MAGEA4-specific T cell receptor

Abstract

Provided herein are recombinant T cell receptors (TCRs) that can selectively recognize the MAGE-A4-derived peptides GVYDGEEHSV or KVEEHVVRV when presented by HLA-A*0201 sufficiently to activate recombinant T cells. The TCRs provided herein have been extensively screened for lack of cross-reactivity and alloreactivity with similar peptides that may be presented by normal cells or tissues.

Description

[Background technology]

[0001] Adoptive T cell therapy offers tremendous opportunities for treating cancer. Chimeric antigen receptor (CAR)-T cell therapy is an approved adoptive T cell therapy for hematological malignancies, but its targeting scope is limited because it recognizes only cell surface antigens, which account for approximately 25% of the genome. Unlike CAR-T cells, TCR-T cells engineered to express T cell receptors (TCRs) specific for tumor antigens can recognize peptide-MHC complexes (pMHC) derived from intracellular proteins, which account for approximately 75% of the genome, thereby enabling a broader range of targets for multiple cancer indications. Intracellular proteins are processed and presented by the major histocompatibility complex (MHC) as pMHC complexes.

[0002] Cancer-testis antigens (CTAs) are attractive targets for cancer immunotherapy, such as TCR-T cell therapy, due to their restricted expression in germ cells, aberrant reactivation in various cancers, and their immunogenicity. Germ cells, such as the testis (an immune-privileged site), typically do not express HLA class I / II molecules, allowing them to evade attack from the immune system. MAGEA4 is a type I MAGE protein, a family of homologous proteins known to bind to E3 RING ligases to regulate protein ubiquitination. Consistent with this behavior, MAGEA4 has recently been shown to contribute to tumorigenesis by promoting translesion synthesis, an error-prone DNA repair method that may contribute to mutation burden and tumorigenesis in cancer tissues, by working in coordination with the E3 ligase RAD18 to promote PCNA ubiquitination.

[0003] TCR-T cells have been shown to be highly potent and sensitive to tumor-specific peptide-MHC targets, while TCRs can recognize multiple peptides. DNA rearrangements required for TCR formation generate a certain number of T cells that recognize self-antigens. During early T cell development, autoreactive T cells are negatively selected and eliminated in the thymic medulla through the promiscuous expression of a wide range of self-antigens in thymic medullary epithelial cells. This negative selection in the thymus serves as a major mechanism of central immune tolerance, shaping the T cell repertoire and preventing autoimmunity. TCRs engineered to enhance their affinity for specific pMHC or to introduce cross-reactivity to multiple pMHC do not benefit from the negative selection that occurs in the thymus. It is noteworthy that affinity-enhanced MAGE-A3 TCR-T cells cause fatal toxicity due to cross-reactivity to titin, which is expressed in cardiac muscle (Cameron et al., Sci Transl Med. 2013 5(197)). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Cameron et al.,Sci Transl Med.2013 5(197) Summary of the Invention [Means for solving the problem]

[0005] Identifying TCR sequences that recognize tumor-specific antigens has proven to be a significant challenge in the art, particularly due to the rarity of tumor-specific T cells in patient blood, the difficulty of expanding even a small number of tumor-specific T cell clones in vitro, and the potential exhaustion or suppression of tumor-specific T cells in tumor-infiltrating lymphocytes (TILs). Despite these challenges, provided herein are TCR sequences specific for two MAGEA4 peptide-MHC (KVLEHVVRV / HLA-A*02:01, GVYDGREHTV / HLA-A*02:01) identified using healthy donor blood and ex vivo stimulation methods. As demonstrated in the Examples herein, exemplary TCR-T cells that recognize tumor-specific MAGEA4 pMHC can be highly potent therapeutic agents for the treatment of MAGEA4 / HLA-A*02:01+ tumors by exerting cytotoxicity and producing cytokines. These TCR-T cell therapies will be important treatment options for various cancer indications, such as non-small cell lung cancer (NSCLC).

[0006] TCR-T cells are the most potent and sensitive in vitro modality for pMHC targets. The TCR-T cells provided herein exhibit high potency even against cells with very low target expression. This high potency of TCR-T cells results from the complex of a transduced TCR and endogenous CD3 subunit. In some embodiments, to enhance in vitro efficacy, exemplary TCR-T cells contain an activation-dependent IL12 payload incorporated into the TCR-T construct. In some embodiments, IL12 expression is controlled by TCR activation under a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter. In these embodiments, IL12 is produced when TCR-T-IL12 cells encounter tumor antigens. This strategy has been shown to enhance the efficacy of adoptive T cell therapy in vivo, thereby potentially reducing the potential clinical dose by 10-100-fold.

[0007] In a first aspect, the present invention is an expression vector comprising nucleic acid sequences encoding a T cell receptor (TCR) alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain are a. a TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 3, 5, and 7, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 4, 6, and 8, respectively; b. A TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 13, 15, and 17, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 14, 16, and 18, respectively; c. A TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 23, 25, and 27, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively; d. a TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 33, 35, and 37, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 34, 36, and 38, respectively; e. a TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 43, 45, and 47, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 44, 46, and 48, respectively; f. a TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 53, 55, and 57, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 54, 56, and 58, respectively; g. A TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 63, 65, and 67, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 64, 66, and 68, respectively; and h. A TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 73, 75, and 77, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 74, 76, and 78, respectively. is selected from the group consisting of:

[0008] In another aspect, the invention is an expression vector comprising nucleic acid sequences encoding a T cell receptor (TCR) α chain and a TCR β chain, wherein the TCR α chain and the TCR β chain are: a. the amino acid sequence set forth in SEQ ID NO: 9 or 10 and the amino acid sequence set forth in SEQ ID NO: 11 or 12; b. the amino acid sequence set forth in SEQ ID NO: 19 or 20 and the amino acid sequence set forth in SEQ ID NO: 21 or 22; c. the amino acid sequence set forth in SEQ ID NO: 29 or 30 and the amino acid sequence set forth in SEQ ID NO: 31 or 32; d. the amino acid sequence set forth in SEQ ID NO: 39 or 40 and the amino acid sequence set forth in SEQ ID NO: 41 or 42; e. the amino acid sequence set forth in SEQ ID NO: 49 or 50 and the amino acid sequence set forth in SEQ ID NO: 51 or 52; f. the amino acid sequence set forth in SEQ ID NO: 59 or 60 and the amino acid sequence set forth in SEQ ID NO: 61 or 62; g. the amino acid sequence set forth in SEQ ID NO: 69 or 70 and the amino acid sequence set forth in SEQ ID NO: 71 or 72; and h. The amino acid sequence set forth in SEQ ID NO: 79 or 80 and the amino acid sequence set forth in SEQ ID NO: 81 or 82 is selected from the group consisting of:

[0009] In another aspect, the invention is a cell expressing a recombinant T cell receptor (TCR), wherein the TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain comprise: a. a TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 3, 5, and 7, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 4, 6, and 8, respectively; b. A TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 13, 15, and 17, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 14, 16, and 18, respectively; c. A TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 23, 25, and 27, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively; d. a TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 33, 35, and 37, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 34, 36, and 38, respectively; e. a TCR α chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 43, 45, and 47, respectively, and a TCR β chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 44, 46, and 48, respectively; f. a TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 53, 55, and 57, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 54, 56, and 58, respectively; g. A TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 63, 65, and 67, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 64, 66, and 68, respectively; and h. A TCR alpha chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 73, 75, and 77, respectively, and a TCR beta chain comprising CDR1, 2, and 3 sequences comprising the amino acid sequences set forth in SEQ ID NOs: 74, 76, and 78, respectively. is selected from the group consisting of:

[0010] In another aspect, the invention is a cell expressing a recombinant T cell receptor (TCR), wherein the TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain comprise: a. the amino acid sequence set forth in SEQ ID NO: 9 or 10 and the amino acid sequence set forth in SEQ ID NO: 11 or 12; b. the amino acid sequence set forth in SEQ ID NO: 19 or 20 and the amino acid sequence set forth in SEQ ID NO: 21 or 22; c. the amino acid sequence set forth in SEQ ID NO: 29 or 30 and the amino acid sequence set forth in SEQ ID NO: 31 or 32; d. the amino acid sequence set forth in SEQ ID NO: 39 or 40 and the amino acid sequence set forth in SEQ ID NO: 41 or 42; e. the amino acid sequence set forth in SEQ ID NO: 49 or 50 and the amino acid sequence set forth in SEQ ID NO: 51 or 52; f. the amino acid sequence set forth in SEQ ID NO: 59 or 60 and the amino acid sequence set forth in SEQ ID NO: 61 or 62; g. the amino acid sequence set forth in SEQ ID NO: 69 or 70 and the amino acid sequence set forth in SEQ ID NO: 71 or 72; and h. The amino acid sequence set forth in SEQ ID NO: 79 or 80 and the amino acid sequence set forth in SEQ ID NO: 81 or 82 is selected from the group consisting of: [Brief explanation of the drawings]

[0011] [Figure 1A] MAGEA4 is a tumor-specific antigen that is broadly expressed in a wide range of solid tumors. (A) TCGA and internal RNA-seq data for MAGEA4 mRNA expression in various cancers. (B) BodyMap RNA-seq data for MAGEA4 mRNA expression in human normal tissues. MAGEA4 expression is highly restricted to the male reproductive system in normal tissues. (C) MAGE-A4 immunohistochemistry (IHC) using OTI1F9 monoclonal Ab shows that MAGE-A4 protein is expressed in the majority of tumor cells within squamous non-small cell lung cancer tumors. Representative IHC staining of squamous non-small cell lung cancer tumors shows 100% MAGE-A4 positive tumor cells and staining intensity 3+. [Figure 1B]MAGEA4 is a tumor-specific antigen that is broadly expressed in a wide range of solid tumors. (A) TCGA and internal RNA-seq data for MAGEA4 mRNA expression in various cancers. (B) BodyMap RNA-seq data for MAGEA4 mRNA expression in human normal tissues. MAGEA4 expression is highly restricted to the male reproductive system in normal tissues. (C) MAGE-A4 immunohistochemistry (IHC) using OTI1F9 monoclonal Ab shows that MAGE-A4 protein is expressed in the majority of tumor cells within squamous non-small cell lung cancer tumors. Representative IHC staining of squamous non-small cell lung cancer tumors shows 100% MAGE-A4 positive tumor cells and staining intensity 3+. [Figure 1C] MAGEA4 is a tumor-specific antigen that is broadly expressed in a wide range of solid tumors. (A) TCGA and internal RNA-seq data for MAGEA4 mRNA expression in various cancers. (B) BodyMap RNA-seq data for MAGEA4 mRNA expression in human normal tissues. MAGEA4 expression is highly restricted to the male reproductive system in normal tissues. (C) MAGE-A4 immunohistochemistry (IHC) using OTI1F9 monoclonal Ab shows that MAGE-A4 protein is expressed in the majority of tumor cells within squamous non-small cell lung cancer tumors. Representative IHC staining of squamous non-small cell lung cancer tumors shows 100% MAGE-A4 positive tumor cells and staining intensity 3+. [Figure 2] RNA-seq and mass spectrometry (MS) of NSCLC specimens to quantify detectable HLA-A*02:01-binding MAGEA4 target peptides. High levels of MAGE-A4 FPKM mRNA expression are generally associated with detectable MAGEA4 target peptide presentation. [Figure 3]Estimation of annual patient population for specific cancer indications. The annual treatable patient population was estimated based on pMHC target frequency multiplied by new cases per year in the US population. pMHC target frequency in each cancer indication was calculated by MAGE-A4 mRNA expression frequency multiplied by the HLA-A*02:01 carrier frequency (0.41) in the US population. MAGE-A4 mRNA levels (>1 FPKM) in various solid tumors were derived from TCGA data. [Figure 4A] Workflow for identifying MAGE-A4 pMHC-specific TCRs from rare T cell clones in healthy HLA-A*02:01+ donor PBMCs. (A) MAGE-A4 pMHC-specific T cells were stimulated and expanded by coculture with autologous APCs pulsed with MAGE-A4 peptide. MAGE-A4 pMHC-specific T cells were sorted for scRNAseq to identify MAGE-A4 pMHC-specific TCR sequences and functionally validated by IFNγ ELISPOT. (B) Representative screen results demonstrate that positive donor A exhibited enriched MAGE-A4 pMHC-specific T cells after multiple ex vivo stimulations, whereas negative donor B had no Dex+ T cells. (C) MAGE-A4 pMHC-specific T cells were sorted and validated for antigen specificity by IFNγ ELISPOT. [Figure 4B]Workflow for identifying MAGE-A4 pMHC-specific TCRs from rare T cell clones in healthy HLA-A*02:01+ donor PBMCs. (A) MAGE-A4 pMHC-specific T cells were stimulated and expanded by coculture with autologous APCs pulsed with MAGE-A4 peptide. MAGE-A4 pMHC-specific T cells were sorted for scRNAseq to identify MAGE-A4 pMHC-specific TCR sequences and functionally validated by IFNγ ELISPOT. (B) Representative screen results demonstrate that positive donor A exhibited enriched MAGE-A4 pMHC-specific T cells after multiple ex vivo stimulations, whereas negative donor B had no Dex+ T cells. (C) MAGE-A4 pMHC-specific T cells were sorted and validated for antigen specificity by IFNγ ELISPOT. [Figure 4C] Workflow for identifying MAGE-A4 pMHC-specific TCRs from rare T cell clones in healthy HLA-A*02:01+ donor PBMCs. (A) MAGE-A4 pMHC-specific T cells were stimulated and expanded by coculture with autologous APCs pulsed with MAGE-A4 peptide. MAGE-A4 pMHC-specific T cells were sorted for scRNAseq to identify MAGE-A4 pMHC-specific TCR sequences and functionally validated by IFNγ ELISPOT. (B) Representative screen results demonstrate that positive donor A exhibited enriched MAGE-A4 pMHC-specific T cells after multiple ex vivo stimulations, whereas negative donor B had no Dex+ T cells. (C) MAGE-A4 pMHC-specific T cells were sorted and validated for antigen specificity by IFNγ ELISPOT. [Figure 5A]Measurement of MAGE-A4 TCR activity by Jurkat activation assay. (A) T2 cells were loaded with target MAGEA4 peptides and co-cultured with TCR / GFP-transfected Jurkat cells. TCR potency was assessed by quantifying the upregulation of CD69 on Jurkat cells in response to the KVLEHVVRV pMHC TCR. (B) Summary of measured TCR potency as determined by T2 peptide titration assay. [Figure 5B] Measurement of MAGE-A4 TCR activity by Jurkat activation assay. (A) T2 cells were loaded with target MAGEA4 peptides and co-cultured with TCR / GFP-transfected Jurkat cells. TCR potency was assessed by quantifying the upregulation of CD69 on Jurkat cells in response to the KVLEHVVRV pMHC TCR. (B) Summary of measured TCR potency as determined by T2 peptide titration assay. [Figure 6A] Transduction of primary human T cells with TCR-IL12 constructs. (A) The TCR-T-IL12 lentiviral construct contains the TCRα and TCRβ chains together with the 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 a minimal IL-2 promoter. (B) Summary of transduction efficiencies of the top eight TCRs determined by flow cytometry analysis. T cell subset frequencies (%) are shown as the average of transduction efficiencies from TCR-T cells generated from two human donors. [Figure 6B]Transduction of primary human T cells with TCR-IL12 constructs. (A) The TCR-T-IL12 lentiviral construct contains the TCRα and TCRβ chains together with the 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 a minimal IL-2 promoter. (B) Summary of transduction efficiencies of the top eight TCRs determined by flow cytometry analysis. T cell subset frequencies (%) are shown as the average of transduction efficiencies from TCR-T cells generated from two human donors. [Figure 7A] Cytotoxic activity of MAGEA4 TCR / IL-12 T cells against peptide-loaded T2 cells. A T2 peptide titration assay was used to test TCR-Ts using primary human T cells. Representative T cell-dependent cytotoxicity (TDCC) assays, T2 / peptide loading assays, are shown for GVYDGREHTV pMHC (A) and KVLEHVVRVV pMHC (B) TCR-Ts. T cells were ranked by cytotoxic potency to identify the top eight candidate TCRs (C). [Figure 7B] Cytotoxic activity of MAGEA4 TCR / IL-12 T cells against peptide-loaded T2 cells. A T2 peptide titration assay was used to test TCR-Ts using primary human T cells. Representative T cell-dependent cytotoxicity (TDCC) assays, T2 / peptide loading assays, are shown for GVYDGREHTV pMHC (A) and KVLEHVVRVV pMHC (B) TCR-Ts. T cells were ranked by cytotoxic potency to identify the top eight candidate TCRs (C). [Figure 7C] Cytotoxic activity of MAGEA4 TCR / IL-12 T cells against peptide-loaded T2 cells. A T2 peptide titration assay was used to test TCR-Ts using primary human T cells. Representative T cell-dependent cytotoxicity (TDCC) assays, T2 / peptide loading assays, are shown for GVYDGREHTV pMHC (A) and KVLEHVVRVV pMHC (B) TCR-Ts. T cells were ranked by cytotoxic potency to identify the top eight candidate TCRs (C). [Figure 8A] The top 20 similar peptides for GVYDGREHTV and KVLEHVVRV were identified and used to assess TCR cross-reactivity. T2 cells were pre-incubated with 10-5 M of the relevant peptide and co-cultured with the corresponding top 8 TCR-T for 48 hours. Representative examples of TCR-T generated from two different donors. [Figure 8B] The top 20 similar peptides for GVYDGREHTV and KVLEHVVRV were identified and used to assess TCR cross-reactivity. T2 cells were pre-incubated with 10-5 M of the relevant peptide and co-cultured with the corresponding top 8 TCR-T for 48 hours. Representative examples of TCR-T generated from two different donors. [Figure 9] Sequence identity between the target MAGEA4 peptides (GVY and KVL) and the homologous MAGEA8 peptide. The KVLEHVVRV peptide sequence is 100% identical in MAGEA4 and MAGEA8. [Figure 10A] Cross-reactivity of GVYDGREHTV-MHC-specific TCRs to MAGEA8 peptide. (A) T2 cells were loaded with the indicated concentrations of MAGEA8 peptide GLYDGREHSV and incubated with TCR-T for 48 hours before TDCC assessment. (B) Summary of TCR potency data. A >1000-fold difference in EC50 was observed between MAGEA4 and MAGEA8 peptides for the top four TCRs. Representative example of an experiment using TCR-T generated from two donors (8316 and 12665). [Figure 10B] Cross-reactivity of GVYDGREHTV-MHC-specific TCRs to MAGEA8 peptide. (A) T2 cells were loaded with the indicated concentrations of MAGEA8 peptide GLYDGREHSV and incubated with TCR-T for 48 hours before TDCC assessment. (B) Summary of TCR potency data. A >1000-fold difference in EC50 was observed between MAGEA4 and MAGEA8 peptides for the top four TCRs. Representative example of an experiment using TCR-T generated from two donors (8316 and 12665). [Figure 11A]TDCC activity of top-ranked TCR-Ts against MAGEA4+HLA-A*02:01+ cancer cell lines. The top eight TCR-Ts identified in the T2 peptide titration potency assay were further evaluated in a cancer cell death assay. Highly potent cytolytic activity, approaching 100% specific cell death, was observed for the MAGEA4+HLA-A*02:01+ cancer cell lines U266B1 (MAGEA4 FPKM 213.85) (A) and SCaBER (MAGEA4 FPKM 172) (B). The evaluated efficacy metrics are summarized and shown. Representative examples of experiments using TCR-Ts generated from two donors are shown (C and D). MAGEA4 expression in each cell line was obtained from the Cancer Cell Line Encyclopedia (CCLE) and is shown as FPKM. [Figure 11B] TDCC activity of top-ranked TCR-Ts against MAGEA4+HLA-A*02:01+ cancer cell lines. The top eight TCR-Ts identified in the T2 peptide titration potency assay were further evaluated in a cancer cell death assay. Highly potent cytolytic activity, approaching 100% specific cell death, was observed for the MAGEA4+HLA-A*02:01+ cancer cell lines U266B1 (MAGEA4 FPKM 213.85) (A) and SCaBER (MAGEA4 FPKM 172) (B). The evaluated efficacy metrics are summarized and shown. Representative examples of experiments using TCR-Ts generated from two donors are shown (C and D). MAGEA4 expression in each cell line was obtained from the Cancer Cell Line Encyclopedia (CCLE) and is shown as FPKM. [Figure 11C]TDCC activity of top-ranked TCR-Ts against MAGEA4+HLA-A*02:01+ cancer cell lines. The top eight TCR-Ts identified in the T2 peptide titration potency assay were further evaluated in a cancer cell death assay. Highly potent cytolytic activity, approaching 100% specific cell death, was observed for the MAGEA4+HLA-A*02:01+ cancer cell lines U266B1 (MAGEA4 FPKM 213.85) (A) and SCaBER (MAGEA4 FPKM 172) (B). The evaluated efficacy metrics are summarized and shown. Representative examples of experiments using TCR-Ts generated from two donors are shown (C and D). MAGEA4 expression in each cell line was obtained from the Cancer Cell Line Encyclopedia (CCLE) and is shown as FPKM. [Figure 11D] TDCC activity of top-ranked TCR-Ts against MAGEA4+HLA-A*02:01+ cancer cell lines. The top eight TCR-Ts identified in the T2 peptide titration potency assay were further evaluated in a cancer cell death assay. Highly potent cytolytic activity, approaching 100% specific cell death, was observed for the MAGEA4+HLA-A*02:01+ cancer cell lines U266B1 (MAGEA4 FPKM 213.85) (A) and SCaBER (MAGEA4 FPKM 172) (B). The evaluated efficacy metrics are summarized and shown. Representative examples of experiments using TCR-Ts generated from two donors are shown (C and D). MAGEA4 expression in each cell line was obtained from the Cancer Cell Line Encyclopedia (CCLE) and is shown as FPKM. [Figure 12A]The top five identified TCRs were evaluated based on their potency in cell death assays of cancer cell lines (A). Expression levels of MAGEA4 and HLA-A in cancer cell lines were obtained from CCLE and are shown as FPKM. In some cell lines, the HLA-A*02:01-binding MAGEA4 peptide KVLEHVVRV was quantified by mass spectrometry and is shown as copies per cell. The top TCR-Ts demonstrated cytolytic activity against a large set of MAGEA4+HLA-A*02:01 cell lines but did not kill the MAGEA4-HLA-A*02:01+ cell line CFPAC1. Potency statistics are summarized and shown in (B). A representative example of an experiment performed with TCR-Ts from three donors. [Figure 12B] The top five identified TCRs were evaluated based on their potency in cell death assays of cancer cell lines (A). Expression levels of MAGEA4 and HLA-A in cancer cell lines were obtained from CCLE and are shown as FPKM. In some cell lines, the HLA-A*02:01-binding MAGEA4 peptide KVLEHVVRV was quantified by mass spectrometry and is shown as copies per cell. The top TCR-Ts demonstrated cytolytic activity against a large set of MAGEA4+HLA-A*02:01 cell lines but did not kill the MAGEA4-HLA-A*02:01+ cell line CFPAC1. Potency statistics are summarized and shown in (B). A representative example of an experiment performed with TCR-Ts from three donors. [Figure 13A] Potency assay of off-target peptides identified by similar peptide screens. Putative cross-reactive peptides for TCR23 and TCR24 were evaluated in a TDCC / T2 peptide titration assay (A-B). Viability of T2 cells loaded with the MAGEA4-targeting GVY peptide (GVY) is shown as a positive control. Peptides with a potency gap cutoff of less than 103-fold higher than the target peptide at EC50 were considered for further risk assessment. No putative cross-reactivity risk was found with KVL-reactive TCRs. Representative example of experiments performed with TCR-Ts from three donors. [Figure 13B]Potency assay of off-target peptides identified by similar peptide screens. Putative cross-reactive peptides for TCR23 and TCR24 were evaluated in a TDCC / T2 peptide titration assay (A-B). Viability of T2 cells loaded with the MAGEA4-targeting GVY peptide (GVY) is shown as a positive control. Peptides with a potency gap cutoff of less than 103-fold higher than the target peptide at EC50 were considered for further risk assessment. No putative cross-reactivity risk was found with KVL-reactive TCRs. Representative example of experiments performed with TCR-Ts from three donors. [Figure 14A] Assessment of TCR reactivity against human normal cells. (A) The top four TCR-T cells (circles) or RFP+IL12 T cell control (squares) were co-cultured with a panel of human normal primary cells or iPSC-derived cell lines (MAGEA4-HLA-A*02:01+) representing vital organs, including human bronchial epithelial cells (hBEpCs), tracheal epithelial cells (hTEpCs), dermal microvascular endothelial cells (HDMECs), keratinocytes, hepatocytes, renal proximal tubule epithelial cells (RPTECs), iPSC-derived astrocytes, cardiomyocytes, and GABAergic neurons. Caspase 3 / 7 activity was measured over time using the Incucyte system. (B,C) While minimal or negligible reactivity was observed against baseline in all cases for TCR2 and TCR23, TCR10 and TCR24 showed clear cytotoxic activity against multiple normal cells. [Figure 14B]Assessment of TCR reactivity against human normal cells. (A) The top four TCR-T cells (circles) or RFP+IL12 T cell control (squares) were co-cultured with a panel of human normal primary cells or iPSC-derived cell lines (MAGEA4-HLA-A*02:01+) representing vital organs, including human bronchial epithelial cells (hBEpCs), tracheal epithelial cells (hTEpCs), dermal microvascular endothelial cells (HDMECs), keratinocytes, hepatocytes, renal proximal tubule epithelial cells (RPTECs), iPSC-derived astrocytes, cardiomyocytes, and GABAergic neurons. Caspase 3 / 7 activity was measured over time using the Incucyte system. (B,C) While minimal or negligible reactivity was observed against baseline in all cases for TCR2 and TCR23, TCR10 and TCR24 showed clear cytotoxic activity against multiple normal cells. [Figure 14C] Assessment of TCR reactivity against human normal cells. (A) The top four TCR-T cells (circles) or RFP+IL12 T cell control (squares) were co-cultured with a panel of human normal primary cells or iPSC-derived cell lines (MAGEA4-HLA-A*02:01+) representing vital organs, including human bronchial epithelial cells (hBEpCs), tracheal epithelial cells (hTEpCs), dermal microvascular endothelial cells (HDMECs), keratinocytes, hepatocytes, renal proximal tubule epithelial cells (RPTECs), iPSC-derived astrocytes, cardiomyocytes, and GABAergic neurons. Caspase 3 / 7 activity was measured over time using the Incucyte system. (B,C) While minimal or negligible reactivity was observed against baseline in all cases for TCR2 and TCR23, TCR10 and TCR24 showed clear cytotoxic activity against multiple normal cells. [Figure 15A]Overview of alloreactivity assessment. TCR-T-IL12 cells were cocultured with each of 34 BLCLs expressing frequent MHC class I alleles. HLA-A*02:01+U266B1 cell lines pulsed with the relevant MAGE-A4 peptide (KVL:KVLEHVVRV or GVY:GVYGDREHTV) served as a positive control for each TCR-T-IL12 cell line. Secreted IFNγ (A), granzyme B (B), TNFα (C), and IL-12p70 (D) were assessed as a measure of potential alloreactivity by comparing them to levels in response to coculture with IL12-RFP control T cells. For mock-transduced T cells, cytokine and granzyme B changes are shown relative to IL12-RFP cells at an effector:target ratio of 10:1. [Figure 15B] Overview of alloreactivity assessment. TCR-T-IL12 cells were cocultured with each of 34 BLCLs expressing frequent MHC class I alleles. HLA-A*02:01+U266B1 cell lines pulsed with the relevant MAGE-A4 peptide (KVL:KVLEHVVRV or GVY:GVYGDREHTV) served as a positive control for each TCR-T-IL12 cell line. Secreted IFNγ (A), granzyme B (B), TNFα (C), and IL-12p70 (D) were assessed as a measure of potential alloreactivity by comparing them to levels in response to coculture with IL12-RFP control T cells. For mock-transduced T cells, cytokine and granzyme B changes are shown relative to IL12-RFP cells at an effector:target ratio of 10:1. [Figure 15C]Overview of alloreactivity assessment. TCR-T-IL12 cells were cocultured with each of 34 BLCLs expressing frequent MHC class I alleles. HLA-A*02:01+U266B1 cell lines pulsed with the relevant MAGE-A4 peptide (KVL:KVLEHVVRV or GVY:GVYGDREHTV) served as a positive control for each TCR-T-IL12 cell line. Secreted IFNγ (A), granzyme B (B), TNFα (C), and IL-12p70 (D) were assessed as a measure of potential alloreactivity by comparing them to levels in response to coculture with IL12-RFP control T cells. For mock-transduced T cells, cytokine and granzyme B changes are shown relative to IL12-RFP cells at an effector:target ratio of 10:1. [Figure 15D] Overview of alloreactivity assessment. TCR-T-IL12 cells were cocultured with each of 34 BLCLs expressing frequent MHC class I alleles. HLA-A*02:01+U266B1 cell lines pulsed with the relevant MAGE-A4 peptide (KVL:KVLEHVVRV or GVY:GVYGDREHTV) served as a positive control for each TCR-T-IL12 cell line. Secreted IFNγ (A), granzyme B (B), TNFα (C), and IL-12p70 (D) were assessed as a measure of potential alloreactivity by comparing them to levels in response to coculture with IL12-RFP control T cells. For mock-transduced T cells, cytokine and granzyme B changes are shown relative to IL12-RFP cells at an effector:target ratio of 10:1. [Figure 16]Cross-reactivity with additional HLA-A*02 alleles could broaden the patient population. The top-ranking TCR-T recognizes target peptides presented on additional HLA-A alleles with high homology to HLA-A*02:01. The homologous HLA-A alleles were overexpressed on HLA-A-C1R cells and subsequently loaded with target KVL or GVY MAGEA4 peptides for use in TDCC assays. Both TCR2 and TCR23 demonstrated cytolytic activity against target peptide-loaded C1R cells expressing HLA-A*02:05 and HLA-A*02:07, suggesting the potential inclusion of HLA-A*02:05 and HLA-A*02:07 patients for these TCR-T cell therapies. DETAILED DESCRIPTION OF THE INVENTION

[0012] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited in the body of this specification are expressly incorporated by reference in their entirety.

[0013] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques may generally be performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et 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 all purposes. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery and treatment of patients.

[0014] T cell receptors (TCRs) are naturally expressed by CD4+ and CD8+ T cells. TCRs are designed to recognize short peptide antigens that are presented on the surface of antigen-presenting cells in complex with major histocompatibility complex (MHC) molecules (in humans, MHC molecules are also known as human leukocyte antigens or HLA) (Davis, et al., (1998), Annu Rev Immunol 16:523-544). CD8+ T cells, also called cytotoxic T cells, specifically recognize peptides bound to MHC class I and are generally involved in identifying and mediating the destruction of infected or cancerous cells.

[0015] Therapeutic TCRs can be used, for example, as soluble targeting agents for the purpose of delivering cytotoxic or immune effector agents to tumors (Lissin, et al., (2013). "High-Affinity Monoclonal T-cell receptor (mTCR) Fusions. Fusion Protein Technologies for Biophamaceuticals: Applications and Challenges". S.R. Schmidt, Wiley; Boulter, et al., (2003), Protein Eng 16(9):707-71 1; Liddy, et al., (2012), Nat Med 8:980-987), or alternatively, can be used to engineer T cells for adoptive therapy (June, et al., (2014), Cancer Immunol Immunother 63(9):969-975). It is desirable for TCRs for immunotherapy applications to be able to potently recognize target antigens, which means that the TCR should have high affinity and / or a long binding half-life for the target antigen in order to exert a potent response. Naturally occurring TCRs typically have low affinity (low micromolar range) for target antigens, and therefore it is often necessary to identify mutations, including but not limited to substitutions, insertions, and / or deletions, that can be made to a given TCR sequence to improve antigen binding. For use as a soluble targeting agent, the TCR antigen-binding affinity is preferably in the nanomolar to picomolar range and has a binding half-life of several hours. It is also desirable for therapeutic TCRs to demonstrate a high level of specificity for the target antigen to reduce the risk of toxicity in clinical applications due to off-target binding. Given the natural degeneracy of TCR antigen recognition, such high specificity can be particularly difficult to obtain (Wooldridge, et al., (2012), J Biol Chem 287(2):1 168-1 177; Wilson, et al., (2004), Mol Immunol 40(14-15):1047-1055). Finally, it is desirable that therapeutic TCRs can be expressed and purified in a highly stable form.

[0016] The variable domain of each chain is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in framework sequences. The CDRs contain the recognition sites for peptide-MHC binding. Several genes encode the α chain variable (Va) region and several genes encode the β chain variable (Vβ) region. These genes are distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. The Va and ν genes are designated by the prefixes TRAV and TRBV, respectively, in the IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1):42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2):83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Similarly, for the α and β chains, there are several joining or J genes called TRAJ' or TRBJ', respectively, and for the β chain, there is a diversity or D gene called TRBD' (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2):107-114; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2):97-106; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). The enormous diversity of α and β variable region sequences arises from combinatorial rearrangements between various V, J, and D genes, including allelic variants, and additional junctional diversity (Arstila, et al., (1999), Science 286(5441):958-961; Robins et al., (2009), Blood 1 14(19):4099-4107). The constant regions or C regions of the TCR α and β chains are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10).

[0017] The TCR sequences defined herein are described with reference to the IMGT nomenclature, which is widely known and available to practitioners in the TCR field. See, for example, LeFranc and LeFranc, (2001), "T cell Receptor Factsbook," Academic Press; Lefranc, (2011), Cold Spring Harb Protoc 2011(6):595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10; and Lefranc, (2003), Leukemia 17(1):260-266. The αβ TCR consists of two disulfide-linked chains. Each chain (α and β) is generally considered to have two domains: a variable domain and a constant domain. A short joining region connects the variable and constant domains and is usually considered part of the variable region. In addition, the β chain usually contains a short diversity region between the variable and joining regions.

[0018] Provided herein are T cell receptor (TCR) alpha and beta chain pairs that bind to the MAGE-A4-derived peptide GVYDGREHTV (SEQ ID NO: 1) or KVLEHVVRV (SEQ ID NO: 2) when presented by an HLA class I molecule. In some embodiments, the HLA class I molecule is HLA-A*02:01. Identification of specific TCR sequences that bind to the GVYDGREHTV HLA-A*02:01 or KVLEHVVRV HLA-A*02:01 complex is advantageous for the development of novel immunotherapies.

[0019] A "TCR alpha and beta chain pair" may also be referred to herein as a "TCR," "a TCR," or "the TCR." When recombinantly expressed in a cell, e.g., a T cell, the TCR binds to a MAGEA4 peptide-HLA complex on the cell, e.g., a cancer cell, and such binding results in activation of the recombinant cell. Activation of the T cell results in the death or destruction of the cancer cell. Methods for determining T cell activation are known in the art and are provided herein in the Examples.

[0020] In a preferred embodiment, the potency or cytolytic activity (cytotoxicity) of the recombinant cells of the present invention is measured by: (1) measuring the number of copies of the recombinant cells in a T cell-dependent cytotoxicity (TDCC) assay, a T2 / peptide challenge assay, or a T cell count of about 100 copies (about 10 -8 M) / cells as defined by 80–100% lysis of peptide-loaded HLA-A*02:01 target cells or (2) 80–100% lysis of natural pMHC target-positive cancer cell lines.

[0021] Each TCR α and β chain contains a variable and a constant domain. Within the variable domain (Vα or Vβ) there are three CDRs (complementarity determining regions): CDR1, CDR2, and CDR3. The various α and β chain variable domains can be distinguished by their frameworks, as well as by portions of their CDR1, CDR2, and CDR3 sequences.

[0022] As used herein and in the claims, the term "TCR alpha (or a) variable domain" refers to the junction of the TRAV and TRAJ regions; the TRAV region only; or the TRAV and partial TRAJ region, and the term TCR alpha (or a) constant domain refers to the extracellular TRAC region or a C-terminal truncated or full-length TRAC sequence. Similarly, the term "TCR beta (or β) variable domain" can refer to the junction of the TRBV and TRBD / TRBJ regions; the TRBV and TRBD regions only; the TRBV and TRBJ regions only; or the TRBV and partial TRBD and / or TRBJ regions, and the term TCR beta (or β) constant domain refers to the extracellular TRBC region or a C-terminal truncated or full-length TRBC sequence.

[0023] In a preferred embodiment, the TCR comprises an alpha chain having a CDR3 set forth in SEQ ID NO: 7, 17, 27, 37, 47, 57, 67 or 77 and a beta chain having a CDR3 set forth in SEQ ID NO: 8, 18, 28, 38, 48, 58, 68 or 78. The CDR3 regions can be determined by commercially available software (e.g., Cellranger; 10X Genomics). The TCR alpha chain can comprise a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence set forth in any of SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79 or 80. The TCR beta chain may comprise a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence set forth in any of SEQ ID NOs: 11, 12, 21, 22, 31, 32, 41, 42, 51, 52, 61, 62, 71, 72, 81 or 82. Methods for determining identity between two sequences, such as BLAST or Geneious, are well known in the art. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues may be truncated or removed at the C- or N-terminus of any of the sequences set forth in any of SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79 or 80 or any of the sequences set forth in any of SEQ ID NOs: 11, 12, 21, 22, 31, 32, 41, 42, 51, 52, 61, 62, 71, 72, 81 or 82. Exemplary TCRs and corresponding α and β chain CDR3s and full length SEQ ID NOs are provided in Table 1A and Table 1B.

[0024] In one embodiment, the TCR1 alpha chain comprises the use of TRAV4*01 and TRAJ9*01 variable region chains. In one embodiment, the TCR1 beta chain comprises the use of TRBV11-2*01, TRBD2*02, and TRBJ1-4*01 variable region chains and the use of a TRBC1*01 constant region chain. In one embodiment, the TCR1 comprises a TCR1 alpha chain comprising the use of TRAV4*01 and TRAJ9*01 variable region chains, and a TCR1 beta chain comprising the use of TRBV11-2*01, TRBD2*02, and TRBJ1-4*01 variable region chains and the use of a TRBC1*01 constant region chain.

[0025] In one embodiment, the TCR2 alpha chain comprises the use of TRAV8-1*01 and TRAJ37*01 variable region chains. In one embodiment, the TCR2 beta chain comprises the use of TRBV2*01, TRBD1*01 and TRBJ2-7*01 variable region chains and the use of a TRBC2*01 constant region chain. In one embodiment, the TCR2 comprises a TCR2 alpha chain comprising the use of TRAV8-1*01 and TRAJ37*01 variable region chains, and a TCR2 beta chain comprising the use of TRBV2*01, TRBD1*01 and TRBJ2-7*01 variable region chains and the use of a TRBC2*01 constant region chain.

[0026] In one embodiment, the TCR3 alpha chain comprises the use of TRAV13-2*01 and TRAJ5*01 variable region chains. In one embodiment, the TCR3 beta chain comprises the use of TRBV5-6*01, TRBD1*01 and TRBJ2-2*01 variable region chains and the use of a TRBC2*01 constant region chain. In one embodiment, the TCR3 comprises a TCR3 alpha chain comprising the use of TRAV13-2*01 and TRAJ5*01 variable region chains, and a TCR3 beta chain comprising the use of TRBV5-6*01, TRBD1*01 and TRBJ2-2*01 variable region chains and the use of a TRBC2*01 constant region chain.

[0027] In one embodiment, the TCR4 alpha chain comprises the use of TRAV4*01 and TRAJ43*01 variable region chains. In one embodiment, the TCR4 beta chain comprises the use of TRBV11-2*01 and TRBJ2-7*01 variable region chains and the use of a TRBC2*01 constant region chain. In one embodiment, the TCR4 comprises a TCR4 alpha chain comprising the use of TRAV4*01 and TRAJ43*01 variable region chains, and a TCR4 beta chain comprising the use of TRBV11-2*01 and TRBJ2-7*01 variable region chains and the use of a TRBC2*01 constant region chain.

[0028] In one embodiment, the TCR5 alpha chain comprises the use of TRAV4*01 and TRAJ9*01 variable region chains. In one embodiment, the TCR5 beta chain comprises the use of TRBV11-2*01, TRBD2*02, and TRBJ1-1*01 variable region chains and the use of a TRBC1*01 constant region chain. In one embodiment, the TCR5 comprises a TCR5 alpha chain comprising the use of TRAV4*01 and TRAJ9*01 variable region chains, and a TCR5 beta chain comprising the use of TRBV11-2*01, TRBD2*02, and TRBJ1-1*01 variable region chains and the use of a TRBC1*01 constant region chain.

[0029] In one embodiment, the TCR6 alpha chain comprises the use of TRAV38-1*01 and TRAJ41*01 variable region chains. In one embodiment, the TCR6 beta chain comprises the use of TRBV28*01, TRBD1*01, and TRBJ2-3*01 variable region chains and the use of a TRBC2*01 constant region chain. In one embodiment, the TCR6 comprises a TCR6 alpha chain comprising the use of TRAV38-1*01 and TRAJ41*01 variable region chains, and a TCR6 beta chain comprising the use of TRBV28*01, TRBD1*01, and TRBJ2-3*01 variable region chains and the use of a TRBC2*01 constant region chain.

[0030] In one embodiment, the TCR7 alpha chain comprises the use of TRAV38-1*01 and TRAJ29*01 variable region chains. In one embodiment, the TCR7 beta chain comprises the use of TRBV6-6*02 and TRBJ2-1*01 variable region chains and the use of a TRBC2*01 constant region chain. In one embodiment, the TCR7 comprises a TCR7 alpha chain comprising the use of TRAV38-1*01 and TRAJ29*01 variable region chains, and a TCR7 beta chain comprising the use of TRBV6-6*02 and TRBJ2-1*01 variable region chains and the use of a TRBC2*01 constant region chain.

[0031] In one embodiment, the TCR8 alpha chain comprises the use of TRAV21*01 and TRAJ31*01 variable region chains. In one embodiment, the TCR8 beta chain comprises the use of TRBV2*01, TRBD2*01, and TRBJ2-7*01 variable region chains and the use of a TRBC2*01 constant region chain. In one embodiment, the TCR8 comprises a TCR8 alpha chain comprising the use of TRAV21*01 and TRAJ31*01 variable region chains, and a TCR8 beta chain comprising the use of TRBV2*01, TRBD2*01, and TRBJ2-7*01 variable region chains and the use of a TRBC2*01 constant region chain.

[0032] In certain embodiments, the variable domains of the TCR alpha or beta chains may be fused to a non-TCR polypeptide. Exemplary alpha and beta chain variable domains may be used to form soluble TCRs capable of binding MAGE-A4-derived peptides in the context of an HLA molecule.

[0033] The TCR of the present invention may be an α-β heterodimer having an α chain TRAC constant domain sequence and a β chain TRBC1 or TRBC2 constant domain sequence. The α and β chain constant domain sequences may be modified by truncation or substitution to delete the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2, and / or the α and / or β chain constant domain sequences may be modified by substitution of cysteine residues, for example, substitution of Thr48 of TRAC and Ser57 of TRBC1 or TRBC2 with cysteines that form a non-native disulfide bond, to form a non-native disulfide bond between the α and β constant domains of the TCR.

[0034] The TCRs of the present invention may be in single chain formats of the Va-L-vβ, vβ-LV, Va-Ca-L-vβ type, where V and vβ are the TCR and β variable regions, respectively, C is the TCR and β constant region, respectively, and L is a linker sequence. Soluble TCRs may be in a single chain format in which the α and β variable domains are linked by a linker. Soluble TCRs may be fused or linked to a therapeutic or imaging agent.

[0035] The TCRs of the present invention may also include one or more conservative substitutions that have a similar amino acid sequence and / or retain the same function. Those skilled in the art recognize that various amino acids have similar properties and are therefore "conservative." One or more such amino acids of a protein, polypeptide, or peptide can often be substituted by one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide. Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, it is preferred to use glycine and alanine to substitute for one another (because they have relatively short side chains) and valine, leucine, and isoleucine to substitute for one another (because they have more hydrophobic aliphatic side chains). Other amino acids that can often be substituted for one another include phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartate and glutamate (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions. Thus, the present invention extends to the use of TCRs that include the amino acid sequences described above, but that have one or more conservative substitutions in the sequence.

[0036] Exemplary TCRs and corresponding sequences are provided in Tables 1a and 1b, respectively.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3]

[0040] [Table 4]

[0041] [Table 5]

[0042] [Table 6]

[0043] [Table 7]

[0044] [Table 8]

[0045] [Table 9]

[0046] The TCR α or β variable domain may comprise a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences set forth in Table 2. The TCR β chain may comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any of the sequences set forth in SEQ ID NOs: 46-56. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues at the C- or N-terminus of any of the sequences set forth in SEQ ID NOs: 35-56 of Table 2 and Table 1B may be truncated or removed.

[0047] Although recognition of the target peptide in the context of an HLA is required for efficacy, for safety purposes, in some embodiments it is preferred that the TCR lack cross-reactivity with structurally similar peptides or other allotypic HLA molecules when presented by HLA-A*02:01. The cross-reactivity and alloreactivity of exemplary TCRs described herein are provided in the Examples. Thus, an exemplary TCR not only recognizes a MAGE-A4 peptide in the context of HLA-A*02:01 expressed on tumor cells and is capable of activating T cells recombinantly expressing the TCR against tumor cells, but also fails to activate or only minimally activates the recombinant T cells when the peptide is presented in the context of HLA-A*02:01 or other HLA molecules expressed on normal tissues.

[0048] Further embodiments of the invention include nucleic acids encoding the TCR alpha variable domains, TCR beta variable domains, or TCR alpha and TCR beta variable domains described herein. In certain embodiments, the nucleic acid encodes one or more of the alpha or beta variable domains set forth in Table 2. In certain embodiments, the nucleic acid encodes both the alpha and beta variable domains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, or TCR8. In preferred embodiments, the nucleic acid encoding the TCR alpha chain variable domains, TCR beta chain variable domains, or TCR alpha and beta chain variable domains is an expression vector, and the TCR alpha chain variable domains, TCR beta chain variable domains, or TCR alpha and beta chain variable domains are operably linked to a promoter.

[0049] The TCR α and β variable domains can be co-transcribed from the same promoter. In embodiments where the α and β variable domains are linked in a fusion protein, these domains can be co-translated in a single polypeptide. In embodiments where the α and β domains are in separate polypeptides, it is useful to include an internal ribosome entry site (IRES) between the α and β variable domain coding regions in the expression vector.

[0050] Also provided herein are nucleic acids encoding the TCR alpha chain, TCR beta chain, or TCR alpha and beta chains described herein. In certain embodiments, the nucleic acid encodes one or more of the alpha or beta chains described in Table 1. The encoded alpha or beta chain can be full-length or mature. If mature, i.e., lacking the natural leader sequence associated with that alpha or beta chain, the nucleic acid encoding a signal or leader sequence is preferably operably linked to the nucleic acid encoding the alpha or beta chain such that, upon translation, the leader sequence directs the alpha or beta chain to the endoplasmic reticulum.

[0051] In certain embodiments, the nucleic acid encodes both the alpha and beta chains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, or TCR8. In preferred embodiments, the nucleic acid encoding the TCR alpha, TCR beta, or TCR alpha and beta chains is an expression vector, and the TCR alpha, TCR beta, or TCR alpha and beta chains are operably linked to a promoter.

[0052] The TCR α and β chains can be co-transcribed from the same promoter, and in such embodiments, it is useful to include an internal ribosome entry site (IRES) between the α and β chain coding regions in the expression vector.

[0053] 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 TCR α and / or β chains. In certain embodiments, the expression vector encodes one or more cytokines. In a preferred embodiment, the cytokine is a T cell growth factor such as IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21, along with combinations thereof. Because cytokines can have systemic effects, when using an expression vector encoding a cytokine 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 to express IL-12 is described in U.S. Pat. No. 8,556,882.

[0054] Provided herein are cells recombinantly expressing exemplary TCRs described herein. The recombinant cells may comprise one or more expression vectors encoding and expressing a TCR alpha chain, a TCR beta chain, a TCR alpha and beta chain, a TCR alpha variable domain, a TCR beta variable domain, or a TCR alpha and beta variable domain. In preferred embodiments, the cells recombinantly express TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, or TCR8. In certain embodiments, the cells further express one or more recombinant cytokines. In preferred embodiments, the cytokine is IL-12 or a variant thereof, and the expression is controlled by an inducible promoter, e.g., an NFAT-driven promoter.

[0055] In certain embodiments, cells are obtained from a sample taken from a cancer patient. Cells, such as T cells or NKT cells, are isolated from the sample and expanded. In certain embodiments, progenitor cells are isolated and matured into the desired cell type. The cells are transfected / transformed with one or more vectors, e.g., lentiviral vectors, encoding components of a TCR along with any additional polypeptides, e.g., IL-12 or a variant thereof. Such cells can be used in adoptive cell therapy for the cancer patient from whom they were derived.

[0056] In other embodiments, the cell line recombinantly expresses a soluble TCR, which may be a fusion protein with an anti-CD3 antigen binding protein, such as an scFv.

[0057] Provided herein are methods for treating diseases or disorders in which cells associated with the disease or disorder express MAGE-A4. In a preferred embodiment, the cells present the MAGE-A4-derived peptides KVLEHVVRV and / or GVYDGREHTV in the context of an HLA class I molecule, preferably HLA-A2, particularly HLA-A*02:01. Exemplary diseases or disorders that can be treated with the soluble TCRs or recombinant cells of the present invention include hematological or solid tumors. Such diseases and disorders include, but are not limited to, lung cancer, ovarian cancer, squamous cell lung cancer, melanoma, breast cancer, gastric cancer, testicular cancer, head and neck cancer, uterine cancer, esophageal cancer, bladder cancer, and cervical cancer. Preferred diseases and disorders include non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), bladder cancer, esophageal cancer, or ovarian cancer.

[0058] In certain treatments, tumor biopsies are tested for expression of MAGE-A4. Tumors can also be tested for expression of the appropriate HLA molecules that are recognized by the TCRs of the invention when presented with MAGE-A4-derived peptides. Patients whose tumors express MAGE-A4 and are of the appropriate HLA haplotype can be administered the soluble TCRs or recombinant cells of the invention. [Example]

[0059] The following examples, both actual and prophetic, are provided for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit its scope.

[0060] Example 1 - MAGE-A4 is expressed across a wide range of solid tumors, with very limited expression in normal tissues TCGA and Applicant's data demonstrate that MAGE-A4 mRNA has a high incidence across a wide range of solid tumors (Figure 1A). Importantly, Applicant's internal body map data demonstrate very restricted normal tissue expression of MAGE-A4, with the exception of the immune-privileged site, the testis (Figure 1B). MAGE-A4 IHC data in squamous non-small cell lung cancer (NSCLC) (also known as squamous cell carcinoma) demonstrate that within tumors, MAGE-A4 protein is expressed in the majority (60-100%) of tumor cells, but not in stromal cells (Figure 1C). Presentation of both target MAGE-A4 peptides was identified in U266B1 cells by MHC MS and confirmed in squamous NSCLC tumors (Figure 2). The MAGE-A4 peptide GVYDGREHTV (SEQ ID NO: 1) corresponds to amino acid residues 230-239 of the MAGE-A4 protein. KVLEHVVRV (SEQ ID NO: 2) corresponds to amino acid residues 286 to 294 of the MAGE-A4 protein.

[0061] MAGE-A4 is expressed in a wide range of cancer types. Solid tumor indications with MAGE-A4 pMHC expression (MAGE-A4-HLA-A*02:01) include, but are not limited to, approximately 24.9% of squamous cell lung cancer (squamous non-small cell lung cancer, LUSC), 17.7% of head and neck squamous cell carcinoma (HNSCC), 14.5% of urothelial bladder cancer (BLCA), 14.3% of esophageal cancer, 14.1% of ovarian cancer, 9.8% of triple-negative breast cancer (TNBC), 7.3% of gastric cancer (STAD), 4.9% of rectal adenocarcinoma (READ), 4.5% of lung adenocarcinoma (LUAD), 2.2% of colon adenocarcinoma (COAD), and 2% of hepatocellular carcinoma (LIHC) (Figure 3). The pMHC target frequency (%) was calculated by multiplying the MAGE-A4 mRNA expression frequency by the HLA-A*02:01 carrier frequency in the United States (0.41). The patient population in a specific cancer indication was estimated based on the pMHC target frequency (%) multiplied by the new cases per year in the US population. Using the TCGA public dataset of RNA-seq data from tumors of interest, the MAGE-A4 mRNA expression frequency in each tumor indication was estimated with a threshold of MAGEA4 >= 1 FPKM (Figure 3). SEER, EPIC Oncology New Patients, or Epiphany / Epic in 2020 were used to estimate disease incidence (new cases per year) in selected tumor indications, thus deriving the estimated treatable patient population range (Figure 3). HLA-A*02:01 is one of the most common MHC class I alleles in the United States. The estimated HLA-A*02:01 haplotype (carrier) frequency in the US population is 0.41 (www.allelefrequencies.net). The largest patient population is squamous non-small cell lung cancer, followed by HNSCC, bladder cancer, esophageal cancer, and ovarian cancer (Figure 3).

[0062] Example 2 – Identification of MAGE-A4 pMHC-specific TCRs The process for identifying and selecting lead clinical TCR candidates is outlined below. First, 101 dominant MAGE-A4 pMHC-specific TCRs targeting MAGEA4 peptide epitopes were identified using 72 healthy HLA-A*02:01+ donors. Using Jurkat activation assays, 10–11 TCR candidates were selected for each target peptide. Based on these TCR sequences, TCR-T cells were generated for each donor by transducing primary pan-T cells isolated from three donors with lentiviruses carrying individual TCRs. These TCR-T cells were further evaluated by various functional assays, including potency (cytotoxicity) tests on T2 cell lines and multiple cancer cell lines pulsed with the target peptide, a cross-reactivity screen with similar peptides, and an alloreactivity screen. Based on these functional data, we further narrowed down the candidates to the top two TCRs. To enhance in vivo efficacy, all TCRs were produced in a TCR-T-IL12 lentiviral construct, in which IL12 payload expression was controlled by TCR activation under an NFAT response element-driven promoter. Thus, IL12 could only be produced when TCR-T cells bound to their pMHC targets in the tumor.

[0063] MAGE-A4 pMHC-specific TCRs can be identified from rare T cell clones in healthy donor PBMCs Difficulties in identifying tumor antigen-specific TCRs have hindered the development of TCR-mediated immunotherapy. Despite these challenges, a TCR discovery platform capable of identifying tumor antigen pMHC-specific TCRs from rare T cell clones in healthy donors is described herein. The frequency of MAGEA4 pMHC-reactive T cells in PBMCs from healthy HLA-A*02:01+ donors was very low, generally approximately 0% dextramer+ T cells. Dextramer (Dex) is a multimer of peptide-MHC complexes that can specifically bind to TCRs and can therefore be used to isolate antigen (pMHC)-specific T cells. To initially expand rare tumor antigen-specific T clones, we used PBMCs from 72 healthy HLA-A*02:01+ donors to isolate T cells and autoantigen-presenting cells (APCs), such as monocyte-derived dendritic cells and activated B cells. When T cells were cocultured with autologous APCs pulsed with target peptides, these T cells underwent multiple stages of ex vivo stimulation, resulting in tumor antigen pMHC-specific priming, restimulation, and expansion of pMHC-specific T cells. After three to four rounds of antigen restimulation, the MAGE-A4 pMHC-specific T cell population was enriched and verified by both dextramer-PE and dextramer-APC staining (Figure 4). Dex+ T cells were then sorted for single-cell RNA sequencing to identify the sequences of TCRα and TCRβ chains. Furthermore, the sorted Dex+ CD8+ T cells were verified for antigen-specific IFNγ production by ELISPOT assay using peptide-loaded T2 cells (Figure 4). This TCR discovery platform identified 101 dominant MAGE-A4 pMHC-specific TCRs from 72 healthy HLA-A*02:01+ donors. Importantly, TCRs identified from the blood of healthy donors, unlike affinity-enhanced TCRs or bispecific antibodies, have undergone thymic natural selection in humans (in the medulla of the thymus) to eliminate autoreactive TCRs. Therefore, the risk of off-target reactivity for the TCRs of the present invention is hypothesized to be quite low, which was confirmed by our safety assessment assays (described below).

[0064] Selection of top MAGEA4 pMHC-specific TCR-T cells From 101 dominant MAGE-A4 pMHC-specific TCRs identified from a screen of 72 healthy HLA-A*02:01+ donors, 20 TCR candidates were selected by Jurkat activation assay (Figure 5). Lentiviruses carrying individual TCRs were transduced into a Jurkat TCR KO reporter cell line expressing Renilla luciferase, controlled by TCR activation under an NFAT response element-driven promoter. The antigen-specific activity of individual TCRs was measured as the fold change in luciferase activity in the presence of MAGE-A4 peptide-loaded T2 cells compared to T2 cells with vehicle alone (Figure 5).

[0065] Example 3 - Verification of the efficacy of TCR-T-IL12 cells We further engineered 20 TCRs (10 targeting the MAGEA4 KVLEHVVRV epitope and 10 targeting the GVYDGREHTV epitope) in a TCR-T-IL12 lentiviral construct, in which IL12 payload expression is controlled by TCR activation under an NFAT response element-driven promoter (Figure 6A). Thus, when TCR-T-IL12 cells bind to pMHC targets within tumors, IL12 is produced upon TCR signaling, which primarily limits IL12 secretion within tumors. The transduction efficiency of TCRs into primary human T cells during TCR-T cell generation was measured by flow cytometry (Figure 6B). These TCR-T cells were further evaluated by various functional assays. First, the potency of each TCR-T was assessed by using a T2 / peptide cytotoxicity assay (MAGE-A4 peptide), including peptide titration and E:T (effector:target cell ratio) titration assay (Figure 7A-C). T2 is a TAP-deficient cell line expressing HLA-A*02:01. Because the T2 cell line lacks the transporter for endogenous peptides restricted to MHC class I for entry into the ER and primarily presents exogenous peptides, specific recognition of peptides (e.g., restricted to HLA-A*02:01) by TCR-T was studied using a T2 / peptide cytotoxicity assay (a cytolytic activity measurement using a T2 cell line loaded with a peptide of interest). The average potency of the top eight TCRs was identified based on EC50 and is shown in Figure 7C. All of the top eight TCR-IL12 cells exhibited an EC90 of 10 or higher in the T2 / peptide cytotoxicity assay. -8 M efficacy criteria were met.

[0066] Example 4 – Evaluation of TCR-T IL12 cell cross-reactivity Extensive in vitro and ex vivo safety evaluation of TCR-T-IL12 cells as a human-specific HLA target precludes the use of animal models. First, for target expression, MAGE-A4 is a cancer-testis antigen with very limited normal tissue expression (expressed only in testis). Target expression was assessed by RNASeq, IHC, and mass spectrometry using normal human and tumor tissues as described above. Second, a critical safety consideration is off-target reactivity, which was assessed by T2 / peptide cytotoxicity assays using 20 similar peptides for each TCR based on their homology to their respective targets. No cross-reactivity was observed for any of the top eight TCRs, potentially supporting the merit of screening naturally occurring TCRs for candidate selection (Figure 8). The top 20 similar peptides for each of the GVYDGREHTV (SEQ ID NO: 1) and KVLEHVVRV (SEQ ID NO: 2) epitopes are shown in Tables 2 and 3, respectively. TCRs were also screened for cross-reactivity to the related CTA MAGEA8. Similar to MAGEA4, MAGEA8 is aberrantly expressed in a variety of tumors, and its expression in healthy tissues is primarily restricted to the male reproductive system (Figure 9).

[0067] [Table 10]

[0068] [Table 11]

[0069] [Table 12]

[0070] MAGEA4 and MAGEA8 share significant sequence homology. In particular, in the region of the target GVYDGREHTV (SEQ ID NO: 1) peptide targeted by TCR-T in this study, the corresponding MAGEA8 peptide GLYDGREHSV (SEQ ID NO: 123) exhibits 80% sequence identity. The KVLEHVVRV (SEQ ID NO: 2) peptide is 100% identical between both MAGEA4 and MAGEA8 proteins and therefore may not provide evidence for differential activity in cognate TCR-T. Screening of GVYDGREHTV-MHC cognate TCRs for differential activity against the two peptide epitopes revealed a >1000-fold difference in reactivity of the top TCRs previously identified (Figure 10). These data demonstrate that the top four GVYDGREHTV-MHC cognate TCRs identified herein can be used to specifically target tumors with little risk of MAGEA8 cross-reactivity. In contrast, the top KVLEHVVRV-MHC cognate TCRs will likely be useful in killing a broad range of cancerous cells that express MAGEA4, MAGEA8, or both.

[0071] Example 5 – Cytotoxicity against MAGEA4+ cancer cell lines The top eight TCRs were evaluated in TDCC assays against cancer cell lines. All eight TCRs demonstrated cytotoxicity against the MAGEA4+ cell lines U266B1 and SCaBER (Figures 11A and 11B). The EC50 values measured against both cell lines identified a consistent set of the top five TCRs, which were selected for further study. Notably, the top five TCR-Ts expressed the TCR on more than 20% of CD8+ T cells after lentiviral transduction. These TCRs were selected based on (1) potent cytotoxicity of the MAGE-A4 pMHC target, as assessed by TDCC against the T2 / MAGEA4 peptide and MAGE-A4+ cancer cell lines; (2) off-target selectivity, showing no cross-reactivity against 20 homologous peptide- and target-negative cancer cell lines; and (3) manufacturability (e.g., good TCR transduction efficiency).

[0072] The potency (cytotoxicity) of the top five TCR-T-IL12s was validated using a larger set of HLA*0201+ cancer cell lines spanning the range of MAGEA4 expression (Figure 12A). All five TCR-T-IL12s exhibited potent cytotoxicity against cancer cell lines with low MAGE-A4 expression, approximately 3.6 FPKM. All four TCRs were similarly potent, making relative ranking of the top TCRs within a single cell line difficult. However, using an aggregate EC50 ranking system against MAGEA4+ cells, TCR2 and TCR10 were the most potent, followed by TCR23, TCR24, and TCR7.

[0073] Representative cancer cell line potency data for the top five TCR-T-IL12 cells are shown in Figure 12B. Ten cancer cell lines were tested with four TCR-T-IL12 cells generated from two to three donors. TCR-T-IL12 cells demonstrated potent cytotoxicity against some cancer cell lines with low E:T EC50s. For example, the E:T EC50 (FPKM = 457.3) of TCR2 TCR-T against NCI-H1755 was an average of 0.01 across experiments using TCR-Ts generated from three different donors, demonstrating that TCR-Ts can exert significant cytotoxicity against tumor cells even when outnumbered by 100:1.

[0074] Example 6 – Non-clinical safety evaluation summary Because human-specific HLA targeting precludes the use of animal models, extensive in vitro and ex vivo safety evaluations were performed on TCR-T-IL12 cells. First, target expression was assessed using various assays, including RNA-Seq, IHC, and mass spectrometry, using normal human and tumor tissues, as described above. Because MAGE-A4 is a cancer-testis antigen, this study demonstrated very limited normal tissue expression (expressed only in testis). Second, off-target reactivity was assessed using two different strategies. The first strategy involved screening to evaluate cytotoxicity against various normal human primary cells. The second strategy involved identifying a panel of similar peptides based on sequence homology matches with the MAGE-A4 target peptide, along with a positional scanning (X-scan motif)-based strategy to identify putative cross-reactive peptides specific to each TCR. To assess potential cross-reactivity against this complete panel of similar peptides, a T2 / peptide TDCC assay was performed. The third safety assessment was alloreactivity, which was evaluated using 34 BLCLs (B lymphoblastoid cell lines) representing HLA class I alleles with high frequency in the US population, including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles.

[0075] Identification of similar peptides based on homology To assess off-target reactivity, an in silico approach was used to identify a list of peptides with high sequence similarity that could potentially cross-react with candidate TCR-T. To accomplish this, a Python script was used to first search a protein database restricted to Homo sapiens (UniProtKB / Swiss-Prot, June 2019) to generate a list of all possible peptides based on sequence identity to the target. This in silico query using GVYDGREHTV was performed using a Python script, and 137,999 decamer peptides were identified based on a 30% homology (identity) match with the target peptide. To further refine this list, criteria such as high homology matches and software such as NetMHCpan and IEDB (Immune Epitope Database) were utilized. NetMHCpan 3.0 software was used to examine the predicted binding affinity of peptides to HLA-A*02:01. We used the IEDB database (June 2019), a manually curated database of experimentally characterized immune epitopes, to examine the probability of peptides being processed and presented by the HLA-A*02:01 allele. The specific criteria used for peptide selection were: (1) all HLA-A*02:01+ peptides in the IEDB with a 40% or greater homology match (identity) to the target peptide (40 peptides), (2) all peptides with a 60% or greater homology match and a predicted HLA-A*02:01 binding affinity (IC50) of less than 5000 nM (51 peptides), and (3) all peptides with a 50% or greater homology match to the target peptide (represented by the HLA-A*02:01 allele) reported in the IEDB (53 peptides). As a result, this homology-based in silico search and filtering criteria of the human proteome database led to the identification of 144 unique peptides for screening of GVYDGREHTV TCR-23 and TCR-24.

[0076] For the MAGEA4 target peptide KVLEHVVRV, the same in silico search resulted in the identification of 155,353 nonamer peptides based on a 30% homology match to the target peptide. To further refine the peptide list, the following criteria were used: (1) all HLA-A*02:01+ peptides in the IEDB with a 40% or greater homology match (identity) to the target peptide (176 peptides), and (2) all peptides with a 60% or greater homology match and a predicted HLA-A*02:01 binding affinity (IC50) of less than 5000 nM (50 peptides). As a result, this homology-based in silico search and filtering criteria of the human proteome database led to the identification of 226 unique peptides for screening of KVLEHVVRV TCR2 and TCR10.

[0077] Identification of TCR-binding motifs using positional scanning (X-scan) and similar peptides based on X-scan-derived motifs As an orthogonal approach to identifying similar peptides, we used a positional scanning method known as X-scan. This assay uses a peptide library created by sequentially mutating each residue in the MAGE-A4 peptide to one of 19 other naturally occurring amino acids, resulting in a total of 171 peptides for the KVLEHVVRV target and 190 peptides for the GVYDGREHTV target. These peptides were synthesized and tested in T2 / peptide TDCC assays to identify X-scan-derived motifs specific to each individual TCR (Table 3). Briefly, T2 cells were pulsed with each of these peptides at a concentration of 10 μM, followed by the addition of TCR-T cells at a 1:1 E:T ratio. Cell viability was determined using the T2 / peptide TDCC assay. Amino acid substitutions were defined as essential for TCR2 / TCR10 binding, and the observed viability was less than 30% and less than 40% for TCR23 / TCR24. Corresponding search motifs were created to represent which amino acids were allowed at each position in the peptide sequence (Table 3). Underlined amino acids represent the natural residues at the corresponding positions in the peptide. Using the generated motifs, an in silico search of the human proteome (Homo sapiens-limited UniProtKB / Swiss-Prot database, including splice variants, June 2019) was performed to identify all decameric (TCR23 / TCR24) or nonameric (TCR2 / TCR10) sequences that fit the resulting motif. This approach yielded additional sets of peptides for off-target screening specific to individual TCRs (Table 3). To determine whether these sequences would result in off-target toxicity when presented on the cell surface, peptide panels for TCR2 (21) and TCR23 (1) were synthesized and tested for cell viability using the single-point T2 / peptide (10 μM) TDCC assay described above. All three donors showed cell viability greater than 70% to the full panel of peptides, indicating no off-target propensity from either TCR2 or TCR23.

[0078] [Table 13]

[0079] Cross-reactivity screen with a full panel of similar peptides A full panel of similar peptides was synthesized and tested in the T2 / peptide TDCC assay to investigate potential off-target reactivity. To identify potentially cross-reactive peptides for each TCR-T-IL12, the full panel of similar peptides was tested using a T2 / peptide TDCC screen at a high peptide concentration (10 μM). Peptides that showed a survival rate of 25% or less in at least one of three donors were considered putative cross-reactive peptides and selected for further efficacy testing.

[0080] Next, a potency screen (dose-dependent screen) was performed using a T2 / peptide titration TDCC assay against the putative cross-reactive peptides identified from the above screen (Table 4) (Figure 13A-B). The EC50 between the target peptide and the putative cross-reactive peptide was 10. 3A efficacy gap of less than 1:1 was considered a cutoff for further risk assessment. This method did not yield any putative cross-reactive peptides for TCR2 or TCR10, but identified multiple putative cross-reactive proteins for both TCR23 (6 peptides) and TCR24 (8 peptides). Possibly due to high sequence homology within the MAGE protein family, both TCR23 and TCR24 were found to have cross-reactivity with multiple type 1 MAGE family proteins, including MAGEA8, MAGEA10, and MAGEA11. While these findings merit further investigation, as they are members of the cancer-testis antigen class, their putative cross-reactivity with these MAGE family-derived peptides was not considered a significant safety risk. However, these assays also identified two potential non-MAGE family proteins with potential cross-reactivity propensity: FA12 and KI13B for TCR23 and NUCL and RBM47 for TCR24. Given the large potency gap (>100-fold) between these cross-reactive similar peptides and the lack of experimentally validated endogenous HLA-A*02:01 presentation, the relevance of these cross-reactivities to the clinical predisposition of TCR23 and TCR24 remains unclear. In conclusion, all TCRs were found to be unresponsive to similar peptides derived from the majority of the proteome. No predisposition was found for TCR2 and TCR10, whereas two putative cross-reactive peptides derived from non-CTA proteins were identified for TCR23 and TCR24. Notably, TCR2 and TCR23 showed minimal caspase 3 / 7 cleavage when cocultured with either human normal primary or iPSC-derived cells tested, and showed no apparent off-target reactivity to any of the normal cells tested, which may present highly diverse peptides (see the section below; Figure 14B,C).

[0081] [Table 14]

[0082] Evaluation of cytotoxicity against primary human normal cells Next, we evaluated the cytotoxicity of TCR-T-IL12 cells against various human primary normal cell types (without MAGE-A4 expression) representing vital organs, which served as target cells, in a T cell-mediated cytotoxicity assay. TCR2, TCR10, TCR23, and TCR24 were tested against a panel of human primary normal cells or iPSC-derived cell lines representing vital organs, including human bronchial epithelial cells (hBEpCs), human tracheal epithelial cells (hTEpCs), human dermal microvascular endothelial cells (HDMECs), keratinocytes, hepatocytes, renal proximal tubule epithelial cells (RPTECs), iPSC-derived astrocytes, cardiomyocytes, and GABAergic neurons (Figure 14A). All primary cells and iPSC-derived cell lines were derived from normal tissues of HLA-A*02:01-positive donors. Importantly, these normal cells can present a wide variety of peptides to HLA-A*02:01, serving as an assay system to evaluate a wide range of off-target effects. Mock (untransduced) T cells or T cells expressing the IL12-RFP construct (without transduced TCR) from the same donor were included as negative control effector cells. Nine normal primary cell types were evaluated for cytotoxicity, as measured by caspase 3 / 7 cleavage assay, in the presence of TCR-T cells or NFAT.IL12 T cell controls. Importantly, TCR2 and TCR23 showed minimal caspase 3 / 7 cleavage when cocultured with either the normal primary cells or iPSC-derived cells tested, compared with the IL12 T cell control, and showed no apparent off-target reactivity against any of the non-cancerous cells tested (Figure 14B, C). In contrast, the measured caspase 3 / 7 responses were dramatically higher in TCR10 and TCR24 TCR-T cells than in control T cells across all non-cancerous cell lines tested (Fig. 14B, C).

[0083] Assessment of alloreactivity using 34 BLCL strains As part of the safety evaluation, alloreactive potential was assessed using 34 BLCL (B lymphoblastoid cell lines) lines representing MHC class I alleles with high frequency (≥11%) in major US ethnicities, including 39 HLA-A, 40 HLA-B, and 23 HLA-C alleles. Alloreactive potential was assessed by cytokine (IFNγ, TNFα, and IL-12p70) and granzyme B production. No significant increases in cytokine or granzyme B responses to the 34 BLCL lines tested were observed in TCR2-transduced T cells (Figure 15A-D). Only low-level threshold responses, defined as a ≥3-fold induction of any one analyte compared to IL12-RFP control cells, were observed in TCR23 T cells in three of the 34 BLCL lines (Figure 15A-D). In contrast, greater alloreactive responses were observed with TCR10 and TCR24, with TCR24 clearly induced cytokine and granzyme B responses detected in 31 of 34 BLCLs. All four TCR-T-IL12 cells demonstrated strong cytokine and granzyme B responses to positive control U266B1 cells (HLA-A*02:01+MAGE-A4+) pulsed with MAGEA4 peptides (GVYDGREHTV or KVLEHVVRV) (Figure 15A-D).

[0084] Overall, these studies identify exemplary TCR candidates that do not present significant safety concerns based on the safety assessments of normal and alloreactivity potential that were performed.

[0085] Example 7. TCRs demonstrate potent cytotoxic activity against homologous HLA-A pMHC. TCR2 and TCR23 TCR-Ts were evaluated for potency using peptide-loaded C1R cells expressing mismatched HLA-A alleles with high sequence identity to HLA-A*02:01 (Figure 16). To generate these allelic variants of C1R, C1R cells from an HLA-A-deficient cell line were transduced to express HLA-A*02:01, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, or HLA-A*02:07. These transduced C1R cells expressing the new HLA-A2 allele were peptide-pulsed with KVLEHVVRV or GVYDGREHTV peptides before use as target cells in TDCC assays. Both TCR2 and TCR23 demonstrated cytotoxic activity against MAGEA4 peptide-loaded C1R cells expressing HLA-A*02:01 and HLA-A*02:05, and TCR23 demonstrated cytotoxic activity against HLA-A*02:06 and HLA-A*02:07, respectively. These studies suggest that the utility of TCRs may not be limited to HLA-A*02:01+ patients.

[0086] Methods and materials used in the above examples Identification of MAGE-A4 pMHC-specific TCRs by healthy donor screen Production of autologous antigen-presenting cells (APCs) Peripheral blood mononuclear cells (PBMCs) from healthy donors with HLA-A*02:01 positivity were obtained from leukopaks from AllCells or PPA. Monocytes were positively selected from PBMCs using human CD14-microbeads (Miltenyi Biotec, 130-050-201). Mature dendritic cells were obtained using the CellXVivo Human Monocyte-Derived Dendritic Cell (DC) Differentiation Kit (R&D, CDK004). Antigen-presenting B cells were generated using CD40L and IL-4 stimulation. B cells were positively selected from PBMCs using human CD19-microbeads (Miltenyi Biotec, 130-050-301). CD19+ cells were then stimulated with 0.125 μg / ml recombinant huCD40L in B cell medium, and 2 × 10 5 Cells were seeded into 24-well plates at 1 ml / well and 1 ml / ml. B cell culture medium consisted of IMDM, GlutaMax supplement medium (Gibco, 31980030) supplemented with 10% human serum (MilliporeSigma H3667-100ML), 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco, 15140-122), 10 μg / ml gentamicin (Gibco, 15750-060), and 200 IU / ml IL-4 (Peprotech, 20004100UG). Fresh B cell medium with 400 IU / ml IL-4 was added to the B cell cultures at 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 5 after B cell activation.

[0087] Ex vivo stimulation and expansion of antigen-specific T cells On day 7 after CD14+ cell isolation, immature dendritic cells were treated with 1 μM MAGE-A4 peptide (Anaspec custom peptide) along with recombinant human TNF-α. On day 9 after CD14+ cell isolation, mature dendritic cells pulsed with MAGE-A4 peptide were harvested, washed, and mixed with CD14-PBMCs at a 1:10 ratio in human T cell medium containing 10 μM MAGE-A4 peptide, 10 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), and 10 ng / ml IL-7 (Peprotech, AF20007100UG). Human T cell complete medium consisted of a 1:1 mixture of CM and AIM-V (Thermo Fisher Scientific, 12055083). CM consisted of RPMI 1640 supplemented with GlutaMAX (Gibco, 61870-036), 10% human serum (MilliporeSigma, H3667), 25 mM HEPES (Gibco, 15630-080), and 10 μg / ml gentamicin (Gibco, 15750-060). MAGE-A4-specific T cells were further expanded by one to two rounds of peptide-pulsed B cell activation every week. HuCD40L-activated B cells were collected, washed, and diluted to 1 × 10 6 B cells were seeded into 6-well plates at 4 ml / ml and 4 ml / well. 1 μM MAGE-A4 peptide was added to the B cells and incubated for 2 hours at 37°C in an incubator. The peptide-pulsed B cells were then mixed with T cells at a 1:10 ratio in human T cell medium containing 10 IU / ml IL-2 and 10 ng / ml IL-7. MAGE-A4 dextramer-positive cells were identified by flow cytometry and then sorted for TCR identification by single-cell RNA sequencing.

[0088] Selection of activated antigen-specific T cells MAGE-A4 peptide-activated antigen-specific T cells were stained with MAGE-A4 dextramer-APC and -PE for 10 minutes at room temperature in the dark, followed by CD3-FITC (Biolengend, 300440) and CD8-BV605 (BD Biosciences, 564116). Dead cell exclusion stain (Sytox blue) was purchased from ThermoFisher (Invitrogen, S34857). Cells were sorted using an Aria Fusion cell sorter (BD Biosciences, San Jose, CA). Data were analyzed after sorting using Flowjo.

[0089] ELISPOT The selected CD3+CD8+Dex+ T cells were tested for antigen-specific IFNγ production by ELISPOT assay (BD, 551849) using peptide-loaded T2 cells. 2 × 10 6 T2 cells were loaded with 10 μM MAGE-A4 peptide in human T cell complete medium at 1 ml / cell and 1 ml / well for 1-2 hours. 150 μl of human T cell complete medium and 50 μl of peptide-loaded T2 cells were added to each well of a precoated ELISPOT plate. CD3+CD8+Dex+ T cells (500 or 1000 cells) were directly sorted into each well of the ELISPOT plate. After 24 hours of incubation in a 37°C incubator, ELISPOT detection was performed. ELISPOT plates were scanned and counted using ImmunoSpot (Cellular Technology Limited, Cleveland, OH).

[0090] Single-cell RNAseq 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 for direct target enrichment, omitting the whole-transcriptome cDNA amplification step. Briefly, cells and beads carrying barcoded oligonucleotides were encapsulated into nanoliter droplets, where cells were lysed and mRNA was reverse transcribed using a poly-T primer and a barcoded template switch oligo. Nested PCR was then performed using primers and template switch oligos targeting the constant region of the human TCR. A second target enrichment PCR was performed using 13–17 cycles according to the manufacturer's suggestions, depending on the estimated cell input number. The final sequencing library was created from the fragmented PCR products ligated to Illumina sequencing adapters. Libraries were sequenced on a NextSeq 550 or MiSeq (Illumina, Inc., San Diego, CA) using 151 paired-end reads (151 × 8 × 0 × 151) to a depth of at least 5,000 reads / cell. Data were demultiplexed and analyzed with cellranger vdj (2.2.0) to obtain full-length paired TCR sequences that could be assigned to individual cells.

[0091] Cloning and transduction of TCR into Jurkat cells Candidate TCRs were produced as gene fragments. To monitor transfection or transduction, each fragment was cloned into a plasmid expression vector consisting of an MSCV promoter and IRES-driven eGFP. Successful transformants were screened by Sanger sequencing, and verified clones were maxiprepped for downstream applications. TCRs were transfected into a Jurkat TCR KO reporter cell line expressing Renilla luciferase under an NFAT-inducible promoter. Briefly, 1.5 μg of plasmid was added to 3E5 cells suspended in 10 μL of Buffer R (Thermo Fisher Scientific). Neon 1000 cells were incubated according to the manufacturer's instructions. TM Using the transfection system, cells were electroporated using a pulse voltage of 1350 V, pulse width of 20 ms, and pulse number of 2. Cells were then electroporated in a 96-well plate containing 15% heat-inactivated FBS, glutaMAX 1000 μg / ml for overnight culture in a 37°C incubator. TM The contents of the electroporation reaction were diluted in 200 μL of RPMI 1640 supplemented with penicillin / streptomycin and 4.5 g / L D-glucose.

[0092] Jurkat activation assay Antigen-presenting T2 cells (ATCC) were loaded with peptides (Anaspec customized) or vehicle alone at a range of concentrations in serum-free medium for 2 hours. After incubation, the loaded T2 cells were washed three times, resuspended in complete medium, and counted. Next, 2E5 peptide-loaded T2 cells were added directly to TCR-transfected Jurkat cells in 100 μL of complete medium. TCR-expressing Jurkat cells were co-cultured in the presence of T2 cells at 37°C for 24 hours. After incubation, the cells were transferred to a 96-well U-bottom plate, and 150 μL of FACS buffer (PBS w / o CaCl2 & MgCl2 (Corning, 21-040-CV) + 5% FBS (Gibco, 10082-147)) was added, followed by centrifugation at 400 × g for 4 minutes. The supernatant was removed, and the cells were resuspended in 50 μL of 1X Fc block in FACS buffer and incubated at 4°C for 20 minutes. αCD69-BV421 or IgG isotype-BV421 was added at a concentration of 1 μg / mL and incubated for 1 hour at 4°C. After staining, cells were washed three times by centrifugation at 400 × g for 4 minutes, followed by aspiration and resuspension. Prior to analysis, cells were suspended in FACS buffer containing Sytox Red, prepared according to the manufacturer's recommendations. Cells were analyzed using either an LSRII or Symphony cytometer (BD Biosciences) with the recommended acquisition settings. Activity of individual TCRs is shown as the percentage of cells expressing CD69 within a population of GFP-expressing Jurkat cells (indicating plasmid expression).

[0093] Generation of MAGE-A4 TCR-T cells using human primary T cells PBMCs from three healthy donors (HLA-A*02:01) were isolated from leukopaks (Allcells) 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 × 10 6The cells were resuspended in human T cell complete medium at 1000 cells / ml and stimulated with CD3 / CD28 dynabeads (Thermo Fisher Scientific, 11131D) at a T cell-to-bead ratio of 2:1 in the presence of 30 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), 10 ng / ml IL-7 (Peprotech, AF20007100UG), and 25 ng / ml IL-15 (Peprotech, AF20015100UG). T cells were then seeded at 1 ml / well in 24-well plates. On the day of TCR transduction, activated T cells (3E5) were seeded per well in a 48-well plate in complete human T cell medium and transduced with lentivirus in the presence of 8 μg / ml polybrene, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. The T cells were then spin-seeded at 32°C for 1.5 hours. After spin-seeding, 380 μl of medium containing 8 μg / ml polybrene, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15 was added to the cells for a total volume of 600 μl per well. Approximately 17–18 hours after transduction, approximately 500 μl of medium was removed without touching the cells to the bottom of the well. Cells from each well of the 48-well plate were transferred to one well of a Grex 24-well plate (WilsonWolf, P / N 80192M) in 3 ml of complete human T cell medium containing 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. Four days after transduction, Dynabeads were removed according to the manufacturer's protocol. Approximately 10 x 10 cells were cultured per well 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. 6 TCR-T cells were seeded into Grex 6-well plates (WilsonWolf, P / N 80240M) using 1000 cells. TCR-T cells were harvested 7 days post-transduction, frozen, and stored in the vapor phase of liquid nitrogen. TCR transduction efficiency was verified by dextramer binding.

[0094] Flow cytometry The following antibodies were used for T cell phenotyping: CD3-FITC (Biolegend: 300440), CD8-BV605 (BD: 564116), CD4-PE (Biolegend: 317410). The following antibodies were used for dendritic cell phenotyping: CD14-percpcy5.5 (Biolegend: 301824), CD11c-PE (Biolegend: 337206), CD1a-APC-cy7 (Biolegend: 300125), CD86-APC (BD: 555660). The following antibodies were used for B cell phenotyping: MHC class I (Biolegend: 311414), MHC class II (Biolegend: 361706), CD83-PE (BD 556855), CD86-APC (BD: 555660), and CD20-FITC (BD: 556632). Dextramer-APC or -PE was purchased from Immudex (customized dextramer). 50 nM PKI dasatinib (Axon Medchem: 1392) was used to prevent TCR internalization. TCR-expressing T cells were incubated with 50 nM PKI dasatinib for 30 minutes at 37°C, followed by dextramer staining on ice for 30 minutes and cell surface marker staining at 4°C for 15 minutes. A dead cell exclusion stain (Sytox blue, ThermoFisher / Invitrogen, S34857) was used. Flow cytometry data were analyzed using Flowjo.

[0095] T2-Luc cell death assay The functionality and cell death specificity of MAGE-A4 TCR-T were determined by T2-luc (a luciferase-expressing T2 cell line) cell death assay. T2-luc cells were harvested, washed, and cultured at 2 × 10 in T2-luc cell death assay medium (RPMI 1640-GlutaMAX, 1 × non-essential amino acid solution (Gibco, 11140-050), 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% heat-inactivated FBS (Gibco, 10082-147). 6 T2-luc cells were pulsed with the indicated peptide concentrations for 2-4 hours at 37°C. The T2-luc cells were then washed and resuspended at 1 × 10 cells / ml and seeded at 1 ml / well in a 24-well plate. 5 The cells were resuspended at 1000 cells / ml and seeded at 25 μl per well in a 384-well plate (Corning, 3570). T2-Luc cells were incubated with 25 μl of TCR-T cells at the indicated ratio of dextramer + TCR-T to T2-luc cells for 48 hours. Luminescence signals were measured by adding 30 μl of Bio-glo (Promega, G7940) and then using a Biostack Neo system (BioTek, Winooski, VT). Prior to the cell death assay, all of the various TCR-T cells were normalized to the same amount of MAGE-A4 dextramer + cells (e.g., 10%) by adding mock (untransduced) T cells. Specific lysis (% specific cell death) was calculated by normalizing TCR-T + T2 / target peptide cell death with either mock T cells + T2 / target peptide cell death or TCR-T + T2 / no peptide cell death. The formula for specific lysis is shown below: Formula for specific lysis (%) Peptide titration (MAGE-A4 peptide and similar peptides): {1 - (TCRT+T2-luc / test peptide RLU) / (TCRT+T2-luc or C1R-luc / no peptide RLU)} x 100 Cancer cell line cell death: {1-(TCRT + cancer cell line RLU) / (cancer cells, cancer cells + mock, or cancer cells + RFP-IL12 T cell control)} x 100

[0096] Cancer cell death assay The cytotoxicity of TCR-T cells against MAGE-A4 positive and negative cancer cell lines was determined by a cancer cell death assay. Cancer cells were harvested, washed, and then cultured at 1 × 10 in cancer cell death assay medium (RPMI 1640-GlutaMAX, 1 × non-essential amino acid solution (Gibco, 11140-050), 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 10% heat-inactivated FBS (Gibco, 10082-147). 5 Cancer cells were then seeded at 25 μl / well in a 384-well plate and incubated with 25 μl of TCR-T cells at the indicated ratio of dextramer + TCR-T to T2-luc cells for 48 hours. After incubation, for adherent cancer cells, the suspension T cells were removed and washed with Ca using a plate washer. 2+ Mg 2+The wells were washed with DPBS (Corning, 21-031-CM) containing 100 μL of PBS. 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. A 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, Carlsbad, CA, USA). 1 × 10 cancer cells were added to serum-free RPMI medium containing Celltrace far red (dilution 1:4000). 6 Cells were resuspended at 1000 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. A dead cell exclusion stain (Sytox blue, ThermoFisher / Invitrogen, S34857) was used. Specific lysis (% specific cell death) was calculated by normalizing TCR-T cell death against the cancer cell line by mock T cell death against the cancer cell line. The formula for specific lysis is shown above.

[0097] Screen for similar peptides The functional specificity of MAGE-A4 TCR-T was determined using a cell death assay induced by T2luc T cells. Peptides, including target and analog peptides, were synthesized by JPT (Berlin, Germany) or AnaSpec (Fremont, CA). T2luc cells were incubated with the target-specific peptides and analog peptides in assay medium (RPMI 1640 supplemented with 5% heat-inactivated FBS (MilliporeSigma), 1x GlutaMax (Gibco), 1x non-essential amino acid solution (Hyclone), 10 mM HEPES (Hyclone), 50 μM 2-β-mercaptoethanol (Gibco), and 1 mM sodium pyruvate (Gibco)) at final peptide concentrations of 1.0E-05M to 6.0E-16M (potency) or 1.0E-05M (single point) for 2 h at 37°C / 5% CO2. Frozen MAGE-A4 TCR-T and mock cells were thawed, washed, and incubated for 3 hours in 50 / 50 RPMI / AIM-V / 5% huAB serum, 1x GlutaMax, 25 mM HEPES, 100 μg P / S (Gibco), and 10 μg / mL gentamicin (Gibco) prior to assay setup. MAGE-A2 TCR-T cells were washed three times in assay medium and resuspended at 2.5E06 cells / mL. Peptide-loaded T2luc cells were added to a white, clear-bottom, 384-well assay plate (Costar) at 2,000 cells / 25 μL using a Bravo liquid handling system (Agilent). MAGE-A4 TCR-T cells were prepared by diluting MAGE-A4 dextramer-positive cells with mock T cells to obtain a target:effector ratio of 10:1; 20,000 cells / 25 μL (final 1:1 Dex + T cells:T2luc). T2luc-pulsed cells and TCR-T cells were incubated at 37°C / 5% CO for 48 hours. T2luc cell viability was determined using the Bio-GLo Luciferase Assay System (Promega, G7940) according to the manufacturer's recommendations. Luminescence was detected using an EnVision Multilable Plate Reader (Perkin Elmer).Viability was calculated using the following formula: % Viability = (raw RLU value of sample / mean DMSO control RLU) x 100. EC50 was determined using GraphPad Prism (nonlinear regression curve fit analysis).

[0098] Normal human primary cell culture The sources of human primary normal cells and iPSC-derived cells are summarized in Table 5. The culture conditions for these cells are also summarized in Table 5. Primary cells were thawed and cultured according to the supplier's instructions, with the following exceptions: cardiomyocytes, astrocytes, GABAergic neurons, and RPTECs were switched to RPMI 1640 medium immediately before the initiation of coculture. Previous optimization studies demonstrated the tolerability and improved cell viability of RPMI 1640 for these cell types. All cells were counted and assessed for viability prior to the assay.

[0099] [Table 15]

[0100] [Table 16]

[0101] [Table 17]

[0102] Cytotoxicity assay using human primary normal cells Target cell cytotoxicity was assessed using a phase-contrast / fluorescence dynamic imaging assay. Fluorescent caspase 3 / 7 cleavage was measured over time using an IncuCyte® live imaging device and overlaid on phase-contrast images capturing cell confluence. Prior to performing the cytotoxicity assay, different plating densities and the tolerance to various culture media were evaluated to achieve adequate confluence without significant cell overlap in 96-well plates. Target cells (100 μl) were added at the densities listed in Table 3 to black 96-well ViewPlates containing 50 μl of MAGE-A4 TCR-T-IL12 cells, IL-12-RFP T cells, or mock T cells at a dextramer-normalized effector:target (E:T) ratio of 1:1, taking into account the dextramer positivity of each TCR-T construct. CellEvent Caspase 3 / 7 Reagent (50 μl) was added according to the manufacturer's instructions (ThermoFIsher, C10423). The assay plate was placed in a 37°C, 5% CO2 incubator equipped with an IncuCyte® S3. Starting at time 0, phase-contrast and fluorescence images (5 fields) using a 10x objective were collected every 4 hours for 44 or 48 hours and analyzed for total integrated caspase 3 / 7 intensity using IncuCyte® 2019B software. Because T cells are generally smaller than target cells, a minimum area filter of 200 μm2 was set in the fluorescence images to exclude signals from apoptotic T cells. Furthermore, because the fluorescence signal within target cells was not uniform, target cells could be recognized as smaller splits and excluded by the area filter. Therefore, a low edge detection sensitivity was also applied during analysis. After 44 or 48 hours, the plate was removed from the incubator, and 50 μL of cell culture medium was removed from each well for cytokine analysis.

[0103] Alloreactivity Screen Alloreactive potential was assessed by co-culturing each of the four TCR-T cells with 34 BLCL (B lymphoblastoid cell lines) lines representing 39 HLA-A, 40 HLA-B, and 23 HLA-C alleles. BLCL were purchased from Fred Hutchinson Cancer Research Institute (Fred Hutch; Seattle, WA) and Astarte Biologics (Cellero; Bothell, WA) as listed in Table 7. BLCL were cultured in 15% FBS-complete RPMI containing L-glutamine, 15% (v / v) HI-FBS, and 1 mM sodium pyruvate in RPMI-1640. MAGE-A4+HLA-A*02:01+ cells were co-cultured with U266B1 cells (ATCC; 10 in medium) as a positive control cell line by incubation at 37°C for 2 hours. 5Cells (1000 cells / ml) were pulsed with 50 μM MAGE-A4 peptide. TCR-T cells from donor 12665 were thawed by adding culture medium, centrifuged at 400 × g for 5 minutes at 4°C, resuspended in 10 ml of culture medium, and counted. TCR-T cells were co-cultured with either BLCL or peptide-pulsed U266B1 cells in a volume of 200 μl. The dextramer-normalized effector:target ratios of the four TCR-T cells ranged from 3:1 to approximately 8:1 depending on their respective dextramer positivity. All co-cultures were performed for 48 hours in 96-well flat-bottom tissue culture plates at 37°C and 5% CO2. After incubation, the 96-well plates were centrifuged at 887 × g for 1 minute at 4°C, and the supernatants were collected into 96-well V-bottom plates for cytokine analysis. Cytokines and granzyme B were assessed by Luminex assay using a custom Milliplex Human Cytokine / Chemokine Kit (Millipore, SRP1885) containing IFNγ, granzyme B, TNFα, and IL-12p70 analytes according to the manufacturer's instructions. Serial dilutions of standard analytes were performed in replicates on each assay plate. Luminex plates were read on a FlexMap 3D instrument (XMAP technologies). Data were exported by xPONENT software and directly analyzed by EMD Millipore's Milliplex Analyst software, which generated standard curves using a five-parameter logistic nonlinear regression fitting curve. Detection limits (minimum and maximum) were calculated by the Milliplex Analyst software as a result of averaging the appropriate replicate standard curve values obtained from each assay plate, indicating the range within which the analyte could be interpolated from the standard. Samples were run at appropriate dilutions to ensure that sample analyte levels were measured within the assay standard curve limits. Cytokine and granzyme B levels were reported in pg / mL or as fold-difference compared to IL12-RFP T cells (control) and graphed with GraphPad Prism software.

[0104] [Table 18]

[0105] Table 19

Claims

1. An expression vector comprising nucleic acid sequences encoding a T cell receptor (TCR) α chain and a TCRβ chain, wherein the TCRα chain and TCRβ chain are a. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 3, 5, and 7, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 4, 6, and 8, respectively; b. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 13, 15, and 17, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 14, 16, and 18, respectively; c. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 23, 25, and 27, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 24, 26, and 28, respectively; d. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 33, 35, and 37, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 34, 36, and 38, respectively; e. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 43, 45, and 47, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 44, 46, and 48, respectively; f. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 53, 55, and 57, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 54, 56, and 58, respectively; g. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 63, 65, and 67, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 64, 66, and 68, respectively; and h. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 73, 75, and 77, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 74, 76, and 78, respectively. An expression vector selected from the group consisting of the following.

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

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 TCRα chain and TCRβ chain are, a. The amino acid sequence described in SEQ ID NO: 9 or 10 and the amino acid sequence described in SEQ ID NO: 11 or 12; b. The amino acid sequence described in SEQ ID NO: 19 or 20 and the amino acid sequence described in SEQ ID NO: 21 or 22; c. The amino acid sequence described in SEQ ID NO: 29 or 30 and the amino acid sequence described in SEQ ID NO: 31 or 32; d. The amino acid sequence described in SEQ ID NO: 39 or 40 and the amino acid sequence described in SEQ ID NO: 41 or 42; e. The amino acid sequence described in SEQ ID NO: 49 or 50 and the amino acid sequence described in SEQ ID NO: 51 or 52; f. The amino acid sequence described in SEQ ID NO: 59 or 60 and the amino acid sequence described in SEQ ID NO: 61 or 62; g. The amino acid sequence described in SEQ ID NO: 69 or 70 and the amino acid sequence described in SEQ ID NO: 71 or 72; and h. The amino acid sequence described in SEQ ID NO: 79 or 80 and the amino acid sequence described in SEQ ID NO: 81 or 82 The expression vector according to claim 1, comprising an amino acid sequence selected from the group consisting of the following.

7. Cells expressing recombinant T cell receptor (TCR), wherein the TCR comprises a TCRα chain and a TCRβ chain, and the TCRα chain and TCRβ chain are a. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 3, 5, and 7, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 4, 6, and 8, respectively; b. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 13, 15, and 17, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 14, 16, and 18, respectively; c. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 23, 25, and 27, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 24, 26, and 28, respectively; d. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 33, 35, and 37, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 34, 36, and 38, respectively; e. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 43, 45, and 47, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 44, 46, and 48, respectively; f. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 53, 55, and 57, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 54, 56, and 58, respectively; g. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 63, 65, and 67, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 64, 66, and 68, respectively; and h. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 73, 75, and 77, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 74, 76, and 78, respectively. Cells selected from a group consisting of the following.

8. The TCRα chain and TCRβ chain are, a. The amino acid sequence described in SEQ ID NO: 9 or 10 and the amino acid sequence described in SEQ ID NO: 11 or 12; b. The amino acid sequence described in SEQ ID NO: 19 or 20 and the amino acid sequence described in SEQ ID NO: 21 or 22; c. The amino acid sequence described in SEQ ID NO: 29 or 30 and the amino acid sequence described in SEQ ID NO: 31 or 32; d. The amino acid sequence described in SEQ ID NO: 39 or 40 and the amino acid sequence described in SEQ ID NO: 41 or 42; e. The amino acid sequence described in SEQ ID NO: 49 or 50 and the amino acid sequence described in SEQ ID NO: 51 or 52; f. The amino acid sequence described in SEQ ID NO: 59 or 60 and the amino acid sequence described in SEQ ID NO: 61 or 62; g. The amino acid sequence described in SEQ ID NO: 69 or 70 and the amino acid sequence described in SEQ ID NO: 71 or 72; and h. The amino acid sequence described in SEQ ID NO: 79 or 80 and the amino acid sequence described in SEQ ID NO: 81 or 82 The cell according to claim 7, comprising an amino acid sequence selected from the group consisting of the following.

9. The cell according to claim 7 or 8, further expressing recombinant IL-12 or a functional variant thereof.

10. A cell comprising the expression vector described in claim 1.

11. A cell that is a T cell, according to any one of claims 7, 8, and 10.

12. The cell according to claim 11, wherein the TCR binds to the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 in relation to HLA-A*0201, and the binding causes the cell to activate the production of IFNγ, TNFα, IL-12, or granzyme B.

13. A pharmaceutical composition comprising a therapeutically effective amount of the cells described in any one of claims 7, 8, and 10.

14. A pharmaceutical composition comprising a therapeutically effective amount of the cells described in Claim 11.

15. A method for producing cells according to any one of claims 7, 8, and 10, comprising introducing an expression vector into cells comprising nucleic acid sequences encoding a TCRα chain and a TCRβ chain, wherein the TCRα chain and the TCRβ chain are a. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 3, 5, and 7, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 4, 6, and 8, respectively; b. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 13, 15, and 17, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs: 14, 16, and 18, respectively; c. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 23, 25, and 27, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 24, 26, and 28, respectively; d. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 33, 35, and 37, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 34, 36, and 38, respectively; e. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 43, 45, and 47, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 44, 46, and 48, respectively; f. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 53, 55, and 57, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 54, 56, and 58, respectively; g. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 63, 65, and 67, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 64, 66, and 68, respectively; and h. TCRα chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 73, 75, and 77, respectively, and TCRβ chains containing CDR1, 2, and 3 sequences containing the amino acid sequences described in SEQ ID NOs. 74, 76, and 78, respectively. A method selected from the group consisting of the following.

16. The TCRα chain and TCRβ chain are, a. The amino acid sequence described in SEQ ID NO: 9 or 10 and the amino acid sequence described in SEQ ID NO: 11 or 12; b. The amino acid sequence described in SEQ ID NO: 19 or 20 and the amino acid sequence described in SEQ ID NO: 21 or 22; c. The amino acid sequence described in SEQ ID NO: 29 or 30 and the amino acid sequence described in SEQ ID NO: 31 or 32; d. The amino acid sequence described in SEQ ID NO: 39 or 40 and the amino acid sequence described in SEQ ID NO: 41 or 42; e. The amino acid sequence described in SEQ ID NO: 49 or 50 and the amino acid sequence described in SEQ ID NO: 51 or 52; f. The amino acid sequence described in SEQ ID NO: 59 or 60 and the amino acid sequence described in SEQ ID NO: 61 or 62; g. The amino acid sequence described in SEQ ID NO: 69 or 70 and the amino acid sequence described in SEQ ID NO: 71 or 72; and h. The amino acid sequence described in SEQ ID NO: 79 or 80 and the amino acid sequence described in SEQ ID NO: 81 or 82 The method according to claim 15, selected from the group consisting of the following.

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

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

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

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

21. The method according to claim 19, wherein the T cell is a primary T cell.

22. The method according to claim 20, wherein the T cell is a primary T cell.

23. The method according to claim 21, wherein the primary T cells are isolated from a cancer patient.

24. The method according to claim 22, wherein the primary T cells are isolated from a cancer patient.

25. A method for treating cancer expressing MAGE-A4 or MAGE-A8, comprising administering a therapeutically effective amount of the cells described in any one of claims 7, 8, and 10 to a cancer patient.

26. The method according to claim 25, wherein the patient is examined before administration to determine the presence of cancer expressing MAGE-A4 or MAGE-A8.

27. The method according to claim 26, wherein nucleic acids encoding MAGE-A4 or MAGE-A8 are detected.

28. The method according to claim 26, wherein MAGE-A4 or MAGE-A8 protein or MAGE-A4-derived or MAGE-A8-derived peptide is detected.

29. The method according to claim 25, wherein the patient is identified as possessing the HLA-A*0201 allele.

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