MAGE-B2-specific T-cell receptors

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, which is incorporated herein by reference in its entirety for all purposes as fully described herein.

[0002] The present invention relates to a T cell receptor that, when recombinantly expressed on the surface of T cells, can recognize peptides sufficient to activate recombinant T cells.

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

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

[0005] Cancer - testicular antigens (CTAs) are attractive targets for cancer immunotherapies such as TCR - T cell therapy due to their restricted expression in germ cells, abnormal re - activation in various cancers, and their immunogenicity. Germ cells such as the testis (an immune - privileged site) usually do not express HLA class I / II molecules and can avoid attack by the immune system. MAGE - B2 and MAGE - A4 are members of the melanoma antigen (MAGE) gene family, and most of them are classified as intracellular cancer - testicular antigens such as MAGE - B2 and MAGE - A4. Recent studies have suggested that the assembly of MAGE with E3 RING ubiquitin ligase acts as a regulator of ubiquitin binding, plays a role in cell proliferation and carcinogenic activity, and regulates the cell stress response. However, the functions of most MAGE genes such as MAGE - B2 and MAGE - A4 are not fully understood.

[0006] It has been found that TCR - T cells are very powerful and sensitive to tumor - specific peptide - MHC targets, and at the same time, TCR can recognize multiple peptides. The DNA rearrangement required for TCR formation produces 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 thymic medulla through the promiscuous expression of a wide range of self - antigens in thymic medullary epithelial cells. This negative selection in the thymus functions as a major mechanism of central immune tolerance, shaping the T - cell repertoire and avoiding autoimmunity. TCRs engineered to enhance their affinity for a specific pMHC or to introduce cross - reactivity to multiple pMHCs do not have the advantage of 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 Documents

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

[0008] The identification of TCR sequences that recognize tumor-specific antigens is particularly challenging in the art due to, among other things, the rarity of tumor-specific T cells in a patient's blood, the difficulty of expanding very small numbers of tumor-specific T cell clones ex vivo, and the potential depletion or suppression of tumor-specific T cells in tumor infiltrating lymphocytes (TIL). Despite these challenges, TCR sequences specific for MAGE-B2 peptide-MHC (GVYDGEEHSV / HLA-A * 02:01) and MAGE-A4 peptide-MHC (GVYDGREHTV / HLA-A * 02:01) identified using healthy donor blood and ex vivo stimulation methods are provided herein. 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 produce cytokines, thereby being very potent therapeutics for the treatment of MAGE-B2+ / HLA-A * 02:01+ and / or MAGE-A4+ / HLA-A * 02:01+ tumors. These TCR-T cell therapies would be an important treatment option for a wide variety of cancer indications.

[0009] TCR-T cells represent the most potent and sensitive mode of response to pMHC targets in vitro. The TCR-T cells provided herein exhibit high efficacy even against cells with very low target expression. This high efficacy of TCR-T cells stems from a complex of transduced TCRs and endogenous CD3 subunits. Furthermore, to enhance in vivo efficacy, exemplary TCR-T cells incorporate an activation-dependent IL12 payload incorporated into the TCR-T construct, where IL12 expression is regulated 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 TCR-T-IL12 cells encounter tumor antigens. As demonstrated in the mouse studies provided in the examples, the IL12 payload can enhance the efficacy of adoptive T cell therapy in vivo and thus reduce potential clinical doses (10-100 times).

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

[0011] Any expression vector in the first embodiment 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 retrovirus or lentiviral vector.

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

[0013] In a particular embodiment of the first aspect, the vector encodes a TCRα chain containing the CDR3 region amino acid sequence described in SEQ ID NO: 14 and a TCRβ chain containing the CDR3 region amino acid sequence described in SEQ ID NO: 25. In a preferred embodiment, the mature TCRα chain contains the amino acid sequence described in SEQ ID NO: 36, and the mature TCRβ chain contains the amino acid sequence described in SEQ ID NO: 47. The expression vector may encode a full-length TCRα chain containing the amino acid sequence described in SEQ ID NO: 58 and a full-length TCRβ chain containing the amino acid sequence described in SEQ ID NO: 69.

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

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

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

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

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

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

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

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

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

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

[0024] In a preferred embodiment of the second aspect, the cells are a. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 35, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 46; b. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 36, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 47; c. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 37, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 48; d. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 38, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 49; e. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 39, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 50; f. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 40, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 51; g. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 41, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 52; h. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 42, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 53; i. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 43, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 54; j. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 44, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 55; or k. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 45, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 56; Recombinant expression of a TCR containing [specific component].

[0025] Cells of the second embodiment may further express recombinant IL-12 or a functional variant thereof.

[0026] In a specific embodiment of the second aspect, the cells include one or more expression vectors of the first aspect.

[0027] The cell could be a T cell, and the TCR is HLA-A * In the 02:01 scenario, it binds to the peptide of SEQ ID NO: 1 or SEQ ID NO: 2, and this binding leads to the activation of IFNγ, TNFα, IL-12, or the production of granzyme B by cells.

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

[0029] In a fourth embodiment, the present invention relates to a method for producing cells of the second embodiment or a pharmaceutical composition of the third embodiment, comprising introducing an expression vector containing nucleic acid sequences encoding TCRα and TCRβ chains into cells, wherein the TCRα and TCRβ chains are: a. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 13, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 24; b. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 14, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 25; c. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 15, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 26; d. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 16, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 27; e. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 17, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 28; f. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 18, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 29; g. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 19, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 30; h. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 20, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 31; i. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 21, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 32; j. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 22, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 33; or k. A TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 23, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 34; The method is selected from the group consisting of the following.

[0030] In a preferred embodiment of the fourth aspect, the TCRα chain and the TCRβ chain are a. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 35, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 46; b. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 36, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 47; c. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 37, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 48; d. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 38, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 49; e. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 39, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 50; f. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 40, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 51; g. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 41, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 52; h. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 42, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 53; i. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 43, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 54; j. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 44, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 55; and k. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 45, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 56; It is selected from the group consisting of the following.

[0031] In a specific embodiment of the fourth aspect, nucleic acid sequences encoding IL-12 or a functional variant thereof are also introduced into cells and may be encoded on an expression vector encoding the α chain and / or β chain, or on separate vectors.

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

[0033] In a fifth embodiment, the present invention provides a method for treating MAGE-B2 or MAGE-A4 expressing cancer, the method comprising administering a therapeutically effective dose to a cancer patient a cell according to the second embodiment, a pharmaceutical composition according to the third embodiment, or a cell produced by the method according to the fourth embodiment. In a specific embodiment of the fifth embodiment, the patient is tested before administration to determine the presence of cancer expressing MAGE-B2 or MAGE-A4. The test may detect MAGE-B2- or MAGE-A4 coding nucleic acids, MAGE-B2 or MAGE-A4 proteins, or MAGE-B2-derived or MAGE-A4-derived peptides. In a preferred embodiment, the patient is HLA-A * It is identified as possessing the 02:01 allele. [Brief explanation of the drawing]

[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 normal human tissue (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that the MAGE-A4 protein is expressed in the majority of tumor cells within squamous cell non-small cell lung cancer tumors. Representative IHC strains of squamous cell non-small cell lung cancer tumors show 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 normal human tissue (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that the MAGE-A4 protein is expressed in the majority of tumor cells within squamous cell non-small cell lung cancer tumors. Representative IHC strains of squamous cell non-small cell lung cancer tumors show 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 normal human tissue (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that the MAGE-A4 protein is expressed in the majority of tumor cells within squamous cell non-small cell lung cancer tumors. Representative IHC strains of squamous cell non-small cell lung cancer tumors show 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 normal human tissue (Amgen Body map RNA-seq data). (C) MAGE-A4 immunohistochemistry (IHC) with OTI1F9 monoclonal Ab shows that the MAGE-A4 protein is expressed in the majority of tumor cells within squamous cell non-small cell lung cancer tumors. Representative IHC strains of squamous cell non-small cell lung cancer tumors show 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 but not in normal tissues. (B) The table shows the MAGE-B2 pMHC frequencies in representative tumors measured by MS. [Figure 2B] (A) Mass spectrometry (MS) data (Immatics) demonstrate that MAGE-B2 peptide-HLA-A*02:01 is expressed in tumors but not in normal tissues. (B) The table shows the MAGE-B2 pMHC frequencies in representative tumors measured by MS. [Figure 3]The patient population for each specified cancer indication was estimated based on the pMHC target frequency multiplied by the annual number of new cases (new patients) in the US population. The pMHC target frequency for each cancer indication was calculated by multiplying the MAGE-B2 and / or MAGE-A4 mRNA expression frequency (TCGA) by the HLA-A*02:01 carrier frequency in the US population (0.41). MAGE-B2 / A4 indicates cancer patients who are MAGE-B2 and / or MAGE-A4 positive. [Figure 4A] Identification of MAGE-B2 pMHC-specific TCRs from healthy human PBMCs. (A) The schematic diagram illustrates the procedure for identifying MAGE-B2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometry identification of MAGE-B2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorescent dyes (PE and APC), following multiple rounds of enrichment by stimulation with MAGE-B2 peptide-loaded autoantigen-presenting cells. Representative screening results showed that positive donor A exhibited enriched MAGE-B2 pMHC-specific T cells after multiple ex vivo stimulations, while negative donor B did not have Dex+ T cells. (C) IFNγ ELISPOT analysis of selected CD8+ Dex+ T cells stimulated with T2 cells pulsed with MAGE-B2 peptide or an unrelated AFP peptide as a negative control. [Figure 4B]Identification of MAGE-B2 pMHC-specific TCRs from healthy human PBMCs. (A) The schematic diagram illustrates the procedure for identifying MAGE-B2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometry identification of MAGE-B2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorescent dyes (PE and APC), following multiple rounds of enrichment by stimulation with MAGE-B2 peptide-loaded autoantigen-presenting cells. Representative screening results showed that positive donor A exhibited enriched MAGE-B2 pMHC-specific T cells after multiple ex vivo stimulations, while negative donor B did not have Dex+ T cells. (C) IFNγ ELISPOT analysis of selected CD8+ Dex+ T cells stimulated with T2 cells pulsed with MAGE-B2 peptide or an unrelated AFP peptide as a negative control. [Figure 4C] Identification of MAGE-B2 pMHC-specific TCRs from healthy human PBMCs. (A) The schematic diagram illustrates the procedure for identifying MAGE-B2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometry identification of MAGE-B2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorescent dyes (PE and APC), following multiple rounds of enrichment by stimulation with MAGE-B2 peptide-loaded autoantigen-presenting cells. Representative screening results showed that positive donor A exhibited enriched MAGE-B2 pMHC-specific T cells after multiple ex vivo stimulations, while negative donor B did not have Dex+ T cells. (C) IFNγ ELISPOT analysis of selected CD8+ Dex+ T cells stimulated with T2 cells pulsed with MAGE-B2 peptide or an unrelated AFP peptide as a negative control. [Figure 5]MAGE-B2 TCR screen using the Jurkat-luciferase activation assay. The activity of individual TCRs was expressed as the mean magnification change in luciferase activity (luminescence) in the presence of T2 cells loaded with MAGE-B2 peptide, compared to T2 cells with only the excipient. Error bars represent the standard error. [Figure 6A] Selection of the top four MAGE-B2 TCR-T cells by various functional assays. (A) Summary of cytotoxicity of MAGE-B2 TCR-T cells in T2 / MAGE-B2 peptide cytotoxicity assays such as peptide titration and E:T titration studies (average EC90 of peptide concentration (M) from 3 donors or E:T). (B) Cytotoxicity studies using T2 / peptide assays at MAGE-B2 peptide concentrations with E:T=1:1. (C) Cytotoxicity studies using T2 / peptide assays along with E:T titrations were performed at a MAGE-B2 peptide concentration of 10⁻⁸M. (Regarding B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T cells against SK-Mel-5 cancer cell lines expressing 27.5 FPKM of MAGE-B2. TCR1●, TCR2■, TCR8☆. (E) Representative data from a cross-reactivity screen for similar peptides based on homology (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide9■, peptide25▲, peptide46,△, peptide75▼. [Figure 6B]Selection of the top four MAGE-B2 TCR-T cells by various functional assays. (A) Summary of cytotoxicity of MAGE-B2 TCR-T cells in T2 / MAGE-B2 peptide cytotoxicity assays such as peptide titration and E:T titration studies (average EC90 of peptide concentration (M) from 3 donors or E:T). (B) Cytotoxicity studies using T2 / peptide assays at MAGE-B2 peptide concentrations with E:T=1:1. (C) Cytotoxicity studies using T2 / peptide assays along with E:T titrations were performed at a MAGE-B2 peptide concentration of 10⁻⁸M. (Regarding B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T cells against SK-Mel-5 cancer cell lines expressing 27.5 FPKM of MAGE-B2. TCR1●, TCR2■, TCR8☆. (E) Representative data from a cross-reactivity screen for similar peptides based on homology (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide9■, peptide25▲, peptide46,△, peptide75▼. [Figure 6C] Selection of the top four MAGE-B2 TCR-T cells by various functional assays. (A) Summary of cytotoxicity of MAGE-B2 TCR-T cells in T2 / MAGE-B2 peptide cytotoxicity assays such as peptide titration and E:T titration studies (average EC90 of peptide concentration (M) from 3 donors or E:T). (B) Cytotoxicity studies using T2 / peptide assays at MAGE-B2 peptide concentrations with E:T=1:1. (C) Cytotoxicity studies using T2 / peptide assays along with E:T titrations were performed at a MAGE-B2 peptide concentration of 10⁻⁸M. (Regarding B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T cells against SK-Mel-5 cancer cell lines expressing 27.5 FPKM of MAGE-B2. TCR1●, TCR2■, TCR8☆. (E) Representative data from a cross-reactivity screen for similar peptides based on homology (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide9■, peptide25▲, peptide46,△, peptide75▼. [Figure 6D] Selection of the top four MAGE-B2 TCR-T cells by various functional assays. (A) Summary of cytotoxicity of MAGE-B2 TCR-T cells in T2 / MAGE-B2 peptide cytotoxicity assays such as peptide titration and E:T titration studies (average EC90 of peptide concentration (M) from 3 donors or E:T). (B) Cytotoxicity studies using T2 / peptide assays at MAGE-B2 peptide concentrations with E:T=1:1. (C) Cytotoxicity studies using T2 / peptide assays along with E:T titrations were performed at a MAGE-B2 peptide concentration of 10⁻⁸M. (Regarding B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T cells against SK-Mel-5 cancer cell lines expressing 27.5 FPKM of MAGE-B2. TCR1●, TCR2■, TCR8☆. (E) Representative data from a cross-reactivity screen for similar peptides based on homology (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide9■, peptide25▲, peptide46,△, peptide75▼. [Figure 6E]Selection of the top four MAGE-B2 TCR-T cells by various functional assays. (A) Summary of cytotoxicity of MAGE-B2 TCR-T cells in T2 / MAGE-B2 peptide cytotoxicity assays such as peptide titration and E:T titration studies (average EC90 of peptide concentration (M) from 3 donors or E:T). (B) Cytotoxicity studies using T2 / peptide assays at MAGE-B2 peptide concentrations with E:T=1:1. (C) Cytotoxicity studies using T2 / peptide assays along with E:T titrations were performed at a MAGE-B2 peptide concentration of 10⁻⁸M. (Regarding B and C: TCR1●, TCR2■, TCR3▲, TCR4▼, TCR6◆, TCR7☆, TCR8□, TCR11◇, TCR12 small●). (D) Cytolytic activity of TCR-T cells against SK-Mel-5 cancer cell lines expressing 27.5 FPKM of MAGE-B2. TCR1●, TCR2■, TCR8☆. (E) Representative data from a cross-reactivity screen for similar peptides based on homology (T2 / peptide cytotoxicity assay). MAGE-B2●, peptide9■, peptide25▲, peptide46,△, peptide75▼. [Figure 7] A schematic diagram of a TCR-T-IL12 lentivirus construct containing TCRα and TCRβ chains, along with an IL12 payload under a composite promoter containing a furin cleavage site-SGSG-T2A linker under the EF1α promoter, and six NFAT (nuclear factor of activated T cells) response elements linked to the minimal IL-2 promoter. [Figure 8] Efficacy validation of TCR-T-IL12 using a T2 / MAGE-B2 peptide cytotoxicity assay. The table shows the EC90 of peptide concentration (M) from T2 / peptide titration studies using four TCR-T-IL12 cells from three HLA-A*02:01 donors. The E:T ratio (dextramer + T cell:T2) was 1:1. TCR1-IL12●, TCR2-IL12■, TCR3-IL12△, TCR4-IL12▼, IL12 RFP◇, Mock T cell●. [Figure 9]TCR4-IL12 cells from three donors showed 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 efficacy of four TCR-T-IL12 cells against MAGE-B2+MAGE-A4 cancer cell lines. All four TCR-T-IL12 cells showed potent cytotoxicity against cancer cell lines. IL12-RFP T cells (NFAT.IL-12.RFP transduced T cells without a transgenic TCR) and mock (untransduced) T cells were used as negative controls. Maximum specific cell death exceeding 50% is highlighted in gray. [Figure 11] Summary of the efficacy of TCR4-IL12 against MAGE-A4+MAGE-B2 cancer cell lines. Maximum specific cell death exceeding 50% is highlighted in gray. [Figure 12] Summary of the efficacy of 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 exceeding 50% is highlighted in gray. [Figure 13] Representative efficacy of four TCR-T-IL12 cells against MAGE-B2+ and / or MAGE-A4+ cancer cell lines. For efficacy validation, approximately 40 cancer cell lines were tested with four TCR-T-IL12 cells produced from 2-3 donors. MAGE-B2 and / or MAGE-A4 mRNA expression levels (FPKM, RNA-seq) 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 validated using MAGE-B2 KO or B2M KO cancer cell lines. (A) For cytotoxicity assays using E:T titration, DAN-G-derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested with TCR2-IL12 and TCR4-IL12. DAN-G WT●, DAN-G MAGE B2 KI(2E9)▲, DAN-G B2M KO▼. (B) For cytotoxicity assays using E:T titration, 8505C-derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested. The same results were confirmed from multiple donors. MAGE-B2 KO efficiency was validated by sequencing. B2M KO efficiency was validated 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 validated using MAGE-B2 KO or B2M KO cancer cell lines. (A) For cytotoxicity assays using E:T titration, DAN-G-derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested with TCR2-IL12 and TCR4-IL12. DAN-G WT●, DAN-G MAGE B2 KI(2E9)▲, DAN-G B2M KO▼. (B) For cytotoxicity assays using E:T titration, 8505C-derived cancer cell lines (WT, MAGE-B2 KO, and B2M KO) were tested. The same results were confirmed from multiple donors. MAGE-B2 KO efficiency was validated by sequencing. B2M KO efficiency was validated by flow cytometry. 8505C WT●, 8505C neg gRNA■, 8505C MAGE B2 KO▲, 8505C B2M KO▼. [Figure 15A]The IL12 payload increased TCR-T cell efficacy against cells with low target expression, enhancing the efficacy of CAR-T cells in vivo. (A) Comparison of TCR-T and TCR-T-IL12 cell efficacy 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 produced from three different donors. (B) Comparison of CAR-T and CAR-T-IL12 cell efficacy in vivo. The efficacy of huEpCAM CAR-T cells with and without the IL12 payload was evaluated in a B16F10-huEpCAM syngeneic mouse tumor model. [Figure 15B] The IL12 payload increased TCR-T cell efficacy against cells with low target expression, enhancing the efficacy of CAR-T cells in vivo. (A) Comparison of TCR-T and TCR-T-IL12 cell efficacy 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 produced from three different donors. (B) Comparison of CAR-T and CAR-T-IL12 cell efficacy in vivo. The efficacy of huEpCAM CAR-T cells with and without the IL12 payload was evaluated in a B16F10-huEpCAM syngeneic mouse tumor model. [Figure 16] A schematic of the cross-reactivity screen for similar peptides in the complete panel. SLC16A10 and KLHDC3 were identified based on X-scan-inducible motifs, while NRXN1 and MAGE-B1 were identified based on sequence homology to the target peptide. MAGE-B1 is another cancer-testis antigen with very limited normal tissue expression (only in the testes). No similar peptides were identified with off-target issues based on cytotoxic assays in cancer cell lines overexpressing full-length or endogenous proteins. [Figure 17]The risk of SLC16A10 putative cross-reactivity peptide was further reduced by TCDD assays using HLA-A*02:01+ cancer cell lines (NCI-H441 and IGR-1) overexpressing SLA16A10 full-length protein (A) and cancer cell lines (LOUCY and MFE-280) expressing SLC16A10 endogenous protein (B). MAGE-B2 full-length protein overexpressing (OE) cancer cell lines were used as a positive control target cell line (A). IL12-RFP T cells were used as negative control T cells (B). [Figure 18] Summary of human normal cell reactivity evaluation. Increased IFNγ and granzyme B production by TCR2-IL12 and TCR4-IL12 cells was not observed compared to HLA-A*02:01+ human normal primary cells. Representative data from four normal cell types are shown: human bronchial epithelial cells (hBEpC), human tracheal epithelial cells (hTEpC), human cutaneous microvascular endothelial cells (HDMEC), and human keratinocytes (Ker.). The table shows the doubling of IFNγ and granzyme B production compared to control IL12-RFP T cells. Similar results were obtained from all nine normal cell types tested, and for IL-12p70 and TNFα. The B-CPAP cancer cell line (MAGE-B2 65.9FPKM) 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 a transgenic TCR) from the same donor were included as negative control cells. Furthermore, target cells without T cells (simply labeled as targets) were used as a negative control for the cytotoxic assay. [Figure 19-1]Summary of alloreactivity assessment: No fourfold or greater increase in cytokine or granzyme B response (compared to IL12-RFP control T cells) was observed in any of the four TCRT-T-IL12 cells to the 34 BLCLs tested. Some low levels of response (more than threefold but less than fourfold compared to IL12-RFP control cells) were observed in TCR1-IL12 and TCR2-IL12. Similar results were obtained for IL-12p70 and TNFα production. Strong cytokine and granzyme B responses were demonstrated from all four TCR-T-IL12 cells to 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 fourfold or greater increase in cytokine or granzyme B response (compared to IL12-RFP control T cells) was observed in any of the four TCRT-T-IL12 cells to the 34 BLCLs tested. Some low levels of response (more than threefold but less than fourfold compared to IL12-RFP control cells) were observed in TCR1-IL12 and TCR2-IL12. Similar results were obtained for IL-12p70 and TNFα production. Strong cytokine and granzyme B responses were demonstrated from all four TCR-T-IL12 cells to positive control U266B1 cells (HLA-A*02:01+MAGE-B2+MAGE-A4+) pulsed with MAGE-B2 peptide. [Modes for carrying out the invention]

[0035] The section titles used herein are for systematization purposes only and should not be interpreted as limiting the subject matter described. All references cited within the text of this specification are incorporated in their entirety by reference.

[0036] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzyme reactions and purification techniques may be carried out according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The following procedures and techniques may generally be carried out according to conventional methods well known in the art and as described in the various general and more specific references described and discussed throughout this specification. For example, see Sambrooket al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference for any purpose. Unless otherwise specified, the nomenclature used in relation to analytical chemistry, organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, 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 patient treatment.

[0037] HLA class I molecules, preferably HLA-A * A pair of T cell receptor (TCR) α and β chains that bind to the MAGE-B2-derived peptide GVYDGEEHSV (SEQ ID NO: 1), as represented by 02:01, is provided herein. The “TCR α and β chain pair” may also be referred herein to as “TCR,” “one TCR,” or “that TCR.” When recombinantly expressed in cells, such as T cells, the TCR binds to the MAGE-B2 peptide-HLA complex on cells, such as cancer cells, and such binding activates recombinant cells, and T cell activation causes cancer cells to die or be destroyed. Methods for determining T cell activation are known in the art and are provided herein by example.

[0038] In a preferred embodiment, the potency or cytolytic activity (cytotoxicity) of the recombinant cells of the present invention is (1) about 100 copies (about 10 -8 M) / cell in a T cell-dependent cytotoxicity (TDCC) assay, T2 / peptide-loading assay, loaded with a peptide on HLA-A * 02:01 target cells lysed 80-100% or (2) defined by 80-100% lysis of a natural pMHC target-positive cancer cell line.

[0039] In certain embodiments, when presented by HLA class I molecules, preferably HLA-A * 02:01, the TCR further binds to the MAGE-A4-derived peptide GVYDGREHTV. Such TCRs include TCR3, TCR4, TCR6, TCR7, and TCR11.

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

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

[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 α-chain or β-chain may be fused to a non-TCR polypeptide. Using the exemplary α-chain and β-chain variable domains, a soluble TCR can be formed that can bind to MAGE-B2 (and in some cases MAGE-A4) derived peptides in the context of HLA molecules. The soluble TCR may be in a single-chain format in which the α and β variable domains are linked by a linker. Disulfide bonds may be introduced between the α-chain and β-chain to increase stability. The soluble TCR may be fused to or bound to a therapeutic agent or contrast agent.

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

[0056] [Table 13]

[0057] The TCRα or β variable domain may contain sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences specified in Table 2. The TCRβ chain may contain sequences that are 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 described in SEQ ID NOs. 46-56 of Table 2. 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 specified in SEQ ID NOs. 35-56 of Table 2 and Table 1B may be truncated or removed.

[0058] Recognition of the target peptide in the context of HLA is necessary for efficacy, but for safety reasons, in some embodiments, HLA-A * It is preferable that the TCR lacks cross-reactivity with structurally similar peptides or with other allotype HLA molecules when presented by 02:01. The cross-reactivity and alloactivity of the exemplary TCRs described herein are provided in the examples. Thus, the exemplary TCRs are HLA-A expressed on tumor cells. * In the situation described in 02:01, it is possible to recognize the MAGE-B2 peptide and activate T cells that recombinantly express the TCR in tumor cells, and also to introduce HLA-A into the recombinant T cells. * When the peptide is presented in the context of 02:01 or other HLA molecules expressed in normal tissue, it is either not activated or only minimally activated.

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

[0060] The TCRα variable domain and the β variable domain can be co-transcribed from the same promoter. In embodiments where the α and β variable domains are ligated within a fusion protein, the domains can be co-translated within a single polypeptide. In embodiments where the α and β domains reside 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] Nucleic acids encoding TCRα chains, TCRβ chains, or TCRα and TCRβ chains as described herein are also provided herein. In certain embodiments, the nucleic acid encodes one or more of the α or β chains listed in Table 1. The encoded α or β chain may be full length or mature. If it is mature, i.e., lacks a native leader sequence with its α or β chain, the nucleic acid encoding the signal or leader sequence is preferably operably ligated to the nucleic acid encoding the α or β chain so that the leader sequence orients the α or β chain relative to the endoplasmic reticulum when translated.

[0062] In certain embodiments, the nucleic acid encodes both the α and β chains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In preferred embodiments, the nucleic acid encoding the TCRα chain, TCRβ chain, or TCRα and β chains is an expression vector in which the TCRα chain, TCRβ chain, or TCRα and β chains are operably linked to a promoter.

[0063] The TCRα and β chains can be co-transcribed from the same promoter. In such embodiments, it is useful to include an internal ribosome entry site (IRES) between the α-coding region and the β-coding region in the expression vector.

[0064] Examples of expression vectors of the present invention include, but are not limited to, retroviral or lentiviral vectors. The expression vector may further encode one or more additional proteins in addition to the TCRα and / or β chains. In certain embodiments, the expression vector encodes one or more cytokines. In preferred embodiments, the cytokines, in combination, are T cell growth factors such as IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21. Since cytokines can have systemic effects, when an expression vector encoding cytokines is used to produce cells for adoptive cell therapy, cytokine expression is preferably 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 for expressing IL-12 is described in U.S. Patent No. 8,556,882.

[0065] Cells that recombinantly express the exemplary TCRs described herein are provided herein. The recombinant cells may comprise one or more expression vectors encoding and expressing a TCRα chain, a TCRβ chain, a TCRα and β chain, a TCRα variable domain, a TCRβ variable domain, or a TCRα and β variable domain. In a preferred embodiment, the cells recombinantly express TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In a particular embodiment, the cells further express one or more recombinant cytokines. In a preferred embodiment, 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, 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 to the desired cell type. The cells are transfected / transformed with one or more vectors, such as lentiviral vectors, encoding components of the TCR along with any further polypeptides, such as IL-12 or its variants. Such cells can be used in adoptive cell therapy for cancer patients from whom they originate.

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

[0068] A method for treating a disease or disorder is provided herein, wherein cells associated with the disease or disorder express MAGE-B2 and / or MAGE-A4. In a preferred embodiment, the cells express an HLA class I molecule, preferably HLA-A2, and particularly HLA-A *In the context of 02:01, we present the MAGE-B2-derived peptide GVYDGEEHSV and / or the MAGE-A4 peptide GVYDGREHTV. Exemplary diseases or disorders that can be treated with the soluble TCR 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 the expression of MAGE-B2 or MAGE-A4. The tumor may also be tested for the expression of appropriate HLA molecules recognized by the TCR of the present invention when presenting MAGE-B2- or MAGE-A4-derived peptides. Patients whose tumors express MAGE-B2 or MAGE-A4 and have an appropriate HLA haplotype may be administered the soluble TCR or recombinant cells of the present invention.

[0070] Various embodiments in this specification are expressed using the word “includes” under various circumstances, but it should be understood that relevant embodiments may also be described using “consist of” or “essentially consisting of.” This disclosure intends embodiments described as “includes” a feature to encompass embodiments that “consist of” or “essentially consisting of” that feature. The terms “one (a)” or “one (an)” mean one or more; the terms “one (a)” (or “one (an)”), “one or more” and “at least one” can be used interchangeably in this specification. The term “or” should be understood to include items, either by substitution or together, unless otherwise explicitly required in the content. The terms “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 together. As used herein, “may be” or “can be” means something conceived by the inventors that is functional and available as part of the subject matter provided.

[0071] The terminology used in this application is standard in the art, and definitions of specific 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-acceptable forms. Numerical ranges described herein encompass and complement the number defining the range and each integer within the defined range. Methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art, and unless otherwise specified, as described in the various general and more specific references described and discussed throughout this specification. All documents, or parts thereof, described herein, 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 whole of this application, including the drawings and detailed description, and all such features are intended as embodiments of the present invention. Similarly, the features of the present invention described herein can be recombined into further embodiments which are also intended as embodiments of the present invention, whether or not the combination of features is specified as an embodiment or aspect of the present invention. Even if the combination of features is not found together in the same sentence, paragraph, or section of this document, it should be understood that 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 intended. Furthermore, only such limitations described herein as important to the present invention should be considered in themselves; variations of the present invention without limitations described herein as important are intended as embodiments 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, and functionally equivalent methods and components are within the scope of the invention. In fact, in addition to the forms shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the accompanying claims. [Examples]

[0074] Both the following embodiments, actual embodiments, and predictive embodiments are provided for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit their scope.

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

[0076] Furthermore, as a pMHC target, 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) was observed from various tumor and normal tissues (Immatics, Tuebingen, Germany). * 02:01) Expression was demonstrated to be highly specific to tumors and undetectable in normal, healthy tissue (Figure 2A). The frequency of MAGE-B2 pMHC in representative cancer types 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, MAGE-A4 pMHC expression in squamous cell NSCLC tumors was confirmed from in-hospital MS data (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 * The indications for solid tumors with 02:01) are not limited to squamous cell carcinoma of the lung (squamous non-small cell lung cancer, LUSC) accounting for 16.2-22.7%, squamous cell carcinoma of the head and neck (HNSCC) accounting for 9.2-15.8%, esophageal carcinoma accounting for 6.2-11.1%, bladder cancer accounting for 4.7-10.4%, and ovarian cancer accounting for 2.1-7.8% (Figure 3). The cancer population for the specified cancer indications was estimated based on the pMHC target frequency (%) multiplied by the number of new cases (new patients) per year in the US population. The pMHC target frequency (%) is based on HLA-A in the US. * The MAGE-B2 and / or MAGE-A4 mRNA expression frequencies were calculated by multiplying them by the 02:01 carrier frequency (0.41). Using the TCGA public dataset of RNA-seq from the target tumors, the MAGE-B2 and / or MAGE-A4 mRNA expression frequencies in each tumor indication were estimated 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. The number of disease occurrences (new cases per year) in the selected tumor indications was estimated using SEER, EPIC Oncology New Patients, or Epiphany / Epic in 2020, and thus the estimated population range of treatable patients was derived (Figure 3). HLA-A * 02:01 is one of the most common MHC class I alleles in the United States. * The estimated frequency of the 02:01 haplotype (carrier) is 0.41 (www.allelefrequencies.net). The US patient population is twice as large when both MAGE-A4 and MAGE-B2 are covered compared to MAGE-B2 alone. The largest patient populations are squamous cell 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, using an ex vivo stimulation and scRNAseq-based TCR discovery platform, healthy HLA-A * Using 52 donors, 40 dominant MAGE-B2 pMHC-specific TCRs were identified. Eleven TCR candidates were selected from the 40 TCRs using a Jarcut activation assay. Based on these 11 TCR sequences, 11 TCR-T cells were produced per donor by transduction of early pan-T cells isolated from three donors using lentiviruses that retained the individual TCRs. These TCR-T cells were further evaluated by various functional assays, including efficacy (cytotoxicity) tests in T2 cell lines pulsed with target peptides and in multiple (approximately 20) cancer cell lines, cross-reactivity screens using similar peptides, and early alloreactivity screens. Based on the functional data, the inventors narrowed down the 11 TCRs to the top four TCR candidates. To further enhance in vivo efficacy and reduce clinical doses, we constructed the top four TCRs in a TCR-T-IL12 lentiviral construct in which IL12 payload expression is induced by TCR activation under an NFAT response-driven promoter. Therefore, IL12 can only be produced when TCR-T cells bind to pMHC targets (MAGE-B2 and / or MAGE-A4) in tumors. TCR-T-IL12 cells produced from three donors were further evaluated by various functional assays, including efficacy studies in T2 cell lines pulsed with target peptides and in multiple (approximately 40) cancer cell lines, cross-reactivity with a full panel of similar peptides, cytotoxicity screening of normal cells, and a full alloreactivity screening. Based on all 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. The difficulty in identifying tumor antigen-specific TCRs has hindered the development of TCR-mediated immunotherapy. Despite these challenges, the inventors have succeeded in developing a TCR discovery platform in 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, generally around 0% dextramer+ T cells. DEXTRAMER® (Dex) is a peptide-MHC complex multimer that can specifically bind to TCRs and can therefore be used to isolate antigen (pMHC)-specific T cells. Initially, to expand rare tumor antigen-specific T clones, we selected 52 healthy HLA-A * Using PBMCs from 02:01+ donors, T cells and autoantigen-presenting cells (APCs), such as monocyte-derived dendritic cells and activated B cells, were isolated. When auto-APCs pulsed with target peptides were co-cultured with T cells, these T cells underwent multiple steps of ex vivo stimulation, resulting in tumor antigen pMHC-specific priming, restorative stimulation, and expansion of pMHC-specific T cells. After multiple antigen restoratives, a population of MAGE-B2 pMHC dextramer+ (Dex+) T cells (MAGE-B2 pMHC-responsive T cells) was detected. After 2–4 rounds of antigen restorative stimulation, the MAGE-B2 pMHC-specific T cell population was further enriched and confirmed by both dextramer-PE and dextramer-APC strains (Figure 4B). Dex+CD8+ T cells were then sorted for single-cell RNA sequencing, and the sequences of the TCRα and TCRβ chains were identified. Table 1 shows the sequence numbers corresponding to the TCRα and TCRβ sequences of the identified representative TCRs. Furthermore, these selected Dex+CD8+ T cells were validated for MAGE-B2 antigen-specific activation by an IFNγ ELISPOT assay using peptide-loaded T2 cells (Figure 4C). This TCR discovery platform was used to identify 52 healthy HLA-A *02:01+ Forty dominant MAGE-B2 pMHC-specific TCRs were identified from donors. Importantly, TCRs identified from the blood of healthy donors, unlike affinity-enhanced TCRs or bispecific antibodies, are eliminated as autoreactive TCRs through thymic selection in the human body (in the thymic medulla). Therefore, the off-target risk to TCRs is considered to be quite low, and this was confirmed by safety evaluation assays (described below).

[0080] Selection of top-tier 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 candidate TCRs were selected by the Jarcut activation assay (Figure 5). Lentiviruses possessing each individual TCR were transduced into a Jarcut TCRKO reporter cell line constitutively expressing CD8a and sea urchin luciferase, regulated by TCR activation under an NFAT response-driven promoter. The activity of each individual TCR was measured as a fold change in luciferase activity in the presence of MAGE-B2 peptide-loaded T2 cells compared to T2 cells with only the excipient (Figure 5).

[0081] Based on these 11 selected TCR sequences, 11 TCR-T cell lines were created per donor by transduction of lentiviruses containing individual TCRs into human early pan-T cells isolated from three donors. These TCR-T cells were further evaluated for various functional activities. First, the potency of each TCR-T was evaluated using T2 / peptide cytotoxicity assays (MAGE-B2 peptide), such as peptide titration and E:T (effector:target cell ratio) titration assays (Figure 6A-6C). T2 cells lacked the antigen processing-associated transporter (TAP) and HLA-A *Because it is a cell line expressing O2, MHC class I-restriction endogenous peptides cannot enter the ER, and the T2 cell line mainly presents exogenous peptides. Therefore, using a T2 / peptide cytotoxicity assay (measuring cell lysis activity using a T2 cell line conjugated with the target peptide), peptides (e.g., HLA-A) are detected by the TCR of T cells. * The specific recognition of (02:01-restriction) was studied. The efficacy of 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 to the MAGE-A4 peptide, which, as mentioned above, is also a cancer-testicular antigen with a high prevalence in a wide range of solid tumors. TCR4-T showed considerably higher efficacy against the MAGE-A4 peptide compared to TCR3-T. Furthermore, cytotoxicity against several (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. Exemplary TCR-Ts exhibited potent cell death activity against cancer cell lines with low MAGE-B2 expression (approximately 1.4 FPKM) or low E:TEC50 (approximately 0.25).

[0082] To evaluate off-target selectivity, TCR-T cells were examined using T2 / peptide cytotoxicity assays with 131 homology-based analogous peptides and target-negative cancer systems. Representative data are shown in Figure 6E. Details of the off-target strategies and the identification of analogous peptides are described below.

[0083] Regarding early alloreactivity, in the US population, the top five most frequent non-HLA-A * 02:01 TCR-T was tested in co-culture with 5 B lymphoblastoid cell lines (BLCLs) representing the 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 initial alloreactivity readouts. Details of alloreactivity are described below.

[0084] Based on various functional studies, including (1) potent cytotoxicity against MAGE-B2 and / or MAGE-A4 pMHC targets using T2 / MAGE-B2 peptide, T2 / MAGE-A4 peptide, and approximately 20 MAGE-B2+ and / or MAGE-A4+ cancer cell lines, (2) off-target selectivity that does not show cross-reactivity to 131 homology-based analog peptides and target-negative cancer cell lines, (3) no initial alloreactivity, and (4) manufacturability (e.g., excellent TCR transduction efficiency), the top four TCRs (TCR1, TCR2, TCR3, TCR4) were selected from 11 TCRs.

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

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

[0087] Example 5 - Cytotoxicity against MAGE-B2+ and / or MAGE-A4+ cancer cell lines The potency (cytotoxicity) of four TCR-T-IL12 was validated using three different categories of cancer cell lines: 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 the MAGE-B2+MAGE-A4- cancer cell line (Figure 10). All four TCR-T-IL12 showed potent cytotoxicity against cancer cell lines with low MAGE-B2 expression of approximately 1.4 FPKM. In a potency ranking assay against the MAGE-B2+MAGE-A4- cancer cell line, TCR2 was the most potent TCR, followed by TCR4, and then TCR1 and TCR3 were nearly equal. TCR2-IL12 and TCR4-IL12 exhibited cytotoxicity at low E:TEC50 values ​​of approximately 0.07 and 0.21, respectively. TCR-T-IL12 showed high efficacy even against MAGE-B2 low-cancerous cell lines such as 8505C (approximately 1.4 FPKM) and AU565HLA-A2hi (approximately 3.7 FPKM). Target-specific cell death against these MAGE-B2 low-cancerous cell lines was validated using MAGE-B2 KO cell lines produced from these low-cancerous cell lines (described below).

[0088] Secondly, the efficacy of TCR4-IL12 against MAGE-A4+MAGE-B2-cancer cell lines was evaluated using a T2 / peptide assay, taking into account the cross-reactivity of this TCR to the MAGE-A4 peptide (Figure 11). TCR4-IL12 showed cytotoxicity against cancer cell lines with low MAGE-A4 expression (approximately 6.3 FPKM) or low E:TEC50 (approximately 0.46).

[0089] Thirdly, efficacy against double-positive, MAGE-B2+MAGE-A4+ cancer cell lines was evaluated (Figure 12). TCR4-IL12 and TCR2-IL12 showed potent cytotoxicity against MAGE-B2+MAGE-A4+ cancer cell lines. In ranking the efficacy against MAGE-B2+MAGE-A4+ cancer cell lines, TCR4 was the most potent TCR, followed by TCR2, and then TCR3 and TCR1 were nearly equal. Notably, due to its high efficacy against both MAGE-B2 and MAGE-A4 peptides, TCR4-IL12 showed the highest efficacy against double-positive MAGE-B2+MAGE-A4+ cancer cell lines. In particular, since TCR2 has no MAGE-A4 cross-reactivity and only MAGE-B2 specificity, some MAGE-B2-low-MAGE-A4-high cancer cell lines (e.g., A375) can distinguish the efficacy of TCR4 and TCR2. TCR4-IL12 demonstrated cytotoxicity against double-positive cancer cell lines with low MAGE-B2 expression (approximately 1.2 FPKM), low MAGE-A4 expression (approximately 44 FPKM), or low E:TEC50 (0.04). TCR2-IL12 showed cytotoxicity against cell lines with low MAGE-B2 expression (approximately 3.5 FPKM) or low E:TEC50 (0.01).

[0090] Figure 13 shows representative cancer cell line efficacy data for four TCR-T-IL12 cells. Approximately 40 cancer cell lines were tested using four TCR-T-IL12 cells produced from 2-3 donors. 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 against B-CPAP, 0.25 against SK-MEL-5, 0.98 against THP-1, and 0.25 against NCI-H1755.

[0091] Example 6 - Peptide-MHC target-specific cytotoxicity validation using MAGE-B2 KO and B2M KO cancer cell lines. The potent cytotoxicity of TCR-T-IL12 against several MAGE-B2+ cancer lines with very low MAGE-B2 expression was confirmed, and it was determined whether this cytotoxicity depends on pMHC target expression. Therefore, the inventors created MAGE-B2 KO (knockout) cell lines and B2M KO cell lines to eliminate expression of MAGE-B2 and B2M, respectively (Figures 14A and 14B). B2M (β2-microglobulin) is an important subunit of MHC class I molecules. Both MAGE-B2 KO and B2M KO resulted in reduced cell death activity mediated by either TCR2-IL12 or TCR4-IL12. Notably, knocking out MAGE-B2 in the 8505C cancer cell line with very low MAGE-B2 mRNA expression (1.4FPKM) resulted in both TCR-T-IL12 cells losing their ability to kill cells, indicating that the cytolytic activity of TCR-T-IL12 cells is dependent on MAGE-B2 target expression, and that TCR-T-IL12 can truly recognize such low target expression (Figure 14B). Similarly, reduced cytotoxicity was observed in the B2M KO line, demonstrating that TCR-T-IL12 activity responds to HLA expression.

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

[0093] Next, the inventors evaluated the effect of the IL12 payload in a human TCR-T system having multiple cancer cell lines. In particular, for the MAGE-B2-low cancer cell line (shown within the wavy rectangle), the IL12 payload could increase TCR-T cell efficacy compared to parental TCR-T without IL12 (Figure 15A). For the MAGE-B2-high or MAGE-A4-high cancer cell lines (shown outside the wavy rectangle), the effect of IL12 was not significant for these cancer cell lines because the efficacy had already reached its maximum limit with the parental TCR-T.

[0094] Example 8 - Summary of Nonclinical Safety Evaluation Because human-specific HLA targets hinder the use of animal models, extensive in vitro and ex vivo safety assessments were performed on TCR-T-IL12 cells. First, target expression was evaluated using various assays, including RNASeq, IHC, and mass spectrometry, in normal human tissues and tumor tissues as described above. Since MAGE-B2 and MAGE-A4 are cancer-testicular antigens, the study showed very limited normal tissue expression (expressed only in the testes). Second, off-target reactivity was evaluated using two different strategies. The first strategy involved evaluating 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 that match the MAGE-B2 target peptide, along with a positional scan (X-scan) based strategy to identify putative cross-reactive peptides specific to each TCR. A T2 / peptide TECC assay was performed to evaluate the potential cross-reactivity of similar peptides to this panel. The third safety assessment involved alloreactivity, evaluating 34 BLCLs representing high-frequency HLA class I alleles in the US population, including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles.

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

[0096] A homology-based strategy was designed using an in silico approach to identify a list of peptides that could potentially cross-react with candidate TCR-T. To achieve this, a protein database query (UniProtKB / Swiss-Prot, June 2019) was first performed to create 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 were identified based on a 30% homology (identity) match to the target peptide. To further refine this list, criteria such as high homology match, as well as software such as NetMHCpan and the IEDB (Immunoepitope Database), were utilized. NetMHCpan 3.0 was used to analyze HLA-A * We investigated the expected binding affinity of peptides to 02:01. Using the IEDB database (June 2019), a selection of experimentally characterized immunoepitope manuals, we examined 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 as follows: (1) all peptides with a homology match (identity) of 60% or more to the target peptide (65 peptides), (2) all peptides (35 peptides) with a homology match of 50% or more and a predicted binding affinity (IC50) of 50 nM or less, and (3) the target peptide (HLA0A) reported in the IEDB. *All peptides having a homology match of 40% or more to (45 peptides) presented by the 02:01 allele. As a result, the inventors identified 131 unique peptides from this homology-based in silico search of the human proteome database.

[0097] Identification of TCR-binding motifs and similar peptides based on X-scan-induced motifs using positional scanning (X-scan). As an orthogonal approach to identifying similar peptides, the inventors used a positional scanning method known as X-scan. The X-scan assay used a peptide library created by sequentially introducing mutations into one of 19 other native amino acids for each residue of the MAGE-B2 peptide, resulting in a total of 190 peptides. These 190 peptides were synthesized and tested in the T2 / peptide TDCC assay to identify X-scan-inducing motifs specific to 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 confirmed viability was less than 20%. Corresponding search motifs were created to indicate which amino acids are acceptable at each position in the peptide sequence (Table 3). Underlined amino acids represent native residues at the corresponding positions in the peptide.

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

[0099] In the case of two TCRs (TCR3 and TCR2), when the number of motif search-based peptides obtained was considerably large, further anchor residue restrictions (at residues 2 and 10) were applied to the derived motif to limit the final selection of cross-reactive peptides (Table 3). Specifically, 2583 decaperated HLA-A peptides obtained from the IEDB database were selected. * 02:01 The sequences of the positive peptides were analyzed to calculate the amino acid frequencies 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), thereby restricting position 2 to amino acids T, M, E, I, V, and L, and position 10 to amino acids Y, I, A, L, and V.

[0100] [Table 14]

[0101] Cross-reactivity screen with similar peptides in a complete panel We synthesized analogous peptides from the complete panel (including sets based on X-scan motifs and sets based on homology) and examined them in the T2 / peptide TDCC assay to investigate the potential for off-target reactivity.

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

[0103] Next, a potency screen (dose-dependent screen) was performed using a T2 / peptide titration TDCC assay on the putative cross-reactive peptides identified from the above screen. The risk of most putative cross-reactive peptides was reduced by this potency screen. The EC50 between the target peptide and the putative cross-reactive peptide was 10 3 A potency gap of less than 1:1 was considered a cutoff for further risk assessment. Results from cross-reactivity screening with full-panel analog peptides against the top four TCR-T-IL12 cells are summarized in Figure 16, and 10 for the target MAGE-B2 peptide. 3 All peptides exhibiting less than double potency are listed. TCR4-IL12 cells did not produce any putative cross-reactive peptides (except MAGE-A4). The other three TCR-T-IL12 cells each possessed one putative cross-reactive peptide identified from this complete panel peptide screen, except for the cancer testis antigen MAGE-B1. All three putative cross-reactive peptides (derived from the proteins SLC16A10, KLHDC3, and NRXN1) further overexpress their respective full-length proteins and HLA-A * Risk was reduced by TDCC assays using 02:01+ cancer cell lines or cancer cell lines expressing endogenous proteins (Figure 17). Cytotoxicity against cancer cell lines overexpressing these putative cross-reactive proteins or endogenous proteins was not confirmed by any of the TCR-T-IL12 cells (TCR1, TCR2, and TCR3), suggesting that these peptides are not processed naturally and are unlikely to be presented from proteins (Figures 16 and 17). In conclusion, no significant cross-reactivity across the complete panel of similar peptides identified by sequence homology and X-scan-induced TCR motifs was demonstrated by any of the four TCR-T-IL12 cell lines.

[0104] Evaluation of cytotoxicity in normal human cells Next, in a T cell-mediated cytotoxicity assay, the cytotoxicity of four MAGE-B2 TCR-T-IL12 cells (TCR1-IL12, TCR2-IL12, TCR3-IL12, and TCR4-IL12) was evaluated against a panel of nine representative normal human early or iPSC-derived cell types that act as target cells in major organs (without MAGE-B2 or MAGE-A4 expression). The panel of nine normal human cells included bronchial epithelial cells (hBEpC), tracheal epithelial cells (hTEpC), cutaneous microvascular endothelial cells (HDMEC), keratinocytes, hepatocytes, renal proximal tubular epithelial cells (RPTEC), iPSC-derived astrocytes, cardiomyocytes, and GABAergic neurons (Figure 18). All normal cells were HLA-A * 02:01 - (HLA-A obtained from a positive donor) * (02:01 expression was confirmed by RNASeq). Importantly, these normal cells were HLA-A * Because it can present a wide variety of peptides in relation to 02:01, it serves as an assay system for evaluating a broad range of off-target effects. MAGE-B2 and HLA-A * B-CPAP cancer cell lines expressing 02:01 were used as positive control target cells. Mock (untransduced) T cells or T cells expressing the IL12-RFP construct (without transgenic TCR) from the same donor were included as negative control effect cells. Cytokine (IFNγ, IL-12p70, TNFα) and granzyme B production, as well as target cell cytotoxicity (measured by caspase 3 / 7 cleavage), were evaluated in co-culture with TCR-T-IL12 cells (Figure 18). When co-cultured with positive control B-CPAP cells, all four TCR-T-IL12 cells induced cytokine production and target cell cytotoxicity (MAGE-B2+HLA-A *02:01+). Importantly, TCR2-IL12 and TCR4-IL12 did not mediate cytokine production or enhance caspase 3 / 7 cleavage when co-cultured with either the tested normal human primary or iPSC-derived cells, and showed no off-target responsiveness to either of the tested normal cells.

[0105] Evaluation of alloreactivity potential using 34 BLCL systems As part of the safety assessment, the potential for alloreactivity was evaluated using a panel of 34 BLCLs (B lymphoblastoma cell lines) representing high-frequency (>11%) MHC class I alleles in major US racial groups, such as 38HLA-A, 40HLA-B, and 24HLA-C alleles. When TCR-T-IL12 cells were co-cultured with each BLCL, the potential for alloreactivity was assessed by the production of cytokines (IFNγ, TNFα, and IL-12p70) and granzyme B. No significant increase in cytokine or granzyme B response (≥4-fold compared to IL12-RFP control T cells) was observed in any of the four TCR-T-IL12 cells for the 34 BLCLs tested (Figure 19). Some low levels of response (≥3-fold but <4-fold compared to IL12-RFP control cells) were observed for TCR1-IL12 and TCR2-IL12. All four TCR-T-IL12 cells pulsed with MAGE-B2 peptide, creating a positive control U266B1 cell (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 exhibit significant safety issues based on the safety assessments of standard and alloreactivity potential performed.

[0107] Methods and materials used in the above examples Identification of MAGE-B2 pMHC-specific TCRs using healthy donor screening. Production of autoantigen-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 using human CD14 microbeads (Miltenyi Biotec, San Diego, CA, 130-050-201). Mature dendritic cells were obtained using the CellXVivo® Human Monocyte-Induced Dendritic Cell (DC) Differentiation Kit (R&D, Minneapolis, MN, CDK004). Antigen-presenting B cells were produced using the CD40L and IL-4 stimulation method. B cells were positively selected from PBMCs using human CD19 microbeads (Miltenyi Biotec, 130-050-301). CD19+ cells were then stimulated with 0.125 ug / ml recombinant huCD40L in B cell medium, resulting in 2 × 10⁶ cells. 5 Cells were seeded into 24-well plates at a rate of cells / ml and 1 ml / well. The B cell medium consisted of IMDM, GlutaMax® supplement medium (Gibco, 31980030), supplemented with 10% heat-inactive human serum (MilliporeSigma H3667-100ML), 100 U / ml penicillin and 100 ug / ml streptomycin (Gibco, 15140-122), 10 μg / ml gentamicin (Gibco, 15750-060), and 200 IU / ml IL-4 (Peprotech, Rock Hill, NJ, 20004100 ug). Fresh B cell medium containing 400 IU / ml IL-4 was added to the B cell culture medium at a rate of 1 ml / well, without disturbing the cells, on day 3 after B cell activation. 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 isolation of CD14+ cells, MAGE-B2 peptide (Anaspec custom peptide, Freemont, CA) was added to immature dendritic cells at a concentration of 1 μM along with recombinant human TNF-α. On day 9 after isolation of CD14+ cells, MAGE-B2 peptide-pulsed mature dendritic cells were collected, washed, and mixed with CD14-PBMCs in a 1:10 ratio in human T cell medium containing 10 μM MAGE-B2 peptide, 10 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), and 10 ng / ml IL-7 (Peprotech, AF20007100UG). The complete human T cell medium consisted of a 1:1 mixture of CM and AIM-V (Trademark) (ThermoFisher, 12055083). The 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 augmented by 1-3 rounds of weekly peptide-pulsed B cell activation (up to 4 T cell antigen-specific stimulations in total). HuCD40L activated B cells were collected, washed, and 1 × 10⁶ cells were extracted. 6 Cells were seeded into 6-well plates at 4 ml / well and 1 μM MAGE-B2 peptide was added to the B cells, and the cells were incubated at 37°C for 2 hours. The peptide-pulsed B cells were then mixed with human T cells in a 1:10 ratio in a human T cell culture containing 10 IU / ml IL-2 and 10 ng / ml IL-7. MAGE-B2 dextramer-positive cells were identified by flow cytometry and then sorted for TCR identification by single-cell RNA sequencing.

[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 minutes at room temperature in the dark, and then stained with CD3-FITC (Biolegend, San Diego, CA, 300440) and CD8-BV605 (BD Biosciences, San Jose, CA, 564116). Dead cell removal stain (Sytox blue) was purchased from ThermoFisher (Invitrogen, S34857). Cells were sorted using an Aria® Fusion cell sorter (BD Biosciences, San Jose, CA). After sorting, data were analyzed using Flowjo.

[0110] ELISPOT Selected CD3+CD8+Dex+ T cells were validated for antigen-specific IFNγ production using the BD® ELISPOT assay (BD Bioscience, San Jose, CA, 551849) with peptide-loaded T2 cells. 2 × 10⁶ cells were collected in a 24-well plate. 6 T2 cells were loaded with 10 μM MAGE-B2 peptide for 1-2 hours in human T cell complete medium at a rate of cells / ml and 1 ml / well. 150 μl of human T cell complete medium and 50 μl of peptide-loaded T2 cells were added to each well of a pre-coated ELISPOT plate. CD3+CD8+Dex+ T cells (500 or 1000 cells) were sorted directly into each well of the ELISPOT plate. After incubation for 24 hours in a 37°C incubator, ELISPOT was detected. The ELISPOT plates were scanned and counted using IMMUNOSPOT® (Cellular Technology Limited, Cleveland, OH).

[0111] Single-cell RNA sequencing For direct targeted enrichment, omitting cDNA amplification of the entire 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, beads containing cells and barcoded oligonucleotides were encapsulated in nanoliter droplets, where the cells were lysed and mRNA was reverse transcribed using poly-T primers and barcoded template-switched oligonucleotides. Nested PCR was then performed using primers and template-switched oligonucleotides in the constant region of the human TCR. A second targeted enrichment PCR was performed using 13–17 cycles, depending on the estimated number of cell inputs, according to the manufacturer's suggestion. The final sequencing library was prepared from fragmented PCR products ligated to an Illumina sequencing adapter. The library was sequenced with 151 paired-end reads (151×8×0×151) on a NextSeq® 550 or MiSeq® (Illumina, Inc., San Diego, CA) at a rate of at least 5,000 reads / cell. The data were multiplexed and analyzed using cellranger vdj (2.2.0) to obtain full-length paired TCR sequences assigned to individual cells.

[0112] Cloning and transduction of TCRs into Jarcut 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 validated clones were maxiprepped for downstream application. When screening candidate TCRs using transduction, the lentiviral vector was packaged in VSV-G pseudotyped virions (Alstem, Richmond, CA). The lentivirus carrying the TCR was transduced into a Jarcut TCR KO reporter cell line constitutively expressing CD8a and Renilla luciferase under an NFAT-inducible promoter. Briefly, 20 μL of lentiviral particles were added to 1,000,000 to 1,000,000 cells in complete medium containing 5 ug / mL polyblen (MilliporeSigma, TR1003G) in a 50 mL conical tube to achieve a MOI of 10. After adding the virus, the cells were rotated at 1200×g for 45 minutes at 32°C. After rotation, the medium was aspirated and replaced with plenty of fresh medium, and the cells were adjusted to a concentration of 500,000 cells / ml before being placed 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 μl of 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 and incubated at 4°C for 20 minutes. MAGE-B2 peptide-MHC (GVYDGEEHSV / HLA-A) was used at the manufacturer's recommended concentration. *A fluorescent dextramer specific to 02:01, Immudex customized, Fairfax, VA) was incubated with transduced cells in the dark at room temperature for 10 minutes. Subsequently, a 2× antibody cocktail containing anti-CD3 (BD ​​Biosciences) was added by volume at 50 μl before another incubation at 4°C for 20 minutes. After staining, the cells were washed three times by centrifugation at 300×g for 3 minutes, followed by aspiration and resuspending. Before analysis, the cells were fixed in 100 μl of fresh 2% formaldehyde solution at 4°C for 20 minutes. The cells were washed twice to remove formaldehyde before final suspension in 200 μl of PBS containing EDTA. The fixed, labeled cells were subjected to LSRII or Symphony® cytometer (BD Biosciences) using the recommended acquisition settings.

[0113] Jarcut Activation Assay Antigen-presenting T2 cells (ATCCs) were loaded with peptides (Anaspec customized) or excipients only at a specified concentration in serum-free medium for 2 hours. After incubation, the loaded T2 cells were washed three times and counted before resuspending in complete medium, and seeded at 15,000 cells / well in half-area 96-well plates (Corning). Transduced jarcut cells were added to a total volume of 100 μL at 30,000 cells / well. TCR-expressing jarcut cells were co-cultured in the presence of T2 cells at 37°C for 24 hours. At the end of this incubation, before collecting half the volume, the plates were briefly centrifuged at 300 × g and stored to characterize cytokine secretion. An equal volume of RENILLAGLO® (Promega) was added to the remaining volume, and the plate was incubated at room temperature for 20 minutes with shaking before luminescence (detectable by ENVISION® (PerkinElmer, Waltham, MA)). Compared to co-culture with T2 cells containing only the excipient, the activity of individual TCRs in the presence of peptide-loaded T2 cells was expressed as a magnification change in luminescence.

[0114] Production of MAGE-B2 TCR-T and TCR-T-IL12 cells using human primary T cells. Three healthy donors (HLA-A * PBMCs from 02:01) were isolated from leukopak (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 the associated manufacturer's protocol. One day before TCR transduction, frozen pan-T cells were thawed and 1 × 10⁶ cells were extracted. 6Cells were resuspended in human T cell complete medium at a concentration of cells / ml and stimulated with CD3 / CD28 Dynabeads (Thermo Fisher, 11131D) 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) at a T cell:bead ratio of 2:1. Subsequently, T cells were seeded in 24-well plates at a concentration of 1 ml / well. On the day of TCR transduction, activated T cells (300,000) were seeded in human T cell complete medium in a 48-well plate, and transduced with lentivirus in the presence of 8 μg / ml polyblen, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. Subsequently, the T cells were spin-inoculated at 32°C at 1500 × g for 1.5 hours. After spin-inoculation, 380 μl of medium containing 8 μg / ml polyblen, 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 from the bottom of the well without touching the cells. Cells from each well of a 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. On day 4 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 in a cell culture medium. On day 7 after transduction, TCR-T cells were collected, frozen, and stored in a liquid nitrogen vapor phase. The efficiency of TCR transduction was validated by dextramer binding. TCR-T-IL12 cells were produced by the process described in the patent application (PCT Publication Application No.: International Publication No. 2021 / 211104, brochure).

[0115] Flow cytometry The following antibodies were used for T-cell phenotyping: CD3-FITC (Biolegend: 300440), CD8-BV605 (BD: 564116), and 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), and CD86-APC (BD: 555660). The following antibodies were used for B-cell phenotyping: MHC class I (Biolegend: 311414), MHC class II (Biolegend: 361706), CD83-PE (BD556855), CD86-APC (BD: 555660), and CD20-FITC (BD: 556632). Dextramers -APC or -PE were purchased from Immudex (customized dextramers). TCR internalization was prevented using 50 nM PKI dasatinib (Axon Medchem: 1392). TCR-expressing T cells were incubated with 50 nM PKI dasatinib at 37°C for 30 minutes, followed by dextramer staining on ice for 30 minutes, and cell surface marker staining at 4°C for 15 minutes. Dead cell removal staining (Sytox blue, ThermoFisher / Invitrogen, S34857) was used. Raw cytometry data was 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 were determined by a T2-luc (firefly luciferase-expressing T2 cell line) cell death assay. T2-Luc cells were collected, washed, and 2 × 10¹⁶ cells were mixed 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), and 5% thermally inactivated FBS (Gibco, 10082-147). 6 The cells were resuspended at a concentration of 1 ml / ml and then seeded in a 24-well plate with 1 ml / well. T2-Luc cells were pulsed at 37°C for 2-4 hours at the indicated peptide concentration. Subsequently, 2-Luc cells were washed and 1 × 10⁶ cells were removed. 5 The cells were resuspended at 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 for 48 hours at the indicated dextramer+TCR-T to T2-luc cell ratio. The luminescence signal was measured by adding 30 μl of Bio-Glo® (Promega, Madison, WI, G7940), followed by measurement of the luminescence signal using the Biostack® Neo System (BioTek, Winooski, VT). Regarding parental TCR-T, not all TCR-T-IL12 cells were normalized by the addition of mock T cells prior to the cell death assay. Different TCR-T cells were normalized to the same amount of MAGE-B2 dextramer+ cells (e.g., 10%) by the addition of mock (non-transduced) T cells. Specific lysis (specific cell death %) was calculated by normalizing TCR-T+T2 / target peptide cell death, either by 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 dissolution (%) 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 peptide): {1-(TCRT+T2-luc / MAGE-B2 peptide RLU) / (Mock T+T2-luc / MAGE-B2 peptide RLU)}×100 Cancer cell line cell death: {1-(TCRT+Cancer cell line RLU) / (Mock T+Cancer cell line RLU)}×100.

[0117] T cell-mediated cytotoxicity assay for cancer cells (cancer cell TCDD assay) The cytotoxicity of TCR-T cells against MAGE-B2-positive and negative cancer cell lines was determined by a cancer cell death assay. Cancer cells were collected, washed, and placed 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), and 1×10 in 10% thermally inactivated FBS (Gibco, 10082-147, ThermoFisher). 5 The cells were resuspended at a concentration of cells / ml. Next, cancer cells were seeded in a 384-well plate at 25 μl / well and incubated with 25 μl of TCR-T cells for 48 hours at the indicated ratio of dextramer + TCR-T to T2-Luc cells. After incubation, the suspension T cells were removed from the adherent cancer cells, and a plate washer was used to remove Ca 2+ Mg 2+The wells were washed with DPBS (Corning, 21-031-CM) containing [a specific substance]. 30 μl of Celltiter Glo (Promega, G7573) was added, 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 × 10⁶ cells were added to serum-free RPMI medium containing Celltrace® far red (dilution 1:4000). 6 Cancer cells were resuspended at a concentration of cells / ml and incubated at 37°C for 10 minutes. The reaction was stopped by adding 30 ml of cell death assay medium and incubating at room temperature for 10 minutes. Live cancer cells were detected by flow cytometry. Dead cell removal staining (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 using 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 containing the target and similar peptides were synthesized by JPT (Berlin, Germany) or AnaSpec (Fremont, CA). T2-Luc cells were incubated for 2 hours at 37°C / 5% CO2 in T2-Luc cell death medium with reactive similar peptides, target-specific peptides, or DMSO controls at a final peptide concentration range of 1.0E-05M to 6.0E-16M (potency) or 1.0E-05M (single point). 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 2.5E-06 cells / mL. Peptide-loaded T2-Luc cells were added to a white, clear-bottomed 384-well assay plate (Costar) at a rate of 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 at 37°C / 5% CO2 for 48 hours. T2-Luc cell viability was determined using a Bio-Glo® luciferase assay system (Promega, G7940) according to the manufacturer's recommendations. Luminescence was detected using an ENVISION® multi-label plate reader (Perkin Elmer, Santa Clara, CA). The survival rate percentage was calculated using the following formula: % survival rate = (untreated RLU value of sample / mean DMSO control RLU) × 100. EC50 was determined using GraphPad Prism (nonlinear regression curve fitting analysis).

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

[0120] [Table 15]

[0121] [Table 16]

[0122] [Table 17]

[0123] Cytotoxic assay using primary normal human cells The cytotoxicity of target cells was evaluated using a phase-contrast / fluorescence dynamics imaging assay. Fluorescent caspase 3 / 7 cleavage was measured over time using an INCUCYTE® live imaging device (Sartorium, Gottingen, Germany) and superimposed on a phase-contrast image incorporating the cell aggregate. Before performing the cytotoxicity assay, tolerability to different plating densities and various culture media was evaluated to achieve appropriate influence without significant cell overlap in a 96-well plate. Taking into account the dextramer positivity of each TCR-T construct, target cells (100 μl) were added to a black 96-well ViewPlate containing 50 μl of MAGE-B2 TCR-T-IL12 cells, IL-12 RFP T cells, or mock T cells at the densities listed in Table 3, with a dextramer-normalized effector:target (E:T) ratio of 1:1. 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 INCUCYTE® S3. Starting at time 0, phase contrast and fluorescence images (field of view 5) were collected every 4 hours with a 10x objective lens for 44 or 48 hours, and the total integrated intensity of caspase 3 / 7 was analyzed using INCUCYTE® 2019B software. At 44 or 48 hours, the plate was removed from the incubator, and 50 μL of cell medium was taken from the wells for cytokine analysis.

[0124] Cytokine assay using primary normal human cells After 44 or 48 hours, cell culture supernatant (50 μL) was collected from the cytotoxicity assay into a 96-well plate. The plate was sealed and stored at -80°C for subsequent cytokine analysis. The supernatant was thawed according to the manufacturer's instructions. The IFNγ and IL-12p70 plates were blocked with blocking buffer (1% w / v in PBS) from the MSD kit for 1 hour while permeating at room temperature. After washing the plate 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 plate was incubated at room temperature for 2 hours with shaking, followed by three washes with PBS / 0.05% Tween-20. Read buffer (2×, 150 μL) was added to each well, and the plates were analyzed using an MSD MESOSECTOR® S600 instrument (Meso Scale Diagnostics, Rockville, MD). A standard curve was created from a standard substance, and this was used to quantify cytokines in the samples using MSD DISCOVERY WORKBENCH® software 4.0.

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

[0126] By incubating at 37°C for 2 hours, MAGE-B2+MAGE-A4+HLA-A *02:01+ U266B1 cells (ATCC) as a positive control cell line; 10 cells in culture medium 5 Cells ( / ml) were pulsed with 50 μM MAGE-B2 peptide. TCR-T cells from donor D160780 were thawed by adding culture medium, centrifuged at 400 × g for 5 minutes at 4°C, resuspended in 10 ml of medium, and counted. 1.923 × 10 5 TCR-T cells are divided into 200 μl volumes, 1 × 10⁶ 4Cells were co-cultured with either BLCL or peptide-pulsed U266B1 cells. The dextramer-normalized effector:target ratio for four TCR-T cells ranged from 3:1 to approximately 8:1, depending on the dextramer positivity of each cell type. 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×g for 1 minute at 4°C, and the supernatant was collected in 96-well V-bottom plates for cytokine analysis. Cytokines and granzyme B were evaluated by LUMINEX® assay using a custom MILLIPLEX® human cytokine / chemokine kit (Millipore, ST Louis, MO, SRP1885) containing IFNγ, granzyme B, TNFα, and IL-12p70 samples, as per the manufacturer's instructions. Serial dilutions of the sample standards were repeated in each assay plate. LUMINEX® plates were read using a FLEXMAP 3D® instrument (XMAP® technologies, Luminex). Data was exported using XPONENT® software (Luminex) and directly analyzed using EMDMillipore's MILLIPLEX® Analyst software (Burlington, MA). A standard curve was created using a 5-parameter logistic nonlinear regression fitting curve. The detection limits (minimum and maximum) were calculated using the MILLIPLEX® Analyst software (Millipore) as the average of appropriate reproducible standard curve values ​​obtained from each assay plate, indicating the range within which the sample can be interpolated from the standard. Samples were run at appropriate dilutions to ensure that sample level measurements were reliably within the assay standard curve limits. Cytokine and granzyme B levels were reported in pg / mL or as fold-differences compared to IL12 T cells (control), and graphed using 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 HLA-A*02:01 configuration, wherein the TCR comprises a TCRα chain containing the amino acid sequence described in SEQ ID NO: 38 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO:

49.

2. The expression vector according to claim 1, further comprising a nucleic acid encoding interleukin-12 (IL-12).

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

4. The expression vector according to claim 3, wherein the viral vector is a retroviral vector.

5. The expression vector according to claim 4, wherein the retroviral vector is a lentiviral vector.

6. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 60, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

71.

7. Cells expressing recombinant T cell receptors (TCRs) capable of binding to MAGE-B2 peptide and MAGE-A4 peptide in the HLA-A*02:01 state, wherein the TCRs comprise a TCRα chain containing the amino acid sequence described in SEQ ID NO: 38 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO:

49.

8. The cell according to claim 7, further expressing recombinant IL-12.

9. A cell comprising the expression vector according to any one of claims 1 to 6.

10. A cell that is a T cell, as described in any one of claims 7 to 9.

11. The cell according to claim 10, wherein the TCR binds to the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 in the HLA-A*02:01 state, and the binding activates the cell's production of IFNγ, TNFα, IL-12, or granzyme B.

12. A pharmaceutical composition comprising a therapeutically effective amount of the cells described in any one of claims 7 to 11.

13. A method for producing cells according to any one of claims 7 to 11 or a pharmaceutical composition according to claim 12, comprising introducing an expression vector into cells that comprises a nucleic acid sequence encoding a T cell receptor (TCR) capable of binding to MAGE-B2 peptide and MAGE-A4 peptide in the HLA-A*02:01 configuration, The method wherein the TCR comprises a TCRα chain containing the amino acid sequence described in SEQ ID NO: 38 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO:

49.

14. The method according to claim 13, wherein the expression vector further comprises a nucleic acid sequence encoding IL-12.

15. The method according to any one of claims 13 to 14, wherein the cell is a T cell.

16. The method according to claim 15, wherein the cells are primary T cells.

17. The method according to claim 16, wherein the primary T cells are isolated from a cancer patient.

18. A pharmaceutical composition for use in a method for treating cancer expressing MAGE-B2 or MAGE-A4, comprising cells according to any one of claims 7 to 11, or cells produced by the method according to any one of claims 13 to 17, wherein the method comprises administering the cells according to any one of claims 7 to 11, or cells produced by the method according to any one of claims 13 to 17, to a cancer patient in a therapeutically effective amount.

19. The pharmaceutical composition according to claim 18, wherein the patient is tested before administration to determine the presence of cancer expressing MAGE-B2 or MAGE-A4.

20. The pharmaceutical composition according to claim 19, wherein nucleic acids encoding MAGE-B2 or MAGE-A4 are detected.

21. The pharmaceutical composition according to claim 19, wherein MAGE-B2 or MAGE-A4 protein, or MAGE-B2-derived or MAGE-A4-derived peptides are detected.

22. The pharmaceutical composition according to any one of claims 18 to 21, wherein the patient is identified to possess the HLA-A*02:01 allele.

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