Targeting MAGEA1 TCR and application thereof

Abstract

The invention discloses a TCR (T cell receptor) targeting to MAGEA1 and application of the TCR. The invention provides a brand new TCR sequence of a high-affinity and high-specificity targeting MAGEA1 antigen, which is beneficial to development of drugs targeting MAGEA1 positive tumors, improves the treatment effect and safety of TCR-T cell therapy, and provides an innovative sequence resource for clinical transformation in the field of tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a TCR targeting MAGEA1 and its application. Background Technology

[0002] T-cell receptors (TCRs) are molecules on the surface of T cells that specifically recognize antigens. 90%-95% of mature peripheral blood T-cell TCRs consist of α and β chains and recognize antigenic peptides presented by the major histocompatibility complex (MHC) on the surface of target cells through their variable regions, thereby activating T cells to generate an immune response. TCR-T-cell therapy involves introducing TCR-encoding genes that target tumor antigens, obtained in vitro, into the patient's T cells through genetic engineering. This endows T cells with the ability to specifically recognize and kill tumor cells, representing a highly promising immunotherapy strategy.

[0003] Melanoma-associated antigen 1 (MAGEA1) belongs to the cancer-testis antigen family and is normally expressed in normal testicular tissue and various tumor tissues, but not in most normal tissues. For example, it is expressed in various malignant tumors such as melanoma, lung cancer, gastric cancer, and esophageal cancer. Studies have shown that aberrant expression of MAGEA1 is closely related to tumor proliferation, invasion, and metastasis, making it a highly promising target for tumor immunotherapy.

[0004] However, current TCR-T cell therapies targeting MAGEA1 still face many challenges in clinical translation, and more TCR resources are urgently needed for development and optimization. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a TCR targeting MAGE-A1 and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a TCR targeting MAGEA1, the TCR comprising an α chain and a β chain, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain are shown in SEQ ID NO:1-3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain are shown in SEQ ID NO:4-6, respectively.

[0007] Furthermore, the variable region of the α chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and the variable region of the β chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0008] Furthermore, the amino acid sequence of the variable region of the α chain of the TCR is shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the β chain of the TCR is shown in SEQ ID NO:8.

[0009] Furthermore, the constant region of the α chain of the TCR has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:9, and the constant region of the β chain of the TCR has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:10.

[0010] Furthermore, the amino acid sequence of the constant region of the α chain of the TCR is shown in SEQ ID NO:9, and the amino acid sequence of the constant region of the β chain of the TCR is shown in SEQ ID NO:10.

[0011] Furthermore, the connection method of the α chain and β chain of the TCR includes direct connection or indirect connection.

[0012] Furthermore, the α chain and β chain of the TCR are connected indirectly.

[0013] Furthermore, indirect connections are made through self-cutting sequences.

[0014] Furthermore, the connection order of the α chain and β chain can be the α chain... Self-cutting sequence β chain, β chain Self-cutting sequence Alpha chain.

[0015] Furthermore, the self-cutting sequence is selected from the P2A sequence.

[0016] A second aspect of the invention provides a nucleic acid encoding the TCR described in the first aspect of the invention.

[0017] A third aspect of the present invention provides a vector comprising the nucleic acid described in the second aspect of the present invention.

[0018] A fourth aspect of the present invention provides an engineered cell that expresses the TCR described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, or the vector described in the third aspect of the present invention.

[0019] Furthermore, the cells include T cells, NK cells, or stem cells.

[0020] A fifth aspect of the present invention provides a TCR conjugate comprising the TCR described in the first aspect of the present invention and other bioactive molecules.

[0021] Furthermore, the other bioactive molecules include antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, and detectable markers.

[0022] A sixth aspect of the present invention provides a multivalent TCR complex comprising the TCR described in the first aspect of the present invention.

[0023] A seventh aspect of the present invention provides a medicament comprising the TCR described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, the engineered cell described in the fourth aspect of the present invention, the TCR conjugate described in the fifth aspect of the present invention, or the multivalent TCR complex described in the sixth aspect of the present invention.

[0024] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0025] The eighth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the engineered cell described in the fourth aspect of the present invention, the TCR conjugate described in the fifth aspect of the present invention, or the multivalent TCR complex described in the sixth aspect of the present invention in the preparation of a medicament for treating MAGE-related diseases.

[0026] Furthermore, the MAGE-related diseases include MAGE-related tumors and MAGE-related autoimmune diseases.

[0027] Furthermore, the MAGE-related diseases are selected from MAGE-related tumors.

[0028] Furthermore, the MAGE-related tumors include melanoma, liver cancer, lung cancer, gastric cancer, esophageal cancer, leukemia, breast cancer, ovarian cancer, colon cancer, and head and neck squamous cell carcinoma.

[0029] Furthermore, the MAGE-related autoimmune diseases include lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome.

[0030] The ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the engineered cell described in the fourth aspect of the present invention, the TCR conjugate described in the fifth aspect of the present invention, or the multivalent TCR complex described in the sixth aspect of the present invention in antigen detection or in the preparation of antigen detection products.

[0031] Furthermore, the antigen is selected from MAGE.

[0032] Furthermore, the MAGE is MAGEA1.

[0033] Furthermore, the antigen detection is for non-diagnostic purposes.

[0034] Advantages and beneficial effects of the present invention: This application provides a novel TCR sequence with high affinity and high specificity targeting the MAGEA1 antigen, which will help in the development of drugs targeting MAGEA1-positive tumors, improve the therapeutic efficacy and safety of TCR-T cell therapy, and provide innovative sequence resources for clinical translation in the field of tumor immunotherapy. Attached Figure Description

[0035] Figure 1 This is a diagram showing the results of co-incubating DC cells loaded with the MAGEA1 antigen peptide (KVLEYVIKV) with T cells to induce the generation of MAGEA1-specific T cells. Figure 2 This is a schematic diagram of the structure of the TCR synthesis plasmid; Figure 3 This is a flow cytometry result of pMHC tetramer staining 24 hours after peripheral blood T cells activated by different MAGEA1 TCR mRNA electrotransduction. Figure 4 The results show the relative expression of the T cell activation marker 4-1BB and the statistical graph of EC50 values ​​after T cells from the same healthy donor were electrotransduced with different MAGEA1 TCR mRNAs and co-incubated overnight with T2 cells loaded with different concentration gradient antigen peptides. Figure 5 This is a flow cytometry result of pMHC tetramer staining after healthy donor T cells were transduced into G54-18 TCRs via lentivirus. Figure 6 This is a diagram showing the response of G54-18 to antigen-positive tumor cell lines; Figure 7 These are images showing the killing effect of G54-18 on tumor cell lines with different antigen expression levels. Among them, 7A is the killing effect on the A375 cell line, 7B is the killing effect on the NCI H1703 cell line, and 7C is the killing effect on the NCI H1299 (HLA-A*02:01) cell line. Figure 8 This is a diagram showing the results of verifying the G54-18 recognition motif using alanine substitution scanning. Figure 9 This is a schematic diagram of the structure of an HLA expression vector; Figure 10 It is an extended identification diagram of the HLA-A*02 subtype for G54-18; Figure 11 This is a diagram showing the recognition results of G54-18 on EBV-transformed B lymphoblast cell lines expressing multiple HLA alleles; Figure 12 These are images showing the anti-tumor effects of G54-18 in animal models. 12A is the tumor volume image, and 12B is the tumor weight image. Detailed Implementation

[0036] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] The present invention provides a TCR targeting MAGEA1, the TCR comprising an α chain and a β chain, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain are shown in SEQ ID NO:1-3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain are shown in SEQ ID NO:4-6, respectively.

[0038] In some implementations, the T cell receptor (TCR) is a protein on the surface of T cells responsible for specifically recognizing antigenic peptides that bind to the MHC (major histocompatibility complex). When a TCR binds to an antigenic peptide and MHC, T lymphocytes are activated through signal transduction and enter the subsequent immune response. There are four TCR genes in the human genome: two encoding light chain TCRs: the TRA gene encodes TCRα, and the TRG gene encodes TCRγ; and two encoding heavy chain TCRs: the TRB gene encodes TCRβ, and the TRD gene encodes TCRδ. Heavy chain TCRs and light chain TCRs form heterodimers to constitute the complete TCR. Two types of TCRs exist in humans: TCRα / β and TCRγ / δ, with 95% of T cells expressing TCRα / β, referred to as αβ T cells; and 5% of T cells expressing TCRγ / δ, referred to as γ / δ T cells.

[0039] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain further include substitutions, deletions, or insertions of one or more amino acids in the amino acid sequences shown in SEQ ID NO: 1-3; the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain further include substitutions, deletions, or insertions of one or more amino acids in the amino acid sequences shown in SEQ ID NO: 4-6. The substitution, deletion, or insertion of one or more amino acids may be, for example, the substitution, deletion, or insertion of one, two, or three amino acids, and such substitution, deletion, or insertion does not affect the function of the amino acid.

[0040] The variable region of the α chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and the variable region of the β chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0041] In some embodiments, 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%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0042] This invention provides a nucleic acid encoding the above-mentioned TCR.

[0043] In some embodiments, nucleic acid or polynucleotide refers to a nucleotide chain of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0044] The present invention provides a vector comprising the above-mentioned nucleic acid.

[0045] In some embodiments, a vector represents a construct 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. A vector may contain a nucleic acid sequence, such as a replication initiation region, that allows the gene or sequence of interest to replicate in the host cell. The vector may also contain one or more optional marker genes and other genetic elements known to those skilled in the art. Preferably, the vector is an expression vector containing a nucleic acid according to this application, said nucleic acid being effectively linked to a sequence that allows said nucleic acid expression.

[0046] The present invention provides an engineered cell that expresses the above-mentioned TCR, the above-mentioned nucleic acid, or the above-mentioned vector.

[0047] In some embodiments, engineered cells refer to cells in which exogenous nucleic acids have been introduced, including the progeny of these cells. Engineered cells include "transfected cells," which include primary transfected cells and their progeny, regardless of passage number. Progeny cells may not be identical to parent cells in terms of nucleic acid content, but may contain mutations. This application includes mutant progeny that have the same function or biological activity as cells screened or selected in the initially transfected cells.

[0048] In some embodiments, the cells are typically eukaryotic cells (such as mammalian cells) and are typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, and are cells of the immune system, such as cells of innate or adaptive immunity, such as bone marrow or lymphocytes, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from the subject and / or isolated from and frozen from the subject. In some embodiments, the cells include one or more subgroups of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and their subgroups, such as those defined by: function, activation state, maturity, potential for differentiation, expansion, recycling, localization and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion characteristics, and / or degree of differentiation. Regarding the subject to be treated, the cells may be allogeneic and / or autologous. In some respects, such as with existing technologies, cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from a subject, preparing, processing, culturing and / or engineering them as described in this application, and reintroducing them into the same patient before or after cryopreservation.

[0049] In some embodiments, among the subtypes and subsets of T cells and / or CD4+ and / or CD8+ T cells, there are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells), tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), α / β T cells, and δ / γ T cells. In some embodiments, the cells are regulatory T cells (Treg).

[0050] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0051] In some embodiments, the cell includes one or more nucleic acids introduced via genetic engineering, thereby expressing a recombinant or genetically engineered product of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not typically present in the cell or a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, for example, said nucleic acid is not typically found in the engineered cell and / or the organism from which such cells are derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature, including nucleic acids comprising a chimeric combination of nucleic acids encoding various domains from multiple different cell types.

[0052] In some embodiments, the preparation of engineered cells includes one or more culture and / or preparation steps. Cells used for engineering can be isolated from a sample, such as a biological sample, for example, one obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated is a subject suffering from a disease or condition, requiring cell therapy, or to whom cell therapy will be administered. In some embodiments, the subject is a person requiring a specific therapeutic intervention, such as adoptive cell therapy, where cells are isolated, processed, and / or engineered.

[0053] The present invention provides a TCR conjugate comprising the above-mentioned TCR and other bioactive molecules.

[0054] The other bioactive molecules include antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, and detectable markers.

[0055] In some embodiments, the antibody includes intact antibodies (e.g., IgG, IgM, or IgA) or fragments thereof (e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; single-chain antibody (e.g., scFv); single-domain antibody); and multispecific antibodies (e.g., bispecific antibodies).

[0056] In some embodiments, the cytokines include interleukins (e.g., IL-2), chemokines (e.g., MIP-1β), and growth factors (e.g., GCSF).

[0057] In some embodiments, the cytotoxic agent is, for example, doxorubicin; the radioactive material is, for example... 157 Gd, 55 Mn,162 Dy、 52 Cr and 56 Fe; the detectable markers include fluorescent markers (fluorescein isothiocyanate, Texas red, rhodamine, etc.); enzymes include horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA.

[0058] The present invention provides a multivalent TCR complex comprising the above-mentioned TCR.

[0059] In some embodiments, the multivalent TCR complex comprises a polymer of two, three, four, or more TCRs associated with each other (e.g., covalently or otherwise linked) by a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, and neutral avidin, each having four binding sites for biotin. Thus, biotinylated TCR molecules can form a polymer of TCRs having multiple TCR binding sites. The number of TCR molecules in the polymer will depend on the amount of TCR associated with the amount of linker molecules used to make the polymer, and also on the presence of any other biotinylated molecules. Preferred polymers are dimer, trimer, or tetramer TCR complexes.

[0060] In some embodiments, a label or additional portion (e.g., a toxic or therapeutic portion) may be included in the multivalent TCR complex of this application. For example, the label or additional portion may be included in a mixed molecular multimer. An example of such a multimer is a tetramer comprising three TCR molecules and one peroxidase molecule. This can be achieved by mixing the TCR and enzyme in a 3:1 molar ratio to generate a tetramer complex and separating the desired complex from any complex that does not contain the correct ratio of molecules. These mixed molecules may comprise any combination of molecules, provided that steric hindrance does not impair or significantly impair the desired function of the molecules. Due to the low likelihood of steric hindrance, the binding site on the streptavidin molecule is well-positioned for the mixed tetramer.

[0061] The present invention provides a drug comprising the above-mentioned TCR, the above-mentioned nucleic acid, the above-mentioned carrier, the above-mentioned engineered cells, the above-mentioned TCR conjugate, or the above-mentioned multivalent TCR complex.

[0062] The drug also includes pharmaceutically acceptable excipients.

[0063] In some embodiments, pharmaceutically acceptable excipients include all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption-retarding agents. The use of these media and solvents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent in a therapeutic composition is considered unless it is incompatible with the active ingredient.

[0064] Examples of pharmaceutically acceptable excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered yarrow; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa soybean oil; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as TWEENS; wetting agents such as sodium dodecyl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.

[0065] In some embodiments, the appropriate dosage of the drug can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate and responsiveness. Skilled physicians can usually easily determine the prescription and the desired dosage that is effective for treatment.

[0066] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0067] Example 1: Induction of MAGEA1-specific T cells Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of patients with HLA-A*02:01 esophageal cancer at a concentration of 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / well in 96-well plates and cultured in X-VIVO 15 medium containing 10% human serum, with the addition of 500-1000 IU / mL GM-CSF and 200-500 IU / mL IL-4. On day 2, 2 μg / mL antigen peptide was added to the mixed culture system for stimulation. On day 3, half the medium was replaced, and the cytokines in the medium were adjusted to 10-50 ng / mL IL-7, 10-50 ng / mL IL-15, and 10-50 IU / mL IL-2 to promote T cell proliferation. On day 6, antigen peptide stimulation was added again, and the culture continued until day 14. Flow cytometry was used to detect specific T cells in the samples. Figure 1 ).

[0068] MAGEA1-specific T cells were sorted into 96-well plates as single clones by flow cytometry. RNA was extracted and cDNA was obtained by reverse transcription. The TCRα and TCRβ genes were amplified by multiplex PCR, and high-frequency clones were selected for plasmid synthesis.

[0069] Example 2: Construction of in vitro transcription plasmid vector expressing MAGEA1-specific TCR and preparation of mRNA The α and β variable region sequences of the MAGEA1-specific TCR obtained by sequencing were fused with the mouse α and β constant regions, respectively. The TCR α and TCR β chains were linked by a P2A sequence, as shown in the schematic diagram below. Figure 2 As shown in Table 1, the TCRα / β gene, after codon optimization, was synthesized and digested with BamHI and SacI restriction endonucleases. It was then cloned into the in vitro transcription (IVT) plasmid vector between the BamHI and SacI restriction sites. A Kozak (GCCACC) sequence was added downstream of the BamHI restriction site, and a double-stop codon (TGATAA) sequence was added upstream of the SacI restriction site. After constructing the in vitro transcription plasmid vector, mRNA was synthesized and temporarily stored at -80°C for in vitro functional verification. The specific sequence of the TCR is shown in Table 1.

[0070] Table 1 MAGEA1 TCR Sequences

[0071] Example 3 Peptide sensitivity detection of MAGEA1-specific TCR Step 1: Preparation of T cells expressing TCR Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy donors (Miaoshun Biotechnology) were thawed and resuspended in X-VIVO15 medium. T cells were purified and isolated using magnetic beads and activated using CD3 / CD28 magnetic beads. Cells were resuspended in X-VIVO15 medium containing 2.5% human serum and 10–50 IU / mL IL-2 and cultured for 3 days. On day 3, the magnetic beads were removed, and culture continued. On day 4, cells were collected, centrifuged at 350g for 5 minutes, washed once with DPBS, and resuspended in R solution at a density of 2 × 10⁶ cells / mL. 7At a concentration of / mL, 100μL of cell suspension was added to a 1.5mL EP tube, along with 5μg of TCR mRNA, and mixed thoroughly. 3mL of E2 solution was added to the electroporation cuvette, which was then placed in the electroporator's reservoir. Using a 100μL electroporator tip, the cell suspension containing mRNA was carefully aspirated (avoiding air bubbles). The electroporator was inserted into the E2 solution in the electroporator. The electroporation parameters were set to 1400V, 10ms, and 3 pulses to complete the transduction of TCR mRNA into activated T cells. Twenty-four hours after electroporation, T cells were stained with MAGEA1 tetramer, and the transduction results were analyzed by flow cytometry. Figure 3 Among them, G54-18 had the highest positive rate after TCR transduction, CD3 + The proportion of Tetramer-positive cells in T cells reached 45.42%.

[0072] Step 2: Prepare T2 cells T2 cells lack antigen processing-associated transporters (TAPs), preventing them from transporting endogenous peptides to MHC loading sites. Furthermore, their HLA type is HLA-A*02:01, making them ideal target cells for TCR peptide sensitivity validation. In the experiment, cultured T2 cells were collected, washed, and resuspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and then cultured at 1×10⁻⁶ ppm. 6 Cells / mL were seeded in 24-well plates. Then, a concentration gradient of 10-10 was added to each well. -12 M to 10 -6 The antigenic peptide of M was set up with 7 concentration gradient groups, and the antigenic peptide loading time was 4 hours.

[0073] Step 3: Results of peptide sensitivity testing Activated T cells expressing TCR were mixed with T2 cells loaded with different concentrations of antigenic peptides at a ratio of 1×10⁻⁶. 5 Cells were seeded at a mixed density in 96-well plates and incubated overnight at 37°C using a CO2 incubator. The following day, cells from each group were collected and stained with flow cytometry antibodies CD3-Violet786, CD8-BV510, CD4-APC-Cy7, and 4-1BB-BV421, and then analyzed. The CD8+ levels in each sample were then analyzed. + The expression level of the T cell activation marker 4-1BB was used to assess the activation effect of target cells loaded with different concentrations of antigenic peptides on T cells. The highest 4-1BB expression level in each TCR group was taken as 100%, and the relative activation levels of different antigenic peptide concentrations were analyzed. The peptide concentration required to achieve half of the maximum effect, i.e., EC50, was calculated. Figure 4The lower the EC50 value, the higher the sensitivity of the TCR to the epitope peptide. Experimental results showed that the EC50 values ​​of G54-12, G54-13, and G54-18 were 1.505 × 10⁻⁶. -8 M, 3.772×10 -9 M, 9.737×10 -10 Among the M, G54-18 has the smallest EC50 value, indicating that it has the best antigen sensitivity.

[0074] Example 4: Detection of the recognition and killing function of MAGEA1-specific TCR G54-18 on antigen-positive tumor cell lines Step 1: Prepare T cells stably expressing TCR via lentiviral transduction. Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors (Miaoshun Biotechnology) were thawed and resuspended in X-VIVO15 medium. T cells were purified and isolated using magnetic beads and activated using CD3 / CD28 magnetic beads for 3 days. Cas9 RNP complexes were introduced into the activated T cells via electroporation to knock out endogenous TCRs. Electroporation parameters were set to 1600V, 10ms, and 3 pulses. Five minutes after electroporation, 8 μg / mL polybrene and 200 μL of concentrated virus were added to the cell suspension, followed by incubation in a CO2 incubator. After overnight culture, the medium was changed to remove polybrene, and the cells were continuously cultured in X-VIVO 15 medium containing 10% FBS and 500 IU / mL IL-2. Five days after viral infection, cells were collected, and the expression of specific TCRs was detected by flow cytometry. The results showed that the proportion of MAGEA1-specific T cells in the sample reached 45.14%. Figure 5 The samples were then cultured on a larger scale to complete a series of functional verifications.

[0075] Step 2: Recognition of antigen-positive tumor cell lines by G54-18 TCR-T cells Multiple antigen-positive tumor cell lines were co-incubated with G54-18 TCR-T cells, including cell lines expressing MAGEA1 antigen but not of the HLA-A*02:01 type, and cell lines conforming to the HLA-A*02:01 type but not expressing MAGEA1 antigen as control groups, to determine the HLA restriction and antigen specificity of G54-18 TCR. Effector cells and target cells were added to 96-well plates at a 1:1 ratio, and after overnight incubation, the culture supernatant was collected. The secretion level of IFN-γ was detected by ELISA. G54-18 recognized naturally MAGEA1-positive tumor cell lines A375, NCI H1299 (HLA-A*02:01 overexpression), and NCI H1703 (…). Figure 6 ).

[0076] Step 3: Detection of the killing function of G54-18 TCR-T cells against antigen-positive tumor cell lines The killing function of G54-18 TCR-T cells against antigen-positive tumor cell lines was monitored using the Maestro Z cell non-invasive real-time monitoring system. Naturally MAGEA1-positive tumor cell lines A375, NCI H1299 (HLA-A*02:01 overexpressing), and NCIH1703 were seeded into 96-well impedance plates, with 1 × 10⁶ cells per well. 4 Tumor cells. 24 hours later, G54-18 TCR-T cells were added to each well, with an effector-to-target ratio of 4:1. The results showed that G54-18 TCR-T cells could specifically kill the three target cell types, with the strongest killing effect against NCI H1703. Figure 7 ).

[0077] Example 5: Recognition motif of MAGEA1-specific TCR G54-18 Alanine scanning is a routine method for identifying specific amino acid sites closely related to TCR function, stability, and conformation. Replacing amino acids at various positions on a peptide with alanine removes the active groups on the side chains and replaces them with small methyl groups without other functional groups, thus having minimal impact on protein structure and being able to distinguish the effect of a specific amino acid on TCR recognition. In the experiment, T2 cells were loaded with various mutant peptides (10... -5 M) was then co-incubated with G54-18 TCR-T cells from three different donors, and the secretion level of IFN-γ in each group was detected by enzyme-linked immunospot (ELISPOT) assay. The ELISPOT procedure was as follows: After washing both effector cells and target cells once, they were resuspended in serum-free ELISPOT medium and the cell density was adjusted to 5 × 10⁶ cells / year. 5 Effector cells and target cells were seeded at a 1:1 ratio into pre-washed ELISPOT-IFN-γ detection plates at a density of / mL. After overnight incubation, color development was performed, and the plates were then placed in a cool, dark place at room temperature to air dry. The immunospots were then imaged and read using an ELISPOT analyzer (CTL S6). The ELISPOT detection results and statistical results of G54-18 transduced T cells are presented below. Figure 8 As shown in the figure, its identification base sequence is -VLEY-IK-.

[0078] Example 6: HLA-A*02 subtype recognized by MAGEA1-specific TCR G54-18 To evaluate whether HLA-A*02 subtypes other than HLA-A*02:01 loaded with antigenic peptides could be recognized by T cells expressing G54-18, K562 cells overexpressing seven HLA-A*02 subtypes were constructed (plasmid maps are available in [link to plasmid map]). Figure 9 In the experiment, seven cell lines were loaded with saturated antigenic peptides for 4 hours, then washed and co-incubated with effector cells at a 1:1 effector-target ratio in 96-well plates. After 24 hours, the culture supernatant was collected and IFN-γ levels were detected by ELISA. The results showed that the potential of G54-18 to recognize MAGEA1 antigenic peptides was not only present on HLA-A*02:01, but also to varying degrees on HLA-A*02:04, HLA-A*02:05, and HLA-A*02:07. However, G54-18 also interacted with unloaded HLA-A*02:05 and HLA-A*02:07 molecules. Figure 10 Subsequent evaluation of the allogeneic reaction of G54-18 revealed that G54-18 did not recognize HLA-A*02:07 (this type is contained in B-LCL5, B-LCL16, B-LCL18, and B-LCL19). Therefore, G54-18's recognition of K562-HLA-A*02:07 in this experiment may be due to the recognition of endogenous presented peptides (K562 expresses MAGEA1).

[0079] Example 7: Allogeneic reaction of MAGEA1-specific TCR G54-18 To evaluate the allotropic response of G54-18, 21 EBV-transfected B lymphoblastic cell lines (B-LCLs) expressing multiple HLA alleles were constructed. T cells expressing G54-18 were co-incubated with B-LCL cells at a 1:1 effector-target ratio for 24 hours. The culture supernatant was collected, and IFN-γ levels were detected using ELISA. The results showed that G54-18 exhibited no allotropic response to any of the tested HLA types.

[0080] Example 8: In vivo antitumor effect of MAGEA1-specific TCR G54-18 G54-18 transduced TCR-T cells were administered via a single intravenous injection to evaluate their tumor-clearing effect in experimental animals. NCI H1703 cells were digested, washed twice with PBS to remove serum components, and resuspended in serum-free medium, adjusting the cell concentration to 1×10⁶. 7Cells / 100 μL. The matrix gel was slowly thawed on ice, and the cell suspension was mixed with the matrix gel at a 1:1 volume ratio, gently pipetting to avoid air bubbles. 100 μL of the mixture was subcutaneously injected into the axilla of 5-week-old NOG female mice. On day 7 post-injection, animals were randomly assigned to experimental groups based on tumor volume, and were injected via tail vein with 100 μL of PBS, untransduced T cells, or G54-18 TCR-transduced T cells, respectively. The number of cells injected was 1 × 10⁻⁶. 7 Each cell / animal. Tumor volume changes were continuously observed and recorded. G54-18TCR-T cells showed significant anti-tumor effects and good safety. Figure 12 ).

[0081] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A TCR targeting MAGEA1, characterized in that, The TCR comprises an α chain and a β chain, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain are shown in SEQ ID NO:1-3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain are shown in SEQ ID NO:4-6, respectively.

2. The TCR according to claim 1, characterized in that, The variable region of the α chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and the variable region of the β chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO:

8. Preferably, the amino acid sequence of the variable region of the α chain of the TCR is shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the β chain of the TCR is shown in SEQ ID NO:8; Preferably, the constant region of the α chain of the TCR has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:9, and the constant region of the β chain of the TCR has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:

10. Preferably, the amino acid sequence of the constant region of the α chain of the TCR is shown in SEQ ID NO:9, and the amino acid sequence of the constant region of the β chain of the TCR is shown in SEQ ID NO:10; Preferably, the connection method of the α chain and β chain of the TCR includes direct connection or indirect connection; Preferably, the α chain and β chain of the TCR are connected indirectly; Preferably, the connection is made indirectly through a self-cutting sequence; Preferably, the connection order of the α chain and the β chain can be α chain. Self-cutting sequence β chain, β chain Self-cutting sequence alpha chain; Preferably, the self-cutting sequence is selected from the P2A sequence.

3. The nucleic acid encoding the TCR of claim 1 or 2.

4. A vector comprising the nucleic acid of claim 3.

5. An engineered cell, characterized in that, The cells express the TCR of claim 1 or 2, the nucleic acid of claim 3, or the vector of claim 4; Preferably, the cells include T cells, NK cells, or stem cells.

6. A TCR conjugate, characterized in that, The TCR conjugate comprises the TCR as described in claim 1 or 2 and other bioactive molecules; Preferably, the other bioactive molecules include antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, and detectable markers.

7. A multivalent TCR complex, characterized in that, The multivalent TCR complex includes the TCR as described in claim 1 or 2.

8. A drug, characterized in that, The drug comprises the TCR of claim 1 or 2, the nucleic acid of claim 3, the carrier of claim 4, the engineered cell of claim 5, the TCR conjugate of claim 6, or the multivalent TCR complex of claim 7; Preferably, the drug further includes pharmaceutically acceptable excipients.

9. The use of the TCR of claim 1 or 2, the nucleic acid of claim 3, the vector of claim 4, the engineered cell of claim 5, the TCR conjugate of claim 6, or the multivalent TCR complex of claim 7 in the preparation of a medicament for treating MAGE-related diseases; Preferably, the MAGE-related disease is selected from MAGE-related tumors; Preferably, the MAGE-related tumors include melanoma, liver cancer, lung cancer, gastric cancer, esophageal cancer, leukemia, breast cancer, ovarian cancer, colon cancer, and head and neck squamous cell carcinoma.

10. The use of the TCR of claim 1 or 2, the nucleic acid of claim 3, the vector of claim 4, the engineered cell of claim 5, the TCR conjugate of claim 6, or the multivalent TCR complex of claim 7 in antigen detection or in the preparation of antigen detection products; Preferably, the antigen is selected from MAGE; Preferably, the MAGE is MAGEA1.

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