T cell receptors, fusion proteins, lentiviruses, TCR-T cells and their applications

Abstract

This invention relates to the field of biomedicine, and particularly to T-cell receptors, fusion proteins, lentiviruses, TCR-T cells, and their applications. This invention provides a T-cell receptor comprising a TCRα chain variable domain and a TCRβ chain variable domain. The AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells constructed in this invention are the first to combine SNIPRs with an AND logic gate, autocrine IL10 secretion, and MAGE-A4-TCR-T. This effectively reduces the off-target activity faced by conventional CAR-T and TCR-T, resulting in higher safety. Simultaneously, the autocrine IL10 promotes TCR-T cell proliferation and effector function. The application of a combination of cytokines IL7, IL15, and IL21 further enhances the durable tumor-killing activity of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to T cell receptors, fusion proteins, lentiviruses, TCR-T cells and their applications. Background Technology

[0002] Currently, cell immunotherapy mainly includes chimeric antigen receptor-engineered T cells (CAR-T), T cell receptor-engineered TCells (TCR-T), and tumor-infiltrating T lymphocytes (TIL), with CAR-T showing particular interest in its significant therapeutic effect on hematologic malignancies.

[0003] However, the efficacy of CAR-T therapy in treating solid tumors has been less than satisfactory. This is mainly due to the following reasons: 1. Most solid tumor-related proteins are also expressed in normal tissues, and there are almost no tumor-specific surface antigens. This easily leads to non-specific targeting (off-target activity) of CAR-T cells, potentially causing significant harm or even death to patients. For example, Morgan et al. reported in a clinical study of CAR-T cells targeting human epidermal growth factor receptor 2 (HER2) that CAR-T cells caused significant toxicity to the patient's lungs half an hour after administration, leading to the patient's death within several days. 2. The persistence of the anti-tumor response is highly correlated with the persistence of CAR-T cells. However, antigen-independent CAR tetanic signal transduction often leads to conventional CAR-T cells prematurely entering a terminal exhaustion state, preventing them from surviving long-term within the tumor to exert tumor-killing activity. 3. Solid tumors typically have an immunosuppressive tumor microenvironment. Once CAR-T cells reach the tumor and recognize antigens, this immunosuppressive tumor microenvironment may inhibit CAR-T cell activity, resulting in poor tumor-killing effects.

[0004] TCR-T and CAR-T therapies share many methodological similarities, but differ significantly in their tumor cell recognition mechanisms. CAR-T relies on artificially designed single-chain antibody fragments, which can only recognize antigens on the surface of tumor cells and activate T cells by transmitting signals through intracellular co-stimulatory molecules. TCR-T relies on natural or affinity-optimized TCRs to recognize antigens presented by tumor MHC molecules and transmit stimulation signals into the cell via the TCR. Its dominant T cell activation is closer to the natural T cell activation process and can recognize tumor intracellular antigens presented by MHC. Compared with CAR-T, TCR-T has several advantages: (1) Due to the limited number of membrane proteins in CAR-T therapy, the targets that can be applied to solid tumors are very limited; while TCR-T therapy has a wider range of targets. (2) Some tumors are caused by oncogenic viruses, and targeting oncogenic virus antigens can also achieve corresponding therapeutic effects. Since most viral antigens are intracellular proteins, TCR-T therapy is also a preferred option in viral tumor cell therapy. (3) Each CAR-T requires more than 1,000 antigens to activate, while each TCR-T only requires 1 to 50 antigens to activate. Therefore, TCR-T can recognize tumor antigens with low abundance. However, in the treatment of solid tumors, TCR-T also faces the same off-target activity and cell exhaustion dilemma as CAR-T. Summary of the Invention

[0005] In view of this, the present invention provides T cell receptors, fusion proteins, lentiviruses, TCR-T cells, and their applications. The AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells constructed in this invention are the first to combine SNIPRs with an AND logic gate, autocrine IL10 secretion, and MAGE-A4-TCR-T. This effectively reduces the off-target activity faced by conventional CAR-T and TCR-T cells, resulting in higher safety. Simultaneously, the autocrine IL10 promotes TCR-T cell proliferation and effector function. The application of a combination of cytokines IL7, IL15, and IL21 further enhances the durable tumor-killing activity of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a T cell receptor, comprising: a TCRα chain variable domain and a TCRβ chain variable domain;

[0008] (1) The amino acid sequences of CDR1, CDR2, and CDR3 of the variable domain of the TCRα chain are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and

[0009] (2) The amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of the TCRβ chain are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0010] In some embodiments of the present invention, the above-mentioned T cell receptor,

[0011] (3) The TCRα chain variable domain has the amino acid sequence shown in SEQ ID NO:30; and

[0012] (4) The TCRβ chain variable domain has an amino acid sequence as shown in SEQ ID NO:31; or

[0013] (5), (3), or (4) amino acid sequences obtained by substitution, deletion, or addition of one or more amino acids, and which have the same or similar function as the amino acid sequences shown in (3) or (4); or

[0014] (6) An amino acid sequence that is at least 80% homologous to the sequence described in (3), (4) or (5).

[0015] In some embodiments of the present invention, the amino acid sequence of the above-mentioned T cell receptor is shown in SEQ ID NO:8.

[0016] The present invention also provides a nucleic acid molecule encoding the above-mentioned T cell receptor.

[0017] In some embodiments of the present invention, the sequence of the nucleic acid molecule encoding the TCRα chain variable domain in the above-described nucleic acid molecule is as shown in SEQ ID NO:32; and

[0018] The sequence of the nucleic acid molecule encoding the variable domain of the TCRβ chain is shown in SEQ ID NO:33.

[0019] In some embodiments of the present invention, the nucleotide sequence of the above-mentioned T cell receptor nucleic acid molecule is shown in SEQ ID NO:7.

[0020] The present invention also provides a fusion protein comprising: the above-mentioned T cell receptor and IL10.

[0021] In some embodiments of the present invention, the fusion protein further includes: a linker protein T2A; the amino acid sequence of the linker protein T2A is shown in SEQ ID NO:18; and the nucleotide sequence of the nucleic acid molecule encoding the linker protein T2A is shown in SEQ ID NO:17.

[0022] In some embodiments of the present invention, the amino acid sequence of IL10 in the above-mentioned fusion protein is shown in SEQ ID NO:16; and the nucleotide sequence of the nucleic acid molecule encoding IL10 is shown in SEQ ID NO:15.

[0023] In some embodiments of the present invention, the amino acid sequence of the above-mentioned fusion protein is shown in SEQ ID NO:54; the sequence shown in SEQ ID NO:54 is (IL10-MAGE-A4-TCR amino acid).

[0024] The present invention also provides a nucleic acid molecule encoding the above-mentioned fusion protein.

[0025]

[0026] The present invention also provides an expression vector group comprising: the above-mentioned nucleic acid molecule and AntiHER2-SNIPRs; the nucleotide sequence of said AntiHER2-SNIPRs is shown in SEQ ID NO:56.

[0027]

[0028] In some embodiments of the present invention, the amino acid sequences of the above-mentioned AntiHER2-SNIPRs are as shown in SEQ ID NO:57: MALPVTALLLPLALLLHAARPGSDIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNYLAWYQQKPGQSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWIGHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQLNS LTSEDSAVYYCVRSGNYEEYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACFMYVAAAAFVLLFFVGCGVLLSKRKRKHMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNN WECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGS.

[0029] In some embodiments of the present invention, in the above-mentioned AntiHER2-SNIPRs, the amino acid sequence of AntiHER2 is shown in SEQ ID NO:10; the nucleotide sequence of the nucleic acid molecule encoding AntiHER2 is shown in SEQ ID NO:9; the amino acid sequence of SNIPRs is shown in SEQ ID NO:12; and the nucleotide sequence of the nucleic acid molecule encoding SNIPRs is shown in SEQ ID NO:11.

[0030] The present invention also provides TCR-T cells transfected with lentiviruses containing the above-described expression vector group.

[0031] The present invention also provides the use of the above-mentioned T cell receptor, the above-mentioned nucleic acid molecule, the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned expression vector group and / or the above-mentioned TCR-T cell in the preparation of a drug for treating tumors; the tumors include one or more of the following: synovial sarcoma, myxoid / round cell liposarcoma, non-small cell lung cancer, head and neck squamous cell carcinoma, ovarian cancer, urothelial carcinoma, melanoma and gastric cancer.

[0032] The present invention also provides a medicament or a composition thereof, comprising: the above-described T cell receptor, the above-described nucleic acid molecule, the above-described fusion protein, the above-described nucleic acid molecule, the above-described expression vector group and / or the above-described TCR-T cells, and pharmaceutically acceptable adjuvants or excipients.

[0033] In this invention, we used single-cell TCR sequencing + transcriptome sequencing technology to screen for natural TCR sequences targeting GVYDGREHTV (MAGE-A4 230-239, HLA-A*02:01), and linked the TCR α and β chains with sequences such as P2A and Furin restriction sites to construct MAGE-A4-TCR-T targeting GVYDGREHTV, and verified its function.

[0034] Furthermore, based on MAGE-A4-TCR-T, we combined SNIPRs AND logic gates (SyNthetic Intramembrane Proteolysis Receptors (SNIPRs)), an IL10 autocrine strategy, and MAGE-A4-TCR-T to ultimately construct AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T. These TCR-T cells use HER2 as the first tumor recognition antigen and GVYDGREHTV (MAGE-A4 230-239, HLA-A*02:01) as the second tumor recognition antigen. Only when tumor cells simultaneously express HER2 and GVYDGREHTV (MAGE-A4 230-239, HLA-A*02:01 will the tumor cells be recognized. When both antigens (230-239, HLA-A*02:01) are present, AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells activate their tumor-killing function. Furthermore, their tumor-killing activity is stronger and more persistent than that of MAGE-A4-TCR-T cells. Simultaneously, due to the use of a combination of cytokines IL7, IL15, and IL21 during TCR-T preparation, AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells exhibit a higher proportion of stem cell-like memory T cells and lower expression of exhaustion markers.

[0035] Overall, our invented AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells are the first to combine SNIPRs with an AND logic gate, autocrine IL10 secretion, and MAGE-A4-TCR-T. This effectively reduces the off-target activity faced by conventional CAR-T and TCR-T cells, resulting in higher safety. Simultaneously, the autocrine IL10 promotes TCR-T cell proliferation and effector function. The application of the cytokine combination of IL7, IL15, and IL21 further enhances the durable tumor-killing activity of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 Schematic diagram of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cell tumor killing;

[0038] Figure 2 This study aims to demonstrate the TCR expression status of MAGE-A4-TCR-T cells and their tumor target cell killing ability; where: A shows the proportion of TCR expression in MAGE-A4-TCR-T cells detected by flow cytometry; B shows the proportion of tumor target cells killed.

[0039] Figure 3 This shows the killing effect of T cells on tumor target cells and TCR expression in each group; where: A shows the lysis of target cells after co-culturing AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T and other T cells with tumor target cells; B shows the flow cytometry detection and SKBR3... HER2+ / MAGE-A4+ The proportion of TCR expression in T cells of each co-cultured group;

[0040] Figure 4 The data on the killing of tumor target cells and the proportion of TCR expression of T cells in each group are statistically analyzed; where: A shows the proportion of target cell lysis after co-culturing T cells with tumor target cells in each group; B shows the proportion of TCR expression of T cells after co-culturing T cells with tumor target cells in each group.

[0041] Figure 5 The proportion of CD137 expression in T cells after co-culturing T cells with tumor target cells in each group is shown.

[0042] Figure 6 The images show the secretion of IFNγ and TNFα by T cells in each group after co-culturing with tumor target cells; where: A shows the secretion of IFNγ by ELISA; B shows the secretion of TNFα by ELISA.

[0043] Figure 7 The image shows the specific recognition and killing ability of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T on tumor lesions expressing different target genes in mice. Among them: A shows a schematic diagram of tumor implantation and tail vein injection of TCR-T in mice; B shows the tumors expressing different target genes after treatment; C shows the tumor growth curve. Detailed Implementation

[0044] This invention discloses T cell receptors, fusion proteins, lentiviruses, TCR-T cells, and their applications.

[0045] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0046] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0047] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0048] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0049] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0050] This invention discovers that the AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T constructed by combining SNIPRs AND logic gates with the IL10 autocrine strategy and MAGE-A4-TCR-T can greatly reduce the off-target risk while possessing sustained and effective tumor-killing activity.

[0051] In the construction of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs and IL10-MAGE-A4-TCR are carried by two different lentiviruses. After co-transfection of CD3-activated T cells with both lentiviruses, AntiHER2 is expressed on T cells. When AntiHER2 recognizes the tumor surface antigen HER2, it activates the SNIPRs signaling molecule, hydrolyzing and releasing the downstream-linked VP64 transcription factor. VP64 enters the cell nucleus and binds to the UAS structure at the front end of the IL10-MAGE-A4-TCR sequence, thereby driving the expression of MAGE-A4-TCR and IL10. When MAGE-A4-TCR recognizes the MAGE-A4 polypeptide (GVYDGREHTV) presented by HLA-A*02:01 on the tumor surface, it mediates T cell activation, secreting tumor-killing factors such as IFNγ, TNFα, perforin, and Granzyme B, ultimately killing the tumor. The specific mechanism is as follows: Figure 1 As shown.

[0052] The technical solution of the present invention is as follows:

[0053] (I) Construction and Functional Verification of MAGE-A4-TCR

[0054] ① HLA-A*02:01 positive DC cell induction → ② DC cells loaded with MAGE-A4 peptide (GVYDGREHTV) → ③ CD8 + T cell purification → ④CD8 + T cells were co-incubated with DC cells loaded with MAGE-A4 peptide (two rounds of incubation, totaling 28 days) → ⑤CD3 + / CD8 + / Pentamer + / CD137 + Four-positive T-cell sorting → ⑥ Single-cell TCR sequencing + transcriptome sequencing → Construct MAGE-A4-TCR-T→ MAGE-A4-TCR-T cell function assay.

[0055] (II) Construction and Functional Verification of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T

[0056] ① Construction of AntiHER2-SNIPRs plasmid and IL10-MAGE-A4-TCR plasmid → ② Plasmid amplification → ③ Plasmid extraction → ④ Lentiviral preparation → ⑤ Isolation and activation of peripheral blood PBMCs → ⑥ Lentiviral transfection of T cells → AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cell expansion → AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cell function assay.

[0057] Specifically, the steps include the following:

[0058] (I) Construction and Functional Verification of MAGE-A4-TCR

[0059] (1) DC induction and peptide loading

[0060] Peripheral blood was collected from HLA-A*02:01 positive volunteers, and PMBCs were isolated using lymphocyte separation medium. PBMCs were resuspended in serum-free AIM-V medium, and the cell concentration was adjusted to 5E6 cells / mL. The cells were then transferred to T25 culture flasks and incubated at 37°C for 1 hour. The flasks were gently shaken, and the suspended cells were aspirated. PBS was added, and the cells were washed twice to remove the suspension. 5 mL of DC cell induction medium (AIM-V + 5% AB + 1% GlutaMAX + 25 mM HEPES + 1000 IU / mL GM-CSF + 500 IU / mL IL-4) was added to the T25 flasks. The flasks were incubated at 37°C with 5% CO2 for 48 hours. The T25 flasks were removed, the cell suspension was collected, centrifuged at 500g for 3 minutes, the supernatant was discarded, and the pellet was resuspended in 5 mL of DC cell induction medium. The pellet was then added back to the T25 culture flasks, and induction culture was continued for another 48 hours. Remove the T25 flask, collect the cell suspension, centrifuge at 500g for 3 min, discard the supernatant, and resuspend the pellet in 5 mL of DC cell maturation medium (AIM-V + 5% AB + 1% GlutaMAX + 25 mM HEPES + 10 ng / mL TNF-α + 10 ng / mL IL-Iβ + 1000 U / mL IL-6 + 1 ug / mL PGE-2). Add the pellet back to the T25 culture flask and continue induction culture for 48 hours. Tap the DC culture flask until most mature DC cells detach, collect the cell suspension, centrifuge at 500g for 3 min, wash twice with physiological saline, and take samples for cell counting, viability, and flow cytometry analysis (CD11c, HLA-DR, CD80, CD83, CD86, etc.).

[0061] Collect DC cells into 15 mL centrifuge tubes and centrifuge at 500 g for 3 min. Resuspend 2E6 DC cells in 2 mL of pure AIM-V medium, add the corresponding peptide at a concentration of 2 μg / mL, and incubate at 37°C for 2 hours, inverting to mix the cell suspension once every 20 min. Collect peptide-loaded DC cells into 15 mL centrifuge tubes and centrifuge at 500 g for 3 min. Resuspend the cells in 1 mL of DC incubation medium (AIM-V + 5% AB + 1% GlutaMAX + 25 mM HEPES + 30 ng / mL IL-21) to a final concentration of 2E6 Cells / mL.

[0062] (2) CD8 + T cells were purified and co-incubated with DC cells loaded with MAGE-A4 peptide.

[0063] Peripheral blood was collected from volunteers, and PMBCs were isolated using lymphocyte separation fluid. CD8 + T cell purification was performed using Tongli Haiyuan CD8 sorting magnetic beads (catalog number: TL-624). Specific operating procedures are detailed in the product instructions. CD8 was collected. + T cells were incubated with DC culture medium to a concentration of 6E6 cells / mL. At a DC:T ratio of 1:3, 1 mL of CD8 was added to each well of a 24-well plate. + T cells were co-incubated with 1 mL of DC cells for 24 hours.

[0064] Collect DC and CD8 + T cell suspension, centrifuged at 500g for 3 min, supernatant discarded. Cells were resuspended in 2 mL TCR selection medium (AIM-V + 5% AB + 1% GlutaMAX + 25 mM HEPES + 10 ng / mL IL-7 + 10 ng / mL IL-15 + 30 IU / mL IL-2) and transferred to 12-well plates. Half the medium was changed every 48 hours. If cell proliferation was significant in the later stages, wells could be separated. After co-culturing for 14 days, the above stimulation steps were repeated and co-incubated for another 14 days.

[0065] (3) CD3 + / CD8 + / Pentamer + / CD137 + Four-positive T-cell sorting, and single-cell TCR sequencing + transcriptome sequencing

[0066] Collect all secondary stimulation CD8s +T cells were co-incubated for the third time with polypeptide-loaded dendritic cells (DCs). After 24 hours, the cell suspension was collected, and the proportion of CD3, CD8, CD137, and Pentamer-positive T cells was determined by flow cytometry. If quadrivalent positive cells were found, they were flow-cytosored and sent for single-cell TCR sequencing and transcriptome sequencing. After screening, the most abundant TCR pairing sequences were obtained, and the three complementarity-determining regions (CDRs) of the variable domains of the TCR α and TCR β chains were as follows:

[0067] Table 1

[0068]

[0069] Group 1:

[0070] The amino acid sequence of the variable domain of the TCRα chain is: MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSWYYGGATNKLIFGTGTLLAVQPN (as shown in SEQ ID NO:30).

[0071] The amino acid sequence of the variable domain of the TCRβ chain is: MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRAGNPYEQYFGPGTRLTVTE (as shown in SEQ ID NO:31).

[0072] Nucleotide sequence of the variable region of TCR α chain: ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAACTTCACCATCACAGCCTCACAAGTCGTGGACTCAGCAGTATACTTCTGTGCTCTGAGCTGGTATTATGGTGGTGCTACAAACAAGCTCATCTTTGGAACTGGCACTCTGCTTGCTGTCCAGCCAAAT (as shown in SEQ ID NO: 32).

[0073] Nucleotide sequence of the variable region of TCR β chain: ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGAGCAGGGAACCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (as shown in SEQ ID NO: 33).

[0074] Group 2:

[0075] The amino acid sequence of the variable domain of the TCRα chain is: MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVKGINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVSDRGGGTALIFGKGTTLSVSSN (as shown in SEQ ID NO:40).

[0076] The amino acid sequence of the variable domain of the TCRβ chain is: MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLGRINEQYFGPGTRLTVTE (as shown in SEQ ID NO:41).

[0077] Nucleotide sequence of the variable domain of the TCR alpha chain: ATGCTCCTGCTGCTCGTCCCAGCGTTTCCAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTC TCCTGAAGTATTTATCAGGATCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCACTTGAGGAAACCCTCAGTCCATATAAGCGACACGGCTGAGTACTTCTGTGCTGTGAGTGATCGGGGAGGAGGAACTGCTCTGATCTTTGGGAAGGGAACCACCTTATCAGTGAGTTCCAAT (as SEQ ID NO:42).

[0078] Nucleotide sequence of the TCRβ chain variable domain: ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCAGAACTCACAGAAGCTGGGAGTTGCCCAGTCTCCCAGATATAAGATTATAGAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCATGCTACCCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTTCT GATTCAGTTTCAGAATAACGGTGTAGTGGATGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACTCCACTCTCAAGATCCAGCCTGCAAAGCTTGAGGACTCGGCCGTGTATCTCTGTGCCAGCAGCTTAGGAAGGATCAACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (as SEQ ID NO:43).

[0079] Group 3:

[0080] The amino acid sequence of the variable domain of the TCRα chain is: MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAERGPSMLTFGGGTRLMVKPH (as shown in SEQ ID NO:50).

[0081] The amino acid sequence of the variable domain of the TCRβ chain is: MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASTRGGETEAFFGQGTRLTVVE (as shown in SEQ ID NO:51).

[0082] Nucleotide sequence of the variable region of TCR α chain: ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACATGAAACAAGACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACTCAGCTATCTACTTCTGTGCAGAGAGAGGGCCGTCCATGCTCACCTTTGGAGGGGGAACAAGGTTAATGGTCAAACCCCAT (as shown in SEQ ID NO:52).

[0083] Nucleotide sequence of the variable region of TCR β chain: ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTTTCCTGGGAGCAAACACCGTGGATGGTGGAATCACTCAGTCCCCAAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCCTGAGTTGTGAACAGAATTTGAACCACGATGCCATGTACTGGTACCGACAGGACCCAGGGCAAGGGCTGAGATTGATCTACTACTCACAGATAGTAAATGACTTTCAGAAAGGAGATATAGCTGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCTCACTGTGACATCGGCCCAAAAGAACCCGACAGCTTTCTATCTCTGTGCCAGTACCCGCGGGGGTGAGACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAG (as shown in SEQ ID NO:53).

[0084] (4)Construction and functional detection of MAGE-A4-TCR-T

[0085] The MAGE-A4-TCR genes were synthesized by Shanghai Jierui Biotechnology Co., Ltd. The MAGE-A4-TCR coding sequence is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8. The synthesized gene sequences were amplified by PCR and constructed into lentiviral shuttle plasmids using homologous recombination. The constructed shuttle plasmids were transformed into competent DH5α cells to prepare single clones. Single clones were picked and sent to Sangon Biotech Co., Ltd. for sequencing confirmation of the target fragment. Single clones of the correctly recombined MAGE-A4-TCR gene were selected and cultured. After collecting the bacterial culture, plasmids were extracted using the Tiangen Plasmid Medium-Quantity Small-Scale Extraction Kit; specific operating procedures were followed according to the product instructions.

[0086] 1.3E7 293T cells were seeded in a T75 culture flask and cultured for 1 day until the cell density reached approximately 90%. Following the instructions of the Lipo3000 transfection reagent kit, the packaging plasmid, cell membrane plasmid, and shuttle plasmid were mixed with the transfection reagent and added to the culture flask, which was then incubated at 37°C. After 8 hours, the culture medium was aspirated, the cells were washed once with physiological saline or PBS, and 10 mL of fresh DMEM medium was added. 48 hours after transfection, the viral supernatant was collected.

[0087] Peripheral blood was collected from volunteers, and PMBCs were isolated using lymphocyte separation medium. Cells were resuspended in 5 mL of AIM-V medium containing 5% AB serum, 1% GlutaMAX, 25 mM HEPES, and 300 IU / mL IL-2. Based on the cell count, CD3 / 28 magnetic beads were added at a cell-to-bead ratio of 1:3, and the mixture was incubated at 37°C for 48 hours. Magnetic beads were then collected using a magnetic rack. + T cells were resuspended in 3 mL of AIM-V medium containing 5% AB serum, 1% GlutaMAX, 25 mM HEPES, and 300 IU / mL IL-2. The magnetic beads and cell suspension were pipetted to remove the beads. The cell concentration was adjusted to 1E6 Cells / mL. A 12-well plate coated with Retrolectin was placed in the plate, and 2 mL of MAGE-A4-TCR virus was added to each well. The plate was centrifuged at 32°C, 2000g, for 90 min. The viral supernatant was removed, and 1 mL of activated T cell suspension was added to each well. The plate was centrifuged at 32°C, 2000g, for 10 min, and then incubated at 37°C for 24 hours. To improve the transfection rate, transfection could be repeated once after 24 hours.

[0088] Collect transfected TCR-T cells and resuspend them in AIM-V medium containing 5% AB serum + 10 ng / mL IL-7 + 10 ng / mL IL-15 + 10 ng / mL IL-21 + 1% GlutaMAX + 25 mM HEPES. Incubate at 37°C for 48 hours. Perform functional experiments and flow cytometry on a portion of the cells.

[0089] (II) Construction and Functional Verification of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T

[0090] The AntiHER2-SNIPRs and IL10-MAGE-A4-TCR genes were synthesized by Shanghai Jierui Biotechnology Co., Ltd. The AntiHER2 coding sequence is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10. The SNIPRs coding sequence is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:12. The VP64 coding sequence is shown in SEQ ID NO:13, and the amino acid sequence is shown in SEQ ID NO:14. The IL10 coding sequence is shown in SEQ ID NO:15, and the amino acid sequence is shown in SEQ ID NO:16. The MAGE-A4-TCR and IL10 sequences are linked by a T2A linker, the T2A coding sequence of which is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18. The IL10-MAGE-A4-TCR sequence has a UAS structure at its front end, forming the UAS-miniCMV promoter region with miniCMV, and its coding sequence is shown in SEQ ID NO:19. The synthesized gene sequences were amplified by PCR and then homologously recombinated into lentiviral shuttle plasmids to construct the AntiHER2-SNIPRs plasmid and the IL10-MAGE-A4-TCR plasmid, respectively. Following the MAGE-A4-TCR-T preparation method, the two viruses were transfected into T cells to prepare the AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells. After amplification, cells were harvested for functional experiments and flow cytometry analysis.

[0091] The sequence involved in this invention is shown below:

[0092]

[0093] SEQ ID NO:8 (MAGE - A4 - TCR - protein): MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSWYYGGATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRAGNPYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG。

[0094] SEQ ID NO:9 (AntiHER2-nucleic acid): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGGATCTGACATTGTGCTGACCCAAACTCCATCCTCCCTACCTGTGTCAGTTGGAGAGAAGGTTACTATGACCTGCAAGTCCAGTCAGACCCTTTTATATAGTAACAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTCCTGGGCATTCACTAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCGGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTCTAACTATCCGTGGACGTTCGGTGGAGGCACCAGGCTGGAAATCAAACGGGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTCCAGCTGCAGCAGTCTGGACCTGAGGTAGTGAAGACTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGTTACTTCATAAACTGGGTCAAGAAGAACTCTGGAAAGAGCCCTGAGTGGATTGGACACATTAGTTCTTCCTATGCTACCTCTACCTACAACCAGAAGTTTAAAAACAAGGCCGCATTTACTGTAGACACATCCTCCAGCACAGCCTTCATGCAGCTTAACAGCCTGACATCTGAGGACTCTGCAGTCTATTATTGTGTTAGAAGTGGTAACTACGAAGAATATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCGTCA。

[0095] SEQ ID NO:10 (AntiHER2 - Protein): MALPVTALLLPLALLLHAARPGSDIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNYLAWYQQKPGQSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWIGHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYAMDYWGQGTSVTVSS。

[0096] SEQ ID NO:11 (SNIPRs - Nucleic Acid): ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCTTTATGTACGTGGCAGCCGCGGCATTTGTACTTCTGTTTTTCGTGGGGTGCGGTGTGCTTCTCAGCAAGAGGAAACGGAAGCACATGAAGCTGCTGAGCAGCATCGAGCAGGCCTGTGACATCTGCCGGCTGAAGAAACTGAAGTGCAGCAAAGAAAAGCCCAAGTGCGCCAAGTGCCTGAAGAACAACTGGGAGTGCCGGTACAGCCCCAAGACCAAGAGAAGCCCCCTGACCAGAGCCCACCTGACCGAGGTGGAAAGCCGGCTGGAAAGACTGGAACAGCTGTTTCTGCTGATCTTCCCACGCGAGGACCTGGACATGATCCTGAAGATGGACAGCCTGCAGGACATCAAGGCCCTGCTGACCGGCCTGTTCGTGCAGGACAACGTGAACAAGGACGCCGTGACCGACAGACTGGCCAGCGTGGAAACCGACATGCCCCTGACCCTGCGGCAGCACAGAATCAGCGCCACCAGCAGCAGCGAGGAAAGCAGCAACAAGGGCCAGCGGCAGCTGACAGTGTCTGCTGCTGCAGGCGGAAGCGGAGGCTCTGGCGGATCT。

[0097] SEQ ID NO: 12 (SNIPRs - Protein): TTTPAPRPPTPAPTIASQPLSLRPEACFMYVAAAAFVLLFFVGCGVLLSKRKRKHMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGS.

[0098] SEQ ID NO: 13 (VP - 64 - Nucleic Acid): GATGCCCTGGACGACTTCGACCTGGATATGCTGGGCAGCGACGCCCTGGATGATTTTGATCTGGACATGCTGGGATCTGACGCTCTGGACGATTTCGATCTCGACATGTTGGGATCAGATGCACTGGATGACTTTGACCTGGACATGCTCGGATCA.

[0099] SEQ ID NO: 14 (VP - 64 - Protein): DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS.

[0100] SEQ ID NO:15 (IL10 - nucleic acid): AGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC。

[0101] SEQ ID NO:16 (IL10 - protein): SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0102] SEQ ID NO:17 (T2A - nucleic acid): GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT。

[0103] SEQ ID NO:18 (T2A - protein): EGRGSLLTCGDVEENPGP。

[0104] SEQ ID NO:19 (UAS-miniCMV-Nucleic Acid): CGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGATCGATAAGCTTGATATCGAATTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCTCGACATTCGTTGGATC。

[0105]

[0106] SEQ ID NO:21 (MAGE-A4 / HLA-A*02:01 - Protein): MARSVTLVFLVLVSLTGLYAGVYDGREHTVGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV。

[0107] SEQ ID NO:22: caccgCGGCACAGACAGTGCGCGTC。

[0108] SEQ ID NO:23: aaacGACGCGCACTGTCTGTGCCGc。

[0109] SEQ ID NO:24: caccgACCGGCACAGACATGAAGCTG。

[0110] SEQ ID NO:25: aaacCAGCTTCATGTCT GTGCCGGTc。

[0111] SEQ ID NO:26: caccgCTGGACATGCTCCGCCACCTCTACCA。

[0112] SEQ ID NO:27: aaacTGGTAGAGGTGGCGGAGCATGTCCAGc。

[0113]

[0114] SEQ ID NO:29 (Luciferase-protein):

[0115] In Examples 1 to 6 and Comparative Examples 1 to 3 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0116] The present invention will be further illustrated below with reference to the embodiments:

[0117] Example 1: Construction of MAGE-A4-TCR plasmid

[0118] The MAGE-A4-TCR gene fragments were synthesized by Shanghai Jereh Biotechnology Co., Ltd. The MAGE-A4-TCR gene fragments and shuttle plasmid framework were amplified by PCR using the Novizan P505-d1 PCR kit.

[0119] The PCR system for P505-d1 is as follows:

[0120] Table 2

[0121]

[0122] The reaction procedure for P505-d1 is as follows:

[0123] Table 3

[0124]

[0125] After PCR amplification, DNA gel electrophoresis is used to check if the product fragment size meets expectations. If it does, the PCR product is purified using the Magen HiPure PCR Pure Kit; refer to the kit's instruction manual for specific procedures.

[0126] After obtaining the purified PCR product, the natural plasmid template was digested using the FastDigest DpnI-FD1703 methyl digestion kit to reduce the false positive rate of single clones in the later stage. For specific operation, please refer to the kit instructions.

[0127] The digested PCR products were subjected to homologous recombination using the Novizan ClonExpress II One Step Cloning Kit. The optimal amount of cloning vector and insert fragment in the recombination reaction system was 0.03 pmol and 0.06 pmol (vector to insert molar ratio of 1:2). The DNA mass corresponding to these molar amounts can be roughly calculated using the following formula:

[0128] Optimal cloning vector usage = [0.02 × number of cloning vector base pairs] ng (0.03 pmol);

[0129] Optimal amount of insert fragment used = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol);

[0130] When the insert fragment length is greater than the cloning vector, the calculation methods for the optimal cloning vector and insert fragment usage should be reversed; that is, the insert fragment should be treated as the cloning vector, and the cloning vector as the insert fragment. The amount of linearized cloning vector used should be between 50 and 200 ng; the amount of insert fragment amplification product used should be between 10 and 200 ng. If the optimal DNA usage calculated using the above formula exceeds this range, simply select the minimum / maximum usage amount.

[0131] The following ClonExpress II One Step Cloning Kit reaction system was prepared on ice:

[0132] Table 4

[0133]

[0134] The amount of carrier and the amount of inserted fragment are calculated using the formula aX / Y.

[0135] Gently pipette the mixture (do not vortex), and briefly centrifuge to collect the reaction solution at the bottom of the tube. Incubate at 37°C for 30 min on a PCR instrument; then cool to 4°C or immediately place on ice. The MAGE-A4-TCR plasmid recombinant product can be stored at -20°C for one week, and can be thawed and transformed when needed.

[0136] Example 2: MAGE-A4-TCR monoclonal antibody and extraction of MAGE-A4-TCR shuttle plasmid

[0137] Thaw two clonal competent cells (DH5α competent cells) on ice. Add 10 μL of MAGE-A4-TCR recombinant plasmid to 100 μL of competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. (The transformation volume of the recombinant product should not exceed 1 / 10 of the volume of competent cells used). After heat shock in a 42°C water bath for 45 s, immediately cool on ice for 2 min. Add 900 μL of LB medium (without antibiotics) and incubate at 37°C for 1 hr. Preheat ampicillin-resistant agar plates to room temperature for 1 hr. Centrifuge at 5000g for 1 min and discard 900 μL of supernatant. Resuspend the bacterial cells in the remaining medium and gently spread them evenly on the ampicillin-resistant plate using a sterile spreader. Incubate upside down at 37°C for 15 hr.

[0138] Once monoclonal strains have formed, five monoclonal strains of each strain are selected and inoculated into 2 mL of LB medium containing ampicillin, and incubated in a shaker at 37°C for 5 hours. 1 μL of suspension from each monoclonal strain is taken and the target fragment is amplified using the Novozymes liquid PCR kit GreenTaq Mix. Monoclonal strains containing the target fragment are identified by DNA electrophoresis.

[0139] The Green Taq Mix reaction system is as follows:

[0140] Table 5

[0141]

[0142] The reaction procedure for Green Taq Mix is ​​as follows:

[0143] Table 6

[0144]

[0145] Take 1 mL of a single-clone bacterial culture containing the target fragment and send it to Sangon Biotech Co., Ltd. for sequencing confirmation of the target fragment. Store the remaining 1 mL at 4°C. After confirming that the MAGE-A4-TCR clone is correct, select a single-clone strain with the correctly recombined MAGE-A4-TCR gene, and inoculate 350 μL of it into 35 mL of LB medium containing ampicillin. Incubate at 37°C on a shaker for 15 hours.

[0146] The plasmid extraction kit used was the Tiangen Plasmid Medium-Quantity Small-Scale Extraction Kit; specific operating procedures were followed according to the product instructions. The obtained shuttle plasmids, after concentration quantification, were stored at -20°C for later use.

[0147] Example 3 Preparation of MAGE-A4-TCR virus

[0148] One day before transfection, 1.3E7 293T cells were seeded in a T75 culture flask and cultured for one day until the cell density reached approximately 90%. One hour before transfection, the medium was changed with 10 mL of DMEM + 10% FBS + 1% penicillin-dextrose antibody + 1% GlutaMAX + 25 mM HEPES medium. Following the instructions of the Lipo3000 transfection reagent kit, a sterile 1.5 mL EP tube was taken, and 1.5 mL of serum-free Opti-MEM and 120 µL of Lipofectamine-3000 Reagent were added. The mixture was thoroughly mixed to prepare solution A. A sterile 5 mL cryovial was taken, and 1.5 mL of serum-free Opti-MEM, 40 µg of total plasmid (pMD2.G:pRSV-Rev:pMDLg / pRRE:pRRLSIN shuttle plasmid = 1:1:1:2) and 80 µL of P-3000 Reagent were added sequentially. The mixture was thoroughly mixed to prepare solution B. Add solution A to solution B in a 1:1 ratio, mixing gently while adding dropwise. Incubate at room temperature for 15 minutes to allow the DNA and transfection reagent to fully combine and form a stable transfection complex. Add the prepared DNA-transfection reagent mixture to a T75 culture flask, mix gently, and incubate at 37°C.

[0149] After 8 hours, the culture medium was aspirated, and the cells were washed once with physiological saline or PBS. 10 mL of fresh DMEM + 10% FBS + 1% GlutaMAX + 25 mM HEPES medium was added, and the cells were incubated at 37°C. 48 hours after transfection, the culture supernatant was collected into 15 mL centrifuge tubes, centrifuged at 4000g for 5 min, and the viral supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane, aliquoted into cryovials, and stored at -80°C.

[0150] Example 4: Preparation of MAGE-A4-TCR-T cells and verification of their function.

[0151] 10 mL of peripheral blood was drawn from the volunteer and mixed with physiological saline at a 1:1 ratio. Two 15 mL centrifuge tubes were used, each containing 5 mL of room temperature Ficoll, followed by 10 mL of the peripheral blood mixture. The tubes were centrifuged at 800 g for 20 min, and the white membrane layer was transferred to a new 15 mL centrifuge tube. Physiological saline was added to 15 mL, the cells were shaken well, and the tubes were centrifuged at 500 g for 5 min, discarding the supernatant. Physiological saline was added to 15 mL again to resuspend the cells, and the tubes were centrifuged at 200 g for 5 min, discarding the supernatant. PBMCs were resuspended in 5 mL of AIM-V medium containing 5% AB serum + 1% GlutaMAX + 25 mM HEPES + 300 IU / mL IL-2 and counted.

[0152] Vortex the CD3 / 28 magnetic beads. Based on the number of PBMC cells, transfer an appropriate amount of magnetic beads to a new sterile EP tube (magnetic beads: PBMC = 3:1). Resuspend the magnetic beads in 1 mL of DPBS buffer containing 2.5% HSA. Place the tube on a magnetic rack to separate the magnetic beads for 1 min and remove the supernatant. Resuspend the magnetic beads in PBMC cell suspension, mix well, and incubate in a CO2 incubator for 48 hours.

[0153] One day before T cell activation was complete, 12-well plates were coated with retrolectin at a concentration of 15 μg / mL and incubated overnight at 4°C in the dark. On the day of transfection, the coated plates were removed, blocked with 2.5% HSA for 30 min, and 2 mL of MAGE-A4-TCR virus was added to each well. The plates were centrifuged at 32°C, 2000g for 90 min. The plates were then removed, the viral load was discarded, and the plates were washed once with 2.5% HSA. Magnetic beads were then collected using a magnetic rack. + T cells were collected, and unbound cell suspension was removed. The magnetic beads and cells were resuspended in 3 mL of AIM-V medium containing 5% AB serum, 1% GlutaMAX, 25 mM HEPES, and 300 IU / mL IL-2. The cell suspension was pipetted, and the magnetic beads were re-adsorbed using a magnetic rack. The beads were then removed, and the cell suspension was counted. The cell concentration was adjusted to 1E6 Cells / mL. After centrifugation, 1 mL of activated T cell suspension was added to each well of the plate. The plate was centrifuged at 32°C, 2000g for 10 min, and then incubated at 37°C for 24 hours. Transfection could be repeated once after this period. Collect transfected MAGE-A4-TCR-T cells, centrifuge at 300g for 5 min, remove supernatant, resuspend in AIM-V medium containing 5% AB serum + 10 ng / mL IL-7 + 10 ng / mL IL-15 + 10 ng / mL IL-21 + 1% GlutaMAX + 25 mM HEPES, and incubate at 37℃ for amplification. On day 10, CD3 was detected by flow cytometry. + / MAGE-A4Pentamer + T cell ratio.

[0154] The target cell line was selected as SKBR3, with MAGE-A4 / HLA-A... * 02:01 was negative, marked as SKBR3 MAGE-A4- Constructing MAGE-A4 / HLA-A * The 02:01 gene, whose coding sequence is shown in SEQ ID NO:20 and amino acid sequence is shown in SEQ ID NO:21, is overexpressed in the SKBR3 cell line and labeled as SKBR3. MAGE-A4+ SKBR3 MAGE-A4- SKBR3 MAGE-A4+ Both cell groups were resuspended in DMEM medium containing 10% FBS, and the concentration was adjusted to 2E5 cells / mL. 100 μL of each cell group was seeded into 96-well plates (2E4 cells / well). After 24 hours, the DMEM medium was removed. The cell concentrations of the NC-T and MAGE-A4-TCR-T groups were adjusted to 5E5 cells / mL. 200 μL of each T cell group was co-cultured with tumor cells, resulting in a final effector-to-target ratio of 5:1. After 24 hours, the lysis rate of tumor target cells was detected using dual-luciferase reporter gene assay.

[0155] like Figure 2 As shown in Figure A, the TCR positivity rate of MAGE-A4-TCR-T was 66.60%. Figure 2 As shown in B, MAGE-A4-TCR-T and SKBR3 MAGE-A4+ After co-culturing with target cells, the lysis rate of tumor target cells reached 84.20%, while that with SKBR3... MAGE-A4- The lysis rate of the target cell co-culture group was only 2.33%.

[0156] Comparative Example 1

[0157] The company synthesized AntiALPPL2-SynNotch and MCAM-CAR plasmids, and constructed AntiHER2-SynNotch and MCAM-CAR retroviruses. Following the Ficoll instructions, PBMCs were isolated and resuspended in X-VIVO 15 basal medium, adjusted to 2E7 cells / mL, and 1 mL of cell suspension was transferred to a centrifuge tube. CD3 / 28 magnetic beads were added at a cell:bead ratio of 1:3, and the tube was incubated at room temperature for 30 min at 20 rpm. The centrifuge tube was then placed on a magnetic rack and allowed to stand for 1 min, discarding any unadsorbed cell suspension. The adsorbed magnetic beads and T cells were collected, and the cells were resuspended in X-VIVO 15 medium containing 5% AB serum + 300 IU / mL IL-2 and activated at 37°C for 24 hours. One day before T cell activation was complete, 12-well plates were coated with 20 μg / mL retroactive cellulose and incubated overnight at 4°C in the dark. On the day of transfection, the coated plate was removed and blocked with 2.5% HSA for 30 min. AntiALPPL2-SynNotch virus and MCAM-CAR virus were added to each well at MOI=5. The plate was centrifuged at 32℃, 2000g for 2 hours. Magnetic beads were collected using a magnetic rack. + T cells were collected, and unbound cell suspension was removed. The magnetic beads and cells were resuspended in X-VIVO 15 medium containing 5% AB serum and 300 IU / mL IL-2. The magnetic beads were then removed using a magnetic rack, and the cell concentration was adjusted to 1E6 Cells / mL. After centrifugation, 1 mL of activated T cell suspension was added to each well of the plate. The plates were centrifuged at 32°C, 2000g for 10 min, and then incubated at 37°C for 48 hours. AntiALPPL2-SynNotch-MCAM-CAR-T cells were harvested for functional assays and experiments.

[0158] Comparative Example 2

[0159] The IL10-AntiCD19-CAR plasmid was synthesized by the company, and the IL10-AntiCD19-CAR retrovirus was constructed. PBMCs were isolated according to the Ficoll instructions and activated for 48 hours in 1640 medium containing 10% FBS, 10 ng / mL IL-2, 10 ng / mL IL-7, and 50 ng / mL CD3 (OKT3). Six-well plates were coated with 15 μg / mL retrovirus and incubated at 4°C for 18 hours. On the day of transfection, the coated plates were removed and blocked with 0.5% FBS for 30 min. 3 mL of AntiCD19-CAR retrovirus was added to each well, and the plates were centrifuged at 32°C, 2000g, for 2 hours. Activated T cells were collected, and the cell concentration was adjusted to 1E6 cells / mL using 1640 medium containing 10% FBS and 10 ng / mL IL-2. Remove the centrifuged plate, add 3 mL of activated T cell suspension to each well, centrifuge at 32°C, 500g for 30 min, and then incubate at 37°C for 48 h. Harvest IL10-AntiCD19-CAR-T cells for functional assays and experiments.

[0160] Comparative Example 3

[0161] The company synthesized the CCL19-IL7-MAGE-A-TCR plasmid and constructed the CCL19-IL7-MAGE-A-TCR lentivirus. Following the Ficoll manual, PBMCs were isolated and screened for CD3 using CD3 / CD28 magnetic beads. + T cells were activated for 24 hours using 1640 medium containing 10% FBS and 50 IU / mL IL-2. Six-well plates were coated with 15 μg / mL retrotronectin and incubated overnight at 4°C in the dark. On the day of transfection, the coated plates were removed, blocked with 2.5% HSA for 30 min, and CCL19-IL7-MAGE-A-TCR lentivirus was added to each well at MOI=1. The plates were centrifuged at 32°C, 2000g for 90 min. The magnetic beads and cells were resuspended in 1640 medium containing 10% FBS and 50 IU / mL IL-2. The magnetic beads were removed using a magnetic rack, and the cell concentration was adjusted to 1E6 Cells / mL. The centrifuged 6-well plates were then incubated with 2 mL of activated T cell suspension in each well, centrifuged at 32°C, 2000g for 10 min, and then incubated at 37°C for 48 hours. CCL19-IL7-MAGE-A-TCR-T cells were harvested for functional testing and experiments.

[0162] Example 5: Comparison of in vitro tumor-specific recognition and killing abilities of four cell groups: MAGE-A4-TCR-T, IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T, and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T.

[0163] Following the preparation method of MAGE-A4-TCR-T, four groups of TCR-T cells were prepared sequentially: MAGE-A4-TCR-T, IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T, and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T.

[0164] The target cells were selected from the SKBR3 cell line, which is naturally HER2-positive and MAGE-A4 / HLA-A. * 02:01 Negative cells, labeled SKBR3 HER2+ / MAGE-A4- Overexpression of MAGE-A4 / HLA-A in native SKBR3 cells * 02:01 gene, obtained SKBR3 HER2+ / MAGE-A4+ MAGE-A4 / HLA-A * The coding sequence for 02:01 is shown in SEQ ID NO:20, and the amino acid sequence is shown in SEQ ID NO:21. The HER2 gene in native SKBR3 cells was knocked out using CRISPR-Cas9 to obtain HER2-negative cells, which were then labeled as SKBR3. HER2- / MAGE-A4- The three pairs of sgRNA sequences used are as follows: sgRNA-1F sequence as shown in SEQ ID NO:22, sgRNA-1R sequence as shown in SEQ ID NO:23, sgRNA-2F sequence as shown in SEQ ID NO:24, sgRNA-2R sequence as shown in SEQ ID NO:25, sgRNA-3F sequence as shown in SEQ ID NO:26, and sgRNA-3R sequence as shown in SEQ ID NO:27. In SKBR3... HER2- / MAGE-A4- Overexpression of MAGE-A4 / HLA-A in cells * 02:01 gene, obtained SKBR3 HER2- / MAGE-A4+ Cells. All four groups of cells carried the Luciferase gene, the coding sequence of which is shown in SEQ ID NO:28, and the amino acid sequence of which is shown in SEQ ID NO:29, to facilitate in vitro detection of tumor-killing activity.

[0165] SKBR3 HER2- / MAGE-A4- SKBR3 HER2+ / MAGE-A4-SKBR3 HER2- / MAGE-A4+ SKBR3 HER2+ / MAGE-A4+ Four cell lines were resuspended in DMEM medium containing 10% FBS and adjusted to a concentration of 2E5 cells / mL. 100 μL of each cell line was seeded into 96-well plates (2E4 cells / well). After 24 hours, the DMEM medium was removed. The cell concentrations of five other cell lines (NC-T, MAGE-A4-TCR-T, IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T, and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T) were adjusted to 5E5 cells / mL. 200 μL of each T cell line was co-cultured with tumor cells, resulting in an effector-to-target ratio of 5:1. After 36 hours, the culture supernatant was collected, and ELISA was used to detect IFNγ and TNFα secretion. Suspension cells were collected, and CD3+ was detected by flow cytometry. + / MAGE-A4 Pentamer + CD137 + The proportion of T cells. Dual-luciferase reporter gene assay for tumor target cell lysis rate.

[0166] like Figure 3 A and Figure 4 As shown in A. In SKBR3 HER2- / MAGE-A4+ In the various co-culture systems, only the tumor target cells in the MAGE-A4-TCR-T and IL10-MAGE-A4-TCR-T wells were lysed (94.08% and 93.00%, respectively). Because HER2 was knocked out in SKBR3, AntiHER2-SNIPRs could not contact the primary antigen HER2, resulting in AntiHER2-SNIPRs-MAGE-A4-TCR-T and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T failing to express MAGE-A4-TCR, ultimately failing to kill tumor target cells. In SKBR3... HER2+ / MAGE-A4+ In all co-culture systems except the NC-T group, tumor target cells in the other four groups (MAGE-A4-TCR-T, IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T, and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T) were lysed (86.23%, 93.07%, 71.33%, and 85.97%, respectively). Furthermore, there were significant differences in target cell lysis between the AntiHER2-SNIPRs-MAGE-A4-TCR-T group (71.33% lysis rate) and the AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T group (85.97% lysis rate).

[0167] like Figure 3 B and Figure 4 As shown in B, in SKBR3 HER2+ / MAGE-A4+ In the co-culture systems of various groups, except for the NC-T group, the T cells in the co-culture wells of the other four groups (MAGE-A4-TCR-T, IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T, and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T) were analyzed by flow cytometry for CD3 content. + / MAGE-A4 Pentamer + All showed high expression levels (56.99%, 52.70%, 54.11%, 51.69%). For example... Figure 4 As shown in A and B, SKBR3 HER2- / MAGE-A4- SKBR3 HER2+ / MAGE-A4- Two groups, due to MAGE-A4 / HLA-A * 02:01 expression was negative, and regardless of whether HER2 was negative or positive, there was no significant lysis of tumor target cells.

[0168] like Figure 5 As shown, in SKBR3 HER2- / MAGE-A4+ In all co-culture systems, only T cells from the MAGE-A4-TCR-T and IL10-MAGE-A4-TCR-T wells highly expressed CD137 (32.42–37.10%). In SKBR3... HER2+ / MAGE-A4+ In the co-culture systems of each group, except for the NC-T group, T cells in the other four groups (MAGE-A4-TCR-T, IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T, and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T) highly expressed CD137 (29.93–40.82%). Specifically, the proportion of CD137 in the IL10-MAGE-A4-TCR-T group was significantly higher than that in the MAGE-A4-TCR-T group; the proportion of CD137 in the AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T group was also significantly higher than that in the AntiHER2-SNIPRs-MAGE-A4-TCR-T group. SKBR3 HER2- / MAGE-A4- SKBR3 HER2+ / MAGE-A4- Two groups, due to MAGE-A4 / HLA-A * 02:01 Negative expression, regardless of whether HER2 is negative or positive, CD137 is not expressed or is expressed at a very low rate.

[0169] like Figure 6 As shown, in SKBR3HER2- / MAGE-A4+ In the co-culture systems of each group, only the MAGE-A4-TCR-T and IL10-MAGE-A4-TCR-T wells showed high concentrations of IFNγ (4891.56~5645.00 pg / mL) and TNFα (1841.70~2639.08 pg / mL) secretion. The AntiHER2-SNIPRs-MAGE-A4-TCR-T and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T groups showed extremely low secretion. In SKBR3... HER2+ / MAGE-A4+ In the co-culture systems of the various groups, except for the NC-T group, the other four groups—MAGE-A4-TCR-T, IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T, and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T—all exhibited high concentrations of IFNγ (2454.91–6643.73 pg / mL) and TNFα (1034.80–2378.30 pg / mL). Among them, the IFNγ and TNFα levels in the AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T group were significantly higher than those in the AntiHER2-SNIPRs-MAGE-A4-TCR-T group, indicating that IL10 enhances the secretion of IFNγ and TNFα by the AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T group. SKBR3 HER2- / MAGE-A4- SKBR3 HER2+ / MAGE-A4- Two groups, due to MAGE-A4 / HLA-A * 02:01 expression was negative, regardless of whether HER2 was negative or positive, and the secretion of IFNγ and TNFα was extremely low. Cytokine secretion levels were consistent with the tumor lysis trend.

[0170] The above results indicate that AntiHER2-SNIPRs have the function of controlling MAGE-A4-TCR expression in both AntiHER2-SNIPRs-MAGE-A4-TCR-T and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T. Only when target cells simultaneously express HER2 and MAGE-A4 / HLA-A... *MAGE-A4-TCR is only expressed at a gene expression level of 02:01, enabling it to recognize and specifically kill tumor target cells. Compared to MAGE-A4-TCR-T and IL10-MAGE-A4-TCR-T, AntiHER2-SNIPRs-MAGE-A4-TCR-T and AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T show significantly higher safety. Furthermore, AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T exhibits a higher tumor lysis rate than AntiHER2-SNIPRs-MAGE-A4-TCR-T, indicating that IL10 enhances the tumor lysis ability of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T. Overall, AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T demonstrates both higher tumor-killing safety and efficacy.

[0171] Example 6: Verification of the in vivo tumor-specific recognition and killing ability of AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells.

[0172] In the in vivo safety assay for AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T, approximately 8-week-old NSG mice were selected and divided into two groups of three mice each. Two xenograft tumors, SKBR3 and SKBR3, were implanted subcutaneously into the left and right posterior abdominal regions, respectively. HER2 - / MAGE-A4+ SKBR3 HER2+ / MAGE-A4- Tumor cells, or SKBR3 HER2- / MAGE-A4+ SKBR3 HER2+ / MAGE-A4+ Tumor cells, the number of tumor cells is 4E6, such as Figure 7 As shown in Figure A. Six days after tumor implantation, 3E6 AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T cells were injected intravenously. Tumor size was measured in all mice using calipers and recorded every 5 days. Tumor volume was calculated using Excel as follows: Tumor volume = Tumor length × (Tumor width^2) × 0.5. Mice were culled at each endpoint, and tissue and blood were harvested. Results are as follows. Figure 7 As shown in B and C: SKBR3 HER2- / MAGE-A4+ and SKBR3 HER2+ / MAGE-A4- The tumors on both sides were similar in size (1733 mm3 ± 119.9 mm and 2021 mm3 ± 103.4 mm). Compared to SKBR3... HER2- / MAGE-A4+ and SKBR3 HER2+ / MAGE-A4- Tumors on the side, AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T only target SKBR3 HER2+ / MAGE-A4+The tumor on one side showed a significant growth inhibition effect (321.3 mm3 ± 49.17 mm).

[0173] The above results indicate that, in mice, only tumors simultaneously express HER2 and MAGE-A4 / HLA-A. * 02:01 gene timing (SKBR3) HER2+ / MAGE-A4+ Only AntiHER2-SNIPRs-IL10-MAGE-A4-TCR-T can recognize and inhibit tumor growth, which is consistent with the trend of in vitro cell experiments.

[0174] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A T cell receptor that specifically binds to the MAGE-A4 antigen, characterized in that, comprises: a TCRa chain variable domain and a TCRP chain variable domain; (1) the amino acid sequences of CDR1, CDR2 and CDR3 of the TCRa chain variable domain are as set forth in SEQ ID NO: 1, as set forth in SEQ ID NO: 2 and as set forth in SEQ ID NO: 3, respectively; and (2) the amino acid sequences of CDR1, CDR2 and CDR3 of the TCRP chain variable domain are as set forth in SEQ ID NO: 4, as set forth in SEQ ID NO: 5 and as set forth in SEQ ID NO: 6, respectively.

2. The T cell receptor of claim 1, wherein (3) the TCRa chain variable domain has an amino acid sequence as set forth in SEQ ID NO: 30 and the TCRP chain variable domain has an amino acid sequence as set forth in SEQ ID NO: 31; or (4) an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of the amino acid sequence of (3) and an amino acid sequence that is functionally identical or similar to the amino acid sequence of (3); or (5) an amino acid sequence that is at least 80% homologous to the sequence of (3) or (4).

3. A nucleic acid molecule encoding the T cell receptor of claim 2.

4. The nucleic acid molecule of claim 3, wherein, the sequence of the nucleic acid molecule encoding the TCRa chain variable domain is as set forth in SEQ ID NO: 32; and the sequence of the nucleic acid molecule encoding the TCRP chain variable domain is as set forth in SEQ ID NO:

33.

5. A fusion protein characterized in that, comprises: the T cell receptor of claim 1 or 2 and IL10.

6. A nucleic acid molecule encoding the fusion protein of claim 5.

7. Set of expression vectors, characterized in that, comprises: the nucleic acid molecule of claim 6 and AntiHER2-SNIPRs; the nucleotide sequence of the AntiHER2-SNIPRs is as set forth in SEQ ID NO:

56.

8. A TCR-T cell characterized in that, a lentivirus transfected with the expression vector set of claim 7.

9. Use of the T cell receptor of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the fusion protein of claim 5, the nucleic acid molecule of claim 6, the expression vector set of claim 7 and / or the TCR-T cell of claim 8 in the manufacture of a medicament for treating a tumor; the tumor is selected from one or more of synovial sarcoma, myxoid / round cell liposarcoma, non-small cell lung cancer, head and neck squamous cell carcinoma, ovarian cancer, urothelial cancer, melanoma and gastric cancer.

10. A medicament or a composition thereof, characterized by, comprises: the T cell receptor of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the fusion protein of claim 5, the nucleic acid molecule of claim 6, the expression vector set of claim 7 and / or the TCR-T cell of claim 8 and a pharmaceutically acceptable adjuvant.

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