T-cell receptors targeting AFP peptides, their preparation methods, and companion diagnostic kits

Abstract

This invention relates to the field of biomedical technology, and in particular to a T-cell receptor targeting the AFP peptide, its preparation method, and a companion diagnostic kit. A T-cell receptor targeting the AFP peptide, wherein the sequence of the AFP peptide is FMNKFIYEI, and the T-cell receptor contains a TCRα chain variable domain and a TCRβ chain variable domain, is provided. The corresponding nucleic acid molecule, vector, and host cell are provided, as well as a method for preparing the T-cell receptor. A companion diagnostic kit containing the cell receptor is further provided. This invention provides a better option for companion diagnostic and therapeutic products for alpha-fetoprotein (AFP) cells.

Description

[0001] This application is a divisional application of application No. 2025106099945, filed on May 13, 2025, entitled "T cell receptor targeting AFP peptide, its preparation method and companion diagnostic kit". TECHNICAL FIELD

[0002] The present application relates to the technical field of biological medicine, in particular to a T cell receptor targeting AFP peptide, its preparation method and companion diagnostic kit. BACKGROUND

[0003] Adoptive T cell therapy (ACT) strategy has become a very important treatment strategy in cancer immunology, and T cell receptor-engineered T cell (TCR-T) therapy and chimeric antigen receptor T cell (CAR-T) therapy are two main methods for redirecting T cell specificity to tumor antigens. Kymriah, a CD19-directed CAR-T cell immunotherapy developed by Novartis, is the first CAR-T therapy approved for marketing by the FDA worldwide. Currently, more than ten CAR-T products have been approved for marketing worldwide. According to the current research situation, although CAR-T therapy has shown significant efficacy in the treatment of B cell malignancies, it has not achieved satisfactory results in the treatment of solid tumors.

[0004] TCR-T is a T cell expressing a tumor antigen-specific receptor, and its alpha and beta chains are generated from high-quality, high-affinity antigen-specific T cell clones. In recent years, TCR-T cell therapy has made a series of progress, mainly used for solid tumor treatment, covering tumor antigen types including tumor mutation neoantigens (e.g., KRAS mutant antigen), tissue differentiation antigens (e.g., gp100), cancer testis antigens (e.g., MAGE-A4), viral antigens (e.g., HPV-E6), overexpressed antigens (e.g., HER2), etc.; Currently, there are more than 150 ongoing clinical trials; On January 31, 2024, Adaptimmune announced that its TCR-T therapy Afami-cel's biological product license application (BLA) was accepted by the FDA and obtained priority review, and it has been approved for marketing on August 1, 2024.

[0005] Compared with chimeric antigen receptor (CAR), T cell receptor (TCR) has some obvious advantages in T cell-based therapy, mainly including: 1. There are more subunits in its receptor structure, more tyrosine-based activation motifs (ITAM) of immune receptors, and less dependence on antigen; 2. There are more costimulatory receptors (CD3, CD4, CD28, etc.), which can make TCRs with low MHC affinity range effectively activate T cells; 3. Since it can target intracellular tumor-specific antigens, TCRs are more potential in the treatment of solid tumors.

[0006] Alpha-fetoprotein (AFP) is a glycoprotein belonging to the albumin family, mainly synthesized by fetal liver cells and yolk sac, which mainly plays a transport role in fetal development, carrying fatty acids, bilirubin and other substances. In addition, it also has immune regulation function, protecting the fetus from the attack of the mother's immune system. The concentration of AFP in the blood of healthy adults is very low, mainly produced in small amounts in the liver. Studies have shown that in about 50% to 80% of hepatocellular carcinoma (HCC) patients, AFP transcription is reactivated and highly expressed, becoming a protein that is highly expressed specifically in liver cancer cells. The most common HBV-infected liver cancer patients in the Chinese population often have high malignancy and high AFP expression levels. Comprehensive current research shows that AFP is a very attractive target for HCC adoptive T cell therapy, especially for patients with high malignancy and limited treatment options.

[0007] In addition, in addition to being used in the field of treatment, TCR technology can also be used for companion diagnosis of tumor immunotherapy, for example: after screening TCR molecules against specific mutant peptides, they can be used for T cell receptor gene clone identification detection on tissue samples of subjects, by performing T cell receptor gene clone identification detection on samples of subjects at different time nodes, comprehensively analyzing indicators such as the clonal proportion and clonal diversity of related TCRs, and monitoring the dynamic changes of sample TCR indicators in real time, such as total number of clones, clone type, diversity index, and clone abundance, to assist in evaluating the clinical effect of immunotherapy and screening patient populations with better responsiveness and higher clinical benefit.

[0008] Therefore, in order to more conveniently diagnose the related companion diagnosis, the TCR receptor protein can be prepared into a kit for companion diagnosis of subjects.

[0009] Currently, there are not many cell companion diagnosis and treatment products targeting alpha-fetoprotein (AFP), and currently only more than ten TCR-T products are under research, all of which are in clinical phase I. Therefore, patients need more diversified and clinically beneficial options for products targeting this target. SUMMARY

[0010] The technical problem to be solved by the present application is that there are few current alpha-fetoprotein (AFP) cell companion diagnostic and therapeutic products.

[0011] Accordingly, the technical solution adopted by the present application to solve its technical problem is to screen for T cell receptors specific to AFP peptides and use them in companion diagnostic kits. Specifically, part of the AFP peptide segment (such as AFP158-166) can bind to specific HLA molecules such as HLA-A*02:01, form an antigen peptide-MHC complex and be recognized by TCR, inducing an anti-tumor immune response. Using engineered AFP-specific TCR proteins, it is possible to detect whether AFP / HLA complexes exist on the surface of tumor tissues or cells, which is expected to be used for screening for AFP-targeted TCR-T cell therapy or AFP vaccine treatment of the appropriate population, as a companion diagnostic tool for related immunotherapy products.

[0012] In a first aspect, a T cell receptor targeting an AFP peptide is provided, the AFP peptide having the sequence FMNKFIYEI (SEQ ID NO: 25), and the T cell receptor comprising a TCR alpha chain variable domain and a TCR beta chain variable domain,

[0013] wherein the TCR alpha chain variable domain comprises:

[0014] a CDR1a having the sequence DSASNY (SEQ ID NO: 1);

[0015] a CDR2a having the sequence IRSNVGE (SEQ ID NO: 2); and

[0016] a CDR3a having the sequence AAPFGGEAGTALI (SEQ ID NO: 3),

[0017] wherein the TCR beta chain variable domain comprises:

[0018] a CDR1b having the sequence DFQATT (SEQ ID NO: 4);

[0019] a CDR2b having the sequence SNEGSKA (SEQ ID NO: 5); and

[0020] a CDR3b having the sequence SARGPLIGPTDTQY (SEQ ID NO: 6).

[0021] In some embodiments, the T cell receptor is soluble.

[0022] In some embodiments, the T cell receptor comprises an artificial disulfide bond between the alpha chain constant region and the beta chain constant region.

[0023] In some embodiments, the TCR alpha chain variable domain has the sequence:

[0024] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDI

[0025] RSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAAPFGGEAGTALIFGKGTTLSVSSN (SEQ ID NO: 19),

[0026] The sequence of the TCR beta chain variable domain is:

[0027] MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSK

[0028] ATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARGPLIGPTDTQYFGPGTRLTVLE (SEQ ID NO: 20).

[0029] In some embodiments, the T cell receptor is a fusion protein.

[0030] In a second aspect, there is provided a synthetic nucleic acid molecule encoding the T cell receptor of the first aspect of the application.

[0031] In a third aspect, there is provided a vector comprising the synthetic nucleic acid molecule of the application.

[0032] In a fourth aspect, there is provided a host cell comprising the vector of the application.

[0033] Alternatively, there is provided a host cell having integrated into its chromosome the synthetic nucleic acid molecule of the application.

[0034] In a fifth aspect, there is provided a method of producing a T cell receptor, comprising:

[0035] (i) culturing the host cell of the application to express the T cell receptor of the first aspect of the application; and

[0036] (ii) isolating the T cell receptor.

[0037] In a sixth aspect, there is provided a companion diagnostic kit for tumour immunotherapy, comprising:

[0038] a T cell receptor protein comprising a TCR alpha chain variable domain and a TCR beta chain variable domain,

[0039] the TCR alpha chain variable domain comprises:

[0040] a CDR1a having the sequence of DSASNY (SEQ ID NO: 1);

[0041] a CDR2a having the sequence of IRSNVGE (SEQ ID NO: 2); and

[0042] a CDR3a having the sequence of AAPFGGEAGTALI (SEQ ID NO: 3),

[0043] wherein the TCR beta chain variable domain comprises:

[0044] a CDR1b having the sequence of DFQATT (SEQ ID NO: 4);

[0045] a CDR2b having the sequence of SNEGSKA (SEQ ID NO: 5); and

[0046] a CDR3b having the sequence of SARGPLIGPTDTQY (SEQ ID NO: 6), the T cell immunoreceptor targets a tumor antigen peptide derived from an AFP gene, the tumor antigen peptide having the sequence of FMNKFIYEI.

[0047] In some embodiments, the kit further comprises a chromogenic system, a positive control, a negative control, a staining buffer, a blocking solution.

[0048] In some embodiments, the sample of the companion diagnostic kit comprises: a tissue sample, peripheral blood, tumor infiltrating TILs.

[0049] In some embodiments, the companion diagnostic kit comprises: an immunofluorescence staining (IHC) kit or a flow cytometry detection kit.

[0050] In some embodiments, the method of using the companion diagnostic kit comprises:

[0051] 1). using a TCR-Fc protein probe to stain tumor tissue sections;

[0052] 2). detecting whether AFP / HLA complexes exist;

[0053] 3). evaluating AFP antigen presentation ability according to the detection result.

[0054] In some embodiments, the method of using the companion diagnostic kit comprises:

[0055] 1). using a TCR protein probe for flow staining;

[0056] 2). detecting whether AFP / HLA complexes are expressed on the cell surface;

[0057] 3). Dynamic immune monitoring by assessing antigen presentation level or AFP expression.

[0058] In a seventh aspect, the present application relates to a composition comprising a fusion polypeptide comprising the aforementioned TCR a chain and / or TCR β chain.

[0059] In some embodiments, the cell is genetically modified by introducing an isolated nucleic acid molecule encoding a polypeptide comprising at least one of the aforementioned TCR a chain and TCR β chain.

[0060] In some embodiments, the cell is an immune cell.

[0061] In some embodiments, the immune cell is selected from the group consisting of an antigen presenting cell, a B cell, a dendritic cell, a macrophage, a Langerhans cell, a T cell, an NK cell, an NK T cell.

[0062] The beneficial effects of the present application are that by screening with AFP peptide, a T cell receptor with high affinity to AFP peptide and stable expression on cell membrane is obtained, which can specifically bind to FMNKFIYEI-HLA-A*02:01 complex, providing a basis for companion diagnosis of tumor immunotherapy. In addition, the T cell receptor exhibits excellent specific killing activity on antigen-positive tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 Figure showing the results of TCR binding affinity experiment to AFP peptide.

[0064] Figure 2 Figure showing the results of TCR stability experiment on cell membrane expression.

[0065] Figure 3 Figure showing the results of TCR overexpressing T cells specific IFN-γ secretion experiment on antigen-positive target cells.

[0066] Figure 4 Figure showing the results of TCR overexpressing T cells specific IL-2 secretion experiment on antigen-positive target cells.

[0067] Figure 5 Figure showing the results of TCR overexpressing T cells CD137 expression experiment.

[0068] Figure 6 Figure showing the results of TCR overexpressing T cells specific killing activity experiment on antigen-positive tumor cells. DETAILED DESCRIPTION

[0069] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described.

[0070] As used herein, "CDR" is defined as the complementarity determining region amino acid sequence of a TCR or TCR chain.

[0071] In the context of the present application, the following common nucleic acid base abbreviations are used. "A" means adenosine, "C" means cytidine, "G" means guanosine, "T" means thymidine, and "U" means uridine.

[0072] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues connected to one another by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids to the sequence that can make up a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids connected to one another by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, e.g., peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art as proteins, which have many types).

[0073] As used herein, "vector" can mean a nucleic acid sequence that contains an origin of replication. A vector can be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

[0074] In some embodiments, the TCR comprises a TCR a chain variable domain and a TCR β chain variable domain, the 3 complementarity determining regions (CDRs) of the TCR a chain variable domain are:

[0075] CDR1a - DSASNY (SEQ ID NO: 1);

[0076] CDR2a - IRSNVGE (SEQ ID NO: 2); and CDR3a - AAPFGGEAGTALI (SEQ ID NO: 3), and the 3 complementarity determining regions (CDRs) of the TCR β chain variable domain are:

[0077] CDR1β - DFQATT (SEQ ID NO: 4);

[0078] CDR2β-SNEGSKA (SEQ ID NO: 5); and CDR3β-SARGPLIGPTDTQY (SEQ ID NO: 6).

[0079] In some embodiments, the TCR comprises a TCR a chain variable domain and a TCR β chain variable domain, the three complementarity determining regions (CDRs) of the TCR a chain variable domain are:

[0080] CDR1a-DPNSYY (SEQ ID NO: 13);

[0081] CDR2a-VFSSTEI (SEQ ID NO: 14); and CDR3a-AVNSNYAQGLT (SEQ ID NO: 15), and the three complementarity determining regions (CDRs) of the TCR β chain variable domain are:

[0082] CDR1β-NSQYPW (SEQ ID NO: 16);

[0083] CDR2β-LRSPGD (SEQ ID NO: 17); and CDR3β-TCSGSGGAETLY (SEQ ID NO: 18).

[0084] In some embodiments, the TCR comprises a TCR a chain variable domain and a TCR β chain variable domain, the three complementarity determining regions (CDRs) of the TCR a chain variable domain are:

[0085] CDR1a-DPNSYY (SEQ ID NO: 13);

[0086] CDR2a-VFSSTEI (SEQ ID NO: 14); and CDR3a-AVNSNYAQGLT (SEQ ID NO: 15), and the three complementarity determining regions (CDRs) of the TCR β chain variable domain are:

[0087] CDR1β-NSQYPW (SEQ ID NO: 16);

[0088] CDR2β-LRSPGD (SEQ ID NO: 17); and CDR3β-TCSGSGGAETLY (SEQ ID NO: 18).

[0089] In some embodiments, the TCR comprises an a chain variable domain amino acid sequence as set forth in SEQ ID NO: 19, and the TCR comprises a β chain variable domain amino acid sequence as set forth in SEQ ID NO: 20.

[0090] In some embodiments, the TCR comprises an alpha chain variable domain amino acid sequence as set forth in SEQ ID NO: 21 and the TCR comprises a beta chain variable domain amino acid sequence as set forth in SEQ ID NO: 22.

[0091] In some embodiments, the TCR comprises an alpha chain variable domain amino acid sequence as set forth in SEQ ID NO: 23 and the TCR comprises a beta chain variable domain amino acid sequence as set forth in SEQ ID NO: 24.

[0092] In some embodiments, the TCR is single chain.

[0093] In some embodiments, the TCR is linked by a peptide linker between the alpha and beta chain variable regions.

[0094] In some embodiments, a cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.

[0095] In some embodiments, a C- or N-terminus of the alpha and / or beta chain of the TCR is conjugated to a conjugate, preferably a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these. In some embodiments, the TCR is a murine TCR, a human-murine chimeric TCR, or a humanized TCR.

[0096] In some embodiments, the vector comprises an expression vector, i.e., a construct that is capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophage, and viral vectors.

[0097] In some embodiments, the viral vector includes, but is not limited to, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector, a baculoviral vector. Preferably, the vector can transfer the nucleotide of the present application into a cell, e.g., a T cell, such that the cell expresses a TCR specific for AFP antigen. The vector should be capable of expressing at a high level and persistently in the T cell.

[0098] In some embodiments, the lentiviral vector can include: lentiviral expression vector pLenti (addgene).

[0099] In some embodiments, the host cell is a mammalian cell. For example, the host cell is a human cell. While the host cell can be a cell of any cell type, can be derived from any type of tissue, and can be a cell of any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0100] In some embodiments, wherein the host cell or associated cell population is administered, the host cell can be a cell that is allogeneic to the mammal or autologous to the mammal. Preferably, the cell is autologous to the mammal.

[0101] In some embodiments, the mammal refers to any mammal, including but not limited to: rodents such as mice and hamsters, and lagomorphs such as rabbits; preferably the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably the mammal is from the order Artiodactyla including bovids (cattle) and suids (pigs), or from the order Perissodactyla including equids (horses); most preferably the mammal is from the order Primates, the order Simiiformes or the suborder Anthropoidea (humans and anthropoids). It is particularly preferred that the mammal is a human.

[0102] The principles and implementations of the present application are described in detail in the specific examples, and the description of the examples is only used to help understand the method of the present application and the central idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

[0103] Example 1: Cloning of antigenic short peptide specific T cells

[0104] Synthetic short peptides (Genescript) AFP (SEQ ID NO: 25) were used to stimulate peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A*02:01, respectively or in combination. The above short peptides were respectively complexed with biotin-labeled HLA-A*02:01 to prepare pHLA haplotype. These haplotypes were combined with PE-labeled streptavidin (BD company) to form PE-labeled tetramer, and the tetramer and anti-CD8-APC double positive cells were sorted using BD Melody flow sorting instrument, and the positive cells were sorted into 96-well plates, one cell per well.

[0105] Example 2: Obtaining TCR gene and vector construction of AFP antigenic short peptide specific T cell clone After the cells obtained in Example 1 were lysed using 0.1% Triton-X (Shenguo), the SMART RACE cDNA amplification kit of clontech was used, and the primer was designed in the C-terminal conserved region of human TCR gene. Downstream primer of C-terminal conserved region of TCR alpha chain: tcagctggaccacagc

[0106] Downstream primer of C-terminal conserved region of TCR beta chain:

[0107] tcagaaatcctttctcttgac (SEQ ID NO: 27). The full-length genes of TCR a chain and β chain were cloned into lentiviral expression vector pCDH (SBI) by overlap PCR, respectively. The specific steps were as follows: the full-length genes of TCR a chain and TCR β chain were connected by overlap PCR to obtain TCR a-2A-TCR β fragment. The lentiviral expression vector and TCR a-2A-TCR β were connected by enzyme digestion to obtain pCDH-TRA-2A-TRB plasmid, and sequencing was performed and three TCR clone plasmids Y2386-5, Y2357-7 and Y24081-2 were obtained (IMGT).

[0108] The sequencing results of CDR1a, CDR2a and CDR3a of the TCR a chain variable domain of Y2386-5 were SEQ ID NOs: 1-3, respectively; the sequencing results of CDR1β, CDR2β and CDR3β of the TCR β chain variable domain were SEQ ID NOs: 4-6, respectively. The sequencing result of the TCR a chain variable domain was SEQ ID NO: 19, and the sequencing result of the TCR β chain variable domain was SEQ ID NO: 20.

[0109] The sequencing results of CDR1a, CDR2a and CDR3a of the TCR a chain variable domain of Y2357-7 were SEQ ID NOs: 7-9, respectively; the sequencing results of CDR1β, CDR2β and CDR3β of the TCR β chain variable domain were SEQ ID NOs: 10-12, respectively. The sequencing result of the TCR a chain variable domain was SEQ ID NO: 21, and the sequencing result of the TCR β chain variable domain was SEQ ID NO: 22.

[0110] The sequencing results of CDR1a, CDR2a and CDR3a of the TCR a chain variable domain of Y24081-2 were SEQ ID NOs: 13-15, respectively; the sequencing results of CDR1β, CDR2β and CDR3β of the TCR β chain variable domain were SEQ ID NOs: 16-18, respectively. The sequencing result of the TCR a chain variable domain was SEQ ID NO: 23, and the sequencing result of the TCR β chain variable domain was SEQ ID NO: 24.

[0111] Afterwards, the 293T was used to package the pseudovirus. Specifically, the above-mentioned plasmids were mixed with VSVG plasmid, RRE plasmid, Rev plasmid (purchased from Addgene company) in the ratio of 4:5:4:10, and 20 μL was diluted into DMEM medium (1.25 mL) as a DNA solution. 20 μL of polyetherimide (PEI 1 μg / μL) was added to DMEM (1.25 mL), and the above-mentioned PEI / DMEM mixed solution was added to the DNA solution prepared, and incubated at room temperature for 20 minutes, then added to 293T cells cultured in a 15 cm dish and mixed. After 6 hours, the fresh DMEM medium was replaced. After 72 hours, the supernatant containing the lentivirus was collected, which was the lentivirus supernatant of each TCR.

[0112] Example 3: Construction of antigen short peptide specific TCR overexpression cell line

[0113] The NFAT-GFP element (Addgene) was inserted into the expression vector pCDH (SBI) by the standard method described in the Molecular Cloning Laboratory Manual, and the fragment was sequenced to confirm that it was correct. Afterwards, the 293T (Pronex CL-0130) was used to package the pseudovirus. The Jurkat cell line was infected with pseudovirus containing the NFAT-GFP element and pseudovirus containing the TCR element, and through limiting dilution and monoclonal amplification, each Jurkat-NFAT-GFP-TCR overexpression cell line was obtained, namely: Jurkat-NFAT-GFP-Y2386-5-TCR, Jurkat-NFAT-GFP-Y2357-7-TCR and Jurkat-NFAT-GFP-Y24081-2-TCR.

[0114] Example 4: TCR binding affinity experiment to mutant peptide and wild type peptide or tumor associated antigen peptide

[0115] (1) Construction of K562CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01

[0116] The HLA-A*02:01 element (IMGT / HLA Acc No: HLA00043) and CD80

[0117] (NP_005182.1) was synthesized and inserted into the expression vector pCDH (SBI), and the fragment was sequenced to confirm that there was no error. Then the 293T cell line was used to package the lentivirus. Specifically, the plasmid containing the CD80-HLA-A*02:01 element was mixed with the VSVG plasmid, the RRE plasmid, and the Rev plasmid (purchased from Addgene) in a ratio of 4:5:4:10, and 20 μL was diluted into DMEM medium (1.25 mL) as a DNA solution. 20 μL of polyetherimide (PEI 1 μg / μL) was added to DMEM (1.25 mL), and the PEI / DMEM mixed solution was added to the DNA solution prepared above, incubated at room temperature for 20 minutes, and then added to 293T cells cultured in a 15 cm dish and mixed well. After 6 hours, the fresh DMEM medium was replaced. The supernatant containing the lentivirus was collected after 72 hours, which was the lentivirus supernatant of CD80-HLA-A*02:01. The K562 or 293T cell line was infected with the lentivirus containing the CD80-HLA-A*02:01 element, and the K562-CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01 overexpression cell lines were obtained by limiting dilution and monoclonal expansion.

[0118] (2) Jurkat-NFAT-GFP-TCR overexpression cell lines were co-cultured with K562-CD80-HLA-A*02:01 The cell lines (Jurkat-NFAT-GFP-Y2386-5-TCR, Jurkat-NFAT-GFP-Y2357-7-TCR, and Jurkat-NFAT-GFP-Y24081-2-TCR) were co-incubated with K562-CD80-HLA-A*02:01 loaded with different concentrations of the antigen peptide (FMNKFIYEI (AFP)) to be tested. Specifically, the K562 was incubated with different concentrations of the target antigen peptide at 37°C for 1 h, and then the cells were resuspended with medium after centrifugation. The Jurkat and K562 cells were counted, and 2 x 10^4 cells of Jurkat and polypeptide-loaded K562 were taken and mixed, and then co-cultured in a 96-well plate. After 24 h of co-culture, the Jurkat cell reporter gene activation level and CD69 cell activation level were detected by flow cytometry.

[0119] The results are shown in Figure 1 It is shown that the TCR-T cells expressing Y2386-5, Y2357-7, and Y24081-2 have strong reactivity and specificity to the target antigen peptide AFP, and the related TCR proteins are suitable for diagnostic and therapeutic use.

[0120] Example 5: Stability experiment of overexpressed TCR on cell membrane expression

[0121] TCR-T cells expressing TCR (Y23191, Y23192, and Y24318) were constructed using TCR elements (TCRα-2A-TCRβ fragments) from Y2386-5, Y2357-7, and Y24081-2, respectively. The specific steps were as follows: (1) Preparation of TCR lentivirus: according to the "Molecular Cloning Laboratory Manual"

[0122] The standard method described in (Molecular Cloning a Laboratory Manual, ISBN 978-1-936113-42-2; Chapter 3 Cloning and Transformation with Plasmid Vectors) synthesized each TCR element and GFP (addgene) and inserted them into the expression vector pCDH (SBI). The fragments were confirmed to be correct after sequencing. Then, pseudoviruses were packaged using 293T (Pronosai CL-0130) according to the specific operating steps of Example 2.

[0123] (2) Construction of TCR-T cells expressing TCR: After thawing PBMCs, use an appropriate amount of...

[0124] Prepare X-VIVO 15 medium containing 100 IU / mL rhIL-2 to a density of 1×10^6 / mL. Add 10 μL of MACS CD3 / CD28 T cell TransAct beads to every 2×10^6 cells in X-VIVO 15 medium containing 100 IU / mL rhIL-2, mix gently, and incubate in a cell culture incubator. After 24 hours, the cells were centrifuged and the supernatant was discarded to remove the magnetic beads. The cells were resuspended in 1 mL of X-VIVO 15 medium containing 100 IU / mL rhIL-2. The target lentivirus (infected at MOI=10) was added based on the total cell count and viral titer. A control group without lentivirus was also included. Culture medium was added to each control group until a final volume of 2 mL of X-VIVO 15 medium containing 100 IU / mL IL-2 was reached. Polyglobulin was added to a final concentration of 10 μg / mL. The cells were centrifuged in a plate basket at 37°C and 2000g for 60 minutes. The infected cells were then cultured in a CO2 incubator for 24 hours. The culture medium was changed regularly, and the cell density was adjusted until day 14.

[0125] GFP was used to detect the expression rate of TCR-T. The results showed (see...). Figure 2 Each TCR can be stably expressed in the cell membrane.

[0126] Example 6: Specific IFN-γ and IL-2 secretion by TCR-T cells against antigen-positive target cells

[0127] 1. Using T cells expressing TCR (same as Example 5) as effector cells, PBMCs without transduction of TCR were used as a parallel control for expansion culture of effector cells.

[0128] 2. K562-CD80-HLA-A*02:01 cells loaded with 10"7M AFP (FMNKFIYEI) short peptide or irrelevant peptide were used as positive target cells (same as Example 4); the E:T effector-to-target ratio was 1:1, and the expression of IL-2 or / and IFN-γ was detected after 24 h of incubation.

[0129] 3. K562-CD80-HLA-A*02:01 cells loaded with 10"10M-10"5M AFP (FMNKFIYEI) short peptide at different concentrations were used as positive target cells (same as Example 4); the E:T effector-to-target ratio was 1:1, and the expression of CD137 was detected after 24 h of incubation.

[0130] The results showed that the TCR-T overexpression group produced IL-2 and IFN-γ and the expression of CD137 was up-regulated in the presence of positive target cells, and the TCR-T overexpression group did not produce IFN-γ or IL-2 against negative target cells. Some of the results of IL-2 and INF-γ expression and secretion are shown in Figure 3 and Figure 4 Some of the results of CD137 up-regulation are shown in Figure 5 The above data show that the relevant T cells overexpressing TCR have specific activation efficacy against antigen-positive target cells, and the TCR protein obtained by the present application is suitable for use for diagnostic and therapeutic purposes.

[0131] Example 7: Specific killing activity of TCR-T cells overexpressing TCR against antigen-positive tumor cells

[0132] 1. Using T cells expressing TCR (same as Example 5) as effector cells, PBMCs without transduction of TCR were used as a parallel control for expansion culture of effector cells.

[0133] 2. Using 293T-CD80-HLA-A*02:01 cells loaded with 10"8M-10"6M AFP (FMNKFIYEI) short peptide as positive target cells (+) as described in Example 5; E:T effector to target ratio is 10:1, the adhesion ability of target cells is detected by RTCA (Real-Time Cell Analyzer) instrument in real time, which is a real-time label-free cell analysis system that integrates microelectronic cell sensor chips into the bottom of the cell detection plate, and can obtain biological information related to cell physiological functions, including cell growth, stretching, morphological changes, death and adhesion, etc. through real-time dynamic electrode impedance detection. Specifically, the instrument collects the cell adhesion ability value (Cell Index) for each well every 15 min, and the data at the last time point before the addition of T cells is used as the normalized value for subsequent data processing. The normalized cell adhesion ability value (Normalized Cell Index) at each time point in each group is calculated; the results show (see Figure 6 ): the TCR-T overexpression group only has significant killing activity against 293T-CD80-HLA-A*02:01 cells loaded with tumor-associated antigen polypeptides, and has no killing effect on 293T-CD80-HLA-A*02:01 cells without polypeptide loading, and Y23191 has better killing effect.

Claims

1. A T cell receptor targeting an AFP peptide, the sequence of which is FMNKFIYEI, and the T cell receptor comprising a TCR a chain variable domain and a TCR β chain variable domain, wherein the TCR a chain variable domain comprising: a CDR1a of sequence DRVSQS; a CDR2a of sequence IYSNGD; and a CDR3a of sequence AVTPGSNYKLT, wherein the TCR β chain variable domain comprises: a CDR1β of sequence SGHNT; a CDR2β of sequence YYREEE; and a CDR3β of sequence ASSPGLAGEQY.

2. The T cell receptor of claim 1, wherein, the T cell receptor is soluble.

3. The T cell receptor of claim 1, wherein, the T cell receptor comprises an artificial disulfide bond between the a chain constant region and the β chain constant region.

4. The T cell receptor of any one of claims 1 to 3, wherein the sequence of the TCR a chain variable domain is: MDESLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRVSQSFFWYRQYSGKS PELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVTPGSNYKLTFGKGTLLTVNP, wherein the sequence of the TCR β chain variable domain is: MGTSLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSWYQQALGQGPQFI FQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYLCASSPGLAGEQYFGPGTRLTVTE.

5. A synthetic nucleic acid molecule encoding the T cell receptor of claim 1.

6. A vector comprising the synthetic nucleic acid molecule of claim 5.

7. A host cell comprising the vector of claim 6.

8. A host cell having integrated into its chromosome the synthetic nucleic acid molecule of claim 5.

9. A method of producing a T cell receptor, comprising: (i) culturing the host cell of claim 7 or 8 to express the T cell receptor of claim 1; and (ii) isolating the T cell receptor.

10. A companion diagnostic kit for tumor immunotherapy, comprising: a T cell receptor protein comprising a TCR a chain variable domain and a TCR β chain variable domain, wherein the TCR a chain variable domain comprises: a CDR1a of sequence DRVSQS; a CDR2a of sequence IYSNGD; and a CDR3a of sequence AVTPGSNYKLT, wherein the TCR β chain variable domain comprises: a CDR1β of sequence SGHNT; a CDR2β of sequence YYREEE; and a CDR3β of sequence ASSPGLAGEQY, ​ The T cell immune receptor targets a tumor antigen peptide derived from an AFP gene, and a sequence of the tumor antigen peptide is: FMNKFIYEI.

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