T cell receptor and application thereof

Abstract

The present invention relates to the field of cancer immunotherapy. In particular, the present application relates, in some aspects, to binding molecules, such as complexes capable of specifically binding a major histocompatibility complex (MHC) molecule and an AFP peptide. The present invention relates to T cell receptors (TCRs), including antigen binding domains thereof, that bind to or recognize such peptide epitopes. The present application further relates to engineered cells comprising such binding molecules, such as TCR double antibody proteins or TCRs, and the like, and their use in cancer immunotherapy.

Description

[0001] This application is a divisional application of patent CN119930796A, which was filed on 2025-04-10, with application number 202510445476.4 and invention titled "A T-cell receptor and its application". Technical Field

[0002] This invention belongs to the field of cancer immunotherapy, specifically relating to a T-cell receptor capable of specifically binding to a complex of major histocompatibility complex (MHC) molecules and AFP peptide, and its application. Background Technology

[0003] Immunotherapy enhances the function of a patient's immune system, using immune cells to eliminate cancer cells in the body, thereby achieving a cure for cancer. Cell therapy is an adoptive therapy that transfers T cells that specifically respond to target cell antigens into the patient, enabling them to act on target cells. The T cell receptor (TCR) is a membrane protein on the surface of T cells that recognizes corresponding antigenic peptides on the surface of target cells. In the immune system, through the binding of the antigen-peptide-specific TCR to the short peptide-major histocompatibility complex (pMHC complex), T cells directly contact antigen-presenting cells (APCs). Then, other cell membrane surface molecules of both T cells and APCs interact, triggering a series of subsequent signal transductions, allowing T cells with different antigen specificities to exert their immune effects on their target cells.

[0004] Engineered T cells containing tumor antigen-specific T-cell receptors (TCRs) can overcome some of the challenges currently faced by follow-up lymphocyte therapy because they can rapidly generate tumor-reactive T lymphocytes with defined antigen specificity. There is a need in this field for TCRs that target different tumor antigens and provide high in vivo anti-tumor efficacy and low off-target effects. Common problems with TCR-T cell therapy include serious adverse events in clinical trials (such as central nervous system toxicity), which may be related to inappropriate target selection (so-called on-target / off-tumor effects) and biased expansion of the T-cell population.

[0005] Alpha-fetoprotein (AFP) is a highly specific protein expressed by liver cancer cells. Many liver cancer patients (70-80%) have high AFP gene expression during the course of the disease. AFP is a germinal protein whose gene is opened and expressed during fetal development, but is basically shut down after two years of age. However, when liver cancer occurs in adults or when the liver regenerates benignly, the AFP gene is reactivated and expressed in large quantities. Clinically, it is considered a classic tumor marker for liver cancer and is therefore regarded as the gold standard for diagnosing liver cancer.

[0006] Human AFP is an alpha globulin, exhibiting electrophoretic movement between albumin and globulin, with a molecular weight of approximately 64,000–72,000 and a precipitation coefficient of 4.5. This protein is composed of about 18 amino acids, with carbohydrates comprising about 4%. Fetal AFP shows uniform electrophoretic movement, while tumor-derived AFP contains four variants or subcomponents, exhibiting slight differences in electrophoretic movement. These differences primarily lie in the carbohydrate content rather than alterations in the peptide chain.

[0007] AFP-specific TCRs and TCR-Ts therefore hold promise as cancer therapies with high anti-tumor efficacy and low off-target effects. Summary of the Invention

[0008] To achieve the above objectives, the first aspect of the present invention discloses a method for identifying HLA-A-restricted AFP. 158-166 T-cell receptors for epitope peptides, comprising α and β chains. The α and β chains contain the following complementarity-determining regions: 1) The amino acid sequence of CDR1 on the α chain is shown in one of SEQ ID NO: 7-12, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 3. The amino acid sequence of CDR1 on the β chain is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 5, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6; or 2) The amino acid sequence of CDR1 on the α chain is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 3. The amino acid sequence of CDR1 on the β chain is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6; or 3) The amino acid sequence of CDR1 on the α chain is shown in SEQ ID NO: 1 or 7, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO: 13. The amino acid sequence of CDR1 on the β chain is shown in one of SEQ ID NO: 4, 15-17, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 5, 14 or 18, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO: 6.

[0009] This invention should also protect polypeptide or protein molecules that have a function similar to the T-cell receptor of this invention, obtained by substitution and / or deletion and / or addition of one or more amino acid residues; or molecules that have more than 90% sequence similarity and recognize HLA-A-restricted AFP. 158-166 Proteins related to the function of epitope peptides.

[0010] The T cell receptor recognizes HLA-A-restricted AFP. 158-166 Epitope peptide, the amino acid sequence of which is shown in SEQ ID NO:19, is as follows: FMNKFIYEI (SEQ ID NO:19).

[0011] Preferably, the HLA typing includes: HLA-A*0203, HLA-A*0201, HLA-A*0206 or HLA-A*0207.

[0012] The second aspect of the present invention discloses a nucleic acid molecule encoding the above-mentioned T cell receptor.

[0013] In this invention, it is well known to those skilled in the art that, due to the degeneracy of protein genetic codons, there is usually more than one codon determining an amino acid. The substitution of the third nucleotide in a triplet codon often does not change the amino acid composition; therefore, genes encoding the same protein can have different nucleotide sequences. Based on known codon tables, those skilled in the art can deduce the nucleotide sequences of genes encoding these proteins from the amino acid sequences disclosed in this invention. These nucleotide sequences can be obtained through biological methods (such as PCR or mutation methods) or chemical synthesis methods. Therefore, all such nucleotide sequences should be included within the scope of protection of this invention.

[0014] A third aspect of the present invention discloses an engineered immune cell that expresses the aforementioned T cell receptor.

[0015] The T cell receptor is introduced into immune cells via viral or non-viral vector transduction, CRISPR / Cas9 system, transposon system or mRNA transfection to obtain engineered immune cells.

[0016] The viral vector transduction can be performed using retroviruses or lentiviruses to transfer the TCR gene into T cells.

[0017] The non-viral vector transduction includes electroporation or nanoparticle technology to introduce the TCR gene into T cells. The engineered immune cells are derived from NK cells or T cells.

[0018] The fourth aspect of this invention discloses a TCR bispecific antibody protein, which is composed of an antibody targeting receptor molecules on the cell membrane surface of immune cells and the extracellular domain of the aforementioned T cell receptor, and simultaneously binds to immune cells and tumor cells; the immune cells include T cells or NK cells; the antibody targeting receptor molecules on the cell membrane surface of immune cells includes scFv or Fab antibody fragments.

[0019] The preferred receptor molecules on the surface of the immune cell membrane are CD3, CD4, CD8, CD16a and / or NKG2D.

[0020] The extracellular region of the T cell receptor recognizes and binds to antigens on tumor cells.

[0021] The fifth aspect of this invention discloses a TCR bispecific antibody mRNA formulation, comprising the aforementioned TCR bispecific antibody protein, specifically expressed in vivo using mRNA technology.

[0022] The sixth aspect of this invention discloses the use of the above-mentioned T cell receptor, nucleic acid molecule, engineered immune cell, TCR bispecific antibody protein or TCR bispecific antibody mRNA preparation in the preparation of a medicament for the prevention and / or treatment of diseases or conditions expressing AFP.

[0023] Diseases or conditions that express AFP include liver cancer, germ cell tumors, hepatitis, and stomach cancer.

[0024] The drug also includes TCR protein drugs; The TCR protein drug is prepared from a multispecific T cell complex, which is prepared from the aforementioned T cell receptor. The multispecific T cell complex includes TCR bispecific antibodies, TCR trispecific antibodies, and TCR tetraspecific antibodies; And multispecific T-cell complexes consisting of T-cell receptors, TCR bispecific antibodies, TCR trispecific antibodies or TCR tetraspecific antibodies, linked to radiopharmaceuticals, toxins, fluorescein and antibody fragments. Attached Figure Description

[0025] The following embodiments are intended purely as examples of this application and should not be construed as limiting the invention in any way. The following embodiments and detailed descriptions are provided by way of illustration rather than limitation.

[0026] Figure 1 HLA restriction assays of T cells expressing the AFP antigen-specific T cell receptor AM14-M1 in the K562 cell line, and in vitro toxicity assays of HepG2.

[0027] Figure 2 HLA restriction assays of T cells expressing the AFP antigen-specific T cell receptor AM14-M2 in the K562 cell line, and in vitro toxicity assays of HepG2.

[0028] Figure 3 HLA restriction assays of T cells expressing the AFP antigen-specific T cell receptor AM14-M3 in the K562 cell line, and in vitro toxicity assays of HepG2.

[0029] Figure 4 HLA restriction assays of T cells expressing the AFP antigen-specific T cell receptor AM14-M4 in the K562 cell line, and in vitro toxicity assays of HepG2.

[0030] Figure 5 HLA restriction assays of T cells expressing the AFP antigen-specific T cell receptor AM14-M5 in the K562 cell line, and in vitro toxicity assays of HepG2.

[0031] Figure 6 HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor AM14-M6 in the K562 cell line.

[0032] Figure 7 HLA restriction assay of T cells expressing the AFP antigen-specific T cell receptor AM14-M7 in the K562 cell line.

[0033] Figure 8 HLA restriction assays of T cells expressing the AFP antigen-specific T cell receptor AM14-M8 in the K562 cell line, and in vitro HepG2 toxicity assays. Figure 9 The AFP-TCR bispecific antibody protein AM14-M7 efficiently induces T cells to recognize the AFP-HLA-A*02:03 complex on the target cell membrane and activates T cells to secrete IFN-γ.

[0034] Figure 10The AFP-TCR bispecific antibody protein AM14-M10-13 efficiently induces T cells to recognize the AFP-HLA-A*02:03 complex on the target cell membrane and activates T cells to express Luciferase.

[0035] Figure 11 The AFP-TCR bispecific antibody protein AM14-M14-18 efficiently induces T cells to recognize the AFP-HLA-A*02:03 complex on the target cell membrane and activates T cells to express Luciferase. Detailed Implementation

[0036] The following embodiments are intended purely as examples of this application and should not be construed as limiting the invention in any way. The following embodiments and detailed descriptions are provided by way of illustration rather than limitation.

[0037] Example 1: Preparation of AFP-specific T cells TCR mRNA was prepared, and CD3-T cells activated by electroporation were generated to prepare TCR-T cells. On day one, PBMCs were resuscitated, and incubated at 4°C for 5 minutes with pan-T sorting magnetic beads, followed by incubation at 4°C for 10 minutes with secondary antibody. The cell suspension was then added to a sorting column, and CD3-T cells were sorted using a magnetic field. The sorted CD3-T cells were activated using T cell transact (10µL / 1E6 cells), and the cell density was adjusted to 1×10⁶ cells using AIM-V medium. 6 Cells / mL. On day 7, activated CD3-T cells were collected, centrifuged at 350g for 5 minutes, the supernatant was removed, and fresh culture medium was added. Cells were counted. Cells were washed with PBS, the supernatant was removed, and electroporation buffer (1E6 cells / 20µL electroporation buffer) was added to resuspend the cells. 20µL of cell suspension was added to EP tubes for a total of 8 samples. 2µL of TCR mRNA (AM14-M1, AM14-M2, AM14-M3, AM14-M4, AM14-M5, AM14-M6, AM14-M7, and AM14-M8 mRNA) was added to each sample, mixed well, and added to the 20µL electroporation cuvette. The cuvette was gently tapped to ensure that the sample covered the bottom. The cuvette was then placed in the electroporator and electroporation was performed according to the set program.

[0038] Wild-type AM14 includes: α chain, CDR1:VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) β-chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0039] AM14-M1 includes: α-chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) β-chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0040] AM14-M2 includes: α-chain, CDR1: SSMLRG (SEQ ID NO: 8) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) β-chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0041] AM14-M3 includes: α-chain, CDR1: STLLRG (SEQ ID NO: 9) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) β-chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0042] AM14-M4 includes: Alpha chain, CDR1: SSLLRG (SEQ ID NO: 10) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0043] AM14-M5 includes: Alpha chain, CDR1: HSLLRG (SEQ ID NO: 11) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0044] AM14-M6 includes: Alpha chain, CDR1: VSLLRG (SEQ ID NO: 12) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQ ID NO: 3) Beta chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0045] AM14-M7 includes: Alpha chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0046] AM14-M8 includes: α chain, CDR1:VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSNSGYALNF (SEQID NO: 3) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2:FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0047] Example 2: Validation of AFP-specific T cell receptor AM14-M1 TCR Expression and Peptide Recognition Assay K562 cells carrying AFP were used with HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07 genotypes respectively. 158-166 The peptides were used to stimulate T cells transfected with T cell receptors in an HLA restriction assay, which was measured using flow cytometry and human IFN-γ ELISA.

[0048] TCR-transduced T cells were collected and washed with 2 mL of PBS, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The TCR-transduced T cells were then resuspended in AIM-V medium (containing 5% AB serum) and adjusted to a concentration of 1 × 10⁶ cells / mL. 6 Cells / mL, 100µL added to 96-well plates. AFP-loaded cells of the same density and number were then added. 158-166 The target cells of the peptide were mixed with tumor-specific T cells, and brefeldin A (final concentration 3 µg / mL) was added to the wells and cultured for 4 hours. After 4 hours, the cells were collected and used for intracellular IFN-γ, surface CD3 and TCR expression staining, which was detected by flow cytometry.

[0049] For ELISA detection of IFN-γ, 100 µL / well loaded with AFP was used. 158-166 K562 cells containing polypeptides (1×10) 6100 µL / well of TCR-transduced T cells (1×10⁻⁶ cells / mL) and 100 µL / well of TCR-transduced T cells (1×10⁻⁶ cells / mL). 6 The mixture (cells / mL) was added to 96-well plates and incubated for 24 hours. After 24 hours, 175 µL of supernatant was collected from each well of the 96-well plate and used for IFN-γ ELISA detection using the IFN-γ ELISA HRP kit. On Day 1, the high protein binding ELISA plate was plated with antibody 1-D1K (IFN-γ ELISA HRP kit) diluted to 2 µg / mL in PBS (pH 7.4) (by adding 100 µL / well) and incubated overnight at 4°C. On Day 2, the plate was washed twice with PBS (200 µL / well). The plate was then blocked by adding 200 µL / well of culture medium and incubated at room temperature (RT) for 1 hour. Prepare human IFN-γ standards (IFN-γ ELISA HRP kit) to a concentration of 0.5 µg / mL in 2 mL PBS (containing 1% BSA) and incubate at RT for 15 minutes, then vortex the tubes. Dilute 100 µL / well of sample or standard in culture medium and incubate at RT for 2 hours. Test samples and standard probes in duplicate. Wash the plate five times with PBS containing 0.05% Tween 20. Add 100 µL / well of antibody 7-B6-1-biotin (IFN-γ ELISA HRP kit) at 1 µg / mL (in PBS), incubate at RT for 1 hour, and wash. Add 100 µL / well of streptavidin-HRP (IFN-γ ELISA HRP kit) diluted 1:1000 in PBS, incubate at RT for 1 hour, and wash. Add 100 µL / well of TMB acceptor solution and incubate in the dark at RT for 15 to 30 minutes, until the solution in the wells turns a visible blue color. Add 100 µL / well of stop solution to terminate the enzyme reaction. The solution color changes from blue to yellow. Measure the light intensity at 450 nm using an ELISA reader.

[0050] like Figure 1 As shown in Figures A and B, T cells transfected with AM14-M1 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptides.

[0051] In vitro toxicity assay T cells transfected with the T-cell receptor AM14-M1 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were HepG2 cells naturally expressing HLA-A*02:01 and AFP proteins. Untransfected T-cell receptor T cells were mixed with target cells at the same ratio as the control group. The killing power of effector cells against target cells in both the experimental and control groups was measured continuously for 20 hours. After 20 hours, the killing plate was removed, centrifuged at 500g, and 175 µL of supernatant was collected from each well. This supernatant was then used for IFN-γ ELISA detection using the IFN-γ ELISA HRP kit.

[0052] like Figure 1 As shown in C and D, T cells transfected with AM14-M1 significantly killed HepG2 target cells that naturally expressed AFP protein and secreted IFN-γ, resulting in the death of target cells. The effector-to-target ratio (E:T) was 5:1. In the control group, untransfected T cells had no killing effect on target cells.

[0053] Example 3: Validation of AFP-specific T cell receptor AM14-M2-8 The AFP-specific T-cell receptor AM14-M2-8 was analyzed in TCR expression and peptide recognition, HLA restriction assays, in vitro toxicity assays, and IFN-γ ELISA detection. The procedure was the same as in Example 2, and the results are as follows: Figure 2-8 As shown.

[0054] Figure 2 A and B indicate that T cells transfected with AM14-M2 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptide; Figure 2 C and D indicate that T cells transfected with AM14-M2 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-to-target ratio (E:T) is 5:1.

[0055] Figure 3 A and B indicate that T cells transfected with AM14-M3 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptide; Figure 3 C and D indicate that T cells transfected with AM14-M3 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-to-target ratio (E:T) is 5:1.

[0056] Figure 4A and B indicate that T cells transfected with AM14-M4 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptide; Figure 4 C and D indicate that T cells transfected with AM14-M4 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-to-target ratio (E:T) is 5:1.

[0057] Figure 5 A and B indicate that T cells transfected with AM14-M5 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptide; Figure 5 C and D indicate that T cells transfected with AM14-M5 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-to-target ratio (E:T) is 5:1.

[0058] Figure 6 A and B indicate that T cells transfected with AM14-M6 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptides.

[0059] Figure 7 A and B indicate that T cells transfected with AM14-M7 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptides.

[0060] Figure 8 A and B indicate that T cells transfected with AM14-M8 can recognize AFP presented by HLA-A*02:03 / HLA-A*02:01 / HLA-A*02:06 / HLA-A*02:07. 158-166 Antigenic peptide; Figure 8 C and D indicate that T cells transfected with AM14-M8 have a significant killing effect on HepG2 target cells that naturally express AFP protein, and the target cells are killed; the effector-to-target ratio (E:T) is 5:1.

[0061] Example 4: Validation of AFP-TCR Bispecific Antibody Protein The purpose of this embodiment is to verify the recognition of target cells and epitopes by AFP-TCR bispecific antibody protein and to detect the function of in vitro induced T cells.

[0062] Ten AFP antigen-specific T-cell receptors, AM14-M7, AM14-M10, AM14-M11, AM14-M12, AM14-M13, AM14-M14, AM14-M15, AM14-M16, AM14-M17 and AM14-M18, were prepared for in vitro functional validation experiments.

[0063] IFN-γ ELISA detection At a 1:1 ratio, activated T cells with the HLA-A*02:03 genotype were used with AFP-loaded cells. 158-166 After HLA-A*02:03 transduced with antigen peptides was used to transduce K562 cells and co-cultured with the target bispecific antibody protein for 48 h, human IFN-γ ELISA was performed.

[0064] On day 1, HLA-A*02:03 transduced K562 cells were collected, centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. The cell clumps were resuspended by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% penicillin-dextrose antibody) and adjusted to approximately 1×10⁻⁶ cells / cm². 6 Add AFP at a concentration of 1 µg / mL per cell / mL. 158-166 Incubate the antigenic peptide for 2 hours. Collect the AFP-loaded peptide. 158-166 HLA-A*02:03 transduced into K562 cells after antigen peptide administration, and HLA-A*02:03 transduced into K562 cells (negative control group), were centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. Cells were washed with 10 mL PBS (containing 2% fetal bovine serum), centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. Cell clumps were resuspended by adding complete AIM-V medium (AIM-V medium + 2% human serum + 1% penicillin-dextrin antibody) and adjusted to a concentration of 1×10⁶ cells / mL. 6 Add cells / mL, 100 µL / well to a 96-well round-bottom plate. Collect activated T cells, centrifuge at 350g for 5 minutes, and completely aspirate the supernatant. Resuspend the cell clumps by adding complete AIM-V medium (AIM-V medium + 2% human serum + 1% penicillin antibiotics) and adjust the volume to 1×10⁶ cells / mL. 6 Add 20 nM of penicillin protein (final concentration after dilution) per cell / mL, mix well, and then add AFP-loaded protein at 100 µL / well. 158-166HLA-A*02:03 transduced into K562 cells after antigen peptide administration was thoroughly mixed and cultured for 48 hours. On the second day, high-protein binding ELISA plates were plated with antibody 1-D1K (IFN-γ ELISA HRP kit) diluted to 2 µg / mL in PBS (pH 7.4) (by adding 100 µL / well) and incubated overnight at 4°C. On the third day, the ELISA plates were washed twice with PBS (200 µL / well). The ELISA plates were then blocked by adding 200 µL / well of culture medium and incubated at room temperature (RT) for 1 hour. Human IFN-γ standard (IFN-γ ELISA HRP kit) was prepared in 2 mL PBS (with 1% BSA) to a concentration of 0.5 µg / mL and placed at RT for 15 minutes, followed by vortexing. Dilute 100 µL / well of sample or standard in culture medium and incubate at RT for 2 hours. Wash the ELISA plate five times with PBS containing 0.05% Tween 20. Add 100 µL / well of antibody 7-B6-1-biotin (IFN-γ ELISA HRP kit) at 1 µg / mL (in PBS), incubate at RT for 1 hour, and wash. Add 100 µL / well of streptavidin-HRP (IFN-γ ELISA HRP kit) diluted 1:1000 in PBS, incubate at RT for 1 hour, and wash. Add 100 µL / well of TMB acceptor solution, incubate at RT in the dark for 15 to 30 minutes, until the solution in the wells turns a visible blue color. Add 100 µL / well of stop solution to terminate the enzyme reaction. The solution color changes from blue to yellow. Measure the light intensity at 450 nm using an ELISA reader. The final measured value is the experimental group's measured value minus the negative control group's measured value.

[0065] like Figure 9 As shown, compared to wild-type AM14, AM14-M7 exhibits a stronger and more pronounced induction of T cell activation and IFN-γ secretion at a TCR dual antibody concentration of 10 nM.

[0066] Jurkat Luciferase Detection Using a load AFP at a 1:1 ratio 158-166 After HLA-A*02:03 transduced K562 cells with antigen peptide and HLA-A*02:03 transduced K562 cells were co-cultured with Jurkat Luciferase cells and the target protein double antibody for 24 h, the expression of luciferase was detected.

[0067] On day 1, HLA-A*02:03 transduced K562 cells were collected, centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. The cell clumps were resuspended by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% penicillin-dextrose antibody) and adjusted to approximately 1×10⁶ cells / mL. 6 Add AFP at a concentration of 1 µg / mL per cell / mL. 158-166 Incubate the antigenic peptide for 2 hours. Collect the AFP-loaded peptide. 158-166 HLA-A*02:03 transduced into K562 cells after antigen peptide administration, and HLA-A*02:03 transduced into K562 cells (negative control group), were centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. Cells were washed with 10 mL PBS (containing 2% fetal bovine serum), centrifuged at 350g for 5 minutes, and the supernatant was completely aspirated. Cell clumps were resuspended by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% penicillin-dextrin antibiotics) and adjusted to approximately 4 × 10⁶ cells / mL. 5 Jurkat Luciferase cells were added at 50 µL / well to 96-well round-bottom plates. The cells were collected and centrifuged at 350g for 5 minutes, with the supernatant completely aspirated. The cell clumps were resuspended by adding complete 1640 medium (1640 medium + 10% fetal bovine serum + 1% penicillin-dextrose antibody) and the culture temperature was adjusted to 4 × 10⁶ cells / mL. 5 Add 2 nM of penicillin protein (final concentration after dilution to 1 nM) per cell / mL, mix well, and then add AFP-loaded protein at a rate of 50 µL / well. 158-166 HLA-A*02:03 transduced into K562 cells after antigen peptide administration and HLA-A*02:03 transduction were thoroughly mixed and cultured for 24 hours. On the second day, the detection reagents in the fluorescent insect luciferase reporter gene assay kit were brought to room temperature beforehand. 60 µl of the co-cultured cell mixture was transferred to a white ELISA plate, and then 60 µl of the detection reagent was mixed with the cell suspension. The mixture was allowed to react for 10-20 minutes, and the fluorescence value was detected using a chemiluminescence immunoassay reader. The final measured value was the experimental group value minus the negative control group value.

[0068] like Figure 10 As shown, compared to wild-type AM14, AM14-M10, AM14-M11, AM14-M12, and AM14-M13 at a TCR dual antibody concentration of 1 nM have a stronger and more obvious function in inducing T cell activation and luciferase expression.

[0069] like Figure 11As shown, compared to AM14-M10, AM14-M14, AM14-M15, AM14-M16, AM14-M17, and AM14-M18 have a stronger and more obvious function of inducing T cell activation and luciferase expression at a TCR double antibody concentration of 1 nM.

[0070] AM14-M7 includes: α chain, CDR1:VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0071] AM14-M10 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1:SEHNR (SEQ ID NO: 4) CDR2: FQNEAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0072] AM14-M11 includes: α chain, CDR1:VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1:LGMNR (SEQ ID NO: 15) CDR2:FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0073] AM14-M12 includes: Alpha chain, CDR1: VSGLRG (SEQ ID NO: 1) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1: YDHNR (SEQ ID NO: 16) CDR2: FQNDYL (SEQ ID NO: 18) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0074] AM14 - M13 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1: LGMNR (SEQ ID NO: 15) CDR2: FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0075] AM14 - M14 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) Beta chain, CDR1: LGMNR (SEQ ID NO: 15) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0076] AM14 - M15 includes: Alpha chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: YDHNR (SEQ ID NO: 16) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0077] AM14-M16 includes: α chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: SEYRW (SEQ ID NO: 17) CDR2: FQNEAQ (SEQ ID NO: 5) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0078] AM14-M17 includes: α chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FIFGAQ (SEQ ID NO: 14) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

[0079] AM14-M18 includes: α chain, CDR1: SRMLRG (SEQ ID NO: 7) CDR2: LYSAGEE (SEQ ID NO: 2) CDR3: CAVQADSWPSYALNF (SEQ ID NO: 13) β chain, CDR1: SEHNR (SEQ ID NO: 4) CDR2: FQNDYL (SEQ ID NO: 18) CDR3: CASSLTGLEQFF (SEQ ID NO: 6).

Claims

1. A T-cell receptor comprising an α-chain and a β-chain, characterized in that, The α and β chains contain the following complementarity-determining regions: 1) The amino acid sequence of CDR1 on the α chain is shown in either SEQ ID NO: 9 or 11, the amino acid sequence of CDR2 on the α chain is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 on the α chain is shown in SEQ ID NO:

3. The amino acid sequence of CDR1 on the β chain is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 on the β chain is shown in SEQ ID NO: 5, and the amino acid sequence of CDR3 on the β chain is shown in SEQ ID NO:

6.

2. The T cell receptor according to claim 1, characterized in that, The T cell receptor recognizes HLA-A-restricted AFP. 158-166 Epitope peptide, the amino acid sequence of which is shown in SEQ ID NO:

19.

3. The T-cell receptor according to claim 2, characterized in that, The HLA typing is HLA-A, and HLA-A is HLA-A*0203, HLA-A*0201, HLA-A*0206 or HLA-A*0207.

4. A nucleic acid molecule encoding the T cell receptor as described in any one of claims 1-3.

5. An engineered immune cell, characterized in that, Expressing the T cell receptor as described in any one of claims 1-3.

6. The engineered immune cell as described in claim 5, characterized in that, The T cell receptor is introduced into immune cells via viral or non-viral vector transduction, CRISPR / Cas9 system, transposon system or mRNA transfection to obtain engineered immune cells; the engineered immune cells include T cells or NK cells.

7. A TCR bispecific antibody protein, characterized in that, The TCR bispecific antibody protein consists of an antibody that targets receptor molecules on the surface of immune cell membranes and the extracellular domain of the T cell receptor as described in any one of claims 1-3, and binds to both immune cells and tumor cells. The immune cells include T cells or NK cells; The antibodies against the receptor molecules on the surface of the immune cell membrane include scFv or Fab antibody fragments.

8. The TCR bispecific antibody protein as described in claim 7, characterized in that, The immune cell membrane surface receptor molecules are CD3, CD4, CD8, CD16a and / or NKG2D.

9. A TCR bispecific antibody protein as described in claim 7, characterized in that, The extracellular region of the T cell receptor recognizes and binds to antigens on tumor cells.

10. A TCR bispecific antibody mRNA formulation, characterized in that, The TCR bispecific antibody protein according to any one of claims 7-9 is expressed in vivo using mRNA technology.

11. The use of the T cell receptor as described in any one of claims 1-3, the nucleic acid molecule as described in claim 4, the engineered immune cell as described in any one of claims 5-6, the TCR bispecific antibody protein as described in any one of claims 7-9, or the TCR bispecific antibody mRNA preparation as described in claim 10 in the preparation of a drug for treating liver cancer.

12. The application as described in claim 11, characterized in that, The drug also includes TCR protein drugs; The TCR protein drug is prepared from a multispecific T cell complex, which is prepared from the T cell receptor according to any one of claims 1-3; and... The multispecific T cell complex includes a TCR bispecific antibody, a TCR trispecific antibody, or a TCR tetraspecific antibody; Alternatively, the multispecific T-cell complex may include a T-cell receptor, a TCR bispecific antibody, a TCR trispecific antibody, or a TCR tetraspecific antibody, and may be a multispecific T-cell complex formed by linking a radiopharmaceutical, a toxin, a fluorescein, and an antibody fragment.

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