homology-based cross-reactivity screen against similar peptides (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide 9■, peptide 25▲, peptide 46,△, peptide 75▼. [Figure 6B]Selection of the top four MAGE-B2 TCR-T by different functional assays. (A) Cytotoxicity summary of MAGE-B2 TCR-T in T2 / MAGE-B2 peptide cytotoxicity assays including peptide titration and E:T titration studies (EC90 average of peptide concentrations (M) from three donors or E:T). (B) Cytotoxicity study using T2 / peptide assay with MAGE-B2 peptide concentration at E:T=1:1. (C) Cytotoxicity study using T2 / peptide assay with E:T titration was performed at MAGE-B2 peptide concentration of 10-8M. (For B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T against SK-Mel-5 cancer cell line with MAGE-B2 expression of 27.5 FPKM. TCR1●, TCR2■, TCR8☆. (E) Representative data from a homology-based cross-reactivity screen against similar peptides (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide 9■, peptide 25▲, peptide 46,△, peptide 75▼. [Figure 6C] Selection of the top four MAGE-B2 TCR-T by different functional assays. (A) Cytotoxicity summary of MAGE-B2 TCR-T in T2 / MAGE-B2 peptide cytotoxicity assays including peptide titration and E:T titration studies (EC90 average of peptide concentrations (M) from three donors or E:T). (B) Cytotoxicity study using T2 / peptide assay with MAGE-B2 peptide concentration at E:T=1:1. (C) Cytotoxicity study using T2 / peptide assay with E:T titration was performed at MAGE-B2 peptide concentration of 10-8M. (For B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T against SK-Mel-5 cancer cell line with MAGE-B2 expression of 27.5 FPKM. TCR1●, TCR2■, TCR8☆. (E) Representative data from a homology-based cross-reactivity screen against similar peptides (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide 9■, peptide 25▲, peptide 46,△, peptide 75▼. [Figure 6D] Selection of the top four MAGE-B2 TCR-T by different functional assays. (A) Cytotoxicity summary of MAGE-B2 TCR-T in T2 / MAGE-B2 peptide cytotoxicity assays including peptide titration and E:T titration studies (EC90 average of peptide concentrations (M) from three donors or E:T). (B) Cytotoxicity study using T2 / peptide assay with MAGE-B2 peptide concentration at E:T=1:1. (C) Cytotoxicity study using T2 / peptide assay with E:T titration was performed at MAGE-B2 peptide concentration of 10-8M. (For B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T against SK-Mel-5 cancer cell line with MAGE-B2 expression of 27.5 FPKM. TCR1●, TCR2■, TCR8☆. (E) Representative data from a homology-based cross-reactivity screen against similar peptides (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide 9■, peptide 25▲, peptide 46,△, peptide 75▼. [Figure 6E]Selection of the top four MAGE-B2 TCR-T by different functional assays. (A) Cytotoxicity summary of MAGE-B2 TCR-T in T2 / MAGE-B2 peptide cytotoxicity assays including peptide titration and E:T titration studies (EC90 average of peptide concentrations (M) from three donors or E:T). (B) Cytotoxicity study using T2 / peptide assay with MAGE-B2 peptide concentration at E:T=1:1. (C) Cytotoxicity study using T2 / peptide assay with E:T titration was performed at MAGE-B2 peptide concentration of 10-8M. (For B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T against SK-Mel-5 cancer cell line with MAGE-B2 expression of 27.5 FPKM. TCR1●, TCR2■, TCR8☆. (E) Representative data from a homology-based cross-reactivity screen against similar peptides (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide 9■, peptide 25▲, peptide 46,△, peptide 75▼. [Figure 7] Schematic diagram of the TCR-T-IL12 lentiviral construct containing the TCRα and TCRβ chains together with a furin cleavage site-SGSG-T2A linker under the EF1α promoter and an IL12 payload under a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter. [Figure 8] Potency validation of TCR-T-IL12 using a T2 / MAGE-B2 peptide cytotoxicity assay. EC90 peptide concentrations (M) from a T2 / peptide titration study with four TCR-T-IL12 from three HLA-A*02:01 donors are shown in the table. The E:T ratio (dextramer+T cells:T2) was 1:1. TCR1-IL12●, TCR2-IL12■, TCR3-IL12△, TCR4-IL12▼, IL12 RFP◇, Mock T cells●. [Figure 9]TCR4-IL12 cells from three donors demonstrated potent cytotoxicity against both MAGE-B2 peptide- and MAGE-A4 peptide-loaded T2 cells in peptide titration studies. MAGE-B2◇ / dashed line, MAGE-A4■. [Figure 10] Summary of the potency of the four TCR-T-IL12 cells against MAGE-B2+MAGE-A4- cancer cell lines. All four TCR-T-IL12 showed potent cytotoxicity against cancer cell lines. IL12-RFP T cells (NFAT.IL-12.RFP transduced T cells without transgenic TCR) and mock (untransduced) T cells were used as negative controls. Maximum specific cell death above 50% is highlighted in grey. [Figure 11] Summary of the efficacy of TCR4-IL12 against MAGE-A4+MAGE-B2- cancer cell lines. Maximum specific cell death above 50% is highlighted in grey. [Figure 12] Summary of the efficacy of the four TCR-T-IL12 cells against MAGE-B2+MAGE-A4+ cancer cell lines. TCR4-IL12 and TCR2-IL12 showed potent cytotoxicity against MAGE-B2+MAGE-A4+ cancer cell lines. Maximum specific cell death of over 50% is highlighted in grey. [Figure 13] Representative potency of the four TCR-T-IL12 cells against MAGE-B2+ and / or MAGE-A4+ cancer cell lines. For potency validation, approximately 40 cancer cell lines were tested with the four TCR-T-IL12 cells generated from 2-3 donors. MAGE-B2 and / or MAGE-A4 mRNA expression levels (FPKM, RNAseq) are shown for each cancer cell line. TCR1-IL12●, TCR2-IL12■, TCR3-IL12△, TCR4-IL12▼, IL12 RFP◇. [Figure 14A]The peptide-MHC target-specific cytotoxicity of TCR-T-IL12 was verified by MAGE-B2 KO or B2M KO cancer cell lines. (A) DAN-G derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested with TCR2-IL12 and TCR4-IL12 for cytotoxicity assays with E:T titration. DAN-G WT●, DAN-G MAGE B2 KI(2E9)▲, DAN-G B2M KO▼. (B) 8505C derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested for cytotoxicity assays with E:T titration. The same results were confirmed from multiple donors. MAGE-B2 KO efficiency was verified by sequencing. B2M KO efficiency was verified by flow cytometry. 8505C WT●, 8505C neg gRNA■, 8505C MAGE B2 KO▲, 8505C B2M KO▼. [Figure 14B] The peptide-MHC target-specific cytotoxicity of TCR-T-IL12 was verified by MAGE-B2 KO or B2M KO cancer cell lines. (A) DAN-G derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested with TCR2-IL12 and TCR4-IL12 for cytotoxicity assays with E:T titration. DAN-G WT●, DAN-G MAGE B2 KI(2E9)▲, DAN-G B2M KO▼. (B) 8505C derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested for cytotoxicity assays with E:T titration. The same results were confirmed from multiple donors. MAGE-B2 KO efficiency was verified by sequencing. B2M KO efficiency was verified by flow cytometry. 8505C WT●, 8505C neg gRNA■, 8505C MAGE B2 KO▲, 8505C B2M KO▼. [Figure 15A]IL12 payload increased TCR-T cell potency against low target expressing cells and enhanced CAR-T cell efficacy in vivo. (A) Comparison of TCR-T and TCR-T-IL12 cell potency in vitro. The mean maximum cell death for TCR-T or TCR-T-IL12 cells was derived from the specific cell death activity of TCR-T and TCR-T-IL12 cells generated from three different donors. (B) Comparison of CAR-T and CAR-T-IL12 cell potency in vivo. The efficacy of huEpCAM CAR-T cells with or without IL12 payload was evaluated in the B16F10-huEpCAM syngeneic mouse tumor model. [Figure 15B] IL12 payload increased TCR-T cell potency against low target expressing cells and enhanced CAR-T cell efficacy in vivo. (A) Comparison of TCR-T and TCR-T-IL12 cell potency in vitro. The mean maximum cell death for TCR-T or TCR-T-IL12 cells was derived from the specific cell death activity of TCR-T and TCR-T-IL12 cells generated from three different donors. (B) Comparison of CAR-T and CAR-T-IL12 cell potency in vivo. The efficacy of huEpCAM CAR-T cells with or without IL12 payload was evaluated in the B16F10-huEpCAM syngeneic mouse tumor model. [Figure 16] Overview of the cross-reactivity screen with the complete panel of similar peptides. SLC16A10 and KLHDC3 were identified based on X-scan derived motifs, and NRXN1 and MAGE-B1 were identified based on sequence homology to the target peptides. MAGE-B1 is another cancer testis antigen with very restricted normal tissue expression (only in testis). No similar peptides were identified with off-target issues based on cytotoxicity assays with cancer cell lines overexpressing the full-length protein or the endogenous protein. [Figure 17]The SLC16A10 putative cross-reactive peptides were further de-risked by TCDD assay using HLA-A*02:01+ cancer cell lines overexpressing SLC16A10 full-length protein (NCI-H441 and IGR-1) (A) and cancer cell lines expressing SLC16A10 endogenous protein (LOUCY and MFE-280) (B). MAGE-B2 full-length protein overexpressing (OE) cancer cell lines were used as positive control target cell lines (A). IL12-RFP T cells were used as negative control T cells (B). [Figure 18] Summary of human normal cell reactivity evaluation. No increase in IFNγ and granzyme B production by TCR2-IL12 and TCR4-IL12 cells was observed against HLA-A*02:01+ human normal primary cells. Representative data from four normal cell types are shown, including human bronchial epithelial cells (hBEpC), human tracheal epithelial cells (hTEpC), human dermal microvascular endothelial cells (HDMEC), and human keratinocytes (Ker.). The fold change in IFNγ and granzyme B production compared to control IL12-RFP T cells is shown in the table. Comparable results were obtained from all nine normal cell types tested and for IL-12p70 and TNFα. The B-CPAP cancer cell line (MAGE-B2 65.9 FPKM) was used as a positive control for MAGE-B2+HLA-A*02:01+ cells. Mock (untransduced) T cells or T cells expressing the IL12-RFP construct (without transduced TCR) from the same donor were included as negative control effector cells. Additionally, target cells without T cells (simply labeled as targets) were used as negative controls for the cytotoxicity assay. [Figure 19-1]Summary of alloreactivity assessment. No 4-fold or greater increase in cytokine or granzyme B responses (compared to IL12-RFP control T cells) against the 34 BLCLs tested was observed for any of the four TCR-T-IL12 cells. Some low-level responses (3-fold or greater but less than 4-fold compared to IL12-RFP control cells) were observed for TCR1-IL12 and TCR2-IL12. Comparable results were obtained for IL-12p70 and TNFα production. All four TCR-T-IL12 cells demonstrated strong cytokine and granzyme B responses against the positive control U266B1 cells (HLA-A*02:01+MAGE-B2+MAGE-A4+) pulsed with MAGE-B2 peptide. [Figure 19-2] Summary of alloreactivity assessment. No 4-fold or greater increase in cytokine or granzyme B responses (compared to IL12-RFP control T cells) against the 34 BLCLs tested was observed for any of the four TCR-T-IL12 cells. Some low-level responses (3-fold or greater but less than 4-fold compared to IL12-RFP control cells) were observed for TCR1-IL12 and TCR2-IL12. Comparable results were obtained for IL-12p70 and TNFα production. All four TCR-T-IL12 cells demonstrated strong cytokine and granzyme B responses against the positive control U266B1 cells (HLA-A*02:01+MAGE-B2+MAGE-A4+) pulsed with MAGE-B2 peptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited within the body of this specification are expressly incorporated by reference in their entirety.

[0036] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques may generally be performed according to conventional methods well known in the art and as described in various general and more specific references described and discussed throughout this specification. See, for example, Sambrooke et al., 2001, Molecular Cloning: A Laboratory Manuel, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature used in connection with analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0037] HLA class I molecules, preferably HLA-A * Provided herein is a T cell receptor (TCR) alpha and beta chain pair that binds to the MAGE-B2 derived peptide GVYDGEEHSV (SEQ ID NO: 1) when represented by 02:01. A "TCR alpha and beta chain pair" may also be referred to herein as a "TCR", "a TCR", or "the TCR". When recombinantly expressed in a cell, e.g., a T cell, the TCR binds to a MAGE-B2 peptide-HLA complex on the cell, e.g., a cancer cell, and such binding activates the recombinant cell, and activation of the T cell kills or destroys the cancer cell. Methods for determining T cell activation are known in the art and are provided herein in the Examples.

[0038] In a preferred embodiment, the potency or cytolytic activity (cytotoxicity) of the recombinant cells of the present invention is measured by: (1) measuring the number of copies of the recombinant cells in a T cell-dependent cytotoxicity (TDCC) assay, a T2 / peptide challenge assay, or a combination of the following: -8 M) / HLA-A loaded with peptides in cells * 02:01 defined by 80-100% lysis of target cells or (2) 80-100% lysis of a natural pMHC target positive cancer cell line.

[0039] In a particular embodiment, HLA class I molecules, preferably HLA-A * When presented by 02:01, TCRs further bind to the MAGE-A4 derived peptide GVYDGREHTV. Such TCRs include TCR3, TCR4, TCR6, TCR7, and TCR11.

[0040] Each TCR α and β chain contains a variable and a constant domain. Within the variable domain (Vα or Vβ) there are three CDRs (complementarity determining regions): CDR1, CDR2, and CDR3. The various α and β chain variable domains can be distinguished by their frameworks as well as by parts of their CDR1, CDR2, and CDR3 sequences.

[0041] In a preferred embodiment, the TCR comprises an α chain having a CDR3 as set forth in SEQ ID NOs: 13-23 and a β chain having a CDR3 as set forth in SEQ ID NOs: 24-34. The CDR3 region can be determined by commercially available software (e.g., Cellranger; 10X Genomics). The TCR α chain can comprise a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in any of SEQ ID NOs: 35-45. The TCR β chain can comprise a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in any of SEQ ID NOs: 46-56. Methods for determining identity between two sequences, such as BLAST or Geneious, are well known in the art. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues at the C-terminus or N-terminus of any of the sequences set forth in any of SEQ ID NOs: 35-45, or any of the sequences set forth in any of SEQ ID NOs: 46-56, may be truncated or removed. Exemplary TCRs, and corresponding α-chain and β-chain CDR3s, and full-length SEQ ID NOs: are shown in Tables 1A and 1B, SEQ ID NOs: 13-56.

[0042] [Table 1]

[0043] [Table 2]

[0044] [Table 3]

[0045] [Table 4]

[0046] [Table 5]

[0047] [Table 6]

[0048] [Table 7]

[0049] [Table 8]

[0050] [Table 9]

[0051] [Table 10]

[0052] [Table 11]

[0053] [Table 12]

[0054] In certain embodiments, the variable domains of the TCR alpha or beta chains may be fused to a non-TCR polypeptide. Exemplary alpha and beta chain variable domains may be used to form soluble TCRs capable of binding MAGE-B2 (and in some cases MAGE-A4) derived peptides in the context of an HLA molecule. The soluble TCR may be in a single chain format in which the alpha and beta variable domains are linked by a linker. A disulfide bond may be introduced between the alpha and beta chains to increase stability. The soluble TCR may be fused or linked to a therapeutic or imaging agent.

[0055] Exemplary TCRs and the corresponding α and β variable regions are shown in Table 2.

[0056] [Table 13]

[0057] The TCR α or β variable domain may comprise a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences set forth in Table 2. The TCR β chain may comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 46-56. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues at the C-terminus or N-terminus of any of the sequences set forth in SEQ ID NOs: 35-56 of Table 2 and Table IB may be truncated or removed.

[0058] Recognition of the target peptide in the context of HLA is required for efficacy, but for safety purposes, in some embodiments, * Preferably, the TCR lacks cross-reactivity with peptides structurally similar when presented by HLA-A1:02:01, or with HLA molecules of other allotypes. Cross-reactivity and alloreactivity of exemplary TCRs described herein are provided in the Examples. Thus, exemplary TCRs lack cross-reactivity with peptides structurally similar when presented by HLA-A1:02:01, or with HLA molecules of other allotypes. * Not only can it recognize the MAGE-B2 peptide in the context of 02:01 and activate T cells recombinantly expressing TCR against tumor cells, but it can also activate the recombinant T cells by binding to HLA-A * When the peptide is presented in the context of 02:01 or other HLA molecules expressed on normal tissues, it is either unable to be activated or is only poorly activated.

[0059] Further embodiments of the invention include nucleic acids encoding the TCR alpha variable domain, the TCR beta variable domain, or the TCR alpha and beta variable domains described herein. In certain embodiments, the nucleic acid encodes one or more of the alpha or beta variable domains described in Table 2. In certain embodiments, the nucleic acid encodes both the alpha and beta variable domains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In a preferred embodiment, the nucleic acid encoding the TCR alpha chain variable domain, the TCR beta chain variable domain, or the TCR alpha and beta chain variable domains is an expression vector, and the TCR alpha chain variable domain, the TCR beta chain variable domain, or the TCR alpha and beta chain variable domains are operably linked to a promoter.

[0060] The TCR α and β variable domains may be co-transcribed from the same promoter. For embodiments in which the α and β variable domains are linked into a fusion protein, the domains may be co-translated into a single polypeptide. In embodiments in which the α and β domains are in separate polypeptides, it is useful to include an internal ribosome entry site (IRES) between the α and β variable domain coding regions in the expression vector.

[0061] Also provided herein are nucleic acids encoding the TCR alpha chain, TCR beta chain, or TCR alpha and beta chains described herein. In certain embodiments, the nucleic acid encodes one or more of the alpha or beta chains described in Table 1. The encoded alpha or beta chain may be full-length or mature. If mature, i.e. lacking the native leader sequence associated with that alpha or beta chain, the nucleic acid encoding a signal or leader sequence is preferably operably linked to the nucleic acid encoding the alpha or beta chain such that, when translated, the leader sequence directs the alpha or beta chain to the endoplasmic reticulum.

[0062] In certain embodiments, the nucleic acid encodes both the alpha and beta chains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In a preferred embodiment, the nucleic acid encoding the TCR alpha, TCR beta, or TCR alpha and beta chains is an expression vector, and the TCR alpha, TCR beta, or TCR alpha and beta chains are operably linked to a promoter.

[0063] The TCR α and β chains may be co-transcribed from the same promoter, in such an embodiment, it is useful to include an internal ribosome entry site (IRES) between the α and β chain coding regions in the expression vector.

[0064] The expression vector of the present invention includes, but is not limited to, a retroviral or lentiviral vector. The expression vector may further encode one or more additional proteins in addition to the TCR alpha and / or beta chain. In certain embodiments, the expression vector encodes one or more cytokines. In a preferred embodiment, the cytokine is a T cell growth factor, such as IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21, along with combinations thereof. Cytokines may have systemic effects, and when using an expression vector encoding a cytokine to produce cells for adoptive cell therapy, it is preferred that cytokine expression is controlled by an inducible promoter. In certain embodiments, the promoter is a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter, and the cytokine is IL-12 or a variant thereof. The use of a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter to express IL-12 is described in U.S. Pat. No. 8,556,882.

[0065] Provided herein are cells recombinantly expressing the exemplary TCRs described herein. The recombinant cells may comprise one or more expression vectors encoding and expressing a TCR alpha chain, a TCR beta chain, a TCR alpha and beta chain, a TCR alpha variable domain, a TCR beta variable domain, or a TCR alpha and beta variable domain. In preferred embodiments, the cells recombinantly express TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In certain embodiments, the cells further express one or more recombinant cytokines. In preferred embodiments, the cytokine is IL-12 or a variant thereof, and the expression is controlled by an inducible promoter, such as an NFAT-driven promoter.

[0066] In certain embodiments, the cells are obtained from a sample taken from a cancer patient. Cells such as T cells, NKT or NK cells are isolated from the sample and expanded. In certain embodiments, progenitor cells are isolated and matured into the desired cell type. The cells are transfected / transformed with one or more vectors, e.g., lentiviral vectors, encoding components of the TCR along with any additional polypeptides, e.g., IL-12 or variants thereof. Such cells can be used in adoptive cell therapy for the cancer patient from whom they were derived.

[0067] In other embodiments, the cell line recombinantly expresses a soluble TCR, which may be a fusion protein with an anti-CD3 antigen binding protein, such as an scFv.

[0068] Provided herein is a method of treating a disease or disorder in which cells associated with the disease or disorder express MAGE-B2 and / or MAGE-A4. In a preferred embodiment, the cells express HLA class I molecules, preferably HLA-A2, particularly HLA-A4. *The MAGE-B2 derived peptide GVYDGEEHSV and / or the MAGE-A4 peptide GVYDGREHTV are presented in the context of 02:01. Exemplary diseases or disorders that may be treated with the soluble TCRs or recombinant cells of the present invention include hematological or solid tumors. Such diseases and disorders include, but are not limited to, lung cancer, ovarian cancer, squamous cell lung cancer, melanoma, breast cancer, gastric cancer, testicular cancer, head and neck cancer, uterine cancer, esophageal cancer, bladder cancer, and cervical cancer. Preferred diseases and disorders include non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), bladder cancer, esophageal cancer, or ovarian cancer.

[0069] In certain treatments, tumor biopsies are tested for expression of MAGE-B2 or MAGE-A4. Tumors may also be tested for expression of the appropriate HLA molecules that are recognized by the TCRs of the invention when presented with MAGE-B2- or MAGE-A4-derived peptides. Patients whose tumors express MAGE-B2 or MAGE-A4 and are of the appropriate HLA haplotype may be administered the soluble TCRs or recombinant cells of the invention.

[0070] Various embodiments herein are described using the term "comprising" under various circumstances, but it should be understood that related embodiments may also be described using "consisting of" or "consisting essentially of". The present disclosure contemplates embodiments described as "comprising" a feature to encompass embodiments "consisting of" or "consisting essentially of" that feature. The term "a" or "an" means one or more; the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. The term "or" should be understood to include items in the alternative or together, unless otherwise clearly required by the context. The term "and / or" should be understood to include each item in a list (individually), combinations of items in a list, and all items in a list taken together. As used herein, "may" or "can" refers to something contemplated by the inventors as functional and available as part of the subject matter provided.

[0071] The terminology used in this application is standard within the art, and definitions of certain terms are provided herein to ensure clarity and certainty in the meaning of the claims. Units, prefixes, and symbols may be expressed in their SI accepted form. Numerical ranges described herein are inclusive of the numbers that define the range, and include and complement each integer within the defined range. The methods and techniques described herein are generally carried out according to conventional methods well known in the art, and as described in various general and more specific references described and discussed throughout this specification, unless otherwise specified. All documents, or portions of documents, described in this application, including but not limited to patents, patent applications, articles, books, and academic papers, are expressly incorporated herein by reference.

[0072] Further features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description, and all such features are intended as aspects of the present invention. Similarly, the features of the present invention described herein can be recombined into further embodiments that are also intended as aspects of the present invention, regardless of whether the combination of features is designated as an aspect or embodiment of the present invention. It should be understood that even if the combination of features is not found together in the same sentence, or paragraph, or section of this document, the entire document is intended to relate to an integrated disclosure, and all combinations of features described herein (even if described in separate sections) are contemplated. Also, only those limitations described herein that are significant to the present invention should be considered as such; variations of the present invention that do not have limitations not described herein that are significant are intended as aspects of the present invention.

[0073] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. EXAMPLES

[0074] The following examples, both actual and prophetic, are provided for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit its scope.

[0075] Example 1 - MAGE-A4 and MAGE-B2 have very restricted normal tissue expression and are expressed across a wide range of solid tumors The Cancer Genome Atlas (TCGA) and Applicant's data demonstrate that MAGE-A4 and MAGE-B2 mRNA have high prevalence across a wide range of solid tumors (Figure 1A). Importantly, Applicant's internal body map data show very restricted normal tissue expression of MAGE-A4 and MAGE-B2 mRNA, with the exception of the immune privileged site, testis (Figure 1B). MAGE-A4 IHC data in squamous non-small cell lung cancer (squamous non-small cell lung cancer or lung squamous cell carcinoma) show that within tumors, MAGE-A4 protein is expressed in the majority (60-100%) of tumor cells, but not in stromal cells (Figure 1C). Similarly, MAGE-B2 ISH data show that within tumors, MAGE-B2 mRN is expressed in the majority (>50%) of NSCLC tumor cells, but not in stromal cells (data not shown).

[0076] In addition, HLA-A * Presentation of MAGE-B2 and MAGE-A4 peptides at 02:01 was confirmed by mass spectrometry (MS). MAGE-B2 peptide-MHC (GVYDGEEHSV / HLA-A) peptides were synthesized from various tumor and normal tissues (Immatics, Tuebingen, Germany). * It was demonstrated that MAGE-B2 pMHC expression is highly specific to tumors and not detectable in normal healthy tissues (Figure 2A). The MAGE-B2 pMHC frequency in representative cancer types as measured by MS is shown in the table (Figure 2B). The MAGE-B2 peptide GVYDGEEHSV (SEQ ID NO: 1) corresponds to amino acid residues 231-240 of the MAGE-B2 protein. Furthermore, in-house MS data confirms MAGE-A4 pMHC expression in squamous NSCLC tumors (data not shown). The MAGE-A4 peptide GVYDGREHTV (SEQ ID NO: 2) corresponds to amino acid residues 230-239 of the MAGE-A4 protein.

[0077] MAGE-A4 and MAGE-B2 are expressed in a wide range of cancer types. MAGE-B2 and / or MAGE-A4 pMHC expression (MAGE-B2 / A4-HLA-A * Solid tumor indications with 100% CI (02:01) include, but are not limited to, lung squamous cell carcinoma (LUSC) at 16.2-22.7%, head and neck squamous cell carcinoma (HNSCC) at 9.2-15.8%, esophageal carcinoma at 6.2-11.1%, bladder carcinoma at 4.7-10.4%, and ovarian carcinoma at 2.1-7.8% (Figure 3). The cancer population in a given cancer indication was estimated based on pMHC target frequency (%) multiplied by new cases (new patients) per year in the US population. pMHC target frequency (%) was calculated based on the HLA-A * Calculated by MAGE-B2 and / or MAGE-A4 mRNA expression frequency multiplied by 02:01 carrier frequency (0.41). Using the TCGA public dataset of RNAseq from tumors of interest, MAGE-B2 and / or MAGE-A4 mRNA expression frequency was estimated in each tumor indication at thresholds of (1) MAGE-B2>=1 FPKM and / or MAGE-A4>=10 FPKM or (2) MAGE-B2>=5 FPKM and / or MAGE-A4>=50 FPKM (Figure 3). Patients positive for both MAGE-B2 and MAGE-A4 targets were not counted twice. SEER, EPIC Oncology New Patients or Epiphany / Epic in 2020 were used to estimate disease incidence (new cases per year) in selected tumor indications and thus derive estimated treatable patient population ranges (Figure 3). HLA-A * 02:01 is one of the most common MHC class I alleles in the United States. * The 02:01 haplotype (carrier) frequency estimate is 0.41 (www.allelefrequencies.net). The US patient population is doubled when both MAGE-A4 and MAGE-B2 are covered compared to MAGE-B2 alone. The largest patient population is in squamous non-small cell lung cancer, followed by HNSCC, bladder cancer, esophageal cancer, and ovarian cancer (Figure 3).

[0078] Example 2 - Identification of MAGE-B2 pMHC-specific TCRs The process for identifying and selecting lead clinical TCR candidates is as follows: First, we used a TCR discovery platform based on ex vivo stimulation and scRNAseq to identify healthy HLA-A * Using 52 02:01+ donors, 40 dominant MAGE-B2 pMHC-specific TCRs were identified. Using Jurkat activation assay, 11 TCR candidates were selected from the 40 TCRs. Based on these 11 TCR sequences, 11 TCR-T cells per donor were generated by transducing initial pan-T cells isolated from three donors with lentiviruses carrying individual TCRs. The TCR-T cells were further evaluated by various functional assays, such as potency (cytotoxicity) testing on T2 cell lines pulsed with target peptides and multiple (approximately 20) cancer cell lines, cross-reactivity screens with similar peptides, and initial alloreactivity screens. Based on the functional data, we narrowed down the 11 TCRs to the top four TCR candidates. To further increase in vivo efficacy and reduce clinical dose, the top four TCRs were produced in a TCR-T-IL12 lentiviral construct in which IL12 payload expression is induced by TCR activation under an NFAT response-driven promoter. Thus, IL12 can only be produced when TCR-T cells bind to pMHC targets (MAGE-B2 and / or MAGE-A4) in tumors. The TCR-T-IL12 cells produced from three donors were further evaluated by various functional assays, such as potency testing in T2 cell lines pulsed with target peptides and multiple (approximately 40) cancer cell lines, cross-reactivity with a full panel of similar peptides, cytotoxicity screens on normal cells, and full alloreactivity screens. Based on all the data from these evaluations, we selected one lead clinical TCR candidate.

[0079] MAGE-B2 pMHC-specific TCRs can be identified from rare T cell clones isolated from healthy donor PBMCs Difficulties in identifying tumor antigen-specific TCRs have hindered the development of TCR-mediated immunotherapy. Despite these challenges, the inventors have succeeded in developing a TCR discovery platform by which tumor antigen pMHC-specific TCRs can be identified from rare T cell clones isolated from healthy donor PBMCs (Figure 4A). * The frequency of MAGE-B2 pMHC-reactive T cells in PBMCs from 02:01+ donors was very low, typically around 0% DEXTRAMER+ T cells. DEXTRAMER® (Dex) is a multimer of peptide-MHC complexes that can specifically bind to TCRs and can therefore be used to isolate antigen (pMHC)-specific T cells. First, to expand rare tumor antigen-specific T clones, we used 52 healthy HLA-A * PBMCs from 02:01+ donors were used to isolate T cells and autologous antigen-presenting cells (APCs), such as monocyte-derived dendritic cells and activated B cells. Upon co-culture of T cells with autologous APCs pulsed with target peptide, these T cells went through multiple stages of ex vivo stimulation, resulting in tumor antigen pMHC-specific priming, restimulation, and expansion of pMHC-specific T cells. After multiple antigen restimulations, a population of MAGE-B2 pMHC dextramer+ (Dex+) T cells (MAGE-B2 pMHC-reactive T cells) was detected. After 2–4 rounds of antigen restimulation, the MAGE-B2 pMHC-specific T cell population became more enriched, as confirmed by both dextramer-PE and dextramer-APC lines (Figure 4B). Dex+CD8+ T cells were then sorted for single-cell RNAseq to identify sequences of TCRα and TCRβ chains. The sequence numbers corresponding to the TCRα and TCRβ sequences of the identified representative TCRs are shown in Table 1. Furthermore, the selected Dex+CD8+ T cells were validated for MAGE-B2 antigen-specific activation by IFNγ ELISPOT assay using peptide-loaded T2 cells (Figure 4C). This TCR discovery platform identified 52 healthy HLA-A TCRs. *40 dominant MAGE-B2 pMHC-specific TCRs from 02:01+ donors were identified. Importantly, the TCRs identified from the blood of healthy donors have been selected in humans through thymic natural selection (in the thymic medulla) and have eliminated autoreactive TCRs, unlike affinity-enhanced TCRs or bispecific antibodies. Therefore, the risk of off-targeting to the TCRs is considered to be quite low, which was confirmed by safety evaluation assays (described below).

[0080] Selection of top MAGE-B2 pMHC-specific TCR-T cells 52 healthy HLA-A * From 40 dominant MAGE-B2 pMHC-specific TCRs identified from the 02:01+ donor screen, 11 TCR candidates were selected by Jurkat activation assay (Figure 5). Lentiviruses carrying individual TCRs were transduced into Jurkat TCRKO reporter cell lines, which constitutively express CD8a and Renilla luciferase controlled by TCR activation under an NFAT response-driven promoter. Activity of individual TCRs was measured as fold change in luciferase activity in the presence of MAGE-B2 peptide-loaded T2 cells compared to T2 cells with vehicle alone (Figure 5).

[0081] Based on these 11 selected TCR sequences, 11 TCR-T cell lines per donor were generated by transducing human primary pan-T cells isolated from three donors with lentiviruses carrying individual TCRs. These TCR-T cells were further evaluated by various functional activities. First, the potency of each TCR-T was evaluated using T2 / peptide cytotoxicity assays (MAGE-B2 peptides), such as peptide titration and E:T (effector:target cell ratio) titration assays (Figures 6A-6C). T2 is defective in the transporter (TAP) associated with antigen processing and HLA-A binding. *Since the T2 cell line expresses HLA-A1, MHC class I-restricted endogenous peptides cannot enter the ER, and the T2 cell line presents mainly exogenous peptides. Therefore, a T2 / peptide cytotoxicity assay (a cytolytic activity assay using a T2 cell line loaded with a peptide of interest) can be used to detect peptides (e.g., HLA-A1) bound to the TCR of T cells. * 02:01-restricted) was studied. The potency of the two TCRs, TCR2-T and TCR4-T, against the T2 / MAGE-B2 peptide was very similar (Figures 6A-6C). Importantly, TCR3-T and TCR4-T were also cross-reactive against the MAGE-A4 peptide, which is also a cancer-testis antigen with high prevalence in a wide range of solid tumors as mentioned above. TCR4-T showed significantly higher potency against the MAGE-A4 peptide compared to TCR3-T. Furthermore, cytotoxicity against multiple (approximately 20) MAGE-B2+ and / or MAGE-A4+ cancer cell lines was evaluated. Representative cytotoxicity against the SK-MEL-5 line is shown in Figure 6D. The exemplary TCR-T displayed potent cell death activity against cancer cell lines with MAGE-B2 expression as low as 1.4 FPKM or E:TEC50 as low as 0.25.

[0082] To assess off-target selectivity, TCR-T cells were examined by T2 / peptide cytotoxicity assay using 131 homology-based analog peptides and target-negative cancer lines. Representative data are shown in Figure 6E. Details of the off-target strategy and identification of analog peptides are described below.

[0083] The top 5 most frequent non-HLA-A allogeneic antibodies in the US population for early alloreactivity * TCR-T was tested in co-culture with five B lymphoblastoid cell lines (BLCL) expressing the 02:01 allele (e.g., HLA-A * 01:01, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 24:02, HLA-A *02:07). IFNγ and granzyme B production were used as readouts for early alloreactivity. Details of alloreactivity are described below.

[0084] The top four TCRs (TCR1, TCR2, TCR3, and TCR4) were selected from the 11 TCRs based on various functional studies, such as (1) potent cytotoxicity against MAGE-B2 and / or MAGE-A4 pMHC targets using T2 / MAGE-B2 peptides, T2 / MAGE-A4 peptides, and approximately 20 MAGE-B2+ and / or MAGE-A4+ cancer cell lines; (2) off-target selectivity with no cross-reactivity against 131 homology-based similar peptides and target-negative cancer cell lines; (3) no early alloreactivity; and (4) manufacturability (e.g., excellent TCR transduction efficiency).

[0085] Example 3 - Potency validation of TCR-T-IL12 cells The top four TCRs selected by the various functional assays mentioned above were further produced in the TCR-T-IL12 lentiviral construct in which IL12 payload expression is regulated by TCR activation under an NFAT response driven promoter (Figure 7). Thus, IL12 is produced upon TCR signaling when TCR-T-IL12 cells bind to pMHC targets (MAGE-B2 and / or MAGE-A4) in the tumor. The TCR-T-IL12 cells produced using the three donors were further evaluated by various functional assays. First, potency validation was performed using a T2 / MAGE-B2 peptide cytotoxicity assay (Figure 8). The potency ranking of the four TCR-T-IL12s remains the same as that of the parental TCR-T (without IL12). TCR2-IL12 showed the highest potency, followed by TCR4-IL12, TCR3-IL12, and then TCR1-IL12, which was the lowest. All four TCR-IL12 cells exhibited EC90 >10 in the T2 / peptide cytotoxicity assay. -8 Efficacy criteria were met at M (peptide concentration).

[0086] Example 4 - Potent cytotoxicity of TCR4-IL12 against both MAGE-B2 and MAGE-A4 peptide-loaded T2 cells Notably, TCR4-IL12 can also recognize the MAGE-A4 peptide MHC with high potency in the T2 / peptide cytotoxicity assay (Figure 9). The potency gap between MAGE-A4 and MAGE-B2 peptides against TCR-T-IL12 was only 2.5-fold at EC50 and approximately 7-fold at EC90. Potency data from three different donors showed that potency against MAGE-B2 and MAGE-A4 peptides was quite similar (graph in Figure 9). Importantly, TCR4-IL12 showed an EC90 of 10 for both MAGE-B2 and MAGE-A4 peptides. -8 Efficacy criteria were met at M (peptide concentration).

[0087] Example 5 - Cytotoxicity against MAGE-B2+ and / or MAGE-A4+ cancer cell lines The potency (cytotoxicity) of the four TCR-T-IL12s was validated using three different categories of cancer cell lines, including MAGE-B2+MAGE-A4-, MAGE-B2-MAGE-A4+, and MAGE-B2+MAGE-A4+ cancer cell lines. First, the potency of TCR-T-IL12 was evaluated using MAGE-B2+MAGE-A4- cancer cell lines (Figure 10). All four TCR-T-IL12s showed strong cytotoxicity against cancer cell lines with low MAGE-B2 expression, about 1.4 FPKM. In the potency ranking assay against MAGE-B2+MAGE-A4- cancer cell lines, TCR2 was the most potent TCR, followed by TCR4, then TCR1 and TCR3, which were almost equal. TCR2-IL12 and TCR4-IL12 showed cytotoxicity with low E:TEC50 of about 0.07 and 0.21, respectively. TCR-T-IL12 showed high potency even against MAGE-B2 low cancer cell lines such as 8505C (about 1.4 FPKM) and AU565HLA-A2hi (about 3.7 FPKM). Target-specific cell death against these MAGE-B2 low cancer cell lines was verified by MAGE-B2 KO cell lines generated from these low cancer cell lines (described below).

[0088] Second, the potency of TCR4-IL12 against MAGE-A4+MAGE-B2- cancer cell lines was evaluated from T2 / peptide assays, taking into account the cross-reactivity of this TCR to MAGE-A4 peptides (Figure 11). TCR4-IL12 showed cytotoxicity against cancer cell lines with low MAGE-A4 expression of about 6.3 FPKM or E:TEC50 of about 0.46.

[0089] Third, the potency against double positive, MAGE-B2+MAGE-A4+ cancer cell lines was evaluated (Figure 12). TCR4-IL12 and TCR2-IL12 showed strong cytotoxicity against MAGE-B2+MAGE-A4+ cancer cell lines. In the potency ranking against MAGE-B2+MAGE-A4+ cancer cell lines, TCR4 was the most potent TCR, followed by TCR2, then TCR3 and TCR1 were almost equal. Notably, due to its high potency against both MAGE-B2 and MAGE-A4 peptides, TCR4-IL12 showed the highest potency against double positive MAGE-B2+MAGE-A4+ cancer cell lines. Notably, some MAGE-B2-low MAGE-A4-high cancer cell lines (e.g., A375) can distinguish the potency of TCR4 from TCR2, since TCR2 only has MAGE-B2 specificity without MAGE-A4 cross-reactivity. TCR4-IL12 demonstrated cytotoxicity against double positive cancer cell lines with MAGE-B2 expression as low as 1.2 FPKM or MAGE-A4 expression as low as 44 FPKM or E:TEC50 as low as 0.04. TCR2-IL12 showed cytotoxicity against cell lines with MAGE-B2 expression as low as 3.5 FPKM or E:TEC50 as low as 0.01.

[0090] Representative cancer cell line potency data of the four TCR-T-IL12 cells are shown in Figure 13. Approximately 40 cancer cell lines were tested with the four TCR-T-IL12 cells produced from 2-3 donors. The TCR-T-IL12 cells demonstrated potent cytotoxicity against some cancer cell lines with low E:T EC50. For example, the E:T EC50 of TCR4-IL12 against cancer cell lines was 0.21 for B-CPAP, 0.25 for SK-MEL-5, 0.98 for THP-1, and 0.25 for NCI-H1755.

[0091] Example 6 - Peptide-MHC target specific cytotoxicity validation with MAGE-B2 KO and B2M KO cancer cell lines The potent cytotoxicity of TCR-T-IL12 against multiple MAGE-B2+ cancer lines with very low expression of MAGE-B2 was confirmed, and it was determined whether this cytotoxicity was dependent on pMHC target expression. Therefore, we generated MAGE-B2 KO (knockout) cell lines and B2M KO cell lines to eliminate expression MAGE-B2 and B2M, respectively (Figures 14A and 14B). B2M (β2 microglobulin) is a critical subunit of MHC class I molecules. Both MAGE-B2 KO and B2M KO resulted in a decrease in cell death activity by either TCR2-IL12 or TCR4-IL12. Remarkably, when MAGE-B2 was knocked out in the 8505C cancer cell line with very low MAGE-B2 mRNA expression (1.4 FPKM), both TCR-T-IL12 cells lost cell death ability, indicating that the cytolytic activity of TCR-T-IL12 cells depends on MAGE-B2 target expression and that TCR-T-IL12 can truly recognize such low expression of the target (Figure 14B).Similarly, a decrease in cytotoxicity was seen in the B2M KO line, demonstrating that TCR-T-IL12 activity is responsive to HLA expression.

[0092] Example 7 - Effect of IL12 payload on TCR-T potency HuEpCAM CAR-T cells with or without an IL12 payload were evaluated in the B16F10-huEpCAM syngeneic mouse tumor model. This mouse study demonstrates that the IL12 payload can enhance T cell efficacy in vivo and reduce potential clinical doses (Figure 15B).

[0093] Next, we evaluated the effect of IL12 payload in human TCR-T system with multiple cancer cell lines. Especially for MAGE-B2-low cancer cell lines (shown in wavy box), IL12 payload can increase TCR-T cell potency compared to parental TCR-T without IL12 (Figure 15A). For MAGE-B2-high or MAGE-A4-high cancer cell lines (shown outside wavy box), IL12 had less effect on those cancer cell lines because potency was already reached to the maximum by parental TCR-T.

[0094] Example 8 - Non-clinical safety evaluation summary As human-specific HLA targeting precludes the use of animal models, extensive in vitro and ex vivo safety evaluations were performed on TCR-T-IL12 cells. First, target expression was assessed by various assays such as RNASeq, IHC, and mass spectrometry using normal human tissues as well as tumor tissues, as described above. As MAGE-B2 and MAGE-A4 are cancer-testis antigens, studies showed very limited normal tissue expression (expressed only in testis). Second, off-target reactivity was evaluated and assessed using two different strategies. The first strategy involved evaluation of cytotoxicity against various normal human primary cell types representative of major organs. The second strategy involved identifying a panel of similar peptides based on sequence homology matching the MAGE-B2 target peptide, along with a positional scan (X-scan)-based strategy to identify putative cross-reactive peptides unique to each TCR. To evaluate potential cross-reactivity against this panel of similar peptides, a T2 / peptide TECC assay was performed. The third safety assessment was alloreactivity, evaluating 34 BLCLs representing frequent HLA class I alleles in the US population, including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles.

[0095] Identification of similar peptides based on homology To assess off-target reactivity, a full panel of similar peptides to the MAGE-B2 target peptide was identified using two different strategies, based on either sequence homology to the target peptide or to X-scan derived motifs.

[0096] A homology-based strategy was designed using an in silico approach to identify a list of peptides that could potentially cross-react with the candidate TCR-T. To accomplish this, a protein database (UniProtKB / Swiss-Prot, June 2019) query was first performed to generate a list of all possible decameric peptides based on amino acid identity matches to the target MAGE-B2 peptide (GVYDGEEHSV). This in silico query was performed using a Python script, and 170,082 peptides based on a 30% homology (identity) match to the target peptide were identified. To further refine this list, criteria such as high homology matches and software such as NetMHCpan software and IEDB (Immune Epitope Database) were utilized. NetMHCpan 3.0 was used to generate a list of all possible decameric peptides based on amino acid identity matches to the target MAGE-B2 peptide (GVYDGEEHSV). * The predicted binding affinity of peptides to 02:01 was examined using the manually curated IEDB database of experimentally characterized immune epitopes (June 2019) against HLA-A * The probability of peptides being processed and presented by the 02:01 allele was examined. The specific criteria used for peptide selection were: (1) all peptides with 60% or more homology match (identity) to the target peptide (65 peptides), (2) all peptides with 50% or more homology match and predicted binding affinity (IC50) of 50 nM or less (35 peptides), and (3) all peptides with 100% or more homology match and 100% or less predicted binding affinity (IC50) to the target peptide reported in the IEDB (HLA0A *All peptides with a homology match of 40% or more to the 02:01 allele (45 peptides). As a result, from this homology-based in silico search of the human proteome database, we identified 131 unique peptides.

[0097] Identification of TCR-binding motifs using positional scanning (X-scan) and similar peptides based on X-scan-derived motifs As an orthogonal approach to identify similar peptides, we used a positional scanning method known as X-scan. The X-scan assay uses a peptide library created by sequentially mutating each residue of the MAGE-B2 peptide to one of the other 19 natural amino acids, resulting in a total of 190 peptides. These 190 peptides were synthesized and tested in a T2 / peptide TDCC assay to identify X-scan-derived motifs specific for each individual TCR (Table 3). Briefly, T2 cells were pulsed with each of these peptides at a concentration of 10 μM or 1 μM, followed by the addition of TCR-T cells at an E:T ratio of 1:1. Cell viability was determined using the T2 / peptide TDCC assay. Amino substitutions were defined as essential for TCR engagement, and viability was confirmed to be less than 20%. Corresponding search motifs were created to represent which amino acids were allowed at each position in the peptide sequence (Table 3). The underlined amino acids represent the natural residues at the corresponding positions in the peptides.

[0098] Using a python script, an in silico search of the UniProtKB / Swiss-Prot database with splice variants was performed (June 2019) to identify all decameric sequences that followed the induced motif. A blast search based on this motif identified unique human peptide matches that followed the specific TCR-T consensus motif.

[0099] In the case of two TCRs (TCR3 and TCR2), when the number of motif search-based peptides obtained was too large, further anchor residue restrictions (at residues 2 and 10) were applied to the derived motifs to limit the final cross-reactive peptide selection (Table 3). * The sequences of 02:01 positive peptides were analyzed to calculate the amino acid frequency at the anchor residue positions. A 3% amino acid frequency cutoff was applied to both anchor residues of the motif (residue 2 and residue 10), whereby position 2 was restricted to the amino acids T, M, E, I, V, and L, and position 10 was restricted to the amino acids Y, I, A, L, and V.

[0100] [Table 14]

[0101] Cross-reactivity screen with a complete panel of similar peptides A complete panel of similar peptides (including an X-scan motif-based set and a homology-based set) was synthesized and tested in the T2 / peptide TDCC assay to investigate potential off-target reactivity.

[0102] To identify potential cross-reactive peptides for each TCR-T-IL12, a full panel of similar peptides was tested using a T2 / peptide TDCC screen at high peptide concentrations (10 μM or 1 μM). Peptides that showed 25% or less viability in at least one of three donors were considered putative cross-reactive peptides and selected for further efficacy testing. All three different donors showed good concordance with peptide responses.

[0103] A potency screen (dose-dependent screen) was then performed using the T2 / peptide titration TDCC assay against the putative cross-reactive peptides identified from the above screen. Most of the putative cross-reactive peptides were de-risked by this potency screen. 3 A potency gap of less than 1-fold was considered as a cutoff for further risk assessment. Results from a cross-reactivity screen with a full panel of similar peptides against the top four TCR-T-IL12 cells are summarized in Figure 16, showing a 10-fold increase in potency against the target MAGE-B2 peptide. 3 All peptides showing less than 1-fold potency are listed. TCR4-IL12 cells did not produce any putative cross-reactive peptides (with the exception of MAGE-A4). The other three TCR-T-IL12 cells each had one putative cross-reactive peptide identified from this full panel peptide screen, with the exception of MAGE-B1, a cancer testis antigen. All three putative cross-reactive peptides (arising from proteins SLC16A10, KLHDC3, and NRXN1) were further identified in HLA-A cells overexpressing the respective full-length proteins. * The risk was reduced by TDCC assays using 02:01+ cancer cell lines or cancer cell lines expressing endogenous proteins (Figure 17). Cytotoxicity against these putative cross-reactive proteins or cancer cell lines overexpressing endogenous proteins was not confirmed by any of the TCR-T-IL12 (TCR1, TCR2, and TCR3) suggesting that these peptides are unlikely to be naturally processed and presented from the protein (Figures 16 and 17). In conclusion, no significant cross-reactivity was demonstrated by any of the four TCR-T-IL12 cells across the full panel of similar peptides identified by sequence homology and X-scan derived TCR motifs.

[0104] Evaluation of cytotoxicity against normal human cells Next, the cytotoxicity of the four MAGE-B2 TCR-T-IL12 cells (TCR1-IL12, TCR2-IL12, TCR3-IL12, and TCR4-IL12) was evaluated against a panel of nine normal human primary or iPSC-derived cell types representative of major organs (without MAGE-B2 or MAGE-A4 expression) serving as target cells in a T cell-mediated cytotoxicity assay. The panel of nine normal human cells included bronchial epithelial cells (hBEpC), tracheal epithelial cells (hTEpC), dermal microvascular endothelial cells (HDMEC), keratinocytes, hepatocytes, renal proximal tubule epithelial cells (RPTEC), iPSC-derived astrocytes, cardiomyocytes, and GABAergic neurons (Figure 18). All normal cells expressed HLA-A * 02:01 - From a positive donor (HLA-A * (02:01 expression was confirmed by RNASeq). Importantly, these normal cells express HLA-A * The large variety of peptides that can be presented to 02:01 serves as an assay system to assess a wide range of off-target effects. * B-CPAP cancer cell line with 02:01 expression was used as positive control target cells. Mock (untransduced) T cells or T cells expressing IL12-RFP construct (without transduced TCR) from the same donor were included as negative control effector cells. Cytokine (IFNγ, IL-12p70, TNFα) and granzyme B production, as well as target cell cytotoxicity (measured by caspase 3 / 7 cleavage) were assessed in co-culture with TCR-T-IL12 cells (Figure 18). All four TCR-T-IL12 cells induced cytokine production and target cell cytotoxicity (MAGE-B2+HLA-A) when co-cultured with positive control B-CPAP cells. *Importantly, TCR2-IL12 and TCR4-IL12 did not mediate cytokine production or enhance caspase 3 / 7 cleavage when co-cultured with any of the normal human primary or iPSC-derived cells tested, and did not display off-target reactivity towards any of the normal cells tested.

[0105] Assessment of alloreactive potential using 34 BLCL lines As part of the safety evaluation, alloreactive potential was assessed using a panel of 34 BLCLs (B-lymphoblastoid cell lines) representing high frequency (>11%) MHC class I alleles in major US racial groups, including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles. Alloreactive potential was assessed by cytokine (IFNγ, TNFα, and IL-12p70) and granzyme B production when TCR-T-IL12 cells were co-cultured with each of the BLCLs. No significant increase (4-fold or greater compared to IL12-RFP control T cells) in cytokine or granzyme B responses to the 34 BLCLs tested was observed for any of the four TCR-T-IL12 cells (Figure 19). Some low-level responses (3-fold or greater but less than 4-fold compared to IL12-RFP control cells) were observed for TCR1-IL12 and TCR2-IL12. All four TCR-T-IL12 cells inhibited the expression of MAGE-B2 peptide-pulsed positive control U266B1 cells (HLA-A * A strong cytokine and granzyme B response was demonstrated against 02:01+MAGE-B2+MAGE-A4+).

[0106] Overall, the four exemplary TCR-T-IL12 candidates did not demonstrate significant safety issues based on the standard and alloreactivity potential safety assessments performed.

[0107] Methods and materials used in the above examples Identification of MAGE-B2 pMHC-specific TCRs by healthy donor screen Generation of autologous antigen-presenting cells (APCs) Fresh or frozen HLA-A * 02:01 positive healthy donor peripheral blood mononuclear cells (PBMCs) were used. Monocytes were positively selected from PBMCs by using human CD14-microbeads (Miltenyi Biotec, San Diego, CA, 130-050-201). Mature dendritic cells were obtained using CellXVivo™ human monocyte-derived dendritic cell (DC) differentiation kit (R&D, Minneapolis, MN, CDK004). Antigen-presenting B cells were generated using CD40L and IL-4 stimulation. B cells were positively selected from PBMCs by using human CD19-microbeads (Miltenyi Biotec, 130-050-301). CD19+ cells were then stimulated with 0.125ug / ml recombinant huCD40L in B cell medium and 2×10 5 Cells were seeded into 24-well plates at 1 ml / ml and 1 ml / well. B cell medium consisted of IMDM, GlutaMax™ supplement medium (Gibco, 31980030) supplemented with 10% heat-inactivated human serum (MilliporeSigma H3667-100ML), 100U / ml penicillin and 100ug / ml streptomycin (Gibco, 15140-122), 10μg / ml gentamicin (Gibco, 15750-060) and 200IU / ml IL-4 (Peprotech, Rock Hill, NJ, 20004100UG). Fresh B cell medium with 400IU / ml IL-4 was added to the B cell cultures at 1 ml / well on day 3 after B cell activation without disturbing the cells. Activated B cells were immediately available for antigen-reactive T cell stimulation on day 6 after B cell activation.

[0108] Ex vivo stimulation and expansion of antigen-specific T cells On day 7 after CD14+ cell isolation, MAGE-B2 peptide (Anaspec custom peptides, Freemont, CA) was added to immature dendritic cells at 1 μM along with recombinant human TNF-α. On day 9 after CD14+ cell isolation, MAGE-B2 peptide-pulsed mature dendritic cells were collected, washed, and mixed with CD14-PBMCs at a ratio of 1:10 in human T cell medium with 10 μM MAGE-B2 peptide, 10 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), and 10 ng / ml IL-7 (Peprotech, AF20007100UG). Human T cell complete medium consisted of a 1:1 mixture of CM and AIM-V™ (ThermoFisher, 12055083). CM consisted of RPMI 1640 supplemented with GlutaMAX™ (Gibco, 61870-036, ThermoFisher), 10% human serum (MilliporeSigma, H3667), 25 mM HEPES (Gibco, 15630-080, ThermoFisher) and 10 μg / ml gentamicin (Gibco, 15750-060, ThermoFisher). MAGE-B2-specific T cells were further expanded by 1 to 3 rounds of weekly peptide-pulsed B cell activation (up to a total of four T cell antigen-specific stimulations). HuCD40L-activated B cells were collected, washed and diluted at 1 × 10 6 Cells were seeded in 6-well plates at 1000 cells / ml and 4 ml / well, and 1 μM MAGE-B2 peptide was added to the B cells and incubated for 2 hours at 37°C in an incubator. Peptide-pulsed B cells were then mixed with T cells at a 1:10 ratio in human T cell media with 10 IU / ml IL-2 and 10 ng / ml IL-7. MAGE-B2 dextramer positive cells were confirmed by flow cytometry and then sorted for TCR identification by single-cell RNAseq.

[0109] Selection of activated antigen-specific T cells MAGE-B2 peptide-activated antigen-specific T cells were stained with MAGE-B2 dextramer-APC and -PE for 10 min at room temperature in the dark, then stained with CD3-FITC (Biolegend, San Diego, CA, 300440) and CD8-BV605 (BD Biosciences, San Jose, CA, 564116). Dead cell exclusion stain (Sytox blue) was purchased from ThermoFisher (Invitrogen, S34857). Cells were sorted using an Aria™ Fusion cell sorter (BD Biosciences, San Jose, CA). Data was analyzed after sorting using Flowjo.

[0110] ELISPOT Sorted CD3+CD8+Dex+ T cells were validated for antigen-specific IFNγ production by BD® ELISPOT assay (BD Bioscience, San Jose, CA, 551849) using peptide-loaded T2 cells. 2×10 6 T2 cells were loaded with 10μM MAGE-B2 peptide in human T cell complete medium at 1ml / cell and 1ml / well for 1-2 hours. 150ul of human T cell complete medium and 50μl of peptide-loaded T2 cells were added to each well in the pre-coated ELISPOT plate. CD3+CD8+Dex+ T cells (500 or 1000 cells) were sorted directly into each well in the ELISPOT plate. ELISPOT was detected after 24 hours of incubation in a 37℃ incubator. ELISPOT plates were scanned and counted by IMMUNOSPOT® (Cellular Technology Limited, Cleveland, OH).

[0111] Single-cell RNAseq For direct target enrichment, which dispenses with cDNA amplification of the whole transcriptome, samples were processed using a Chromium™ Controller (10X Genomics, Pleasanton, CA) with the V(D)J Single Cell Human T Cell Enrichment Kit (PN-1000006, PN-1000005, PN-120236, PN-120262) according to the manufacturer's instructions. Briefly, cells and beads with barcoded oligonucleotides were encapsulated in nanoliter droplets, where cells were lysed and mRNA was reverse transcribed using a poly-T primer and a barcoded template switch oligo. A nested PCR was then performed using primers in the constant region of the human TCR and a template switch oligo. A second target enrichment PCR was performed using 13-17 cycles depending on the estimated cell input number, according to the manufacturer's suggestions. Final sequencing libraries were generated from the fragmented PCR products ligated to Illumina sequencing adapters. Libraries were sequenced with 151 paired-end reads (151 x 8 x 0 x 151) on a NextSeq™ 550 or MiSeq™ (Illumina, Inc., San Diego, CA) with at least 5,000 reads / cell. Data were demultiplexed and analyzed with cellranger vdj (2.2.0) to obtain full-length paired TCR sequences that could be assigned to individual cells.

[0112] Cloning and transduction of TCR into Jurkat cells Candidate TCRs were produced as gene fragments. To monitor transfection or transduction, each fragment was cloned into a lentiviral expression vector consisting of an MSCV promoter and IRES-driven eGFP. Successful transformants were screened by Sanger sequencing, and verified clones were maxiprepped for downstream applications. When using transduction to screen candidate TCRs, lentiviral vectors were packaged into VSV-G pseudotyped virions (Alstem, Richmond, CA). Lentivirus carrying the TCRs was transduced into Jurkat TCR KO reporter cell line, which constitutively expresses CD8a and Renilla luciferase under an NFAT-inducible promoter. Briefly, 20 μL of lentiviral particles was added to 1,000,000 to 1,000,000 cells in complete medium containing 5 ug / mL polybrene (MilliporeSigma, TR1003G) in a 50 mL conical tube to achieve a multiplicity of infection (MOI) of 10. After adding the virus, the cells were spun at 1200×g for 45 minutes at 32° C. After spinning, the medium was aspirated and replaced with enough fresh medium to adjust the cells to a concentration of 500,000 cells / ml before placing in a 37° C. incubator. Approximately 72 hours after transduction, the cells were analyzed by flow cytometry. 50 μl of cells were transferred to a 96-well U-bottom plate and 150 ul FACS buffer (PBS w / o CaCl2 & MgCl2 (Corning, Corning, NY, 21-040-CV) + 5% FBS (Gibco, 10082-147)) was added before centrifugation at 300×g for 3 minutes. The supernatant was removed and the cells were resuspended in 50 μl of 1× Fc block in FACS buffer, which was incubated at 4° C. for 20 minutes. MAGE-B2 peptide-MHC (GVYDGEEHSV / HLA-A) was incubated at 4° C. for 20 minutes using the manufacturer's recommended concentrations. *Fluorescent dextramers specific for CD4+ (02:01, Immudex Customized, Fairfax, VA) were incubated with the transduced cells for 10 minutes at room temperature in the dark. A 2x antibody cocktail containing anti-CD3 (BD ​​Biosciences) was then added in a volume of 50ul before another incubation at 4°C for 20 minutes. Cells were washed three times after staining by centrifugation at 300xg for 3 minutes, followed by aspirating and resuspension. Before analysis, cells were fixed in 100μl of fresh 2% formaldehyde solution for 20 minutes at 4°C. Cells were washed twice to remove formaldehyde before final suspension in 200μl of PBS with EDTA. Fixed, labeled cells were run on an LSRII or Symphony™ cytometer (BD Biosciences) using the recommended acquisition settings.

[0113] Jurkat activation assay Antigen-presenting T2 cells (ATCC) were loaded with peptides (Anaspec customized) or vehicle alone at a range of concentrations for 2 hours in serum-free medium. After incubation, the loaded T2 cells were washed three times, counted, and seeded at 15,000 cells / well in half-area 96-well plates (Corning) before being resuspended in complete medium. Successfully transduced Jurkat cells were added at 30,000 cells / well in a total volume of 100 μL. TCR-expressing Jurkat cells were co-cultured in the presence of T2 cells at 37°C for 24 hours. At the end of this incubation, plates were briefly centrifuged at 300×g before half the volume was collected and stored for characterization of cytokine secretion. To the remaining volume, an equal volume of RENILLAGLO® (Promega) was added and the plates were incubated for 20 minutes at room temperature with shaking before luminescence (detected by ENVISION® (PerkinElmer, Waltham, Mass.)). Activation of individual TCRs was expressed as fold change in luminescence in the presence of peptide-loaded T2 cells compared to co-culture with vehicle-only T2 cells.

[0114] Generation of MAGE-B2 TCR-T and TCR-T-IL12 cells using primary human T cells Three healthy donors (HLA-A * PBMCs from the 02:01 mice were isolated from leukopaks (Allcells, Alameda, CA) using Ficoll-Paque gradient centrifugation, and further T cell isolation was performed using a CD3 negative selection kit (Miltenyi Biotec, 130-096-535) and associated manufacturer's protocol. One day prior to TCR transduction, frozen pan-T cells were thawed and diluted at 1 × 10 6The cells were resuspended in human T cell complete medium at 100 cells / ml and stimulated with CD3 / CD28 Dynabeads™ (Thermo Fisher, 11131D) at a T cell:bead ratio (2:1) in the presence of 30 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), 10 ng / ml IL-7 (Peprotech, AF20007100UG) and 25 ng / ml IL-15 (Peprotech, AF20015100UG). T cells were then seeded at 1 ml / well in 24-well plates. On the day of TCR transduction, activated T cells (300,000) were seeded in 48-well plates in human T cell complete medium and transduced with lentivirus in the presence of 8 μg / ml polybrene, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. T cells were then spin-seeded at 1500×g for 1.5 hours at 32°C. After spin-seeding, 380 ul of medium with 8 μg / ml polybrene, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15 was added to the cells to a total volume of 600 μl / well. 17-18 hours after transduction, approximately 400 μl of medium was removed without touching the cells at the bottom of the well. Cells from each well of the 48-well plate were transferred to one well of a G-REX® 24-well plate (WilsonWolf, St Paul, MN, P / N 80192M) in 3 ml of human T cell complete medium containing 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. Four days after transduction, the Dynabeads were removed according to the manufacturer's protocol. Approximately 10 × 10 cells were transferred to a G-REX® 6-well plate (WilsonWolf, P / N 80240M) in 30 ml of medium in the presence of 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. 6TCR-T cells were seeded with cells. On the 7th day after transduction, TCR-T were collected, frozen, and stored in liquid nitrogen vapor phase. TCR transduction efficiency was validated by dextramer binding. TCR-T-IL12 cells were produced by the process described in the patent application (PCT Publication No.: WO 2021 / 211104).

[0115] Flow cytometry The following antibodies were used for T cell phenotyping: CD3-FITC (Biolegend: 300440), CD8-BV605 (BD: 564116), CD4-PE (Biolegend: 317410). The following antibodies were used for dendritic cell phenotyping: CD14-PerCP / Cy5.5 (Biolegend: 301824), CD11c-PE (Biolegend: 337206), CD1a-APC-cy7 (Biolegend: 300125), CD86-APC (BD: 555660). The following antibodies were used for B cell phenotyping: MHC class I (Biolegend: 311414), MHC class II (Biolengend: 361706), CD83-PE (BD556855), CD86-APC (BD: 555660), CD20-FITC (BD: 556632). Dextramer-APC or -PE were purchased from Immudex (customized dextramers). 50 nM PKI dasatinib (Axon Medchem: 1392) was used to prevent TCR internalization. TCR-expressing T cells were incubated with 50 nM PKI dasatinib for 30 min at 37°C, followed by dextramer staining for 30 min on ice and cell surface marker staining for 15 min at 4°C. A dead cell exclusion stain (Sytox blue, ThermoFisher / Invitrogen, S34857) was used. Flow cytometry data were analyzed using Flowjo.

[0116] T cell-mediated T2-luc / peptide cytotoxicity assay (T2 / peptide TDCC assay) The functionality and cell death specificity of MAGE-B2 TCR-T was determined by T2-luc (a T2 cell line expressing firefly luciferase) cell death assay. T2-Luc cells were harvested, washed, and diluted at 2 × 10 in T2-Luc cell death assay medium (RPMI 1640-GlutaMAX™, 1 × non-essential amino acid solution (Gibco, 11140-050, ThermoFisher, Waltham, MA), 10 mM HEPES (Gibco, 15630-080), 50 μM 2-β-mercaptoethanol (Gibco, 21985-023), 1 mM sodium pyruvate (Gibco, 11360-070), 100 U / ml penicillin-streptomycin (Gibco, 15140-122), 5% heat-inactivated FBS (Gibco, 10082-147). 6 T2-Luc cells were pulsed with the indicated peptide concentrations for 2-4 hours at 37°C. T2-Luc cells were then washed and diluted with 1 × 10 5 The cells were resuspended at 1000 cells / ml and seeded at 25 μl / well in a 384-well plate (Corning, 3570). T2-Luc cells were incubated with 25 μl of TCR-T cells at the indicated ratio of dextramer+TCR-T to T2-luc cells for 48 hours. Luminescence signals were measured by adding 30 μl of Bio-Glo™ (Promega, Madison, WI, G7940) and then using a Biostack™ Neo system (BioTek, Winooski, VT). For parental TCR-T, not all of the TCR-T-IL12 cells were normalized by adding mock T cells before cell death assay. Different TCR-T were normalized to the same amount of MAGE-B2 dextramer+ cells (e.g. 10%) by adding mock (non-transduced) T cells. Specific lysis (% specific cell death) was calculated by normalizing TCR-T+T2 / target peptide cell death with either mock T cells+T2 / target peptide cell death, or TCR-T+T2 / no peptide cell death. The specific lysis formula is shown below: Formula for specific lysis (%) Peptide titration (MAGE-B2 / A4 peptides and similar peptides): {1-(TCRT+T2-luc / test peptide RLU) / (TCRT+T2-luc / no peptide RLU)}×100 E:T titration (MAGE-B2 / A4 peptides): {1-(TCRT+T2-luc / MAGE-B2 peptide RLU) / (Mock T+T2-luc / MAGE-B2 peptide RLU)} x 100 Cancer cell line cell death: {1-(TCRT+Cancer cell line RLU) / (Mock T+Cancer cell line RLU)}×100.

[0117] T cell-mediated cancer cell cytotoxicity assay (cancer cell TCDD assay) The cytotoxicity of TCR-T cells against MAGE-B2 positive and negative cancer cell lines was determined by cancer cell death assay. Cancer cells were harvested, washed, and diluted to 1 × 10 in Cancer Cell Death Assay Medium (RPMI 1640-GlutaMAX™, 1 × Non-essential amino acid solution (Gibco, 11140-050, ThermoFisher), 10 mM HEPES (Gibco, 15630-080, ThermoFisher), 50 μM 2-β-mercaptoethanol (Gibco, 21985-023, ThermoFisher), 1 mM sodium pyruvate (Gibco, 11360-070, ThermoFisher), 100 U / ml penicillin-streptomycin (Gibco, 15140-122, ThermoFisher), 10% heat-inactivated FBS (Gibco, 10082-147, ThermoFisher). 5 Cancer cells were then seeded at 25 μl / well in 384-well plates and incubated with 25 μl of TCR-T cells at the indicated ratio of dextramer+TCR-T to T2-Luc cells for 48 hours. After incubation, for adherent cancer cells, the suspension T cells were removed and washed with Ca using a plate washer. 2+ Mg 2+The wells were washed with DPBS (Corning, 21-031-CM) containing 10 μl of Celltiter Glo (Promega, G7573) and the luminescence signal was measured. For suspension luciferase-labeled cancer cells, 30 μl of Bio-Glo™ (Promega, G7940) was added and the luminescence signal was measured. The Biostack™ neo system was used to measure luminescence. For suspension cancer cells without luciferase labeling, the cancer cells were labeled with Celltrace far red (Invitrogen, C34572, Carlsbad, CA, USA). 1 × 10 cells were added to serum-free RPMI medium containing Celltrace™ far red (dilution 1:4000). 6 Cancer cells were resuspended at 1000 cells / ml and incubated at 37°C for 10 min. The reaction was stopped by adding 30 ml of cell death assay medium and incubating at room temperature for 10 min. Live cancer cells were detected by flow cytometry. A dead cell exclusion stain (Sytox™ blue, ThermoFisher / Invitrogen, S34857) was used. Specific lysis (% specific cell death) was calculated by normalizing TCR-T cell death against the cancer cell line with either mock T cell death or IL12-RFP T cell death against the cancer cell line. The specific lysis formula is shown above.

[0118] Similar peptide screens The functional specificity of MAGE-B2 TCR-T was determined using a T2-Luc / peptide directed cell death assay. Peptides including target and analogous peptides were synthesized by JPT (Berlin, Germany) or AnaSpec (Fremont, CA). T2-Luc cells were incubated with reactive analogous peptides, target-specific peptides or DMSO control in T2-Luc cell death medium at 37°C / 5% CO2 at a final peptide concentration range of 1.0E-05M to 6.0E-16M (potency) or 1.0E-05M (single point) for 2 hours. Frozen MAGE-B2 TCR-T and mock T cells were thawed, washed and rested in human T cell medium for 3 hours before assay setup. MAGE-B2 TCR-T cells were washed three times in assay medium and resuspended at 2.5E06 cells / mL. Peptide-loaded T2-Luc cells were added to white clear-bottom 384-well assay plates (Costar) at 2,000 cells / 25 μL using a Bravo liquid handling system (Agilent, Santa Clara, CA). MAGE-B2 TCR-T cells were prepared by diluting MAGE-B2 dextramer positive cells with mock T cells to obtain a target:effector ratio of 10:1; 20,000 cells / 25 μL (final Dex+T cells:T2-Luc 1:1). T2-Luc pulsed cells and TCR-T cells were incubated for 48 hours at 37°C / 5% CO2. T2-Luc cell viability was determined using the Bio-Glo™ Luciferase Assay System (Promega, G7940) according to the manufacturer's recommendations. Luminescence was detected using an ENVISION™ Multilabel Plate Reader (Perkin Elmer, Santa Clara, CA). Percent viability was calculated using the following formula: % Viability = (Sample Untreated RLU Value / Mean DMSO Control RLU) x 100. EC50 was determined using GraphPad Prism (nonlinear regression curve fit analysis).

[0119] Normal human primary cell cultures The sources of human primary normal cells and iPSC-derived cells are summarized in Table 4. The culture conditions of these cells are summarized in Table 5. Primary cells were thawed and cultured according to the supplier's instructions, except for the following: cardiomyocytes, astrocytes, GABA neurons, and RPTECs, which were converted to RPMI 1640 medium immediately before the start of co-culture. Previous optimization studies demonstrated the tolerability and improved cell viability of RPMI 1640 for these cell types. All cells were counted and assessed for viability prior to the assay.

[0120] [Table 15]

[0121] [Table 16]

[0122] [Table 17]

[0123] Cytotoxicity assay using human primary normal cells Cytotoxicity of target cells was evaluated using a phase contrast / fluorescence dynamic imaging assay. Fluorescent caspase 3 / 7 cleavage was measured over time with an INCUCYTE® live imaging device (Sartorium, Göttingen, Germany) and overlaid on phase contrast images of cell populations. Prior to performing the cytotoxicity assay, different plating densities and tolerance to various culture media were evaluated to achieve adequate fluence without significant cell overlap in 96-well plates. Target cells (100 μl) were added at the densities listed in Table 3 to black 96-well ViewPlates containing 50 μl of MAGE-B2 TCR-T-IL12 cells, IL-12 RFP T cells, or mock T cells at a 1:1 dextramer-normalized effector:target (E:T) ratio by taking into account the dextramer positivity of each TCR-T construct. CellEvent™ Caspase 3 / 7 Reagent (50 μl) was added according to the manufacturer's instructions (ThermoFisher, C10423). The assay plate was placed in a 37° C., 5% CO2 incubator equipped with an INCUCYTE® S3. Starting at time 0, phase contrast and fluorescence images (5 fields) with a 10x objective were collected every 4 hours for 44 or 48 hours and analyzed for total integrated intensity of Caspase 3 / 7 using INCUCYTE® 2019B software. At 44 or 48 hours, the plate was removed from the incubator and 50 μL of cell culture medium was removed from the wells for cytokine analysis.

[0124] Cytokine assay using primary human normal cells At 44 or 48 hours, cell culture supernatants (50 μL) were harvested from the cytotoxicity assay into 96-well plates. Plates were sealed and stored at -80°C for cytokine analysis at a later date. Supernatants were thawed according to the manufacturer's instructions. IFNγ and IL-12p70 plates were blocked with blocking buffer (1% w / v in PBS) from the MSD kit for 1 hour at room temperature with shaking. After washing the plates three times with PBS / 0.05% Tween-20, standards and samples (25 μL undiluted) were added according to the plate layout. Detection antibody (25 μL) was added and the plates were incubated for 2 hours at room temperature with shaking, followed by washing three times with PBS / 0.05% Tween-20. Read buffer (2x, 150 μL) was added to each well and the plate was analyzed on an MSD MESOSECTOR® S600 instrument (Meso Scale Diagnostics, Rockville, MD). A standard curve was generated from the standards and used to quantitate cytokines in the samples using MSD DISCOVERY WORKBENCH® software 4.0.

[0125] Alloreactivity Screen Alloreactive potential was assessed by co-culturing each of the four TCR-T-IL12 cells with each of 34 BLCL lines (B-lymphoblastoid cell lines) representing 39 HLA-A, 40 HLA-B, and 23 HLA-C alleles. BLCL were purchased from Fred Hutchinson Cancer Research Institute (Seattle, WA) and Cellero (Bothell, WA) as shown in Table 6. BLCL were cultured in 15% FBS complete RPMI containing RPMI-1640 with L-glutamine, 15% (v / v) HI-FBS, and 1 mM sodium pyruvate.

[0126] MAGE-B2+MAGE-A4+HLA-A were incubated at 37°C for 2 hours. *02:01 + U266B1 cells (ATCC; 10 5 TCR-T cells from donor D160780 were thawed by addition of medium, centrifuged at 400×g for 5 min at 4° C., resuspended in 10 ml of medium, and counted. 5 TCR-T cells were cultured at 1 × 10 in a volume of 200 μl. 4The TCR-T cells were co-cultured with either BLCL or peptide-pulsed U266B1 cells. The dextramer-normalized effector:target ratios for the four TCR-T cells ranged from 3:1 to approximately 8:1 depending on the respective dextramer positivity. All co-cultures were performed for 48 hours at 37°C and 5% CO2 in 96-well flat-bottom tissue culture plates. After incubation, the 96-well plates were centrifuged at 887 x g for 1 minute at 4°C and the supernatants were collected in 96-well V-bottom plates for cytokine analysis. Cytokines and granzyme B were assessed by LUMINEX® assay using a custom MILLIPLEX® Human Cytokine / Chemokine Kit (Millipore, ST Louis, MO, SRP1885) including IFNγ, granzyme B, TNFα and IL-12p70 analytes according to the manufacturer's instructions. Serial dilutions of analyte standards were performed in duplicate on each assay plate. LUMINEX® plates were read on a FLEXMAP 3D® instrument (XMAP® technologies, Luminex). Data was exported by XPONENT® software (Luminex) and directly analyzed by EMD Millipore's MILLIPLEX® Analyst software (Burlington, MA) to generate standard curves using a 5-parameter logistic nonlinear regression fitting curve. Detection limits (minimum and maximum) were calculated by MILLIPLEX® Analyst software (Millipore) as the average of the appropriate replicate standard curve values ​​from each assay plate, indicating the range within which analytes could be interpolated from the standards. Samples were run at appropriate dilutions to ensure that sample analyte level measurements were within the assay standard curve limits. Cytokine and granzyme B levels were reported in pg / mL or as fold-difference compared to IL12 T cells (control) and graphed with GraphPad Prism software (GraphPad, San Diego, CA).

[0127] [Table 18]

Claims

1. An expression vector comprising a nucleic acid sequence encoding a T cell receptor (TCR) capable of binding to MAGE-B2 peptide and MAGE-A4 peptide in the context of HLA-A*02:01, wherein the TCR comprises a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 16, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO:

27.

2. The expression vector of claim 1, further comprising a nucleic acid encoding interleukin-12 (IL-12) or a functional variant thereof.

3. The expression vector according to claim 1 or 2, which is a viral vector.

4. The expression vector described in claim 3, wherein the viral vector is a retroviral vector.

5. The expression vector described in claim 4, wherein the retroviral vector is a lentiviral vector.

6. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 38 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

49.

7. The expression vector of claim 6, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 60, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

71.

8. A cell expressing a recombinant T cell receptor (TCR) capable of binding to MAGE-B2 peptide and MAGE-A4 peptide in the context of HLA-A*02:01, wherein the TCR comprises a TCR alpha chain having the amino acid sequence set forth in SEQ ID NO: 16, and a TCR beta chain having the amino acid sequence set forth in SEQ ID NO:

27.

9. The cell of claim 8 , wherein the TCR comprises a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO:

49.

10. The cell according to claim 8 or 9, which further expresses recombinant IL-12 or a functional variant thereof.

11. A cell comprising the expression vector according to any one of claims 1 to 7.

12. The cell according to any one of claims 8 to 11, which is a T cell.

13. The TCR is HLA-A ＊ 13. The cell of claim 12, which binds to the peptides of SEQ ID NO:1 and SEQ ID NO:2 in the context of SEQ ID NO:02:01, and said binding results in activation of IFNγ, TNFα, IL-12, or granzyme B production by said cell.

14. A pharmaceutical composition comprising a therapeutically effective amount of the cells according to any one of claims 8 to 13.

15. A method for producing a cell according to any one of claims 8 to 13 or a pharmaceutical composition according to claim 14, comprising introducing into a cell an expression vector comprising a nucleic acid sequence encoding a T cell receptor (TCR) capable of binding to a MAGE-B2 peptide and a MAGE-A4 peptide in the context of HLA-A*02:01, The method, wherein the TCR comprises a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 16, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO:

27.

16. The method described in claim 15, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 38, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

49.

17. The method of claim 15 or 16, wherein the expression vector further comprises a nucleic acid sequence encoding IL-12 or a functional variant thereof.

18. The method of any one of claims 15 to 17, wherein the cell is a T cell.

19. 20. The method of claim 18, wherein the cells are primary T cells.

20. 20. The method of claim 19, wherein the primary T cells are isolated from a cancer patient.

21. A pharmaceutical composition for use in a method of treating a MAGE-B2 or MAGE-A4 expressing cancer, comprising a cell according to any one of claims 8 to 13, or a cell produced by a method according to any one of claims 15 to 20, said method comprising administering to a cancer patient a therapeutically effective amount of a cell according to any one of claims 8 to 13, or a cell produced by a method according to any one of claims 15 to 20.

22. 22. The pharmaceutical composition of claim 21, wherein the patient is tested prior to administration to determine the presence of a MAGE-B2 or MAGE-A4 expressing cancer.

23. The pharmaceutical composition of claim 22, wherein a nucleic acid encoding MAGE-B2 or MAGE-A4 is detected.

24. 23. The pharmaceutical composition of claim 22, wherein MAGE-B2 or MAGE-A4 protein or a MAGE-B2-derived or MAGE-A4-derived peptide is detected.

25. The patient is HLA-A ＊ The pharmaceutical composition of any one of claims 21 to 24, which is identified as carrying the 02:01 allele.

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