T cell receptor engineering modification method and use thereof

Abstract

Provided in the present invention is a T cell receptor engineering modification method, which comprises the steps of: obtaining CDR regions of a given T cell receptor sequence by means of a database, mutating one or more amino acid residues in the CDR regions into histidine, and establishing a first T cell receptor mutation library. The T cell receptor engineering modification method provided by the present invention is based on a histidine scanning method, realizes TCR engineering modification independent of three-dimensional structures, overcomes the disadvantages of high affinity and realizes the modification of TCRs with low affinity and high activation, thereby providing more options for clinical use. The T cell receptor provided by the present invention comprises the following six CDR regions, CDR1α, CDR2α and CDR3α in a TCRα chain, and CDR1β, CDR2β and CDR3β in a TCRβ chain, the amino acid sequences of which are shown as SEQ ID No. 1-6, respectively. In the present invention, engineering modification of a wild-type MAGE-A3 TCR molecule is achieved by means of a catch bond to obtain an efficient and non-toxic TCR targeting MAGE-A3, which is free of toxic and side effects caused by affinity maturation. Moreover, the engineered TCR is applied to TCR-T cell preparation for solid tumor treatment.

Description

A method for engineering T-cell receptors and its application Technical Field

[0001] This invention relates to the fields of protein engineering and biomedicine, and in particular to a method for engineering T-cell receptors and its application. Background Technology

[0002] In tumor immunotherapy, T cells play a central role. Through their surface T cell receptors (TCRs), T cells specifically recognize tumor antigen peptide-MHC complexes (pMHC) on the surface of cancerous cells, enabling targeted killing of tumor cells and immune surveillance. However, when tumor cells develop tumor antigen mutations / decreased or absent antigen expression levels, T cells cannot effectively recognize and kill them, allowing the tumor to escape and gain a growth advantage. Therefore, engineering TCRs to enhance their recognition and killing of tumor target antigens and tumor target cells is of great significance for tumor treatment.

[0003] In the engineering of tumor receptors (TCRs), affinity maturation methods are often used to enrich TCR variants with high affinity for tumor target antigens. While the specific activation levels of some high-affinity TCR variants can indeed be enhanced, excessively high affinity may lead to off-target recognition of other antigens, particularly normal autoantigens in healthy tissues and vital organs, causing severe side effects and even patient death in clinical practice.

[0004] Recent studies have revealed no significant correlation between T cell activation levels and affinity; rather, the strength of activation is determined by the catch bond level between the TCR and pMHC. Catch bonds are non-covalent interactions between protein macromolecules induced by external forces, including hydrogen bonds and salt bridges. Under piezoelectric shear forces, the bond lifetime of these interactions initially increases and then decreases with increasing applied force, peaking around 10 pN. Current techniques, based on the three-dimensional structure of TCR-pMHC, have used library design to target amino acid residues at specific distances, discovering some TCRs with low affinity but high activation levels, offering a potential solution to the off-target toxicity of high-affinity TCRs. However, for most TCR-pMHC combinations, their three-dimensional structures are unavailable due to low affinity. Therefore, there is a need to develop an engineering method for low-affinity, high-activation TCRs that is independent of three-dimensional structure.

[0005] Cancer-testis antigens (CT antigens), also known as tumor-testis antigens, are antigens that are not expressed in normal tissues other than the testes and placenta, but are frequently expressed in various tumors, exhibiting high tumor specificity and strong immunogenicity. Among these, MAGE-A, MAGE-B, MAGE-C, and NY-ESO-1 family proteins have been extensively studied. Currently, CT antigens are a major target in TCR-T cell research.

[0006] MAGE-A3 is expressed in the tumor tissues of more than 76% of melanoma patients and some lung cancer patients. In addition, except for the placenta and testes, MAGE-A3 is not expressed in normal human cells. This provides a theoretical basis for tumor immunotherapy targeting MAGE-A3 and makes it an ideal target for TCR-T therapy. Summary of the Invention

[0007] This invention is made to solve the above-mentioned problems. The purpose of this invention is to provide a method for engineering T-cell receptors and their applications, a T-cell receptor targeting MAGE-A3 and its uses, in order to solve the problems in the prior art.

[0008] To achieve the above and other related objectives, the present invention provides a method for engineering T-cell receptors, the method comprising the following steps:

[0009] S1: Obtain the CDR region of a given T cell receptor sequence from the database, mutate one or more amino acid residues in the CDR region to histidine, and establish the first T cell receptor mutant library.

[0010] S2: Express the mutant T cell receptor from the first T cell receptor mutant library on T cells;

[0011] S3: Mix the T cell line expressing the mutant T cell receptor in S2 with the antigen, and determine the potential mutation hotspot residues in the CDR region of the T cell receptor based on the expression level of marker molecules for lymphocyte activation.

[0012] S4: Randomly mutate potential mutation hotspot residues in S3 into polar or charged amino acids to establish a second T cell receptor mutant library;

[0013] S5: Express the mutant T cell receptor from the second T cell receptor mutant library on T cells;

[0014] S6: Mix the T cells expressing the mutant T cell receptor in S5 with the antigen, and enrich the T cells according to the staining results of pMHC tetramer and lymphocyte activation marker molecules. The T cells have higher activation than the cells expressing the given T cell receptor in S1. The engineered T cell receptor can be obtained through the highly activated T cells.

[0015] Preferably, the database in step S1 can be selected from the IMGT database, NCBI database, Ensembl database, or UNIPROT database.

[0016] The present invention also provides the application of the aforementioned T cell receptor engineering modification method in screening mutant T cell receptors.

[0017] The present invention also provides engineered T-cell receptors obtained by the aforementioned T-cell receptor engineering modification method.

[0018] This invention also provides a T-cell receptor targeting MAGE-A3, wherein the T-cell receptor comprises the following six CDR regions: CDR1α, CDR2α, and CDR3α in the TCRα chain, and CDR1β, CDR2β, and CDR3β in the TCRβ chain: wherein,

[0019] 1) The amino acid sequence of CDR1α is shown in SEQ ID No. 1;

[0020] 2) The amino acid sequence of CDR2α is shown in SEQ ID No. 2;

[0021] 3) The amino acid sequence of CDR3α is shown in SEQ ID No. 3;

[0022] 4) The amino acid sequence of CDR1β is shown in SEQ ID No. 4;

[0023] 5) The amino acid sequence of CDR2β is shown in SEQ ID No. 5;

[0024] 6) The amino acid sequence of CDR3β is shown in SEQ ID No. 6.

[0025] Wherein, the first X in SEQ ID No. 1 is selected from D or H; and / or, the second X in SEQ ID No. 1 is selected from S or H; and / or, the third X in SEQ ID No. 1 is selected from A or H; and / or, the fourth X in SEQ ID No. 1 is selected from N or H.

[0026] Wherein, the first X in SEQ ID No. 2 is selected from S or H; and / or, the second X in SEQ ID No. 2 is selected from R or H.

[0027] In SEQ ID No. 3, X is selected from F or H.

[0028] Wherein, the first X in SEQ ID No. 5 is selected from F or H; and / or, the second X in SEQ ID No. 5 is selected from T or H.

[0029] In SEQ ID No. 6, the first X is selected from S or H; and / or, the second X is selected from P or H; and / or, the third X is selected from Y or H.

[0030] The present invention also provides an antibody drug comprising the aforementioned T-cell receptor.

[0031] The present invention also provides an isolated polynucleotide that encodes the aforementioned T-cell receptor.

[0032] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide and plasmid backbone.

[0033] The present invention also provides a viral vector containing the aforementioned polynucleotides.

[0034] The present invention also provides an isolated T cell containing the aforementioned T cell receptor, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector.

[0035] The present invention also provides the use of the aforementioned T-cell receptor, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector in the preparation of tumor treatment products.

[0036] The present invention also provides a tumor treatment product comprising the aforementioned T-cell receptor, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector, and pharmaceutically acceptable excipients.

[0037] As described above, the T-cell receptor engineering method, the MAGE-A3-targeting T-cell receptor, and their applications provided by this invention have the following beneficial effects:

[0038] The T-cell receptor engineering method provided by this invention is based on histidine scanning, which realizes TCR engineering modification independent of three-dimensional structure, and overcomes the drawback of high affinity, realizing the modification of TCR with low affinity and high activation, providing more options for clinical applications.

[0039] Furthermore, this invention utilizes reverse-locking engineering to modify wild-type MAGE-A3 TCR molecules, obtaining a highly efficient and non-toxic TCR targeting MAGE-A3, thus avoiding the toxic side effects caused by affinity maturation. This engineered TCR is then applied to TCR-T cell preparation for the treatment of solid tumors. Attached Figure Description

[0040] Figure 1 shows a schematic diagram of the histidine scanning method in this invention.

[0041] Figure 2 shows the MAGE TCR mutation hotspots discovered by histidine scanning in this invention.

[0042] Figure 3 shows a schematic diagram of the library design based on histidine hotspot residues in this invention.

[0043] Figure 4 shows the screening results of the TAK1 TCR display library based on histidine hotspot residues in this invention.

[0044] Figure 5 shows the TAK1 TCR function screening results based on histidine hotspot residues in this invention.

[0045] Figure 6 shows the affinity detection results of TAK1 TCR discovered by histidine scanning method in this invention.

[0046] Figure 7 shows the results of the in vitro cytokine secretion experiment of MAGE TCR, which was discovered based on histidine scanning in this invention.

[0047] Figure 8 shows the results of the in vitro cell killing experiment of MAGE TCR, which was discovered based on histidine scanning in this invention.

[0048] Figure 9 shows the CDR information of the wild-type MAGE-A3 T cell receptor in this invention.

[0049] Figure 10 shows the results of the mutant MAGE-A3 T cell receptor modification in this invention.

[0050] Figure 11 shows the verification results of the reverse-locking property of the mutant MAGE-A3 T cell receptor in this invention.

[0051] Figure 12 shows the flow cytometry results of T cell receptor expression levels in MAGE-A3 TCR-T cells in this invention.

[0052] Figure 13 shows the in vivo tumor killing detection results of MAGE-A3 TCR-T cells in this invention.

[0053] Figure 14 shows the results of co-receptor dependence detection of MAGE-A3 TCR-T cells in this invention.

[0054] Figure 15(a) shows the X-scan heatmap of MAGE-A3 TCR-T cells in this invention, and Figure 15(b) shows the cross-reactivity detection results of MAGE-A3 TCR-T cells in this invention.

[0055] Figure 16 shows the results of allogeneic reaction detection of MAGE-A3 TCR-T cells in this invention. Detailed Implementation

[0056] This invention provides a method for engineering T-cell receptors, the method comprising the following steps:

[0057] S1: Obtain the CDR region of a given T cell receptor sequence from the database, mutate one or more amino acid residues in the CDR region to histidine, and establish the first T cell receptor mutant library;

[0058] S2: Express the mutant T cell receptor from the first T cell receptor mutant library on T cells;

[0059] S3: Mix T cells expressing mutant T cell receptors from S2 with the antigen, and identify potential mutation hotspot residues in the CDR region of the T cell receptor based on the expression level of marker molecules for lymphocyte activation.

[0060] S4: Randomly mutate potential mutation hotspot residues in S3 into polar or charged amino acids to establish a second T cell receptor mutant library;

[0061] S5: Express the mutant T cell receptor from the second T cell receptor mutant library on T cells;

[0062] S6: T cells expressing the mutant T cell receptor from S5 are mixed with the antigen. Based on the staining results of pMHC tetramer and marker molecules for lymphocyte activation, T cells are enriched. These T cells exhibit higher activation compared to cells expressing the given T cell receptor from S1. The engineered T cell receptor can be obtained from these highly activated T cells. Here, "high activation" can be understood as: T cells containing the engineered T cell receptor exhibit a higher level of activation compared to T cells containing wild-type T cell receptors or T cells containing a given fixed-sequence T cell receptor.

[0063] In existing technologies, the analysis of TCR activation sites typically involves determining the distance after TCR-pMHC binding within a certain range. The search involves looking near the amino acid residues on the left and right sides of the T cell receptor. Therefore, in existing technologies, the crystal structure of the TCR is usually determined first when engineering T cell receptors. This invention provides a method for engineering T cell receptors based on histidine scanning, which can obtain engineered T cell receptors with the target effect without relying on the three-dimensional structure of the TCR, simply by establishing a mutant library of the CDR region.

[0064] In some specific embodiments, the highly activated T cells in step S6 are either high-affinity or low-affinity T cells. Low affinity can be understood as less than 10 times the affinity of the given T cell receptor cells expressed in S1; high affinity can be understood as more than 10 times the affinity of the given T cell receptor cells expressed in S1.

[0065] In some specific embodiments, the marker molecule for lymphocyte activation may be selected from one or more of CD69, CD25, CD38, CD71, CD44, CD62L, CD45RO, NFAT, NF-κB, AP1, or HLA-DR (human class II major histocompatibility complex antigen).

[0066] In some specific embodiments, the first T cell receptor mutation library and / or the second T cell receptor mutation library are single point mutation libraries or multi-site mutation libraries.

[0067] In some specific implementations, the database in step S1 can be selected from the IMGT database, NCBI database, Ensembl database, or UNIPROT database.

[0068] In some specific embodiments, the T cells in step S2 are T cells lacking T cell receptors. Specifically, the T cells lacking T cell receptors can be selected from any of SKW-3 cells, 58- / - cells, Jurkat T cells, TCRP1A CD8 T cells, OT-1CD8 T cells, or LLC-OVA cells.

[0069] In some specific embodiments, the antigen in step S3 can be selected from short antigen peptides, antigen proteins, or antigen-presenting cells. Preferably, the antigen-presenting cells can be selected from dendritic cells, macrophages, or artificial antigen-presenting cells. Specifically, the artificial antigen-presenting cells can be selected from 293 cells or 293T cells.

[0070] Furthermore, the artificial antigen-presenting cells can be obtained by infecting 293 cells or 293T cells with a lentiviral vector carrying a specific HLA subtype that can simultaneously express a fusion fluorescent protein tag and TCR recognition.

[0071] In some specific embodiments, the polar or charged amino acid in step S4 is selected from one or more of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, threonine, or serine. Preferably, the polar or charged amino acid is selected from one or more of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, or serine.

[0072] In some specific embodiments, the T cells in step S5 are T cells lacking the T cell receptor. Preferably, the T cells lacking the T cell receptor can be selected from any one of SKW-3 cells, 58- / - cells, Jurkat T cells, TCRP1A CD8 T cells, OT-1CD8 T cells, or LLC-OVA cells.

[0073] In some specific embodiments, the antigen in step S6 can be selected from short antigen peptides, antigen proteins, or antigen-presenting cells. Preferably, the antigen-presenting cells can be selected from dendritic cells, macrophages, or artificial antigen-presenting cells. Specifically, the artificial antigen-presenting cells can be selected from 293 cells or 293T cells.

[0074] The present invention also provides the application of the aforementioned T cell receptor engineering modification method in screening mutant T cell receptors.

[0075] The present invention also provides engineered T-cell receptors obtained by the aforementioned T-cell receptor engineering modification method.

[0076] The present invention also provides a T-cell receptor (TCR) targeting MAGE-A3, wherein the T-cell receptor comprises the following six CDR regions: CDR1α, CDR2α and CDR3α in the TCRα chain, and CDR1β, CDR2β and CDR3β in the TCRβ chain.

[0077] In this invention, the TCR targeting MAGE-A3 is highly efficient and non-toxic, avoiding the toxic side effects caused by affinity maturation.

[0078] In some specific embodiments, the amino acid sequence of CDR1α is shown in SEQ ID No. 1.

[0079] In some specific embodiments, the amino acid sequence of CDR2α is shown in SEQ ID No. 2.

[0080] In some specific embodiments, the amino acid sequence of CDR3α is shown in SEQ ID No. 3.

[0081] In some specific embodiments, the amino acid sequence of CDR1β is shown in SEQ ID No. 4.

[0082] In some specific embodiments, the amino acid sequence of CDR2β is shown in SEQ ID No. 5.

[0083] In some specific embodiments, the amino acid sequence of CDR3β is shown in SEQ ID No. 6.

[0084] Wherein, the first X in SEQ ID No. 1 is selected from D or H; and / or, the second X in SEQ ID No. 1 is selected from S or H; and / or, the third X in SEQ ID No. 1 is selected from A or H; and / or, the fourth X in SEQ ID No. 1 is selected from N or H.

[0085] Wherein, the first X in SEQ ID No. 2 is selected from S or H; and / or, the second X in SEQ ID No. 2 is selected from R or H.

[0086] In SEQ ID No. 3, X is selected from F or H.

[0087] Wherein, the first X in SEQ ID No. 5 is selected from F or H; and / or, the second X in SEQ ID No. 5 is selected from T or H.

[0088] In SEQ ID No. 6, the first X is selected from S or H; and / or, the second X is selected from P or H; and / or, the third X is selected from Y or H.

[0089] In some specific embodiments, the amino acid sequence of the α-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No. 7; and / or, the amino acid sequence of the β-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No. 8.

[0090] Wherein, the first X in SEQ ID No. 7 is selected from D or H; and / or, the second X in SEQ ID No. 7 is selected from S or H; and / or, the third X in SEQ ID No. 7 is selected from A or H; and / or, the fourth X in SEQ ID No. 7 is selected from N or H; and / or, the fifth X in SEQ ID No. 7 is selected from S or H; and / or, the sixth X in SEQ ID No. 7 is selected from R or H; and / or, the seventh X in SEQ ID No. 7 is selected from F or H.

[0091] Wherein, the first X in SEQ ID No. 8 is selected from F or H; and / or, the second X in SEQ ID No. 8 is selected from T or H; and / or, the third X in SEQ ID No. 8 is selected from S or H; and / or, the fourth X in SEQ ID No. 8 is selected from P or H; and / or, the fifth X in SEQ ID No. 8 is selected from Y or H.

[0092] In some specific embodiments, the α-chain variable region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 7; the β-chain variable region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 8.

[0093] In some specific embodiments, the T cell receptor targeting MAGE-A3 further includes a TCRα chain constant region or a TCRβ chain constant region.

[0094] In some specific embodiments, the amino acid sequence of the constant region of the TCRα chain is shown in SEQ ID No. 9; or, the amino acid sequence of the constant region of the TCRβ chain is shown in SEQ ID No. 10.

[0095] In some specific embodiments, the TCRα chain constant region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 9; the TCRβ chain constant region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 10.

[0096] In some specific embodiments, the amino acid sequence of the TCRα chain is as shown in any of SEQ ID No. 11 or 13-19; and / or, the amino acid sequence of the TCRβ chain is as shown in any of SEQ ID No. 12 or 20-24.

[0097] Further, the amino acid sequence of the α chain of the TCR is as shown in SEQ ID No. 11, and the amino acid sequence of the β chain is as shown in any one of SEQ ID No. 20-24; or, the amino acid sequence of the α chain of the TCR is as shown in SEQ ID No. 13, and the amino acid sequence of the β chain is as shown in SEQ ID No. 20; or, the amino acid sequence of the α chain of the TCR is as shown in any one of SEQ ID No. 13-19, and the amino acid sequence of the β chain is as shown in SEQ ID No. 12.

[0098] In some specific embodiments, when having the aforementioned α and β chain variable region CDR sequences, the TCR α chain may further be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 11 or 13-19; the TCR β chain may further be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 11 or 13-19; Peptides having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in No. 12 or 20-24.

[0099] In some specific embodiments, the TCRα chain is selected from human TCRα chain, humanized TCRα chain, chimeric TCRα chain, or mouse TCRα chain; the TCRβ chain is selected from human TCRβ chain, humanized TCRβ chain, chimeric TCRβ chain, or mouse TCRβ chain. Here, "chimeric TCRα chain" or "chimeric TCRβ chain" refers to a TCRα chain or TCRβ chain containing sequences derived from more than one species, such as sequences derived from humans and mice.

[0100] In some specific embodiments, the T-cell receptor targeting MAGE-A3 binds to the MAGE-A3 antigen but not to the TITIN antigen.

[0101] In some specific embodiments, the MAGE-A3 antigen comprises the following amino acid sequence: SEQ ID No. 26: peptide of EVDPIGHLY; and / or, the TITIN antigen comprises the following amino acid sequence: SEQ ID No. 27: peptide of ESDPIVAQY.

[0102] The present invention also provides an antibody drug comprising the aforementioned T-cell receptor targeting MAGE-A3.

[0103] In some specific embodiments, the antibody drug may be a TCR-BiTE (Bispecific T-cell Engagers) antibody protein drug.

[0104] Furthermore, the TCR-BiTE antibody protein drug comprises the aforementioned T cell receptor targeting MAGE-A3, and a single-chain variable fragment (scFv) against any one of CD3, CD4, CD5, CD6, CD7, CD8, CD28, or 4-1BB.

[0105] The present invention also provides an isolated polynucleotide that encodes the aforementioned T-cell receptor targeting MAGE-A3.

[0106] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide and plasmid backbone.

[0107] In some specific embodiments, the plasmid backbone is a viral, adenovirus, or adeno-associated virus plasmid backbone.

[0108] The present invention provides a viral vector containing the aforementioned polynucleotides.

[0109] In some specific embodiments, the viral vector is selected from lentiviruses, adenoviruses, or adeno-associated viruses.

[0110] The present invention provides an isolated T cell containing the aforementioned T cell receptor targeting MAGE-A3, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector.

[0111] In some specific embodiments, the T cells are TCR-T cells.

[0112] In some specific embodiments, the T cells are selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed-type hypersensitivity T cells, native T cells, or memory T cells. Helper T cells are T cells that assist in humoral and cellular immunity; suppressor T cells are T cells that suppress cellular and humoral immunity; effector T cells are T cells that release lymphokines; cytotoxic T cells are T cells that kill target cells; delayed-type hypersensitivity T cells are T cells that participate in type IV hypersensitivity reactions and can act on helper and suppressor T cells to amplify the immune effect; native T cells are undifferentiated T cells; and memory T cells are T cells that remember specific antigen stimulation.

[0113] The present invention also provides the use of the aforementioned T-cell receptor targeting MAGE-A3, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned viral vector, or the aforementioned T cell in the preparation of tumor treatment products.

[0114] In some specific embodiments, the tumor is selected from one or more of the following: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, lymphoid tumor, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

[0115] The present invention also provides a tumor treatment product comprising the aforementioned T-cell receptor targeting MAGE-A3, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned viral vector or the aforementioned T-cell and pharmaceutically acceptable excipients.

[0116] In some specific embodiments, the excipients include various excipients and diluents, which are not essential active ingredients and do not cause excessive toxicity after application. The excipients contain sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When the excipients are used in an aqueous solution for injection, they are selected from physiological saline, isotonic glucose solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or nonionic surfactants (Tween 80 or HCO-50). In the tumor treatment product provided by this invention, the aforementioned TCR, polynucleotide, nucleic acid construct, or T cell is a single effective ingredient, or it can be combined with one or more other active components useful for tumor treatment to form a combined formulation. The active components are various other drugs used for tumor treatment. The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient in the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, or the aforementioned T cell and tumor treatment product depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the aforementioned tumor treatment product as an active ingredient is 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0117] The present invention also provides a method for tumor treatment, wherein the method comprises administering the aforementioned T cell receptor MAGE-A3, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned viral vector, or the aforementioned T cells to a tumor patient.

[0118] In some specific embodiments, the dosage is 1-1000 mg / kg / day. Specifically, the dosage is 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or 500 mg-1000 mg / kg / day.

[0119] In some specific embodiments, the object of the method can be a mammal; preferably, the object of the method is a human.

[0120] In this invention, the T-cell receptor (TCR) is a molecule present on the surface of T cells that is responsible for recognizing peptide-MHC complexes. In some embodiments, the TCR is a truncated or full-length TCR. In some embodiments, the TCR is a heterodimer composed of α and β chains. In some embodiments, the TCR may also be a single-chain TCR (scTCR).

[0121] In this invention, the term "CDR" stands for complementarity determining region, which is part of the antibody. It is usually composed of hypervariable regions in the variable regions of the heavy and light chains of the antibody. It is responsible for complementing the epitope structure of a specific antigen, thereby achieving specific binding between the antibody and the antigen. The sequence diversity of the CDR region is the key to the specific recognition of antigen by the antibody. Alternatively, it can refer to structurally equivalent hypervariable regions in the T cell receptor, which exist on the α and β chains respectively, forming the binding site between the T cell receptor and the antigen. The α chain usually has three CDR regions, starting from the N-terminus of the α chain, with the three CDR regions being amino acid residues 22-38, 45-63, and 84-110, respectively. The β chain usually has three CDR regions, starting from the N-terminus of the β chain, with the three CDR regions being amino acid residues 20-37, 42-60, and 85-110, respectively. These regions are often complementary to antigenic determinants in terms of spatial structure, so they are also called complementarity determining regions (CDRs). The α-chain variable region usually includes three complementarity determining regions, namely CDRα1, CDRα2 and CDRα3, and the β-chain variable region usually includes three complementarity determining regions, namely CDRβ1, CDRβ2 and CDRβ3.

[0122] In some specific embodiments, the α-chain variable region and the β-chain variable region may further include a framework region, which may be located between complementarity-determining regions or at both ends of the complementarity-determining regions. In some specific embodiments of the present invention, the framework region sequence is a human monoclonal antibody variable region or a mouse monoclonal antibody variable region framework region sequence obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0123] In this invention, the term "CD69" refers to a type II transmembrane glycoprotein belonging to the C-type lectin receptor family. It is the first protein expressed after lymphocytes are activated and is therefore often used as a marker molecule for lymphocyte activation.

[0124] In this invention, the term "TCRα chain constant region" or "TCRβ chain constant region" comprises an extracellular constant region, a transmembrane region, and an intracellular constant region connected in sequence. The extracellular constant region may include the hinge regions of the TCRα and TCRβ chains, participating in the formation of disulfide bonds between the TCRα and TCRβ chains. The transmembrane region is also a constant region, and its functions include participating in the cell membrane anchoring of the TCRα and TCRβ chains and interacting with the CD3 subunit to form the TCR-CD3 complex. The possible functions of the intracellular constant region include participating in the conformational change of the TCR-CD3 complex and signal transduction after TCR signal transduction.

[0125] In this invention, the term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., the α-chain or β-chain of a TCR) involved in the binding of an immunoglobulin superfamily binding protein (e.g., a TCR) to an antigen. The variable domains (Vα and Vβ, respectively) of the α-chain and β-chain of a native TCR typically have similar structures, each containing four conserved frame regions (FRs) and three core regions (CDRs). The Vα domain is encoded by two independent DNA segments, a variable gene segment and a linker gene segment (VJ); the Vβ domain is encoded by three independent DNA segments, namely, a variable gene segment, a diversity gene segment, and a linker gene segment (VDJ). A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs binding to specific antigens can be isolated from antigen-binding TCRs using either the Vα or Vβ domain to screen libraries of complementary Vα or Vβ domains, respectively.

[0126] In this invention, the term "antigen" refers to a cell surface molecule or an intracellular molecule presented by MHC molecules or MHC-like molecules that can be bound by antibodies or T-cell receptors (TCRs), including but not limited to polypeptide antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), or polysaccharide antigens (e.g., CA199, CA72-4, and CA125). The antigen may be a tumor antigen, such as a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0127] In this invention, the term "isolated" refers to material that has been removed from its natural state or otherwise artificially manipulated, such as the α-chain, β-chain, T-cell receptor, and nucleic acid described herein. The isolated material may be substantially or substantially free of the components normally accompanying it in its natural state, or may be manipulated into an artificial state in conjunction with the components normally accompanying it in its natural state. The isolated material may be in a natural, chemically synthesized, or recombinant form. The isolated material may also, or alternatively, be in an enriched, partially purified, or purified form.

[0128] In this invention, the term "polynucleotide" is also called "nucleotide" or "nucleic acid" and refers to a chain of nucleic acids consisting of deoxyribonucleic acid, ribonucleic acid, modified nucleic acids or bases, and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0129] In this invention, the term "nucleic acid construct," also known as "vector," refers to a device capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, granules, or phage vectors.

[0130] In this invention, the term "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of these cells.

[0131] In this invention, to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. The amino acid residues or nucleic acids at the corresponding amino acid or nucleic acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleic acid at the corresponding position in the second sequence, the molecules are identical at that position.

[0132] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleic acid sequences. The particularly preferred set of parameters (and a set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0133] Alternatively, the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleic acid sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0134] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0135] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0136] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0137] The sequence information used in this invention is as follows:

[0138] SEQ ID No.1

[0139] SEQ ID No.2

[0140] SEQ ID No. 3

[0141] SEQ ID No. 4

[0142] SEQ ID No. 5

[0143] SEQ ID No. 6

[0144] SEQ ID No.7

[0145] SEQ ID No. 8

[0146] SEQ ID NO.9

[0147] SEQ ID NO.10

[0148] SEQ ID No.11 κκτανν

[0149] SEQ ID No.12 β-layer

[0150] SEQ ID NO.13 MAGEα-D28H

[0151] SEQ ID No.14 MAGEα-S29H

[0152] SEQ ID NO.15 MAGEα-A30H

[0153] SEQ ID NO.16 MAGEα-N33H

[0154] SEQ ID NO.17 MAGEα-S54H

[0155] SEQ ID NO.18 MAGEα-R56H

[0156] SEQ ID NO.19 MAGEα-F105H

[0157] SEQ ID NO.20 MAGEβ-F51H

[0158] SEQ ID NO.21 MAGEβ-T54H

[0159] SEQ ID NO.22 MAGEβ-S94H

[0160] SEQ ID NO.23 MAGEβ-P96H

[0161] SEQ ID NO.24 MAGEβ-Y103H

[0162] SEQ ID No. 25

[0163] Example 1: Histidine Scanning and Discovery of Hotspot Residues

[0164] For a given TCR sequence, the CDR region location can be predicted using the IMGT database (https: / / www.imgt.org / IMGT_vquest / input). Subsequently, mutation primers were designed to mutate each amino acid residue in the CDR region of both the α and β chains to histidine, obtaining the corresponding mutant TCR sequence (Figure 1). The wild-type TCR sequence and a series of histidine single-point mutation sequences were cloned into the lentiviral vector pHR plasmid. The resulting plasmid, along with two other plasmids required for lentiviral packaging, psPAX and pMD2.G, were co-transfected into lentiviral packaging cells Lenti-X. Forty-eight hours after transfection, the supernatant was harvested, containing lentiviral particles. The supernatant containing lentiviral particles was centrifuged and filtered, then added to the TCR-deficient T cell line SKW-3. Forty-eight hours after infection, flow cytometry was used to ensure the TCR positivity rate on the cell surface, ultimately obtaining SKW-3 cell lines expressing either wild-type or mutant TCR.

[0165] Next, the activation levels of these TCR mutants were tested using a co-culture method with SKW-3 cells and antigen-presenting cells 293T. Specific HLA typing with a GFP tag, recognized by TCR, was overexpressed in 293T cells via the aforementioned lentiviral infection method. Specific HLA typing-positive cells were screened using the GFP tag to construct specific HLA antigen-presenting cell lines. The synthesized antigen peptides were dissolved in DMSO to a concentration of 10... -1 M was then subjected to a series of 10-fold serial dilutions to obtain a concentration of 10. -1 ~10 -11 M antigen peptide solution. Pre-lay 5×10⁶ cells in 96-well U-type plates. 4 One antigen-presenting cell was given an antigen peptide solution at a concentration of 1000:1000. -4 ~10 -14 M peptide pulsing. After 3 hours of treatment, centrifuge and discard the supernatant, then add 5 × 10⁻⁶ peptides to each well. 4 TCR wild-type or mutant SKW-3 cells were cultured at 37 degrees Celsius for 12–14 hours. CD69 expression levels were analyzed by flow cytometry. Dose-response curves were plotted based on CD69 MFI to compare the activation levels of mutant and wild-type cells. The mutants with the highest activation levels were selected as the hotspots for modification (Figure 2).

[0166] Example 2: Design of a T-cell display library based on histidine hotspot residues

[0167] The degenerate codon VRW can encode the vast majority of polar and charged amino acids (Arg, Asn, Asp, Gln, Glu, His, Lys, Ser). For the hotspot residues obtained in Example 1, VRW degenerate primers were designed to simultaneously replace them with the codon VRW (Figure 3). The resulting plasmid library was electroporated into competent MegaX cells of *E. coli*, yielding a diversity of up to 10. 7 The TCR library plasmid was then packaged into lentivirus using the aforementioned method and used to infect 10... 8 SKW3 cells were selected to achieve a positivity rate of approximately 10%, ensuring that each cell was infected by only one viral particle.

[0168] Example 3 Construction of cDNA mutant library

[0169] Antigen-presenting cells were subjected to 10 -5 M peptide pulses were then co-cultured with SKW-3 cells containing a TCR library, 10 cells per well. 5 SKW3 cells, totaling 10 8 After co-culturing for 12–14 hours, cells from each well were collected and mixed for pMHC tetramer and CD69 staining to characterize the affinity and activation level of TCRs in the library, respectively. Low-affinity, highly activated T cell populations compared to wild-type cells were enriched by flow cytometry. After further culture, this enrichment process was repeated 3–5 times (Figure 4).

[0170] After 3-5 rounds of enrichment, the T cell population was seeded as single-cell clones through extreme dilution. Once the clones had grown, the activation experiment in Example 1 was repeated to screen for single clones with high activation levels. The genome of the candidate single clones was extracted, and the integrated TCR sequence was amplified by PCR. After integration into the pHR vector, single clones were selected for sequencing to obtain candidate TCR mutant sequences.

[0171] SKW-3 cell lines with candidate TCR mutants were constructed, and the activation experiment in Example 1 was repeated to verify the activation level of the TCR mutants (Figure 5).

[0172] The TCR mutant sequence was integrated into the protein expression vector pD649, and the pairing and expression of the TCR were stabilized using acidic and basic zippers. After obtaining a sufficient amount of protein, the affinity of the TCR mutant was verified by SPR experiments on a Biacore 8K instrument (Figure 6).

[0173] Example 4: Functional Verification of TCR Variants

[0174] For the low-affinity, high-activation TCRs obtained from library screening, the activation effect of TCR mutants can be verified in human primary T cells. The TCR α and β chains were cloned into the same pHR vector and linked via P2A. The obtained plasmids, along with two other plasmids required for lentiviral packaging, pVSVG and pCMV-dR8.91, were co-transfected into Lenti-X lentiviral packaging cells. The supernatant was harvested 48 hours after transfection, and the lentiviral particles were found in the supernatant. Human PBMCs were revived and activated using CD3 / CD28 magnetic beads. Centrifugation infection was performed 48 hours later, and the centrifugation infection was repeated the next day. The TCR positivity rate on the cell surface was detected by flow cytometry 2–4 days later. The magnetic beads were removed before in vitro experiments. (1) Primary T cells expressing wild-type TCR or mutant TCR were co-cultured with tumor cells expressing specific pMHC, with an effector cell / target cell (E / T) ratio of 1:1, and BFA was added to block the secretion of cytokines. After co-culturing for 6 hours, membrane proteins (CD8, TCR) and intracellular cytokines (TNF, IFNg) were stained, and the positive rate of the cytokine secretion population was detected by flow cytometry (Figure 7). (2) Primary T cells expressing wild-type TCR or mutant TCR were co-cultured with tumor cells expressing specific pMHC and carrying luciferase, with E / T ratios of 10:1, 5:1, and 1:1, respectively. After co-culturing for 24 hours, the cells were lysed, and luciferin, the substrate of luciferase, was added. The number of tumor cells was quantified by enzyme-linked immunosorbent assay (ELISA) to verify the killing effect of T cells on tumor cells (Figure 8).

[0175] Example 5: Screening of T-cell receptors targeting MAGE-A3

[0176] The known wild-type MAGE-A3 TCR sequence is:

[0177] TCRαchain:

[0178] TCRβchain:

[0179] Among them, the CDR region (αchain and βchain) of the corresponding pMHC (MAGE-A3 antigenic epitope peptide-HLA-A*01:01) is shown in Figure 9.

[0180] Based on the CDR prediction results, suitable amino acid sites in the α-chain and β-chain CDR regions were selected and mutated to histidine (H). The modified sequence was then integrated into the lentiviral vector pHR via molecular cloning. Subsequently, the MAGE-A3 TCR sequence was introduced into the genome of the human leukemia T cell line SKW-3 via lentiviral infection, enabling stable expression of the corresponding TCR molecules: MAGE-A3 WT (wild-type), MAGE-A3 A3A (high affinity), and MAGEα / β-mutants (histidine mutants).

[0181] Subsequently, a series of SKW-3 cells overexpressing TCR were co-incubated with HEK-293T cells (expressing HLA-A*01:01) loaded with MAGE-A3 (EVDPIGHLY) and TITIN (ESDPIVAQY) antigenic epitope peptides in 96-well plates at a 1:1 ratio for 14 hours. The activation status of SKW-3 cells (CD69 MFI) was detected to identify the effect of the mutation modification. The detection data are shown in Figure 10.

[0182] Based on the test results, TCR molecules that can be activated by the MAGE-A3 antigenic epitope peptide and do not respond to the TITIN antigenic epitope peptide (a highly efficient and non-toxic qualitative indicator) were screened. Two effective mutant TCR molecules were obtained: MAGEα-D28H (amino acid D at position 28 of the αchain is mutated to H, including SEQ ID No. 13 and 12) and MAGEβ-F51H (amino acid F at position 51 of the βchain is mutated to H, including SEQ ID No. 11 and 20). Furthermore, combining these two molecules yielded a dual-modified TCR molecule (MAGEα-D28H & β-F51H, including SEQ ID No. 13 and 20).

[0183] Example 6: Optical tweezers detection of the bond lifetime between screened TCR molecules and pMHC

[0184] In this embodiment, optical tweezers technology is used to detect the bond lifetime of the bond formed between the screened MAGE-A3 TCR histidine mutant and pMHC, thereby determining whether a reverse-locking bond is formed between the two.

[0185] First, a biotinylation site was added to the MAGE-A3 pMHC protein sequence, and the pMHC protein was cloned and constructed into the pD649 plasmid. This plasmid was then transfected with Expi293F to produce the pMHC protein. After purification with Ni-NTA, the pMHC protein was biotinylated using BirA and purified by molecular sieve chromatography to obtain pure biotinylated pMHC protein (amino acid sequence shown in SEQ ID No. 25). Subsequently, SKW-3 cells expressing the TCR molecule to be detected and the pMHC protein were loaded onto an M-Trap optical tweezers instrument. The bond lifetime duration under different stress conditions was measured to obtain bond lifetime curves. The bond lifetime curves are shown in Figure 11.

[0186] According to the test results, the bond lifetime between the two TCR mutants, MAGEα-D28H and MAGEβ-F51H, and the pMHC protein first increases and then decreases with the increase of external force, which is consistent with the reverse-locked bond property.

[0187] Example 7 Preparation of MAGE-A3 TCR-T cells

[0188] The MAGE-A3 TCR sequence was introduced into the genome of human primary T cells via lentiviral infection, enabling stable expression of the corresponding TCR molecule. The results of TCR expression level detection are shown in Figure 12.

[0189] Based on the flow cytometry results of the above experiments, the TCR positivity rate of each MAGE-A3 TCR-T cell line was approximately 95%. The high positivity rate and small differences indicate that these cells can be used for subsequent in vitro experiments.

[0190] Example 8: In vitro functional activity verification of MAGE-A3 TCR-T cells

[0191] MAGE-A3 TCR-T cells were co-incubated with target cells HCT-116 (human colon cancer cell line) at a 1:1 ratio in 96-well plates for 6 h and 48 h, respectively. At 6 h, cell samples were collected and flow cytometry was used to detect the release of inflammatory cytokines (IFNγ, TNF) from TCR-T cells. At 48 h, cell samples were collected and flow cytometry was used to detect the depletion status (PD-1) of TCR-T cells. The results are shown in Figure 7.

[0192] According to flow cytometry results, the anti-locked TCR-T cells exhibited better activation than wild-type TCR-T cells (MAGE WT), with the dual anti-locked TCR-T cells (MAGEα-D28H & β-F51H) showing activation effects approaching those of high-affinity TCR-T cells (A3A). Furthermore, the exhaustion level of the anti-locked TCR-T cells was significantly lower than that of the high-affinity TCR-T cells, similar to that of wild-type TCR-T cells. These results indicate that anti-locked TCR-T cells, especially the dual anti-locked cells, possess excellent target cell recognition and activation capabilities, while effectively avoiding cell exhaustion caused by over-activation.

[0193] Example 9: Detection of Target Cell Killing Efficiency

[0194] MAGE-A3 TCR-T cells were co-incubated with target cells HCT-116Luc (an HCT-116 cell line overexpressing luciferase) at different ratios (10:1, 5:1, and 1:1) in 96-well plates for 24 h. After co-incubation, luciferin (luciferin, a substrate for luciferase degradation) solution was added for further incubation. The luminescence intensity (quantitative substrate degradation, allowing calculation of the number of surviving target cells) was detected using an Envision chemiluminescence analyzer to assess the target cell killing efficiency. The results are shown in Figure 8, indicating that the mutant TCR cell line was more effective at killing target cells than the wild-type line.

[0195] Example 10 In vivo tumor killing detection

[0196] Prepare 6-week-old NSG immunodeficient mice, 5 mice per group, and subcutaneously inject 5×10 6 HCT-116 cells were used to create a subcutaneous tumor model, and tumor volume was measured every 2-3 days. The tumor was allowed to grow to 100-200 mm. 3 At approximately 7 days, administer 5×10 via tail vein injection. 6 Treatment with TCR-T cells included a saline group and five MAGE TCRs (MAGE WT, MAGEα-D28H, MAGEβ-F51H, MAGEα-D28H&β-F51H, and A3A). Tumor volume was measured every 2–3 days, and tumor growth curves were plotted. The results are shown in Figure 13. Compared with the saline group and the wild-type group, the tumor growth rate of MAGEα-D28H&β-F51H was significantly slowed.

[0197] Example 11 Co-receptor dependence detection

[0198] Primary TCR-T cells expressing wild-type or mutant TCRs were co-cultured with MAGE-A3 or hCMVpp50. Specifically, wild-type or mutant TCRs (MAGEα-D28H & β-F51H) were transduced into purified CD8+ cells using viral vectors. + T cells and CD4 + In T cells, the activation of the TCR was determined to be dependent on the co-receptors CD4 or CD8 by detecting the secretion levels of cytokines (such as TNF) in CD4+ and CD8+ T cells. As shown in Figure 14, the results showed that wild-type TCR or mutant TCR (MAGEα-D28H&β-F51H) was specifically activated by MAGE-A3, and the MAGE TCR (i.e., MAGE WT) was a CD8-dependent TCR. The modified mutant TCR (i.e., MAGEα-D28H&β-F51H) still exhibited CD8 dependence.

[0199] Example 12 Off-target toxicity verification (cross-reactivity) detection

[0200] By replacing each residue of the target antigen peptide with 19 additional amino acids, 171 mutant peptides were synthesized to activate T cells containing both wild-type and mutant TCRs. The activation levels of the mutant peptides were divided by the activation levels of the wild-type peptides to create an X-scan heatmap of the TCRs, as shown in Figure 15(a). Mutation sites and residues representing more than 10% of the mutations in the heatmap were included in the predicted motifs. For example, the motif corresponding to the mutant MAGEα-D28H & β-F51H is E-[AILMVGHNQSTYC]-DP-[ILMVDTC]-[AILMFVPGWRHKDENQSTY]-[AILMFVPGWHKDENQSTYC]-[AILMFVPGWRHKDENQSTYC]-[AILMFVPGKY]. This motif was then input into the ScanProsite website to predict peptides in the human proteome that overlap with this motif. These overlapping peptides are potential off-target peptides that can activate mutant TCRs. The results, as shown in Figure 15(b), indicate that the mutant MAGEα-D28H&β-F51H does not cross-react with these peptides, meaning that the mutant MAGEα-D28H&β-F51H has a low risk of off-target toxicity.

[0201] Example 13 Off-target toxicity verification (allo-reactivity) detection

[0202] Tumor cell lines and B-LCLs with different HLA typing and antigen expression profiles were selected and co-cultured with T cells containing wild-type and mutant TCRs. Specifically, three scenarios were set up: HLA-A*0.1:0.1+MAGEA3 peptide / MAGEA3, HLA-A*0.1:0.1 only, and MAGEA3 peptide / MAGEA3 only. The results are shown in Figure 16. Only antigen-presenting cells with correct HLA typing and expressing the target antigen (i.e., HLA-A*0.1:0.1+MAGEA3 peptide / MAGEA3) could activate the mutant TCR (i.e., MAGEα-D28H & β-F51H), thus excluding allogeneic reactions.

[0203] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for engineering T-cell receptors, characterized in that, The method for engineering T-cell receptors includes the following steps: S1: Obtain the CDR region of a given T cell receptor sequence from the database, mutate one or more amino acid residues in the CDR region to histidine, and establish the first T cell receptor mutant library. S2: Express the mutant T cell receptor from the first T cell receptor mutant library on T cells; S3: Mix T cells expressing mutant T cell receptors from S2 with the antigen, and identify potential mutation hotspot residues in the CDR region of the T cell receptor based on the expression level of marker molecules for lymphocyte activation. S4: Randomly mutate potential mutation hotspot residues in S3 into polar or charged amino acids to establish a second T cell receptor mutant library; S5: Express the mutant T cell receptor from the second T cell receptor mutant library on T cells; S6: Mix the T cells expressing the mutant T cell receptor in S5 with the antigen, and enrich the T cells according to the staining results of pMHC tetramer and lymphocyte activation marker molecules. The T cells have higher activation than the cells expressing the given T cell receptor in S1. The engineered T cell receptor can be obtained through the highly activated T cells.

2. The method for engineering T-cell receptors according to claim 1, characterized in that, In step S6, the highly activated T cells are either high-affinity or low-affinity T cells.

3. The method for engineering T-cell receptors according to claim 1, characterized in that, In step S1, the database is selected from IMGT database, NCBI database, Ensembl database or UNIPROT database.

4. The T cell receptor engineering method of claim 1, wherein, In step S2 or step S5, the T cells are T cells lacking the T cell receptor.

5. The T cell receptor engineered method of claim 4, wherein, The T cells lacking the T cell receptor were selected from SKW-3 cells and 58 cells. - / - The T cells are any one of Jurkat T cells, TCRP1A CD8 T cells, OT-I CD8 T cells, or LLC-OVA cells; preferably, the T cells lacking the T cell receptor are SKW-3 cells.

6. The method for engineering T-cell receptors according to claim 1, characterized in that, In step S3 or S6, the antigen is a short peptide, a protein, or an antigen-presenting cell.

7. The method for engineering T-cell receptors according to claim 6, characterized in that, The antigen-presenting cells are selected from dendritic cells, macrophages, or artificial antigen-presenting cells; preferably, the artificial antigen-presenting cells are selected from 293 cells or 293T cells.

8. The T cell receptor engineering method of claim 1, wherein, In step S4, the polar or charged amino acid is selected from one or more of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, or threonine.

9. The application of the T-cell receptor engineering method according to any one of claims 1-8 in screening mutant T-cell receptors.

10. The engineered T-cell receptor obtained by the T-cell receptor engineering method according to any one of claims 1-8.

11. A T-cell receptor targeting MAGE-A3, characterized in that, The T cell receptor comprises the following six CDR regions: CDR1α, CDR2α, and CDR3α in the TCRα chain, and CDR1β, CDR2β, and CDR3β in the TCRβ chain: wherein, 1) The amino acid sequence of CDR1α is shown in SEQ ID No. 1; 2) The amino acid sequence of CDR2α is shown in SEQ ID No. 2; 3) The amino acid sequence of CDR3α is shown in SEQ ID No. 3; 4) The amino acid sequence of CDR1β is shown in SEQ ID No. 4; 5) The amino acid sequence of CDR2β is shown in SEQ ID No. 5; 6) The amino acid sequence of CDR3β is shown in SEQ ID No. 6; Wherein, the first X in SEQ ID No. 1 is selected from D or H; and / or, the second X in SEQ ID No. 1 is selected from S or H; and / or, the third X in SEQ ID No. 1 is selected from A or H; and / or, the fourth X in SEQ ID No. 1 is selected from N or H; And / or, the first X in SEQ ID No. 2 is selected from S or H; and / or, the second X in SEQ ID No. 2 is selected from R or H; And / or, in SEQ ID No. 3, X is selected from F or H; And / or, the first X in SEQ ID No. 5 is selected from F or H; and / or, the second X in SEQ ID No. 5 is selected from T or H; And / or, the first X in SEQ ID No. 6 is selected from S or H; and / or, the second X in SEQ ID No. 6 is selected from P or H; and / or, the third X in SEQ ID No. 6 is selected from Y or H.

12. The T cell receptor according to claim 11, characterized in that, The amino acid sequence of the α-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No. 7; and / or, the amino acid sequence of the β-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No.

8.

13. The T-cell receptor according to claim 11, characterized in that, The amino acid sequence of the TCRα chain is as shown in any of SEQ ID No. 11 or 13-19; and / or, the amino acid sequence of the TCRβ chain is as shown in any of SEQ ID No. 12 or 20-24.

14. An antibody drug, characterized by, The antibody drug comprises the T-cell receptor as described in any one of claims 11-13.

15. The antibody drug according to claim 14, characterized in that, The antibody drug is a TCR-BiTE antibody protein drug.

16. An isolated polynucleotide, characterized in that, The polynucleotide encodes the T-cell receptor according to any one of claims 11-13.

17. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the polynucleotide and plasmid backbone as described in claim 16.

18. A viral vector, characterized in that, The viral vector contains the polynucleotide as described in claim 16.

19. An isolated T cell, wherein, The T cell contains the T cell receptor of any one of claims 11-13, the polynucleotide of claim 16, the nucleic acid construct of claim 17, or the viral vector of claim 18.

20. Use of the T-cell receptor of any one of claims 11-13, the antibody drug of claim 14 or 15, the polynucleotide of claim 16, the nucleic acid construct of claim 17, or the viral vector of claim 18 in the preparation of tumor therapeutic products.

21. A tumor treatment product, characterized in that, The tumor treatment product comprises the T-cell receptor of any one of claims 11-13, the antibody drug of claim 14 or 15, the polynucleotide of claim 16, the nucleic acid construct of claim 17, or the viral vector of claim 18, and pharmaceutically acceptable excipients.

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