Magea4-specific t cell receptor

Abstract

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

Description

Background Art

[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 has a limited target range because it only recognizes cell surface antigens that make up approximately 25% of the genome. Unlike CAR-T cells, TCR-T cells, which are engineered to express T cell receptors (TCRs) specific for tumor antigens, can utilize a wider range of targets for multiple cancer indications because TCR-T cells can recognize peptide-MHC complexes (pMHC) derived from intracellular proteins that make up approximately 75% of the genome. Intracellular proteins are processed by the major histocompatibility complex (MHC) and presented as pMHC complexes.

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

[0003] Although TCR-T cells have been shown to be a very effective and sensitive mode for tumor-specific peptide-MHC targets, TCR can recognize a variety of peptides. The DNA rearrangement required for TCR formation will produce a certain number of T cells that recognize autoantigens. During early T cell development, autoreactive T cells are negatively selected and eliminated in the medulla of the thymus by haphazardly expressing a variety of autoantigens in thymic medullary epithelial cells. This negative selection in the thymus acts as the main mechanism of central tolerance and shapes the T cell library to avoid autoimmunity. TCRs engineered to increase affinity for certain pMHCs or to introduce cross-reactivity to multiple pMHCs cannot benefit from negative selection occurring in the thymus. It is worth noting that due to the cross-reactivity to titin expressed in the myocardium, affinity-enhanced MAGE-A3 TCR-T cells cause fatal toxicity (Cameron et al., Sci Transl Med. [Science Translational Medicine] 2013 5 (197)). Summary of the invention

[0004] The identification of TCR sequences that recognize tumor-specific antigens has been shown to be very challenging in the art, particularly due to the rarity of tumor-specific T cells in patient blood, the difficulty of ex vivo amplification of very small amounts of tumor-specific T cell clones, and the potential exhaustion or suppression of tumor-specific T cells in tumor-infiltrating lymphocytes (TILs). Despite these challenges, TCR sequences specific for two MAGEA4 peptide-MHCs (KVLEHVVRV / HLA-A*02:01, GVYDGREHTV / HLA-A*02:01) identified by healthy donor blood and ex vivo stimulation methods are provided herein. As demonstrated in the examples herein, exemplary TCR-T cells that recognize tumor-specific MAGEA4pMHC can be highly effective therapeutic agents for treating MAGEA4 / HLA-A*02:01+ tumors by exerting cytotoxicity and producing cytokines. These TCR-T cell therapies will be important treatment options for a variety of cancer indications, such as non-small cell lung cancer (NSCLC).

[0005] TCR-T cells are the most effective and sensitive mode for pMHC targets in vitro. TCR-T cells provided herein also show high efficacy for even very low target expression cells. This high efficacy of TCR-T cells comes from the complex of transduced TCR and endogenous CD3 subunits. In certain embodiments, in order to enhance efficacy in vivo, exemplary TCR-T cells include activation-dependent IL12 payloads integrated into TCR-T constructs. In certain embodiments, IL12 expression is regulated by TCR activation under a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements connected to a minimal IL-2 promoter. In these embodiments, when TCR-T-IL12 cells encounter tumor antigens, IL12 is produced. This strategy has been shown to enhance the efficacy of adoptive T cell therapy in vivo, and therefore the potential clinical dose can be reduced to 1 / 100 to 1 / 10.

[0006] In a first aspect, the present invention is an expression vector comprising a nucleic acid sequence encoding a T cell receptor (TCR) α chain and a TCR β chain, wherein the TCR α chain and the TCR β chain are selected from the group consisting of:

[0007] a. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 3, 5 and 7, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 4, 6 and 8, respectively;

[0008] b. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 13, 15 and 17, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 14, 16 and 18, respectively;

[0009] c. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 23, 25 and 27, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 24, 26 and 28, respectively;

[0010] d. a TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown in SEQ ID NOs: 34, 36 and 38, respectively;

[0011] e. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 43, 45 and 47, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 44, 46 and 48, respectively;

[0012] f. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown in SEQ ID NOs: 54, 56 and 58, respectively;

[0013] g. a TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown in SEQ ID NOs: 64, 66 and 68, respectively; and

[0014] h. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:73, 75 and 77, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:74, 76 and 78, respectively.

[0015] In another aspect, the present invention is an expression vector comprising a nucleic acid sequence encoding a T cell receptor (TCR) α chain and a TCR β chain, wherein the TCR α chain and the TCR β chain are selected from the group consisting of:

[0016] a. the amino acid sequence shown in SEQ ID NO: 9 or 10, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 11 or 12;

[0017] b. the amino acid sequence shown in SEQ ID NO: 19 or 20, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 21 or 22;

[0018] c. the amino acid sequence shown in SEQ ID NO: 29 or 30, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 31 or 32;

[0019] d. the amino acid sequence shown in SEQ ID NO: 39 or 40, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 41 or 42;

[0020] e. the amino acid sequence shown in SEQ ID NO: 49 or 50, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 51 or 52;

[0021] f. the amino acid sequence shown in SEQ ID NO: 59 or 60, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 61 or 62;

[0022] g. the amino acid sequence shown in SEQ ID NO: 69 or 70, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 71 or 72; and

[0023] h. The amino acid sequence shown in SEQ ID NO:79 or 80, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO:81 or 82.

[0024] In another aspect, the invention is a cell expressing a recombinant T cell receptor (TCR), the TCR comprising a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain are selected from the group consisting of:

[0025] a. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 3, 5 and 7, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 4, 6 and 8, respectively;

[0026] b. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 13, 15 and 17, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 14, 16 and 18, respectively;

[0027] c. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 23, 25 and 27, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 24, 26 and 28, respectively;

[0028] d. a TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown in SEQ ID NOs: 34, 36 and 38, respectively;

[0029] e. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 43, 45 and 47, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 44, 46 and 48, respectively;

[0030] f. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown in SEQ ID NOs: 54, 56 and 58, respectively;

[0031] g. a TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown in SEQ ID NOs: 64, 66 and 68, respectively; and

[0032] h. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:73, 75 and 77, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:74, 76 and 78, respectively.

[0033] In another aspect, the invention is a cell expressing a recombinant T cell receptor (TCR), the TCR comprising a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain are selected from the group consisting of:

[0034] a. the amino acid sequence shown in SEQ ID NO: 9 or 10, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 11 or 12;

[0035] b. the amino acid sequence shown in SEQ ID NO: 19 or 20, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 21 or 22;

[0036] c. the amino acid sequence shown in SEQ ID NO: 29 or 30, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 31 or 32;

[0037] d. the amino acid sequence shown in SEQ ID NO: 39 or 40, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 41 or 42;

[0038] e. the amino acid sequence shown in SEQ ID NO: 49 or 50, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 51 or 52;

[0039] f. the amino acid sequence shown in SEQ ID NO: 59 or 60, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 61 or 62;

[0040] g. the amino acid sequence shown in SEQ ID NO: 69 or 70, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 71 or 72; and

[0041] h. The amino acid sequence shown in SEQ ID NO:79 or 80, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO:81 or 82. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1. MAGEA4 is a tumor-specific antigen widely expressed in a wide range of solid tumors. (A) TCGA and internal RNA-seq data of MAGEA4 mRNA expression in various cancers. (B) Body atlas RNA-seq data of MAGEA4 mRNA expression in normal human tissues. MAGEA4 expression is extremely limited to the male reproductive system in normal tissues. (C) MAGE-A4 immunohistochemistry (IHC) performed by OTI1F9 monoclonal Ab shows that within squamous NSCLC tumors, MAGE-A4 protein is expressed in most tumor cells. Representative IHC staining of squamous NSCLC tumors showed 100% of MAGE-A4 positive tumor cells and 3+ strong staining.

[0043] Figure 2 RNA-seq and mass spectrometry (MS) of NSCLC samples quantitated detectable HLA-A*02:01-bound MAGEA4 target peptide. High levels of MAGE-A4 FPKM mRNA expression were generally associated with detectable MAGEA4 target peptide presentation.

[0044] Figure 3 .Estimates of the annual patient population in the specified cancer indications. The annual treatable patient population was estimated based on the pMHC target frequency in the US population × annual new cases. The pMHC target frequency in each cancer indication was calculated by the MAGE-A4 mRNA expression frequency in the US population × the HLA-A*02:01 carrier frequency (0.41). The MAGE-A4 mRNA levels in various solid tumors (>1 FPKM) were derived from TCGA data.

[0045] Figure 4. Workflow for identification of MAGE-A4 pMHC-specific TCRs from rare T cell clones from PBMCs of healthy HLA-A*02:01+ donors. (A) MAGE-A4 pMHC-specific T cells were stimulated and expanded by co-culture with autologous APCs pulsed with MAGE-A4 peptides. 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 screening results demonstrated that positive donor A showed enriched MAGE-A4 pMHC-specific T cells after multiple ex vivo stimulations, while negative donor B was devoid of Dex+ T cells. (C) MAGE-A4 pMHC-specific T cells were sorted and antigen specificity was validated by IFNγ ELISPOT.

[0046] Figure 5. Measurement of MAGE-A4 TCR activity by Jurkat activation assay. (A) T2 cells were loaded with target MAGEA4 peptide and co-cultured with TCR / GFP transfected Jurkat cells. For KVLEHVVRV pMHC TCR, TCR potency was evaluated by quantifying CD69 upregulation on Jurkat cells. (B) Summary of measured TCR potency determined by T2 peptide titration assay.

[0047] Fig. 6. Transduction of TCR-IL12 constructs into primary human T cells. (A) TCR-T-IL12 lentiviral constructs contain TCR α chain and TCR β chain with furin cleavage site-SGSG-T2A linker under EF1α promoter, and IL12 payload under a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements connected to a minimal IL-2 promoter. (B) Summary of transduction efficiency of the first eight TCRs determined by flow cytometry analysis. T cell subset frequency (%) is expressed as the mean value of the transduction efficiency of TCR-T generated from two human donors.

[0048] Figure 7. Cytotoxic activity of MAGEA4 TCR / IL-12 T cells against peptide-loaded T2 cells. TCR-T using primary human T cells was tested using the T2 peptide titration assay. Representative T cell-dependent cytotoxicity (TDCC) assays, T2 / peptide loading assays for GVYDGREHTV pMHC (A) and KVLEHVVRVV pMHC (B) TCR-Ts are shown. T cells were graded by cytotoxic potency to identify the top 8 candidate TCRs (C).

[0049] Figure 8. The top 20 similar peptides of GVYDGREHTV and KVLEHVVRV were identified and used to evaluate TCR cross-reactivity. -5 Pre-incubated with the relevant peptides of M and co-cultured with the corresponding first 8 TCR-T for 48 hours. Representative of TCR-T generated from 2 different donors.

[0050] Fig. 9 . Sequence identity between target MAGEA4 peptides (GVY and KVL) and homologous MAGEA8 peptides. The KVLEHVVRV peptide sequence is 100% identical in MAGEA4 and MAGEA8.

[0051] Figure 10. Cross-reactivity of GVYDGREHTV-MHC specific TCR against MAGEA8 peptide. (A) Before evaluating TDCC, T2 cells were loaded with a specified concentration of MAGEA8 peptide GLYDGREHSV and incubated with TCR-T for 48h. (B) Summary of TCR efficacy data. For the first four TCRs, it was observed that the EC50 difference between MAGEA4 and MAGEA8 peptides was greater than about 1000 times. Representative of experiments performed with TCR-T generated from two donors (8316 and 12665).

[0052] Figure 11. TDCC activity of the top TCR-T against MAGEA4+HLA-A*02:01+ cancer cell lines. The top 8 TCR-T identified by T2 peptide titration potency assay were further evaluated in cancer cell killing assays. For MAGEA4+HLA-A*02:01+ cancer cell lines U266B1 (MAGEA4 FPKM 213.85) (A) and SCaBER (MAGEA4 FPKM 172) (B), highly effective cytolytic activity close to 100% specific killing was observed. The evaluated potency metrics are summarized and presented. Representatives of the experiments are shown using TCR-T generated from two donors (C and D). The MAGEA4 expression in each cell line is derived from the Cancer Cell Line Encyclopedia (CCLE) and is expressed in FPKM.

[0053] Figure 12. The top five identified TCRs were evaluated based on their potency in cancer cell line killing assays (A). Expression levels of MAGEA4 and HLA-A in cancer cell lines were derived from CCLE and expressed in FPKM. In some cell lines, the MAGEA4 peptide KVLEHVVRV bound to HLA-A*02:01 was quantified by mass spectrometry and expressed in copies per cell. The top TCR-Ts showed cytolytic activity against a large panel 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 presented in (B). Representative of experiments performed with TCR-Ts from three donors.

[0054] Figure 13. Potency determination of off-target peptides identified by similar peptide screening. Putative cross-reactive peptides (AB) of TCR23 and TCR24 were evaluated in the TDCC / T2 peptide titration assay. The viability of T2 cells loaded with the MAGEA4 target GVY peptide (GVY) was represented as a positive control. The potency gap cutoff <10 relative to the target peptide EC50 was set. 3 Peptides were considered for further risk assessment. No putative cross-reactivity risk was found in KVL-reactive TCRs. Representative of experiments performed with TCR-T from three donors.

[0055] Figure 14. Evaluation of TCR reactivity against human normal cells. (A) The first 4 TCR-T (circles) or RFP+IL12 T cell controls (squares) were co-cultured with a panel of human normal primary cells or iPSC-derived cell lines (MAGEA4-HLA-A*02:01+) representing important organs, including bronchial epithelial cells (hBEpC), tracheal epithelial cells (hTEpC), skin microvascular endothelial cells (HDMEC), keratinocytes, hepatocytes, renal proximal tubule epithelial cells (RPTEC), iPSC-derived astrocytes, cardiomyocytes, and GABA neurons. Caspase 3 / 7 activity was measured over time using the Incucyte system. (B,C) In all cases, minimal or negligible reactivity relative to baseline was observed for TCR2 and TCR23, while TCR10 and TCR24 showed significant cytotoxic activity against a variety of normal cells.

[0056] Figure 15. Summary of alloreactivity assessment. TCR-T-IL12 cells were co-cultured with each of 34 BLCLs representing high frequency MHC class I alleles. HLA-A*02:01 pulsed with the relevant MAGE-A4 peptide + U266B1 cell lines (KVL: KVLEHVVRV or GVY: GVYGDREHTV) served as positive controls for each TCR-T-IL12 cell. Secreted IFNγ (A), granzyme B (B), TNFα (C), and IL-12p70 (D) were evaluated as a measure of potential alloreactivity by comparison with levels in response to co-culture with IL12-RFP control T cells. For mock-transduced T cells, cytokine and granzyme B changes compared to IL12-RFP cells are shown at an effector: target ratio of 10:1.

[0057] Fig.16 Cross-reactivity with additional HLA-A*02 alleles can broaden the patient population. The front TCR-T recognizes target peptides presented on additional HLA-A alleles with high homology to HLA-A*02:01. - C1R cells were overexpressed with cognate HLA-A alleles and subsequently loaded with target KVL or GVY MAGEA4 peptides for TDCC assays. Both TCR2 and TCR23 exhibited cytolytic activity against HLA-A*02:05 and HLA-A*02:07-expressing C1R cells loaded with target peptides, suggesting that HLA-A*02:05 and HLA-A*02:07 patients could potentially be included in these TCR-T cell therapies. DETAILED DESCRIPTION

[0058] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.All references cited in the text of this specification are expressly incorporated by reference in their entirety.

[0059] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as generally implemented in the art or as described herein. The following procedures and techniques can be generally carried out according to conventional methods well known in the art and as described in different general and more specific references cited and discussed in this specification in its entirety. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York], which is incorporated herein by reference for any purpose. Unless a specific definition is provided, the nomenclature and laboratory processes and techniques used in connection with analytical chemistry, organic chemistry, and medicine and pharmaceutical chemistry described herein are those well known in the art and commonly used. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery, and the treatment of patients.

[0060] T cell receptor (TCR) is naturally expressed by CD4+ and CD8+ T cells. TCR is designed to recognize short peptide antigens, which are compounded with major histocompatibility complex (MHC) molecules (in humans, MHC molecules are also called human leukocyte antigens or HLA) and displayed on the surface of antigen presenting cells (Davis et al., (1998), Annu Rev Immunol [Annuals of Immunology] 16: 523-544.). CD8+ T cells, also known as cytotoxic T cells, specifically recognize peptides bound to MHC class I, and are generally responsible for finding infected cells or cancer cells and mediating the destruction of infected cells or cancer cells.

[0061] 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. SR Schmidt, Wiley; Boulter et al., (2003), Protein Eng 16(9):707-711; Liddy et al., (2012), Nat Med 8:980-987), or alternatively, they can be used to engineer T cells for adoptive therapy (June et al., (2014), Cancer Immunol Immunother 63(9):969-975). It is expected that the TCR for immunotherapy purposes can strongly recognize the target antigen, which means that the TCR should have high affinity and / or long binding half-life to the target antigen in order to exert a strong response. Since the TCR present in nature usually has a low affinity (low micromolar range) to the target antigen, it is usually necessary to identify the mutation that can be performed on a given TCR sequence, including but not limited to substitution, insertion and / or deletion, in order to improve antigen binding. For use as a soluble targeting agent, it is preferred that the TCR antigen binding affinity in the nanomolar to picomolar range and the binding half-life of several hours. It is also expected that the therapeutic TCR shows a high level of specificity to the target antigen to mitigate the risk of toxicity due to off-target binding in clinical applications. Achieving this high specificity can be particularly challenging given the natural degeneracy of TCR antigen recognition (Wooldridge et al., (2012), J Biol Chem 287(2):1 168-1177; Wilson et al., (2004), Mol Immunol 40(14-15):1047-1055). Finally, it is desirable that therapeutic TCRs be expressed and purified in a highly stable form.

[0062] The variable domain of each chain is located at the N-terminus and contains three complementary determining regions (CDRs) embedded in the framework sequence. The CDRs contain recognition sites for peptide-MHC binding. There are several genes encoding the α chain variable (Va) region and several genes encoding the β chain variable (\ / β) region. These genes differ in their framework, CDR1 and CDR2 sequences and partially determined CDR3 sequences. The Va and v genes are represented in the IMGT nomenclature by the prefixes TRAV and TRBV, respectively (Folch and Lefranc, (2000), Exp Clin Immunogenet [Experimental and Clinical Immunogenetology] 17(1):42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet [Experimental and Clinical Immunogenetology] 17(2):83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook [T cell receptor facts manual], Academic Press [Academic Press]). Similarly, there are several joining genes or J genes called TRAJ' or TRBJ' for the α and β chains, respectively, and for the β chain, there is a diversity gene or D gene called TRBD' (Folch and Lefranc, (2000), Exp Clin Immunogenet [Experimental and Clinical Immunogenet] 17(2): 107-1 14; Scaviner and Lefranc, (2000), Exp Clin Immunogenet [Experimental and Clinical Immunogenet] 17(2): 97-106; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook [T cell receptor facts manual]", Academic Press [Academic Press]). The great diversity of α and β variable region sequences is generated by 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 called TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10).

[0063] TCR sequences defined herein are described with reference to the IMGT nomenclature, which is well known and available to practitioners in the TCR field. See, for example, LeFranc and LeFranc, (2001). "T cell Receptor Factsbook", Academic Press; Lefranc, (201 1), Cold Spring Harb Protoc 201 1 (6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10; and Lefranc, (2003), Leukemia 17 (1): 260-266. αβTCRs consist 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 connecting region connects the variable domains to the constant domains and is generally considered to be part of the variable region. In addition, the beta chain generally contains a short diversity region between the variable region and the connecting region.

[0064] 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. Identifying specific TCR sequences that bind to the GVYDGREHTV HLA-A*02:01 or KVLEHVVRV HLA-A*02:01 complex is advantageous for developing novel immunotherapies.

[0065] 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 this binding results in activation of the recombinant cell. Activation of T cells results in the death or destruction of cancer cells. Methods for determining T cell activation are known in the art and are provided with examples herein.

[0066] In preferred embodiments, the potency or cytolytic activity (cytotoxicity) of the recombinant cells of the invention is defined as follows: (1) loading with about 100 copies (about 10 -8(M) 80%-100% lysis of HLA-A*02:01 target cells with peptides expressed / cell; or (2) 80%-100% lysis of native pMHC target-positive cancer cell lines.

[0067] Each TCR α chain and β chain comprises a variable domain and a constant domain. There are three CDRs (complementarity determining regions) in the variable domain (Vα or Vβ): CDR1, CDR2 and CDR3. Various α chain and β chain variable domains can be distinguished by their frameworks and their CDR1, CDR2 and a portion of their CDR3 sequences.

[0068] In the present specification and claims, the term "TCR α (or a) variable domain" refers to the tandem of the TRAV region and the TRAJ region; only the TRAV region; or the TRAV region and a portion of the TRAJ region, and the term TCR α (or a) constant domain refers to the extracellular TRAC region, or to a C-terminal truncated or full-length TRAC sequence. Similarly, the term "TCR β (or β) variable domain" may refer to the tandem of the TRBV region and the TRBD / TRBJ region; only the TRBV region and the TRBD region; only the TRBV region and the TRBJ region; or to the TRBV region and a portion of the TRBD region and / or the TRBJ region, and the term TCR β (or β) constant domain refers to the extracellular TRBC region, or to a C-terminal truncated or full-length TRBC sequence.

[0069] In a preferred embodiment, the TCR comprises an alpha chain having a CDR3 as set forth in SEQ ID No: 7, 17, 27, 37, 47, 57, 67 or 77 and a beta chain having a CDR3 as set forth in SEQ ID No: 8, 18, 28, 38, 48, 58, 68 or 78. The CDR3 region can be determined by commercially available software (e.g., Cellranger; 10X Genomics). The TCR alpha chain may comprise a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of the sequences set forth in SEQ ID No: 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 shown in any one of SEQ ID No: 11, 12, 21, 22, 31, 32, 41, 42, 51, 52, 61, 62, 71, 72, 81 or 82. Methods for determining the identity between two sequences are well known in the art, for example, BLAST or Geneious. In certain embodiments, the C-terminal or N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of any of the sequences shown in any one 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 shown in any one of SEQ ID Nos: 11, 12, 21, 22, 31, 32, 41, 42, 51, 52, 61, 62, 71, 72, 81 or 82 may be truncated or removed. Exemplary TCRs and corresponding α and β chain CDR3s and full-length SEQ ID Nos are provided in Tables 1A and 1B.

[0070] 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 TRBC1*01 constant region chains. In one embodiment, 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 TRBC1*01 constant region chains.

[0071] 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 TRBC2*01 constant region chains. In one embodiment, 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 TRBC2*01 constant region chains.

[0072] 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 TRBC2*01 constant region chains. In one embodiment, TCR3 comprises a TCR3 alpha chain and a TCR3 beta chain, wherein the TCR3 alpha chain comprises the use of TRAV13-2*01 and TRAJ5*01 variable region chains, and the TCR3 beta chain comprises the use of TRBV5-6*01, TRBD1*01 and TRBJ2-2*01 variable region chains and the use of TRBC2*01 constant region chains.

[0073] 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 TRBC2*01 constant region chains. In one embodiment, TCR4 comprises a TCR4 alpha chain and a TCR4 beta chain, the TCR4 alpha chain comprises the use of TRAV4*01 and TRAJ43*01 variable region chains, the TCR4 beta chain comprises the use of TRBV11-2*01 and TRBJ2-7*01 variable region chains and the use of TRBC2*01 constant region chains.

[0074] 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 TRBC1*01 constant region chains. In one embodiment, TCR5 comprises a TCR5 alpha chain and a TCR5 beta chain, wherein the TCR5 alpha chain comprises the use of TRAV4*01 and TRAJ9*01 variable region chains, and the TCR5 beta chain comprises the use of TRBV11-2*01, TRBD2*02 and TRBJ1-1*01 variable region chains and the use of TRBC1*01 constant region chains.

[0075] 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 TRBC2*01 constant region chains. In one embodiment, 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 TRBC2*01 constant region chains.

[0076] 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 TRBC2*01 constant region chains. In one embodiment, 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 TRBC2*01 constant region chains.

[0077] 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 TRBC2*01 constant region chain. In one embodiment, TCR8 comprises a TCR8 alpha chain and a TCR8 beta chain, wherein the TCR8 alpha chain comprises the use of TRAV21*01 and TRAJ31*01 variable region chains, and the TCR8 beta chain comprises the use of TRBV2*01, TRBD2*01 and TRBJ2-7*01 variable region chains and the use of TRBC2*01 constant region chain.

[0078] In certain embodiments, the variable domain of a TCR alpha chain or beta chain can be fused to a non-TCR polypeptide. Exemplary alpha and beta chain variable domains can be used to generate soluble TCRs capable of binding to MAGE-A4 derived peptides in the context of HLA molecules.

[0079] The TCR of the present invention can 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 chain and β chain constant domain sequences can be modified by truncating or replacing to delete the natural disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2, and / or the α chain and / or β chain constant domain sequences can be modified by replacing cysteine ​​residues to form a non-natural disulfide bond between the α constant domain and the β constant domain of the TCR; for example, Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 are replaced with cysteine, which forms a non-natural disulfide bond.

[0080] The TCR of the present invention can be a single-chain form of Va-L-vβ, vβ-LV, Va-Ca-L-vβ type, wherein V and vβ are TCR and β variable regions, respectively, C and are TCR and β constant regions, respectively, and L is a linker sequence. Soluble TCR can be in a single-chain form, wherein the α variable domain and the β variable domain are connected by a linker. Soluble TCR can be fused or connected to a therapeutic agent or imaging agent.

[0081] The TCR of the present invention may also include one or more conservative substitutions with similar amino acid sequences and / or retaining the same function. It is known to those skilled in the art that a variety of amino acids have similar properties and are therefore "conservative". One or more such amino acids of a protein, polypeptide or peptide may generally be substituted by one or more other such amino acids without eliminating the desired activity of the protein, polypeptide or peptide. Therefore, the amino acids glycine, alanine, valine, leucine and isoleucine may generally replace each other (amino acids with aliphatic side chains). Among these possible substitutions, it is preferred that glycine and alanine are substituted with each other (because they have relatively short side chains), and valine, leucine and isoleucine are substituted with each other (because they have larger hydrophobic aliphatic side chains). Other amino acids that are generally substituted with each other include: phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); lysine, arginine and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (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 comprising the above amino acid sequences but having one or more conservative substitutions in that sequence.

[0082] Exemplary TCRs and corresponding sequences are provided in Tables 1a and 1b, respectively.

[0083] Table 1A

[0084]

[0085]

[0086] Table 1B

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] The TCR alpha or beta variable domain may comprise a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences specified in Table 2. The TCR beta chain may comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID Nos: 46-56. In certain embodiments, the C-terminal or N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues of any one of the sequences specified in Table 2 and Table 1B SEQ ID NOs: 35-56 may be truncated or removed.

[0099] Although recognition of the target peptide in the context of HLA is essential for efficacy, for safety purposes, in some embodiments, it is preferred that the TCR lack cross-reactivity with structurally similar peptides when presented by HLA-A*02:01 or presented with HLA molecules of other allotypes. The cross-reactivity and alloreactivity of the exemplary TCRs described herein are provided in the Examples. Thus, the exemplary TCRs are not only able to recognize MAGE-A4 peptides in the context of HLA-A*02:01 expressed on tumor cells and activate T cells recombinantly expressing the TCR against tumor cells, but also fail to activate or have minimal activation when the recombinant T cells are presented with peptides in the context of HLA-A*02:01 or other HLA molecules expressed on normal tissues.

[0100] Other embodiments of the present invention include nucleic acids encoding TCR alpha variable domains, TCR β variable domains, or TCR alpha variable domains and TCR β variable domains as described herein. In a specific embodiment, one or more of the α or β variable domains shown in nucleic acid encoding table 2. In certain embodiments, nucleic acid encoding α and β variable domains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7 or TCR8. In a preferred embodiment, the nucleic acid encoding TCR alpha chain variable domains, TCR β chain variable domains, or TCR alpha chain variable domains and β chain variable domains is an expression vector, wherein TCR alpha chain variable domains, TCR β chain variable domains, or TCR alpha chain variable domains and β chain variable domains are operably connected to a promoter.

[0101] TCR α variable domain and β variable domain can be co-translated from the same promoter. For embodiments in which the α variable domain and β variable domain are linked within a fusion protein, these domains can also be co-translated within a single polypeptide. In embodiments in which the α domain and β domain are within separate polypeptides, it is useful to include an internal ribosome entry site (IRES) between the α variable domain and β variable domain coding regions within the expression vector.

[0102] Also provided herein are nucleic acids encoding TCR alpha chains, TCR beta chains, or TCR alpha and TCR beta chains as described herein. In a specific embodiment, the nucleic acid encodes one or more of the alpha or beta chains shown in Table 1. The encoded alpha or beta chains can be full-length or mature. When mature, i.e., lacking a natural leader sequence associated with the alpha or beta chain, it is preferred that the nucleic acid encoding the signal or leader sequence is operably connected to the nucleic acid encoding the alpha chain or beta chain so that when translated, the leader sequence directs the alpha or beta chain to the endoplasmic reticulum.

[0103] In certain embodiments, the nucleic acid encodes the α and β chains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7 or TCR8. In a preferred embodiment, the nucleic acid encoding TCR α chain, TCR β chain, or TCR α chain and β chain is an expression vector, wherein TCR α chain, TCR β chain, or TCR α chain and β chain are operably connected to a promoter.

[0104] The TCR α chain and β chain can be co-transformed from the same promoter. In such embodiments, it is useful to include an internal ribosome entry site (IRES) between the α chain and β chain coding regions within the expression vector.

[0105] The expression vector of the present invention includes but is not limited to retrovirus or lentiviral vector. In addition to TCR α chain and / or β chain, the expression vector may further encode one or more additional proteins. 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, and a combination thereof. Since cytokines can have systemic effects, when the expression vector encoding the cytokine is used to produce cells for adoptive cell therapy, it is preferred that cytokine expression is controlled by an inducible promoter. In certain embodiments, the promoter is a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements connected to a minimal IL-2 promoter, and the cytokine is IL-12 or a variant thereof. The use of a composite promoter expressing IL-12 containing six NFAT (nuclear factor of activated T cells) response elements connected to a minimal IL-2 promoter is described in U.S. Patent No. 8,556,882.

[0106] Provided herein are cells recombinantly expressing exemplary TCRs described herein. The recombinant cell may include one or more encoding and expression vectors of TCR alpha chains, TCR beta chains, TCR alpha and beta chains, TCR alpha variable domains, TCR beta variable domains, or TCR alpha and beta variable domains. In a preferred embodiment, the cell recombinantly expresses TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7 or TCR8. In certain embodiments, the cell further expresses one or more recombinant cytokines. In a preferred embodiment, the cytokine is IL-12 or a variant thereof, and the expression is controlled by an inducible promoter (e.g., a promoter driven by NFAT).

[0107] In certain embodiments, the cell is derived from a sample taken from a cancer patient. Cells, such as T cells or NKT cells, are isolated from the sample and amplified. In certain embodiments, progenitor cells are separated and matured into the desired cell type. Cells are transfected / transformed with one or more vectors (e.g., lentiviral vectors) encoding TCR and any additional polypeptide (e.g., IL-12 or its variants). Such cells can be used for adoptive cell therapy of cancer patients from which they are derived.

[0108] In other embodiments, the cell line recombinantly expresses a soluble TCR. The soluble TCR can be a fusion protein with an anti-CD3 antigen binding protein such as a scFv.

[0109] Provided herein are methods for treating a disease or condition in which a cell associated with the disease or condition expresses MAGE-A4. In a preferred embodiment, the cell presents 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 conditions that can be treated with the soluble TCR or recombinant cells of the present invention include hematological tumors or solid tumors. Such diseases and conditions 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 conditions include non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), bladder cancer, esophageal cancer, or ovarian cancer.

[0110] For certain treatments, tumor biopsies are tested for expression or MAGE-A4. Tumors can also be tested for expression of 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 have the appropriate HLA haplotype can be administered the soluble TCRs or recombinant cells of the invention.

[0111] Examples

[0112] The following examples (both actual and predicted) are provided to illustrate specific embodiments or features of the invention and are not intended to limit the scope thereof.

[0113] Example 1 - MAGE-A4 is expressed in a wide range of solid tumors with highly restricted normal tissue expression

[0114] Data from TCGA and the applicant demonstrate that MAGE-A4 mRNA has a high prevalence in a wide range of solid tumors ( Figure 1A Importantly, Applicants' internal body mapping data showed that normal tissue expression of MAGE-A4 was extremely limited, with the exception of the testis, which is an immune privileged site ( Figure 1B MAGE-A4 IHC data in squamous NSCLC (squamous non-small cell lung cancer or lung squamous cell carcinoma) showed that within the tumor, MAGE-A4 protein was expressed in the majority of tumor cells (60%-100%) but not in stromal cells ( Figure 1C ). Presentation of two target MAGE-A4 peptides was identified by MS on MHC in U266B1 cells 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 286-294 of the MAGE A4 protein.

[0115] 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 lung squamous cell carcinoma (squamous NSCLC, LUSC), approximately 17.7% of head and neck squamous cell carcinoma (HNSCC), approximately 14.5% of urothelial bladder cancer (BLCA), approximately 14.3% of esophageal cancer, approximately 14.1% of ovarian cancer, approximately 9.8% of triple-negative breast cancer (TNBC), approximately 7.3% of gastric cancer (STAD), approximately 4.9% of rectal adenocarcinoma (READ), approximately 4.5% of lung adenocarcinoma (LUAD), approximately 2.2% of colon adenocarcinoma (COAD), and approximately 2% of hepatocellular carcinoma (LIHC) ( Figure 3 ). In the United States, the pMHC target frequency (%) was calculated by MAGE-A4 mRNA expression frequency × HLA-A*02:01 carrier frequency (0.41). The patient population in the specified cancer indication was estimated based on the pMHC target frequency (%) in the US population × new cases per year. The TCGA public dataset of RNAseq from the target tumor was used to estimate the MAGE-A4 mRNA expression frequency in each tumor indication at the threshold of MAGEA4>=1 FPKM ( Figure 3 ). Use 2020 SEER, EPIC Oncology New Patients, or Epiphany / Epic to estimate incidence (new cases per year) in selected oncology indications and, therefore, derive an 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 frequency of the HLA-A*02:01 haplotype (carrier) in the U.S. population is 0.41 (www.allelefrequencies.net). The largest patient population is squamous NSCLC, followed by HNSCC, bladder cancer, esophageal cancer, and ovarian cancer ( Figure 3 ).

[0116] Example 2 - Identification of TCRs specific for MAGE-A4 pMHC

[0117] The identification and selection process of the lead clinical TCR candidate 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 the Jurkat activation assay, 10-11 TCR candidates were selected for each target peptide. Based on these TCR sequences, primary pan-T cells isolated from 3 donors were transduced with lentivirus carrying a single TCR to generate TCR-T cells for each donor. These TCR-T cells were further evaluated by various functional assays, including efficacy (cytotoxicity) tests of T2 cell lines and various cancer cell lines pulsed with target peptides, cross-reactivity screening with similar peptides, and allogeneic reactivity screening. Based on those functional data, we further narrowed down to 2 leading TCR candidates. To enhance in vivo efficacy, all TCRs were manufactured in a TCR-T-IL12 lentiviral construct, in which expression of the IL12 payload was regulated by TCR activation under a promoter driven by an NFAT response element. Thus, IL12 could be produced only when the TCR-T cells engaged pMHC targets in the tumor.

[0118] MAGE-A4 pMHC-specific TCRs can be identified from rare T cell clones derived from PBMCs of healthy donors

[0119] The difficulty in identifying tumor antigen-specific TCRs has hampered the development of TCR-mediated immunotherapy. Despite these challenges, this article describes a TCR discovery platform by which tumor antigen pMHC-specific TCRs can be identified from rare T cell clones from healthy donors. The frequency of MAGEA4 pMHC-reactive T cells in PBMCs from healthy HLA-A*02:01+ donors is extremely low, typically about 0% dextramer+T cells. Dextramer (Dex) is a polymer of peptide-MHC complexes that can specifically bind to TCRs and can therefore be used to isolate antigen (pMHC)-specific T cells. First, to expand rare tumor antigen-specific T clones, we used PBMCs from 72 healthy HLA-A*02:01+ donors to isolate T cells and autologous antigen-presenting cells (APCs), such as monocyte-derived dendritic cells and activated B cells. When T cells were co-cultured with autologous APCs pulsed with target peptides, these T cells underwent multiple ex vivo stimulation steps, in which tumor antigen pMHC-specific priming, restimulation of pMHC-specific T cells, and expansion occurred. After 3-4 rounds of antigen restimulation, the MAGE-A4 pMHC-specific T cell population was enriched and verified by dextramer-PE and dextramer-APC staining (Figure 4). Dex+ T cells were then sorted for single-cell RNAseq to identify the sequences of TCRα and TCRβ chains. In addition, antigen-specific IFNγ production of those sorted Dex+ CD8+ T cells was verified by ELISPOT assay using peptide-loaded T2 cells (Figure 4). This TCR discovery platform led to the identification of 101 dominant MAGE-A4 pMHC-specific TCRs from 72 healthy HLA-A*02:01+ donors. Importantly, unlike affinity-enhanced TCRs or bispecific antibodies, the TCRs identified from the blood of healthy donors have already undergone natural thymic selection in vivo (in the thymic medulla) to eliminate autoreactive TCRs. Therefore, it is hypothesized that the risk of off-target reactivity of our TCRs is quite low, which was confirmed by our safety assessment assays (described below).

[0120] Selection of MAGEA4 pMHC-specific TCR-T cells

[0121] Among the 101 dominant MAGE-A4pMHC specific TCRs identified from the screening of 72 healthy HLA-A*02:01+ donors, 20 TCR candidates were selected by Jurkat activation assay (Fig. 5). The lentivirus carrying a single TCR was transduced into the Jurkat TCR KO reporter cell line expressing Renilla luciferase, which was regulated by TCR activation under the promoter driven by the NFAT response element. The antigen-specific activity of a single TCR was measured as the luciferase activity in the presence of T2 cells loaded with MAGE-A4 peptides compared to the change multiple (Fig. 5) of T2 cells containing only vehicle.

[0122] Example 3-Efficacy verification of TCR-T-IL12 cells

[0123] Twenty TCRs (ten targeting the MAGEA4KVLEHVVRV and ten targeting the GVYDGREHTV epitope) were further made in the TCR-T-IL12 lentiviral construct, in which expression of the IL12 payload was regulated by TCR activation under a promoter driven by the NFAT response element. Fig. 6A ). Therefore, when TCR-T-IL12 cells bind to pMHC targets in tumors, IL12 is produced upon TCR signaling, which limits IL12 secretion mainly within tumors. The transduction efficiency of TCR to primary human T cells during TCR-T generation was measured by flow cytometry ( Figure 6B Those 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 assays. Fig. 7A -C). T2 is a TAP-deficient cell line expressing HLA-A*02:01. Since the T2 cell line lacks the transporter for endogenous peptides restricted by MHC class I to enter the ER and mainly presents exogenous peptides, the T2 / peptide cytotoxicity assay (using the T2 cell line loaded with the target peptide for cytolytic activity measurement) was used to study the specific recognition of TCR-T for peptides (e.g., HLA-A*02:01 restricted peptides). The average potency of the top 8 TCRs was identified based on EC50 and is shown in Figure 7C All top eight TCR-IL12 cells met the EC90 of 10 -8 The effectiveness standard of M.

[0124] Example 4-Evaluation of cross-reactivity of TCR-TIL12 cells

[0125] The extensive in vitro and ex vivo safety assessments of TCR-T-IL12 cells as human-specific HLA targets exclude 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 using normal human tissues as described above and tumor tissues by RNASeq, IHC and mass spectrometry. Secondly, key safety considerations are off-target reactivity, which is evaluated by T2 / peptide cytotoxicity assays, which use 20 similar peptides based on homology for each TCR for its respective target. No cross-reactivity was observed for any of the first 8 TCRs, which potentially supports the advantage (Fig. 8) of screening naturally occurring TCRs for candidate selection. The first 20 similar peptides of each of GVYDGREHTV (SEQ ID NO: 1) and KVLEHVVRV (SEQ ID NO: 2) epitopes are shown in Tables 2 and 3, respectively. TCR was also screened for the cross-reactivity of the relevant CTA MAGEA8. Similar to MAGEA4, MAGEA8 is aberrantly expressed in a variety of tumors, and its expression in healthy tissues is largely restricted to the male reproductive system ( Fig. 9 ).

[0126] Table 2 - Top 20 similar peptides used in cross-reactivity screening of the GVYDGREHTV MAGEA4 epitope.

[0127]

[0128]

[0129] Table 3 - Top 20 similar peptides used in cross-reactivity screening of the KVLEHVVRV MAGEA4 epitope.

[0130]

[0131]

[0132] MAGEA4 and MAGEA8 have significant sequence homology. It is noteworthy that 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) showed 80% sequence identity. The KVLEHVVRV (SEQ ID NO: 2) peptide is 100% identical between MAGEA4 and MAGEA8 proteins and therefore will not provide a basis for differential activity in homologous TCR-T. Screening of the differential activity of the GVYDGREHTV-MHC homologous TCRs against the two peptide epitopes revealed that the reactivity of the previously identified top TCRs differed by more than about 1000-fold (Figure 10). These data demonstrate that the top 4 GVYDGREHTV-MHC homologous TCRs identified here can be used to specifically target tumors with little risk of MAGEA8 cross-reactivity. In contrast, the top KVLEHVVRV-MHC homologous TCRs will likely be useful in killing a large class of cancer cells expressing MAGEA4, MAGEA8, or both.

[0133] Example 5 - Cytotoxicity against MAGEA4+ cancer cell lines

[0134] The top 8 TCRs were evaluated in the TDCC assay against cancer cell lines. All 8 TCRs showed cytotoxicity against the MAGEA4+ cell lines U266B1 and SCaBER ( Fig.11A A and B). The measured EC50s against both cell lines identified a consistent set of top 5 TCRs that were selected for subsequent studies. Notably, the top 5 TCR-Ts expressed the TCRs in greater than 20% of CD8+ T cells following lentiviral transduction. In summary, these TCRs were selected based on: (1) potent cytotoxic MAGE-A4 pMHC targets against T2 / MAGEA4 peptides and MAGE-A4+ cancer cell lines as evaluated by TDCC; (2) off-target selectivity that showed no cross-reactivity against 20 similar peptides based on homology and target-negative cancer cell lines; and (3) manufacturability (e.g., good TCR transduction efficiency).

[0135] The potency (cytotoxicity) of the first five TCR-T-IL12s was validated using a larger panel of HLA*0201+ cancer cell lines spanning the range of MAGEA4 expression ( Fig. 12A). All five TCR-T-IL12s showed potent cytotoxicity against cancer cell lines with MAGE-A4 expression as low as about 3.6 FPKM. All four TCRs had similar potency, making it challenging to relatively rank the top TCRs in a cell line. However, using the overall EC50 ranking system for MAGEA4+ cells, TCR2 and TCR10 were the most potent, followed by TCR23, TCR24, and TCR7.

[0136] Representative cancer cell line potency data for the first five TCR-T-IL12 cells are shown in Fig. 12B In. 10 cancer cell lines were tested with four TCR-T-IL12 cells generated from 2-3 donors. TCR-T-IL12 cells showed potent cytotoxicity against some cancer cell lines, but the E:T EC50 was low. For example, the E:T EC50 of TCR2 TCR-T against NCI-H1755 (FPKM=457.3) averaged 0.01 in experiments using TCR-T generated from three different donors, indicating that TCR-T can show significant cytotoxicity against tumor cells even at over 100:1.

[0137] Example 6 - Overview of Nonclinical Safety Evaluations

[0138] TCR-T-IL12 cells were extensively evaluated for safety in vitro and ex vivo, as human-specific HLA targets preclude the use of animal models. First, target expression was assessed using normal human tissues as described above as well as tumor tissues by various assays (including RNASeq, IHC, and mass spectrometry). Since MAGE-A4 is a cancer-testis antigen, our studies show that normal tissue expression is extremely limited (expressed only in the testis). Secondly, two different strategies were used to assess off-target reactivity. The first strategy involves screening to evaluate cytotoxicity against various normal human primary cells. The second strategy involves identifying a group of similar peptides based on sequence homology matching with the MAGE-A4 target peptide and a site scanning (X-scanning motif)-based strategy to identify putative cross-reactive peptides unique to each TCR. In order to obtain potential cross-reactivity with the entire group of similar peptides, a T2 / peptide TDCC assay was performed. The third safety assessment was alloreactivity, which was assessed using 34 BLCLs (B lymphoblastoid cell lines) representing high-frequency HLA class I alleles in the U.S. population (including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles).

[0139] Identification of similar peptides based on homology

[0140] 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 the candidate TCR-T. To achieve this, a protein database restricted to Homo sapiens (UniProtKB / Swiss-Prot, June 2019) was first queried using a Python script 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 to the target peptide. To further refine this list, criteria such as high homology matches were utilized, as well as software such as NetMHCpan and IEDB (Immune Epitope Database). NetMHCpan3.0 software was used to consider the predicted binding affinity of the peptides for HLA-A*02:01. The probability of peptides being processed and presented by the HLA-A*02:01 allele was considered using the IEDB database (June 2019), which is a manually curated database of experimentally characterized immune epitopes. The specific criteria used for peptide selection were as follows: (1) all HLA-A*02:01+ peptides in the IEDB with ≥40% homology match (identity) to the target peptide (40 peptides); (2) all peptides with ≥60% homology match and predicted HLA-A*02:01 binding affinity (IC50) <5000nM (51 peptides); and (3) all peptides with ≥50% homology match to the target peptide reported in the IEDB (presented by the HLA-A*02:01 allele) (53 peptides). Thus, this homology-based in silico search of the human proteome database along with the screening criteria resulted in the identification of 144 unique peptides for screening against GVYDGREHTV TCR-23 and TCR-24.

[0141] For the MAGEA4 target peptide KVLEHVVRV, the same computer search resulted in the identification of 155,353 nonamer peptides based on a 30% homology match to the target peptide. To further refine the list of peptides, the following criteria were used: (1) all HLA-A*02:01+ peptides in the IEDB with ≥40% homology match (identity) to the target peptide (176 peptides); and (2) all peptides with ≥60% homology match and predicted HLA-A*02:01 binding affinity (IC50) <5000nM (50 peptides). Thus, this homology-based computer search of the human proteome database and screening criteria resulted in the identification of 226 unique peptides for screening KVLEHVVRV TCR2 and TCR10.

[0142] Use site scanning (X-scanning) to identify TCR binding motifs and similar peptides based on X-scan-derived motifs

[0143] As an orthogonal method for identifying similar peptides, we used a site scanning method, called X-scanning. This assay uses a peptide library generated by mutating each residue of the MAGE-A4 peptide to one of the other 19 naturally occurring amino acids in turn, thereby generating a total of 171 peptides for the KVLEHVVRV target and 190 peptides for the GVYDGREHTV target. These peptides were synthesized and tested in the T2 / peptide TDCC assay to identify the motifs (Table 3) with specific X-scanning sources for each individual TCR. In brief, T2 cells were pulsed with each of these peptides at a concentration of 10 μM, and then TCR-T was added with an E:T ratio of 1:1. Cell viability was determined using T2 / peptide TDCC assays. In the case where the observed vigor was less than 30% (and less than 40% for TCR23 / TCR24), amino acid substitutions were defined as being necessary for TCR2 / TCR10 engagement. Corresponding search motifs were constructed to express which amino acids are tolerated at each position in the peptide sequence (Table 3). The underlined amino acids represent the native residues at the corresponding positions in the peptide. Using the generated motifs, an in silico search of the human proteome (UniProtKB / Swiss-Prot database limited to Homo sapiens with splice variants, June 2019) was performed to identify all decamer (TCR23 / TCR24) or nonamer (TCR2 / TCR10) sequences that matched the derived motifs. This approach generated an additional set of peptides for off-target screening that were specific for a single TCR-T (Table 3). To determine whether these sequences would lead to off-target toxicity if presented on the cell surface, a set of peptides for TCR2 (21) and TCR23 (1) were synthesized and tested for cell viability using the single-point T2 / peptide (10 μM) TDCC assay as described above. All three donors showed greater than 70% cell viability against the full set of peptides, indicating no off-target propensity from TCR2 or TCR23.

[0144] Table 3

[0145]

[0146] Cross-reactivity screening of the entire panel of similar peptides

[0147] A full set of similar peptides was synthesized and tested in the T2 / peptide TDCC assay to investigate the possibility of off-target reactivity. To identify potential cross-reactive peptides for each TCR-T-IL12, the full set of similar peptides was tested using the T2 / peptide TDCC screening with a high peptide concentration (10 μM). Peptides that showed less than or equal to 25% activity in at least one of the three donors were considered putative cross-reactive peptides and were selected for further efficacy testing.

[0148] Next, the T2 / peptide titration TDCC assay was used to screen the Fig.13A -B) were screened for potency (dose-dependent screening) with putative cross-reactive peptides identified in Table 4. The target peptide had a 10% affinity to the putative cross-reactive peptide. 3 The EC50 efficacy gap of 100 times is considered as the cutoff value of future risk assessment. This method does not produce putative cross-reactive peptides for TCR2 and TCR10, while multiple putative cross-reactive proteins are identified for both TCR23 (6 peptides) and TCR24 (8 peptides). It may be due to the high sequence homology within the MAGE protein family, it is found that TCR23 and TCR24 have cross-reactivity to multiple type 1 MAGE family proteins (such as MAGEA8, MAGEA10 and MAGEA11). Although these findings are worthy of further study, because they belong to the cancer testis antigen category, these putative cross-reactivity for these MAGE family source peptides is not considered as a significant safety risk. However, these assays also identify two potential non-MAGE family proteins as potential cross-reactivity tendencies: FA12 and KI13B are for TCR23, and NUCL and RBM47 are for TCR24. Given the large disparity in potency (>100-fold) between these cross-reactive similar peptides and the lack of experimentally confirmed endogenous HLA-A*02:01 presentation, the relevance of these cross-reactivities with respect to the clinical tropism of TCR23 and TCR24 is unclear. In summary, all TCRs were found to be unreactive to the vast majority of proteome-derived similar peptides. No tropism was found for TCR2 and TCR10, while two putative cross-reactive peptides derived from non-CTA proteins were identified for TCR23 and TCR24. Notably, TCR2 and TCR23 exhibited minimal caspase 3 / 7 cleavage when co-cultured with any of the tested normal human primary cells or iPSC-derived cells, suggesting that there was no significant off-target reactivity against any of the tested normal cells, which can present a highly diverse set of peptides (see the following section; Fig. 14B , C).

[0149] Table 4. Peptides identified from similar peptide screening

[0150]

[0151]

[0152] Evaluation of cytotoxicity against primary human normal cells

[0153] Next, the cytotoxicity of TCR-T-IL12 cells to various human primary normal cell types representing vital organs (without MAGE-A4 expression) acting as target cells was evaluated in a T cell-mediated cytotoxicity assay. TCR2, TCR10, TCR23, and TCR24 were tested against a group of human normal primary cells or iPSC-derived cell lines representing vital organs, including bronchial epithelial cells (hBEpC), tracheal epithelial cells (hTEpC), skin microvascular endothelial cells (HDMEC), keratinocytes, hepatocytes, renal proximal tubule epithelial cells (RPTEC), iPSC-derived astrocytes, cardiomyocytes, and GABA neurons. ( Fig.14A ). All primary cells and iPSC-derived cell lines were derived from normal tissues of HLA-A*02:01-positive donors. Importantly, since those normal cells can present highly diverse peptides on HLA-A*02:01, this serves 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 transgenic TCR) from the same donor were included as negative control effector cells. In the presence of TCR-T cells or NFAT.IL12 T cell controls, the cytotoxicity of nine normal primary cell types was assessed by a caspase 3 / 7 cleavage assay. Importantly, TCR2 and TCR23 exhibited minimal caspase 3 / 7 cleavage when co-cultured with any normal primary cells or iPSC-derived cells tested compared to the IL12 T cell controls, indicating that there was no significant off-target reactivity against any non-cancerous cells tested ( Fig. 14B , C). In contrast, in all non-cancer cells tested, the measured caspase 3 / 7 responses were significantly higher for TCR10 and TCR24 TCR-T cells than for control T cells ( Fig. 14B , C).

[0154] Evaluation of alloreactivity using 34 BLCL lines

[0155] As part of the safety assessment, alloreactivity potential was evaluated using 34 BLCL lines (B lymphoblastoid cell lines) representing high-frequency (≥11%) MHC class I alleles in major American ethnicities, including 39 HLA-A, 40 HLA-B, and 23 HLA-C alleles. Alloreactivity potential was evaluated by cytokine (IFNγ, TNFα, and IL-12p70) and granzyme B production. For TCR2-transduced T cells, no significant increase in cytokine or granzyme B responses was observed against the 34 BLCL lines tested ( Fig.15AFor TCR23 T cells in 3 / 34 BLCL, only low-level threshold responses were observed, defined as greater than or equal to 3-fold induction in any one analyte compared with IL12-RFP control cells ( Fig.15A -D). In contrast, greater alloreactive responses were observed for TCR10 and TCR24, with significant induction of cytokine and granzyme B responses detected for TCR24 in 31 / 34 BLCLs. All four TCR-T-IL12 cells exhibited robust cytokine and granzyme B responses against positive control U266B1 cells (HLA-A*02:01+MAGE-A4+) pulsed with MAGEA4 peptide (GVYDGREHTV or KVLEHVVRV). Fig.15A -D).

[0156] In summary, these studies identified exemplary TCR candidates that did not exhibit significant safety issues based on the potential safety assessments performed for both normal and alloreactivity.

[0157] Example 7. TCR displays potent cytotoxic activity against cognate HLA-A pMHC.

[0158] The potency of TCR2 and TCR23 TCR-T was evaluated using peptide-loaded C1R cells expressing a mismatched HLA-A allele with high sequence identity to HLA-A*02:01 ( Fig.16 ). To generate these allelic variants of C1R, an HLA-A deficient cell line, C1R cells 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 novel HLA-A2 alleles were pulsed with KVLEHVVRV or GVYDGREHTV peptides prior to use as target cells in the TDCC assay. Both TCR2 and TCR23 exhibited cytotoxic activity against MAGEA4 peptide-loaded C1R cells expressing HLA-A*02:01 and HLA-A*02:05, in addition, TCR23 exhibited cytotoxic activity against HLA-A*02:06, and TCR2 exhibited cytotoxic activity against HLA-A*02:07. These studies suggest that the application of TCRs may not be limited to HLA-A*02:01+ patients.

[0159] Methods and Materials Used in the Examples

[0160] Identification of MAGE-A4 pMHC-specific TCRs by healthy donor screening

[0161] Generation of autologous antigen presenting cells (APCs)

[0162] HLA-A*02:01 positive healthy donor peripheral blood mononuclear cells (PBMC) were obtained from AllCells or PPA's leukopak. Monocytes were positively selected from PBMCs by using human CD14-microbeads (Miltenyi Biotec, 130-050-201). Mature dendritic cells were obtained by using the CellXVivo human monocyte-derived dendritic cell (DC) differentiation kit (R&D, CDK004). Antigen-presenting B cells were generated by using CD40L and IL-4 stimulation methods. B cells were positively selected from PBMCs by using human CD19-microbeads (Miltenyi Biotec, 130-050-301). CD19+ cells were then stimulated with 0.125ug / ml recombinant huCD40L in B cell culture medium and 2×10 5 Cells / ml and 1ml / well are seeded in 24-well plates. B cell culture medium includes IMDM, supplemented with 10% human serum (MilliporeSigma, H3667-100ML), 100U / ml penicillin and 100ug / ml streptomycin (Gibco, 15140-122), 10μg / ml gentamicin (Gibco, 15750-060) and 200IU / ml IL-4 (Peprotech, 20004100UG) GlutaMax supplemented culture medium (Gibco, 31980030). On the 3rd day after B cell activation, fresh B cell culture medium containing 400IU / ml IL-4 is added to B cell culture with 1ml / well without interfering with cells. On the 5th day after B cell activation, activated B cells are prepared for antigen-reactive T cell stimulation.

[0163] Ex vivo stimulation and expansion of antigen-specific T cells

[0164] On the 7th day after CD14+ cell separation, MAGE-A4 peptide (Anaspec custom peptide) was added to immature dendritic cells at 1 μM together with recombinant human TNF-α. On the 9th day after CD14+ cell separation, mature dendritic cells pulsed with MAGE-A4 peptide were collected, washed, and mixed with CD14-PBMC at a ratio of 1 to 10 in human T cell culture medium containing 10 μM MAGE-A4 peptide, 10 IU / ml IL-2 (Miltenyi Biotech, 130-097-745) and 10 ng / ml IL-7 (Peptech, AF20007100UG). Human T cell complete culture medium consists of a 1 to 1 mixture of CM and AIM-V (Thermo Fisher, 12055083). CM consisted of RPMI 1640 supplemented with GlutaMAX (Gibco, 61870-036), 10% human serum (Millipore Sigma, 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 per week. HuCD40L-activated B cells were collected, washed, and cultured at 1×10 6 Cells / ml and 4ml / well were seeded in 6-well plates, 1μM MAGE-A4 peptide was added to B cells and incubated in an incubator at 37°C for 2 hours. Peptide-pulsed B cells were then mixed with T cells at a ratio of 1:10 in human T cell medium containing 10IU / ml IL-2 and 10ng / ml IL-7. MAGE-A4 dextramer-positive cells were confirmed by flow cytometry and then sorted for TCR identification by single-cell RNAseq.

[0165] Isolation of activated antigen-specific T cells

[0166] Antigen-specific T cells activated by MAGE-A4 peptide were stained with MAGE-A4 dextramer-APC and -PE at room temperature in the dark for 10 min, and then stained by CD3-FITC (Biolengend, 300440) and CD8-BV605 (BD Biosciences, 564116). Dead cell exclusion dye (Sytox blue) was purchased from Thermo Fisher Scientific (Invitrogen, S34857). Cells were sorted using an Aria fusion cell sorter (BD Biosciences, San Jose, CA). Post-sorting data analysis was performed using Flowjo.

[0167] ELISPOT

[0168] Antigen-specific IFNγ production by sorted CD3+CD8+Dex+ T cells was verified by ELISPOT assay using peptide-loaded T2 cells (BD, 551849). 10 μM MAGE-A4 peptide in complete human T cell culture medium was added at 2×10 6 Cells / ml and 1ml / well were loaded onto T2 cells in a 24-well plate for 1-2 hours. 150ul of complete human T cell culture medium and 50μl of peptide-loaded T2 cells were added to each well in the pre-coated ELISPOT plate. CD3+CD8+Dex+T cells (500 or 1000 cells) were sorted directly into each well in the ELISPOT plate. ELISPOT was detected after incubation for 24 hours in a 37°C incubator. The ELISPOT plate was scanned and counted by ImmunoSpot (Cellular Technology Limited, Cleveland, Ohio).

[0169] Single-cell RNAseq

[0170] Using Chromium Controller (10X Genomics, Pleasanton, California), V (D) J single cell human T cell enrichment kit (PN-1000006, PN-1000005, PN-120236, PN-120262) was used to directly perform target enrichment according to the manufacturer's instructions, skipping the cDNA amplification step of the whole transcriptome to process the sample. In brief, cells and beads with barcoded oligonucleotides were encapsulated in nanoliter droplets, where cells were lysed and mRNA was reverse transcribed with poly-T primers and barcoded template switching oligonucleotides. Nested PCR was then performed with primers and template switching oligonucleotides in the constant region of human TCR. According to the manufacturer's recommendations, 13-17 cycles were used to perform the second target enrichment PCR according to the estimated cell input number. The final sequencing library was generated by the fragmented PCR product connected to the Illumina sequencing adapter. The libraries were sequenced with 151 paired-end reads (151×8×0×151) at a depth of at least 5,000 reads per cell on a NextSeq 550 or MiSeq (Illumina, Inc., San Diego, CA). The data were demultiplexed and analyzed with cellranger vdj (2.2.0) to obtain full-length paired TCR sequences assigned to individual cells.

[0171] Cloning and transduction of TCR into Jurkat cells

[0172] Candidate TCRs were generated as gene fragments. Each fragment was cloned into a plasmid expression vector consisting of the MSCV promoter and IRES-driven eGFP for monitoring transfection or transduction. Successful transformants were screened by Sanger sequencing and validated clones were maximally pre-treated for downstream applications. TCRs were transfected into the Jurkat TCR KO reporter cell line expressing Renilla luciferase under the 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 TM Transfection system, according to the manufacturer's conditions, the cells were electroporated using a pulse voltage of 1350 V, a pulse width of 20 ms, and a pulse number of 2. The contents of the electroporation reaction were then diluted into 96-well plates supplemented with 15% heat-inactivated FBS, glutaMAX TM , penicillin / streptomycin and 4.5 g / LD-glucose in 200 μL RPMI 1640 for overnight culture in a 37°C incubator.

[0173] Jurkat activation assay

[0174] Antigen presenting T2 cells (ATCC) are loaded with peptides (customized by Anaspec) or vehicles only in a certain concentration range in serum-free medium for two hours. After incubation, the T2 cells loaded are washed three times, then resuspended in complete medium and cells are counted. The T2 cells loaded with 2E5 peptides are then directly added to the Jurkat cells transfected with TCR in 100uL complete medium. Jurkat cells expressing TCR are co-cultured at 37C for 24 hours in the presence of T2 cells. After incubation, cells are transferred to 96-well U-shaped bottom plates, and 150ul FACS buffer (PBS, without CaCl and MgCl (Corning, 21-040-CV) + 5% FBS (Gibco, 10082-147)) is added, then centrifuged at 400xg for 4min. The supernatant was removed and the cells were resuspended in 50 μl 1X Fc block in FACS buffer which was incubated for 20 min at 4C. αCD69-BV421 or IgG isotype-BV421 was added at a concentration of 1 μg / mL and incubated for 1 hr at 4°C. After staining, the cells were washed three times by centrifugation at 400xg for 4 min followed by aspiration and resuspension. Prior to analysis, the cells were suspended in FACS buffer containing Sytox Red prepared according to the manufacturer's recommendations. Cells were analyzed using an LSRII or Symphony cytometer (BD Biosciences) using the recommended acquisition settings. The activity of individual TCRs is expressed as the percentage of cells expressing CD69 in a population of Jurkat cells expressing GFP (indicating plasmid expression).

[0175] MAGE-A4 TCR-T cell production using human primary T cells

[0176] PBMCs from three healthy donors (HLA-A*02:01) were isolated from leukopak (Auscell) using Ficoll-Paque gradient centrifugation, and additional T cell isolation was performed by using the CD3 negative selection kit (Miltenyi Biotec, 130-096-535) and the relevant manufacturer's protocol. One day before TCR transduction, frozen pan T cells were thawed and cultured at 1 × 10 6Cells / ml were resuspended in complete human T cell medium and stimulated by CD3 / CD28 dynabead (Thermo Fisher Scientific, 11131D) in the presence of 30 IU / ml IL-2 (Miltenyi Biotech, 130-097-745), 10 ng / ml IL-7 (Peptech, AF20007100UG) and 25 ng / ml IL-15 (Peptech, AF20015100UG), with a T cell to bead ratio of 2: 1. T cells were then seeded in 24-well plates at 1 ml / well. On the day of TCR transduction, activated T cells (3E5) were seeded in complete human T cell medium per well in a 48-well plate 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. Then T cells were inoculated with rotation at 32°C for 1.5 hours. After the rotation, 380ul of culture medium containing 8μg / ml polybrene, 100IU / ml IL-2, 10ng / ml IL-7 and 25ng / ml IL-15 was added to the cells so that the total volume was 600μl / well. 17-18 hours after transduction, about 500μl culture medium was removed without contacting the cells at the bottom of the well. In 3ml human T cell complete culture medium containing 100IU / ml IL-2, 10ng / ml IL-7 and 25ng / ml IL-15, the cells in each well of the 48-well plate were transferred to a well of a Grex 24-well plate (WilsonWolf, P / N 80192M). On the 4th day after transduction, dynabead was removed according to the manufacturer's protocol. TCR-T cells were cultured at a rate of approximately 10 × 10 cells / well in 30 ml of culture medium in the presence of 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. 6 Cells were seeded into Grex 6-well plates (Wilson Wolfe, P / N80240M). On day 7 after transduction, TCR-Ts were harvested, frozen, and stored in liquid nitrogen vapor phase. TCR transduction efficiency was verified by dextramer binding.

[0177] Flow cytometry

[0178] The following antibodies were used for T cell phenotype analysis: CD3-FITC (Baijin: 300440), CD8-BV605 (BD: 564116), CD4-PE (Baijin: 317410). The following antibodies were used for dendritic cell phenotype analysis: CD14-percpcy5.5 (Baijin: 301824), CD11c-PE (Baijin: 337206), CD1a-APC-cy7 (Baijin: 300125), CD86-APC (BD: 555660). The following antibodies were used for B cell phenotype analysis: MHC class I (Baijin: 311414), MHC class II (Baijin: 361706), CD83-PE (BD 556855), CD86-APC (BD: 555660), CD20-FITC (BD: 556632). Dextramers-APC or -PE were purchased from Immudex (custom dextramer). 50 nM PKI dasatinib (Axon Medchem: 1392) was used to prevent TCR internalization. T cells expressing TCR were incubated with 50 nM PKI dasatinib at 37 ° C for 30 min, then dextramer staining was performed on ice for 30 min, and cell surface marker staining was performed at 4 ° C for 15 min. Dead cell exclusion dye (Sytox blue, Thermo Fisher Scientific / Invitrogen, S34857) was used. Flow cytometry data were analyzed using Flowjo.

[0179] T2-Luc killing assay

[0180] The functionality and killing specificity of MAGE-A4 TCR-T were determined by T2-luc (T2 cell line expressing luciferase) killing assay. T2-luc cells were collected, washed, and plated at 2×10 6 Cells were resuspended in T2-luc killing 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), 5% heat-inactivated FBS (Gibco, 10082-147)) and then seeded in 24-well plates at 1 ml / well. T2-Luc cells were pulsed with the indicated peptide concentrations for two to four hours at 37°C. T2-luc cells were then washed and incubated at 1×10 5The cells / ml were resuspended and inoculated in 384-well plates (Corning, 3570) at 25 μl / well. T2-Luc cells were incubated with 25 μl TCR-T cells for 48 hours with the dextramer+TCR-T and T2-luc cell ratios shown. The luminescent signal was measured by adding 30 μl Bio-glo (Promega, G7940), and then by using the Biostack neo system (BioTek, Winooski, Vermont). Before the killing assay, all different TCR-Ts were normalized relative to the same amount of MAGE-A4 dextramer+ cells (e.g., 10%) by adding simulated (untransduced) T cells. TCR-T+T2 / target peptide killing was normalized by simulating T cells+T2 / target peptide killing or by TCR-T+T2 / no peptide killing, and specific lysis (specific killing %) was calculated. The specific lysis formula is as described below.

[0181] Formula for specific lysis (%)

[0182] Peptide titration (MAGE-A4 peptide and similar peptides):

[0183] {1-(TCRT+T2-luc / test peptide RLU) / (TCRT+T2-luc or C1R-luc / no peptide RLU)}×100

[0184] Cancer cell line killing:

[0185] {1-(TCRT+cancer cell line RLU) / (cancer cell, cancer cell+mock or cancer cell+RFP-IL12 T cell control)}×100

[0186] Cancer cell killing assay

[0187] The cytotoxicity of TCR-T cells against MAGE-A4 positive and negative cancer cell lines was determined by cancer cell killing assay. Cancer cells were collected, washed, and plated at 1 × 10 5Cells / ml were resuspended in cancer cell killing assay medium (RPMI 1640–GlutaMAX, 1x 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), 10% heat-inactivated FBS (Gibco, 10082-147)). Cancer cells were then seeded at 25 μl / well in 384-well plates and incubated with 25 μl TCR-T cells at the indicated ratio of dextramer+TCR-T to T2-luc cells for 48 hours. After incubation, for adherent cancer cells, remove the suspended T cells and wash with Ca2+ using a plate washer. 2+ Mg 2+ The wells were washed with DPBS (Corning, 21-031-CM). The luminescent signal was measured by adding 30 μl Celltiter Glo (Promega, G7573). For suspended luciferase-labeled cancer cells, the luminescent signal was measured by adding 30 μl Bio-glo (Promega, G7940). Luminescence measurement was performed using the Biostack neo system. For suspended cancer cells without luciferase labeling, cancer cells were labeled by Celltrace far red (Invitrogen, Carlsbad, CA, USA). Cancer cells were plated at 1×10 6 Cells / ml were resuspended in serum-free RPMI medium containing Celltrace far red (1:4000 dilution) and incubated at 37°C for 10 min. The reaction was terminated by adding 30 ml of killing assay medium and incubating at room temperature for 10 min. Live cancer cells were detected by flow cytometry. Dead cell exclusion dye (Sytox blue, Thermo Fisher Scientific / Invitrogen, S34857) was used. TCR-T killing against cancer cell lines was normalized by simulated T cell killing against cancer cell lines, and specific lysis (specific killing %) was calculated. The specific lysis formula is as described above.

[0188] Similar peptide screening

[0189] The functional specificity of MAGE-A4 TCR-T was determined using T2luc T cell directed killing assay. Peptides including target peptides and similar peptides were synthesized by JPT (Berlin, Germany) or AnaSpec (Fremont, California). At 37 ° C / 5% CO2, T2luc cells were incubated with reactive similar peptides and target-specific peptides in assay medium (RPMI 1640 supplemented with 5% heat-inactivated FBS (Millipore Sigma), 1x GlutaMax (Gibco), 1x non-essential amino acid solution (Hyclone), 10mM HEPES (Hyclone), 50μM 2-β-mercaptoethanol (Gibco), 1mM sodium pyruvate (Gibco), and the final peptide concentration range was 1.0E-05M to 6.0E-16M (efficacy) or 1.0E-05M (single point)) for 2 hours. Frozen MAGE-A4 TCR-T and mock cells were thawed, washed, and left to stand for 3 hr in 50 / 50RPMI / AIM-V / 5% huAB serum, 1x GlutaMax, 25mM HEPES, 100u P / S (Gibco), 10ug / mL gentamicin (Gibco) before assay setup. MAGE-A2 TCR-T cells were washed 3 times in assay culture medium and resuspended at 2.5E06 cells / mL. T2luc cells loaded with peptides were added to a white transparent 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 simulated 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% CO2 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 the EnVision multifunctional microplate reader (PerkinElmer). The percentage of viability was calculated using the following formula: % viability = (sample original RLU value / average DMSO / control RLU) × 100. EC50 was determined using GraphPad Prism (non-linear regression curve fitting analysis).

[0190] Human primary normal cell culture

[0191] The sources of human primary normal cells and iPSC-derived cells are summarized in Table 5. The culture conditions of these cells are summarized in Table 5. Primary cells were thawed and cultured according to the supplier's instructions, except for the following: cardiomyocytes, astrocytes, GABA neurons, and RPTECs that were converted to RPMI 1640 medium just before co-culture. Previous optimization studies have demonstrated the tolerance of RPMI 1640 and the improvement of cell viability of these cell types. Prior to the assay, all cells were counted and their viability was assessed.

[0192] Table 5. Sources of human normal primary cells and iPSC-derived cells

[0193]

[0194] Table 6. Culture media and methods for normal human cells

[0195]

[0196]

[0197]

[0198] Cytotoxicity assay using primary human normal cells

[0199] Target cell cytotoxicity was assessed using phase contrast / fluorescence kinetic imaging assays. The live imaging device measured fluorescent caspase 3 / 7 cleavage over time and superimposed on phase contrast images of captured cell confluence. Prior to implementing the cytotoxicity assay, different plating densities and tolerance to various media were evaluated to achieve appropriate confluence without significant cell overlap in 96-well plates. Target cells (100 μl) were added to a black 96-well ViewPlate at the density listed in Table 3, containing 50 μl of MAGE-A4 TCR-T-IL12 cells, IL-12-RFP T cells, or mock T cells, with a dextramer-normalized effector:target (E:T) ratio of 1:1 by considering the dextramer positivity of each TCR-T construct. CellEvent caspase 3 / 7 reagent (50 μl) was added according to the manufacturer's instructions (Thermo Fisher Scientific, C10423). The assay plate was placed in a 96-well plate equipped with S3 was placed in a 37°C, 5% CO2 incubator. Phase contrast and fluorescence images (5 fields) were collected every 4 hours starting at 0 hours for 44 or 48 hours using a 10x objective. 2019B software analyzed caspase 3 / 7 total integrated intensity. Since T cells are usually smaller than target cells, the minimum area filter was set to 200 μm2 in the fluorescence image to exclude signals from apoptotic T cells. In addition, since the fluorescence signal in the target cells was not uniform, the target cells might be identified as smaller fragments and excluded by the area filter. Therefore, low edge detection sensitivity was also applied during the analysis. After 44 or 48 hours, the plates were removed from the incubator and 50 μL of cell culture medium was removed from the wells for cytokine analysis.

[0200] Alloreactivity screening

[0201] Alloreactivity potential was assessed by co-culturing each of the four TCR-T cells with 34 BLCL lines (B-type lymphoblastoid cell 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, Washington) and Astarte Biologics (Cellero; Bothell, Washington) as listed in Table 7. BLCL were cultured in 15% FBS complete RPMI containing the following ingredients: RPMI-1640 containing L-glutamine, 15% (v / v) HI-FBS, and 1 mM sodium pyruvate.

[0202] U266B1 cells (ATCC; 10 μM in culture medium) as a MAGE-A4+HLA-A*02:01+ positive control cell line were treated with 50 μM MAGE-A4 peptide by incubation at 37°C for 2 hours. 5Cells / ml) were pulsed. TCR-T cells from donor 12665 ​​were thawed by adding culture medium, centrifuged at 400xg for 5min at 4°C, resuspended in 10ml culture medium, and counted. TCR-T cells were co-cultured with BLCL or peptide-pulsed U266B1 cells in a volume of 200μl. The dextramer-normalized effector: target ratios of the four TCR-T cells ranged from 3:1 to about 8:1, depending on the respective dextramer positivity. All co-cultures were performed in 96-well flat-bottom tissue culture plates at 37°C, 5% CO2 for 48 hours. After incubation, the 96-well plates were centrifuged at 887xg for 1min at 4°C, and the supernatants were collected in 96-well V-bottom plates for cytokine analysis. According to the manufacturer's instructions, using a customized Milliplex human cytokine / chemokine kit (Millipore, SRP1885), cytokines and granzyme B, including IFNγ, granzyme B, TNFα and IL-12p70 analytes, are evaluated by Luminex assay. Serial dilutions of analyte standards are run in duplicate on each assay plate. Luminex plates are read on FlexMap 3D instruments (XMAP Technologies). Data are exported by xPONENT software and directly analyzed by the Milliplex Analyst software of EMD Millipore, thereby generating a standard curve using a 5-parameter logistic nonlinear regression fitting curve. Detection limits (minimum and maximum values) are calculated by Milliplex Analyst software as the result of the average value of the appropriate repeated standard curve values ​​obtained from each assay plate, and the range in which the analyte can be interpolated from the standard is indicated. Samples are run at appropriate dilutions to ensure that the measured values ​​of the sample analyte levels are within the limits of the assay standard curve. Cytokine and granzyme B levels are reported in pg / mL or as fold difference from IL12-RFP T cells (control) and plotted in GraphPad Prism software.

[0203] Table 6. BLCL lines used for alloreactivity screening

[0204]

[0205]

Claims

1. An expression vector comprising a nucleic acid sequence encoding a T cell receptor (TCR) α chain and a TCR β chain, wherein the TCR α chain and the TCR β chain are selected from the group consisting of: a. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 3, 5 and 7, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 4, 6 and 8, respectively; b. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 13, 15 and 17, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 14, 16 and 18, respectively; c. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 23, 25 and 27, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown 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 shown in SEQ ID NOs: 34, 36 and 38, respectively; e. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 43, 45 and 47, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown 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 shown 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 shown 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 shown in SEQ ID NOs: 64, 66 and 68, respectively; and h. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:73, 75 and 77, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:74, 76 and 78, respectively.

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

3. The expression vector of claim 1 or claim 2, wherein the expression vector is a viral vector. The expression vector of claim 3 , wherein the viral vector is a retroviral vector. The expression vector of claim 4 , wherein the retroviral vector is a lentiviral vector.

6. The expression vector of any one of claims 1 to 5, wherein the TCR α chain and the TCR β chain comprise an amino acid sequence selected from the group consisting of: a. the amino acid sequence shown in SEQ ID NO: 9 or 10, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 11 or 12; b. the amino acid sequence shown in SEQ ID NO: 19 or 20, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 21 or 22; c. the amino acid sequence shown in SEQ ID NO: 29 or 30, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 31 or 32; d. the amino acid sequence shown in SEQ ID NO: 39 or 40, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 41 or 42; e. the amino acid sequence shown in SEQ ID NO: 49 or 50, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 51 or 52; f. the amino acid sequence shown in SEQ ID NO: 59 or 60, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 61 or 62; g. the amino acid sequence shown in SEQ ID NO: 69 or 70, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 71 or 72; and h. The amino acid sequence shown in SEQ ID NO:79 or 80, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO:81 or 82.

7. 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 are selected from the group consisting of: a. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 3, 5 and 7, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 4, 6 and 8, respectively; b. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 13, 15 and 17, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 14, 16 and 18, respectively; c. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 23, 25 and 27, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown 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 shown in SEQ ID NOs: 34, 36 and 38, respectively; e. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 43, 45 and 47, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown 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 shown 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 shown 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 shown in SEQ ID NOs: 64, 66 and 68, respectively; and h. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:73, 75 and 77, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:74, 76 and 78, respectively.

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

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

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

11. The cell of claims 7-10, wherein the cell is a T cell.

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

13. A pharmaceutical composition comprising a therapeutically effective amount of the cell according to any one of claims 7 to 12.

14. A method for preparing the cell according to claims 7-12 or the pharmaceutical composition according to claim 13, the method comprising: Introducing into the cell an expression vector comprising a nucleic acid sequence encoding a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain are selected from the group consisting of: a. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 3, 5 and 7, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 4, 6 and 8, respectively; b. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 13, 15 and 17, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 14, 16 and 18, respectively; c. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 23, 25 and 27, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown 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 shown in SEQ ID NOs: 34, 36 and 38, respectively; e. A TCR alpha chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs: 43, 45 and 47, respectively, and a TCR beta chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown 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 shown 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 shown 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 shown 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 shown in SEQ ID NOs: 64, 66 and 68, respectively; and h. A TCRα chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:73, 75 and 77, respectively, and a TCRβ chain comprising CDR1, 2 and 3 sequences comprising the amino acid sequences shown in SEQ ID NOs:74, 76 and 78, respectively.

15. The method of claim 14, wherein the TCR alpha chain and the TCR beta chain are selected from the group consisting of: a. the amino acid sequence shown in SEQ ID NO: 9 or 10, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 11 or 12; b. the amino acid sequence shown in SEQ ID NO: 19 or 20, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 21 or 22; c. the amino acid sequence shown in SEQ ID NO: 29 or 30, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 31 or 32; d. the amino acid sequence shown in SEQ ID NO: 39 or 40, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 41 or 42; e. the amino acid sequence shown in SEQ ID NO: 49 or 50, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 51 or 52; f. the amino acid sequence shown in SEQ ID NO: 59 or 60, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 61 or 62; g. the amino acid sequence shown in SEQ ID NO: 69 or 70, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO: 71 or 72; and h. The amino acid sequence shown in SEQ ID NO:79 or 80, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO:81 or 82.

16. The method according to claim 14 or 15, wherein the expression vector further comprises a nucleic acid sequence encoding IL-12 or a functional variant thereof.

17. The method of claims 14-16, wherein the cell is a T cell.

18. The method of claim 17, wherein the T cell is a primary T cell.

19. The method of claim 18, wherein the primary T cells are isolated from a cancer patient.

20. A method for treating a cancer expressing MAGE-A4 or MAGE-A8, the method comprising administering to a cancer patient a therapeutically effective amount of the cell of any one of claims 7-12, the pharmaceutical composition of claim 13, or the cell prepared by the method of any one of claims 14-19.

21. The method of claim 20, wherein the patient is tested prior to administration to determine the presence of a cancer expressing MAGE-A4 or MAGE-A8.

22. The method of claim 21, wherein a nucleic acid encoding MAGE-A4 or MAGE-A8 is detected.

23. The method of claim 21, wherein MAGE-A4 or MAGE-A8 protein or a MAGE-A4-derived peptide or a MAGE-A8-derived peptide is detected.

24. The method of any one of claims 20-23, wherein the patient is identified as carrying the HLA-A*0201 allele.

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