T cell receptors recognizing a mage a4 antigen hla-c0702-restricted epitope and uses thereof

Abstract

The present application provides a T cell antigen receptor specifically recognizing MAGE-A4 antigen, which can recognize MAGE-A4 antigen complex presented by HLA-C*07:02. The present application also provides an engineered immune cell expressing the TCR specifically recognizing MAGE-A4 antigen. The present application also provides a nucleic acid and a recombinant expression vector encoding the T cell antigen receptor specifically recognizing MAGE-A4 antigen. The present application also provides use of the TCR and / or the engineered immune cell in the preparation of a medicament for preventing or treating cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to T cell receptors, immune cells, and their applications that specifically recognize the MAGE A4 antigen HLA-C0702 restriction epitope. Background Technology

[0002] Melanoma-associated antigen A4 (MAGE-A4) is an important member of the tumor testis antigen family, exhibiting a strictly tumor-specific expression pattern: it is almost not expressed in normal tissues other than the testes and placenta, but is highly expressed in a variety of solid tumors, including synovial sarcoma, ovarian cancer, head and neck cancer, esophageal cancer, non-small cell lung cancer, and gastric cancer. Due to its unique expression profile and high immunogenicity, MAGE-A4 has become one of the most promising targets for TCR-T cell therapy of solid tumors.

[0003] Adoptive T-cell transfer can enhance the immune system's elimination of tumor cells and is a novel, specific, and non-toxic cancer therapy that has gained significant attention in recent years. In adoptive T-cell transfer, T-cell antigen receptor (T-cell antibody or TCR) gene transfer, as a rapidly developing immunotherapy method, can generate a large number of T cells with known antigen specificity and functional affinity in vitro, which can be applied to adoptive cell immunotherapy for malignant tumors. The generation of TCR-engineered T cells (TCR-T) begins with screening and cloning tumor-specific TCR genes, followed by transfection of T cells with these genes to confer antigen specificity, thereby obtaining genetically engineered antigen-specific T cells. Finally, TCR-transfected T cells are reinfused into the patient to reconstruct the T-cell immune response against antigen-positive tumors.

[0004] There are existing reports in this field of using TCR-T therapy to treat cancers caused by MAGE-A4. The FDA has approved the world's first MAGE-A4 TCR-T therapy, Afamitresgene autoleucel (trade name: Tecelra), for the treatment of patients with unresectable or metastatic synovial sarcoma who are HLA-A02:01 positive and MAGE-A4 positive. This therapy specifically recognizes the MAGE-A4 230-239 epitope (GVYDGREHTV) presented by HLA-A02:01.

[0005] Despite the promising clinical prospects of MAGE-A4 TCR-T therapy, current technologies still have significant limitations and shortcomings. HLA restriction severely limits the applicable population. Most MAGE-A4 TCR-T therapies focus on the HLA-A02:01 subtype. However, the frequency of HLA-A02:01 in the global population is only about 40%, and about 30-35% in East Asian populations. This means that more than 60% of patients with MAGE-A4-positive tumors cannot benefit from existing therapies. Furthermore, HLA-C is an important component of human leukocyte antigen class I molecules and exhibits high polymorphism in the population. Among them, HLA-C07:02 is one of the most widely distributed HLA-C subtypes globally, with a frequency of about 15-20% in European populations and about 10-15% in East Asian populations. However, HLA-C subtype-restricted TCR resources are extremely scarce. Furthermore, MAGE-A4 TCR-T therapy has limited epitope coverage, primarily focusing on a few epitopes, such as GVYDGREHTV (HLA-A*02:01) and SESLKMIF (HLA-B37). The MAGE-A4 protein is 317 amino acids long, theoretically capable of generating a large number of different HLA-restricted epitopes. This limited epitope coverage not only restricts the applicable population but may also lead to immune escape by tumors through epitope loss. To enhance the killing ability against tumor cells expressing low-level antigens, current technologies typically modify TCRs to increase affinity. However, the balance between TCR affinity and specificity is difficult to control; excessive affinity enhancement may cause TCRs to recognize homologous peptides expressed in normal tissues, leading to severe off-target toxicity. For example, a high-affinity TCR targeting MAGE-A3 has resulted in patient death due to accidental recognition of cardiac titin.

[0006] There is also a need in the art for new T cell receptors and engineered immune cells targeting MAGE-A4, particularly T cell receptors and engineered immune cells capable of recognizing MAGE-A4 antigens presented by specific HLA-subtypes, and the use of said T cell receptors and engineered immune cells in the treatment and prevention of diffuse midline gliomas. Summary of the Invention

[0007] This invention provides a T-cell receptor and engineered immune cells targeting the melanoma-associated antigen A4 (MAGE-A4).

[0008] Specifically, the present invention provides a T-cell antigen receptor that can specifically recognize the epitope YEFLWGPRAL complex of melanoma-associated antigen A4 presented by HLA-C*07:02.

[0009] In one aspect of the invention, a T-cell antigen receptor (TCR) that specifically recognizes MAGE-A4 is provided, characterized in that the α-chain variable region VH of the TCR includes complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR3 is as shown in SEQ ID NO:3. In another aspect of the invention, the amino acid sequences of CDR1, CDR2, and CDR3 of the α-chain variable region VH of the provided TCR are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0010] In another aspect of the invention, the amino acid sequence of the α-chain variable region VH of the TCR that specifically recognizes MAGE-A4 is as shown in SEQ ID NO: 7, or has at least 90%, 95%, 98% or 99% identity with SEQ ID NO: 7.

[0011] In one aspect of the invention, the β-chain variable region VL of the TCR that specifically recognizes MAGE-A4 includes complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR3 is as shown in SEQ ID NO:6. In another aspect of the invention, the amino acid sequences of CDR1, CDR2, and CDR3 of the β-chain variable region VL of the provided TCR are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0012] In another aspect of the invention, the amino acid sequence of the β-chain variable region VL of the TCR that specifically recognizes MAGE-A4 is as shown in SEQ ID NO: 8, or has at least 90%, 95%, 98% or 99% identity with SEQ ID NO: 8.

[0013] In one embodiment of the present invention, the α-chain variable region VH of the TCR that specifically recognizes MAGE-A4 includes complementarity-determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR3 is as shown in SEQ ID NO:3, and its β-chain variable region VL includes complementarity-determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR3 is as shown in SEQ ID NO:6.

[0014] In one embodiment of the present invention, the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain variable region VH of the TCR that specifically recognizes MAGE-A4 are as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain variable region VL are as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0015] In one embodiment of the present invention, the amino acid sequence of the α-chain variable region VH of the TCR that specifically recognizes MAGE-A4 is as shown in SEQ ID NO: 7, or has at least 90%, 95%, 98% or 99% identity with SEQ ID NO: 7, and the amino acid sequence of its β-chain variable region VL is as shown in SEQ ID NO: 8, or has at least 90%, 95%, 98% or 99% identity with SEQ ID NO: 8.

[0016] Melanoma-associated antigen A (MAGE) A), belonging to the type I MAGE family, is a type of cancer. An important member of the testis-associated antigen group. MAGE The A gene family is located in the q28 region of the X chromosome and has 12 members (MAGE). A1 to MAGE A12). All MAGE Member A all share the same MAGE homology domain (MHD), which is a highly conserved domain consisting of approximately 170 amino acids and exhibits high sequence homology with each other.

[0017] Melanoma-associated antigen 4 protein (MAGE) A4) is MAGE Members of the A gene family. MAGE A4 is involved in cell cycle progression, transcriptional control, and cell survival or apoptosis. In malignant tumors, overexpression of MAGE... A4 may be associated with tumor growth and metastasis, as well as poor patient prognosis. As an intracellular protein, MAGE... A4 can be degraded in the proteasome and, after processing, presented on the cell surface via major histocompatibility complex I (MHC I). It is then recognized by the T cell receptor (TCR) and induces an immune response, specifically killing tumor cells.

[0018] In this invention, the MAGE-A4 antigen recognizable by the TCR provided by this invention includes any polypeptide or protein containing the amino acid sequence of the epitope YEFLWGPRAL, such as naturally occurring endogenous MAGE-A4 protein or its naturally processed product, or artificially synthesized and genetically engineered or prepared proteins or portions thereof. In this invention, the MAGE-A4 antigen recognizable by the TCR provided by this invention also includes naturally occurring or artificially prepared immunogenic peptides containing the amino acid sequence of the epitope YEFLWGPRAL and of sufficient length to be presented by MHC molecules and activate T cells. In some aspects of this invention, the length of said immunogenic peptide is generally 8-11 mere or longer.

[0019] In this invention, the term "MHC" refers to "major histocompatibility antigen." In humans, MHC genes are referred to as HLA ("human leukocyte antigen") genes. While there is no universally accepted convention, some literature uses HLA to refer to HLA protein molecules and MHC to refer to genes encoding HLA proteins. Therefore, when used herein, the terms "MHC" and "HLA" are equivalent. The human HLA system has an equivalent in mice, the H2 system. The most thoroughly studied HLA genes are the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. In humans, the MHC is divided into three regions: class I, class II, and class III. Genes A, B, and C belong to MHC class I, while the six D genes belong to class II. MHC class I molecules consist of a single polymorphic chain containing three domains (α1, 2, and 3) that associates with β2 microglobulin on the cell surface. Class II molecules consist of two polymorphic chains, each containing two chains (α and β).

[0020] Class I MHC molecules are expressed on almost all nucleated cells. Peptide fragments presented in the context of class I MHC molecules are recognized by CD8 T lymphocytes (cytotoxic T lymphocytes or CTLs). CD8+ T cells or lymphocytes frequently mature into cytotoxic effector cells, which can lyse cells carrying stimulating antigens. Class II MHC molecules are primarily expressed on activated lymphocytes and antigen-presenting cells. CD4+ T lymphocytes (helper T lymphocytes or HTLs) are activated by recognizing unique peptide fragments presented by class II MHC molecules typically present on antigen-presenting cells such as macrophages or dendritic cells. CD4+ T lymphocytes proliferate and secrete cytokines that support antibody-mediated responses by producing IL-4 and IL-10, or cell-mediated responses by producing IL-2 and IFNγ.

[0021] In one aspect of the invention, an isolated nucleic acid is also provided, which is a nucleic acid encoding the aforementioned T-cell antigen receptor that specifically recognizes MAGE-A4.

[0022] In one aspect of the invention, a recombinant expression vector comprising nucleic acid encoding the aforementioned T-cell antigen receptor (TCR) that specifically recognizes MAGE-A4 is also provided. In one aspect of the invention, the recombinant expression vector of the present invention is a plasmid. In yet another aspect of the invention, the recombinant expression vector of the present invention is a plasmid for packaging viral vectors, such as plasmids for packaging baculovirus expression vectors, adenovirus vectors, retroviral vectors, herpesvirus vectors, or lentiviral vectors.

[0023] In one aspect of the invention, the recombinant expression vector of the present invention is a vector for expressing proteins in mammals. In another aspect of the invention, the recombinant expression vector is a viral vector, such as a baculovirus expression vector, adenovirus vector, retrovirus vector, herpesvirus vector, or lentiviral vector. Preferably, the recombinant expression vector of the present invention is a lentiviral vector. In the viral vector, a foreign DNA insert is ligated into the viral genome and can infect target cells (e.g., T cells) and carry the foreign DNA insert into / integrate into the target cell genome, and express the protein it encodes.

[0024] In one aspect of the invention, engineered immune cells expressing the T-cell antigen receptor (TCR) as described above are also provided. In this invention, immune cells include, for example, leukocytes (white blood cells), lymphocytes (T cells, B cells, natural killer (NK) cells) derived from hematopoietic stem cells (HSCs) generated in the bone marrow, and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). T cells include all types of immune cells expressing CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Tregs), and γδ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses.

[0025] In another aspect of the invention, the engineered immune cells provided include T cells, NK cells, NKT cells, or stem cell-derived immune cells. Preferably, the engineered immune cells provided by the present invention are CD4+ T cells or CD8+ T cells.

[0026] The TCR substances of the present invention can be formulated into pharmaceutical compositions. In this regard, embodiments of the present invention provide pharmaceutical compositions comprising any TCR, its functional portion or functional variant, nucleic acid, expression vector, host cells (including populations thereof), and antibodies (including their antigen-binding portions), as well as pharmaceutically acceptable carriers. Pharmaceutical compositions of the present invention containing any TCR substance of the present invention may comprise more than one TCR substance of the present invention, such as a TCR and a nucleic acid, or two or more different TCRs. Optionally, the pharmaceutical composition may comprise the TCR substance of the present invention in combination with other pharmaceutically active agents or drugs such as chemotherapeutic agents. In a preferred embodiment, the pharmaceutical composition comprises engineered cells or populations thereof of the present invention.

[0027] Therefore, the present invention provides pharmaceutical compositions comprising any of the TCRs, nucleic acids, expression vectors, and engineered immune cells described above. The present invention also provides the use of any of the TCRs, nucleic acids, expression vectors, and engineered immune cells described above in the preparation of medicaments for the treatment or prevention of MAGE-A4-related diseases.

[0028] Regarding pharmaceutical compositions, pharmaceutically acceptable carriers can be any conventionally used carriers and are limited only by chemical-physical considerations, such as solubility and lack of reactivity with the active agent, and the route of administration. The pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. Preferred are pharmaceutically acceptable carriers that are chemically inert to the active agent and that do not cause harmful side effects or toxicity under the conditions of use.

[0029] For the purposes of the method of the present invention, when the target population of cells or cell population is given, the cells may be allogeneic cells of mammals or cells from mammals themselves. Preferably, the cells are from mammals themselves.

[0030] The pharmaceutical compositions provided by this invention are applicable to mammals and humans. As used herein, the term mammal includes, but is not limited to, rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. Mammals may be from the order Carnivora, including felines (cats) and canines (dogs). Mammals may be from the order Artiodactyla, including bovids (cattle) and suidae (pigs), or perissodactyls, including equines (horses). Mammals may be from the orders Primates, apes, or monkeys (monkeys) or the suborder Anthropoidea (humans and apes). In one aspect of this invention, mammals as used herein also include humans.

[0031] The TCR substances and pharmaceutical compositions provided by this invention can be used to treat MAGE-A4-related diseases, particularly MAGE-A4-related cancers. Non-limiting examples of MAGE-A4-related cancers include synovial sarcoma, non-small cell lung cancer, esophageal cancer, urothelial carcinoma, oral cancer, sarcoma, head and neck cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, melanoma, ovarian cancer, colorectal cancer, and breast cancer. In one aspect of this invention, MAGE-A4-related diseases are particularly melanoma. Attached Figure Description

[0032] Figure 1 The results show the TCR positivity rate of recombinant TCR on the surface of engineered T cells as detected by flow cytometry.

[0033] Figure 2 Preparation of 293T cells expressing the HLA C*0702-MAGE-A4 polypeptide complex. Figure 2 A shows the structure of the insert fragment of the pHLA C*0702-MAGE-A4 polypeptide construct used for transfection of 293T cells. Figure 2 B shows the results of flow cytometry detection of HLA C*0702-MAGE-A4 polypeptide complex expression, indicating that 293T cells stably express the HLA C*0702-MAGE-A4 polypeptide complex.

[0034] Figure 3 This study demonstrates the specific activation ability of the MAGE-A4 antigen on CD8+ recombinant TCR-T cells provided by this invention. The TCR-T cells of this invention were co-incubated with pA4-293T target cells stably expressing the pHLA C*0702-MAGE-A4 complex. The proportion of CD8+ T cells expressing the cytokine IFNg was detected by flow cytometry to analyze the specific activation ability of the MAGE-A4 antigen on CD8+ recombinant TCR-T cells. Detailed Implementation

[0035] The following will further illustrate the essence and beneficial effects of the present invention with reference to embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0036] Example 1: Obtaining TCRs that specifically recognize the MAGE-A4 epitope YEFLWGPRAL Based on literature review and prediction results, a peptide library containing the MAGE-A4 antigenic epitope was designed and synthesized and produced by Sangon Biotech (Shanghai) Co., Ltd. The peptide with the predicted HLA subtype HLA-C*07:02 and the amino acid sequence YEFLWGPRAL (SEQ ID NO. 9) was used to stimulate peripheral blood mononuclear cells (PBMCs) from an anonymous donor, inducing the expansion of antigen-specific T cell clones. After monitoring and enriching the antigen-specific T cell clones using flow cytometry and enzyme-linked immunospot assay, single-cell transcriptome sequencing and TCR sequencing were performed on the enriched cells; HLA typing of the donor cells was then performed based on the transcriptome data.

[0037] The selected TCRs were sequenced, and the information obtained is shown in Table 1 below.

[0038] Table 1. Structure and amino acid sequence of TCR clones that specifically recognize the MAGE-A4 epitope YEFLWGPRAL

[0039] Example 2 Preparation of recombinant TCR and TCR-T cells New TCRs and TCR-T cells were constructed based on the TCR sequence obtained in Example 1. Specific steps and methods included: Vector construction design: The MP71 reverse transcription vector construct was constructed using standard molecular biology techniques, in which nucleotide inserts encoding the following functional structures were synthesized: HGH SS (human growth hormone signal peptide) - TCRalpha chain variable region (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR) - mCa (mouse TCRalpha chain constant region) - P2A (2A self-cleaving peptide) - TCRbeta chain variable region (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) - mCb (mouse TCRbeta chain constant region) - WPRE (marmot hepatitis virus post-transcriptional regulatory element).

[0040] The amino acid sequences of each functional structural fragment are shown below: HGH SS:MATGSRTSLLLAFGLLCLPWLQEASA; The variable region of the TCR alpha chain (i.e., the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 of the TCR obtained in Example 1): QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYS NGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVTSGTYKYIFGTGTRLKVLA (SEQ ID NO. 7); mCa: DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLD MKAMDSKSNGAIAWSNQTSFTCQDIFKETNACYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS; P2A: RAKRGSGATNFSLLKQAGDVEENPGP; The variable region of the TCR beta chain (i.e., the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 of the TCR obtained in Example 1): KVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFGQGGSPLHFGNGTRLTVT (SEQ ID NO. 8); mCb:EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVN GKEVHSGVSTDPQAYKESNYSYCLSSRLRVCATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS.

[0041] Cell lines and culture media: HEK-293T cells were purchased from ATCC. Peripheral blood mononuclear cells (PBMCs) from anonymous donors were purchased from Shanghai Junxing Biotechnology Co., Ltd. Cells were cultured in DMEM + 10% FBS or X-Vivo + 5% human serum A / B + 1% HEPES + 1% GlutaMAX.

[0042] Preparation of retroviruses: Retroviral vectors and viral packaging vectors were co-transfected into 293T cells using a standard calcium phosphate precipitation transfection method. Viral supernatant was harvested 48 hours later and used for T cell transduction.

[0043] T cell transduction and culture: Before retrovirus transduction, PBMCs were activated for 2 days using T cell stimulation beads. For transduction, freshly harvested retrovirus supernatant was centrifuged at 32°C for 2 hours and transferred to 24-well plates (Clontech) coated with 15 μg RetroNectin per well. Activated PBMCs were loaded onto the plates and centrifuged at 600g for 30 minutes at 32°C. T cells were incubated at 37°C and 5% CO2. Culture medium was replenished every 2 days.

[0044] TCR staining: All antibodies were purchased from Biolegend. 72 hours after cell transfection, cells were stained with antibody against the TCR β chain, followed by flow cytometry to detect recombinant TCR expression. Results are as follows: Figure 1 As shown, flow cytometry results indicated that after transduction of MAGE-A4 TCR, some CD8-positive and CD8-negative T cells expressed recombinant TCR, while no recombinant TCR expression was detected in the untransduced control group CD8-positive and CD8-negative T cells. These results demonstrate that the MAGE-A4 TCR described in this invention is effectively recombinantly expressed on the surface of T cells via a retroviral transduction system.

[0045] Example 3: Detection of Recombinant TCR-T Activity like Figure 2 As shown, 293T cells were edited using retroviruses to stably express the pHLA C*0702-MAGE-A4 complex. The resulting target cells, pA4-293T, were used as antigen-presenting cells for testing MAGE-A4 TCR-T.

[0046] Vector construction design: The MP71 reverse transcription vector construct was built using standard molecular biology techniques, in which nucleotide inserts encoding the following functional structures were synthesized (e.g., Figure 2 (As shown in A): SS (signal peptide) - MAGE-A4 peptide (YEFLWGPRAL) - G4S linker peptide - microglobulin B2M - HLA-C0702 - WPRE (marmot hepatitis virus post-transcriptional regulatory element).

[0047] Preparation of retroviruses: Retroviral vectors and viral packaging vectors were co-transfected into 293T cells using a standard calcium phosphate precipitation transfection method. Viral supernatant was harvested 48 hours later and used for transduction into target 293T cells.

[0048] Target cell 293T cell transduction and culture: Freshly harvested retroviral supernatant was centrifuged at 32°C for 2 hours and transferred to 24-well plates (Clontech) coated with 15 μg RetroNectin per well. 293T cells were loaded onto the plates and centrifuged at 600g for 30 minutes at 32°C. The 293T cells were incubated at 37°C and 5% CO2. After 2 days, the cells were digested and expanded, and culture medium was added.

[0049] G4S staining: All antibodies were purchased from Cell Signaling. 72 hours after cell transfection, cells were stained with an antibody against the G4S linker peptide, followed by flow cytometry to detect the expression of the pHLA C*0702-MAGE-A4 complex. Results are as follows: Figure 2 B shows that the pHLA C*0702-MAGE-A4 complex can be effectively expressed on the surface of target 293T cells via a retroviral transduction system.

[0050] The recombinant TCR-T cells prepared in Example 2 were co-incubated with pA4-293T target cells stably expressing the pHLA C*0702-MAGE-A4 complex at 37°C for 6 h in a 5% CO2 incubator. After incubation, cells were collected, and intracellular cytokine IFNg staining was performed on the co-incubated cells. The proportion of IFNg-expressing cytokine in CD8+ T cells was detected by flow cytometry to analyze the specific activation ability of the MAGE-A4 antigen on CD8+ recombinant TCR-T cells.

[0051] The results are as follows Figure 3 As shown, transfected negative control T cells were not activated by pA4-293T cells, and MAGE-A4 TCR-T cells were not activated by unedited wild-type 293T cells; co-incubation of MAGE-A4 TCR-T and pA4-293T cells led to upregulation of intracellular cytokine IFNg expression levels. The results indicated that recombinant TCR-T cells were specifically activated by pA4-293T cells that presented the MAGE-A4 peptide YEFLWGPRAL via HLA-C*07:02.

[0052] This invention provides a TCR that specifically recognizes the HLA-C07:02 molecule-presented MAGE-A4 epitope YEFLWGPRAL, filling the gap in HLA-C07:02 subtype MAGE-A4 TCR-T therapy and expanding the applicable population for MAGE-A4 TCR-T therapy. This invention can provide treatment opportunities for approximately 10-20% of MAGE-A4-positive tumor patients, complementing existing HLA-A02:01-restricted therapies and significantly improving the overall patient coverage of the MAGE-A4 target. The MAGE-A4 epitope YEFLWGPRAL recognized by this invention is completely different from epitopes recognized by existing therapies (such as GVYDGREHTV). Combining this invention's TCR-T with existing HLA-A02:01-restricted TCR-T can simultaneously target multiple different epitopes of the MAGE-A4 protein, effectively preventing immune escape caused by tumor epitope loss and improving treatment durability and cure rate.

[0053] The applicant has thus provided methods and compositions for treating diseases caused by MAGE-A4, particularly cancer, and developed new treatments for individuals affected by MAGE-A4.

[0054] The foregoing description of the present invention should not be construed as limiting it. Unless otherwise indicated, the present invention will be practiced using conventional techniques such as organic chemistry, polymer chemistry, and biotechnology, and it is obvious that the invention can be implemented in other ways besides those specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art. Many changes and variations are possible based on the teachings of the present invention, and therefore fall within the scope of the invention.

Claims

1. A T-cell antigen receptor (TCR) that specifically recognizes the MAGE-A4 epitope YEFLWGPRAL complex presented by the HLA-C*07:02 molecule.

2. The T-cell antigen receptor according to claim 1, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the α-chain variable region VH of the TCR are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or The amino acid sequences of CDR1, CDR2 and CDR3 of the β chain variable region VL of the TCR are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

3. The T-cell antigen receptor according to claim 2, wherein the amino acid sequence of the α-chain variable region VH of the TCR is as shown in SEQ ID NO: 7, or has at least 90%, 95%, 98% or 99% identity with SEQ ID NO:

7.

4. The T-cell antigen receptor according to claim 2, wherein the amino acid sequence of the β-chain variable region VL of the TCR is as shown in SEQ ID NO: 8, or has at least 90%, 95%, 98% or 99% identity with SEQ ID NO:

8.

5. An isolated nucleic acid encoding a T-cell antigen receptor (TCR) that specifically recognizes MAGE-A4, wherein the TCR is defined as in any one of claims 1-4.

6. A recombinant expression vector comprising nucleic acid encoding a T-cell antigen receptor (TCR) that specifically recognizes MAGE-A4, said vector being a vector for expressing a protein in mammals.

7. An engineered immune cell expressing a T-cell antigen receptor (TCR) as defined in any one of claims 1-4, wherein the immune cell is selected from T cells, NK cells, NKT cells or stem cell-derived immune cells, preferably CD4+ T cells or CD8+ T cells.

8. A pharmaceutical composition comprising the TCR of any one of claims 1-4, the nucleic acid of claim 5, the expression vector of claim 6, and the engineered immune cell of claim 7, the pharmaceutical composition being used to treat or prevent MAGE-A4-related diseases.

9. The pharmaceutical composition according to claim 8, wherein the MAGE-A4-related disease is cancer, such as synovial sarcoma, non-small cell lung cancer, esophageal cancer, urothelial carcinoma, oral cancer, sarcoma, head and neck cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, melanoma, ovarian cancer, colorectal cancer, and breast cancer.

10. Use of the TCR of any one of claims 1-4, the nucleic acid of claim 5, the expression vector of claim 6, and the engineered immune cell of claim 7 in the preparation of a medicament for the treatment or prevention of MAGE-A4-related diseases.

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