T cell receptor targeting PRAME peptide and preparation method thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to T cell receptors targeting the PRAME peptide and methods for preparing the same. Specifically, the present application also provides T cell receptors, corresponding nucleic acid molecules, vectors, and host cells, as well as methods for preparing T cell receptors. The TCR-T cells obtained in the present application can effectively treat melanoma-specific antigen (PRAME)-positive tumors, providing patients with more treatment options.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a T cell receptor targeting a PRAME peptide and a preparation method thereof. Background Art

[0002] TCR-T cells express tumor antigen-specific receptors, whose α and β chains are produced by high-quality, high-affinity antigen-specific T cell clones. In recent years, TCR-T cell therapy has made a series of advances, primarily for the treatment of solid tumors. The types of tumor antigens covered include tumor mutation neoantigens (e.g., KRAS mutant antigens), tissue differentiation antigens (e.g., gp100), cancer testis antigens (e.g., MAGE-A4), viral antigens (e.g., HPV-E6), and overexpressed antigens (e.g., HER2). Currently, over 150 clinical trials are underway. On January 31, 2024, Adaptimmune announced that its Biologics License Application (BLA) for the TCR-T therapy Afami-cel was accepted by the FDA and granted priority review designation. Afami-cel was approved for marketing on August 1, 2024.

[0003] Compared with chimeric antigen receptors (CARs), T cell receptors (TCRs) have some obvious advantages in T cell-based therapies, mainly including: 1. There are more subunits in its receptor structure, more immunoreceptor tyrosine-based activation motifs (ITAMs), and less dependence on antigens; 2. It has more co-stimulatory receptors (CD3, CD4, CD28, etc.), which enables TCRs with a low MHC affinity range to effectively activate T cells; 3. Because it can target tumor-specific antigens in cells, TCRs have greater potential in the treatment of solid tumors.

[0004] PRAME (preferentially expressed antigen of melanoma) was identified in 1997 by Ikeda's team while analyzing the specificity of tumor-reactive T cell clones from patients with metastatic cutaneous melanoma. This type of intracellular antigen, when broken down intracellularly, produces peptide fragments that bind to HLA and are presented on the cell surface. These peptides are recognized by T cells and trigger effector T cell responses, making them an important target for the development of TCR (T-cell receptor)-targeted therapies. Multiple studies have shown that PRAME promotes tumor growth by inhibiting retinoic acid receptor signaling and is highly expressed in melanoma, lung cancer, breast cancer, and ovarian cancer. Current pipeline candidates include IMA203, TK-6302, and MDG1011. IMA203 is the most advanced, with recent clinical data showing an overall response rate of 52.5% in 40 patients, and a 70% overall response rate in the high-dose treatment group. Multiple studies have shown that PRAME is an ideal target for tumor immunotherapy for the following reasons: 1. It is widely and highly expressed in most solid tumors and hematological tumors; 2. It is almost not expressed in normal tissues and has an excellent safety window; 3. It has clear antigen peptide-MHC presentation ability, which facilitates TCR targeting.

[0005] As of now, there are not many cell therapy products targeting PRAME. Currently, there are only more than ten TCR-T products under development. Among them, only IMA203 is in clinical phase III, and most of the others are in clinical phase I or even preclinical stages. Therefore, patients need more diverse options for cell therapy products targeting this target that can obtain greater clinical benefits. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that there are currently few cell therapy products for melanoma-specific antigen (PRAME).

[0007] Accordingly, the present invention addresses this technical problem by screening for T cell receptors that specifically target the PRAME peptide and using them in tumor treatment. Specifically, certain PRAME peptide segments (such as PRAME 100-108) can bind to specific HLA molecules, such as HLA-A*02:01, to form antigenic peptide-MHC complexes that are recognized by TCRs and induce anti-tumor immune responses.

[0008] In a first aspect, the present invention provides a T cell receptor targeting the PRAME peptide, wherein the sequence of the PRAME peptide is VLDGLDVLL (SEQ ID NO: 17), and the T cell receptor comprises a TCR α chain variable domain and a TCR β chain variable domain.

[0009] The variable domain of the TCRα chain contains:

[0010] CDR1α with the sequence SSYSPS (SEQ ID NO: 1);

[0011] CDR2α of the sequence YTSAATLV (SEQ ID NO: 2); and

[0012] CDR3α with the sequence VVDVGNDMR (SEQ ID NO: 3),

[0013] The variable domain of the TCRβ chain contains:

[0014] CDR1β with the sequence SEHNR (SEQ ID NO: 4);

[0015] CDR2β having the sequence FQNEAQ (SEQ ID NO: 5); and

[0016] The CDR3β sequence is ASSKGTERYSPLH (SEQ ID NO: 6).

[0017] In some embodiments, the T cell receptor is soluble.

[0018] In some embodiments, the T cell receptor comprises an artificial disulfide bond between the α chain constant region and the β chain constant region.

[0019] In some embodiments, the sequence of the TCR alpha chain variable domain is:

[0020] AQSVTQLDSHHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLLTKPSAHMSDAAEYFCVVDVGNDMRFGAGTRLTVKPN (SEQ ID NO: 13),

[0021] The sequence of the TCRβ chain variable domain is:

[0022] DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSKGTERYSPLHFGNGTRLTVTE (SEQ ID NO: 14).

[0023] In some embodiments, the T cell receptor is a fusion protein.

[0024] In a second aspect, a synthetic nucleic acid molecule is provided, which encodes the T cell receptor of the first aspect of the present invention.

[0025] In a third aspect, a vector is provided, which contains the synthetic nucleic acid molecule of the present invention.

[0026] In a fourth aspect, a host cell is provided, wherein the host cell contains the vector of the present invention.

[0027] Alternatively, a host cell is provided that has integrated into its chromosome a synthetic nucleic acid molecule of the invention.

[0028] A fifth aspect provides a method for preparing a T cell receptor, comprising:

[0029] (i) culturing the host cell of the present invention to express the T cell receptor of the first aspect of the present invention; and

[0030] (ii) Isolation of T cell receptors.

[0031] In a sixth aspect, the present invention relates to a composition comprising a fusion polypeptide comprising the aforementioned TCRα chain and / or TCRβ chain.

[0032] 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 alpha chain and TCR beta chain.

[0033] In some embodiments, the cell is an immune cell.

[0034] In some embodiments, the immune cell is selected from the group consisting of: antigen presenting cells, B cells, dendritic cells, macrophages, Langerhans cells, T cells, NK cells, NK T cells.

[0035] In a seventh aspect, the present invention relates to a method for preparing specific CTLs targeting PRAME-positive tumors, comprising the following steps:

[0036] 1) Prepare a peptide mixture by using the PRAME (VLDGLDVLL) short peptide;

[0037] 2) adding DCs to the polypeptide mixture of step 1) and culturing them to obtain antigen-presenting cells;

[0038] 3) Co-culturing the antigen-presenting cells in step 2) and peripheral blood mononuclear cells to obtain specific CTLs targeting PRAME-positive tumors.

[0039] In some embodiments, the method for preparing specific CTLs targeting PRAME-positive tumors comprises:

[0040] 1) Prepare a peptide mixture by using the PRAME (VLDGLDVLL) short peptide;

[0041] 2) DCs, the peptide obtained in step 1) and peripheral blood mononuclear cells are directly mixed and co-cultured to obtain specific CTLs targeting PRAME-positive tumors.

[0042] In some embodiments, the method for preparing specific CTLs targeting PRAME-positive tumors comprises:

[0043] 1) Transfect mRNA encoding PRAME amino acids into DC cells;

[0044] 2) The DCs obtained in step 1) are mixed with peripheral blood mononuclear cells and co-cultured to obtain specific CTLs targeting PRAME-positive tumors.

[0045] In an eighth aspect, the present invention relates to a pharmaceutical composition comprising the aforementioned T cell receptor, or the aforementioned fusion protein, or the aforementioned nucleic acid, or the aforementioned vector, or the aforementioned host cell, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0046] The beneficial effect of the present invention is that by screening using the PRAME short peptide, a T cell receptor with high affinity for the PRAME peptide and capable of being stably expressed on the cell membrane is obtained. The T cell receptor can specifically bind to the VLDGLDVLL-HLA A0201 complex and exhibits excellent specific killing activity against antigen-positive tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A diagram showing the results of TCR binding affinity experiments for the PRAME peptide;

[0048] Figure 2 A diagram showing the experimental results of the stability of TCR expression on the cell membrane;

[0049] Figure 3 Graph showing the experimental results of the effect of TCR-overexpressing T cells on the specific IFN-γ secretion of antigen-positive target cells;

[0050] Figure 4 A diagram showing the experimental results of the effect of TCR-overexpressing T cells on the specific IL-2 secretion of antigen-positive target cells;

[0051] Figure 5 A diagram showing the experimental results of the effect of TCR-overexpressing T cells on CD137 expression;

[0052] Figure 6 The figure shows the experimental results of the specific killing activity of T cells overexpressing TCR against antigen-positive tumor cells. DETAILED DESCRIPTION

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0054] As used herein, "CDR" is defined as the complementarity determining region amino acid sequence of a TCR or TCR chain.

[0055] In the context of the present invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0056] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can make up a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art as proteins, of which there are many types).

[0057] As used herein, "vector" may refer to a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

[0058] In some embodiments (Y2358-21), the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, and the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:

[0059] CDR1α-SSYSPS (SEQ ID NO:1);

[0060] CDR2α- YTSAATLV (SEQ ID NO: 2); and

[0061] CDR3α- VVDVGNDMR (SEQ ID NO: 3), and

[0062] The three complementarity determining regions (CDRs) of the TCRβ chain variable domain are:

[0063] CDR1β-SEHNR (SEQ ID NO:4);

[0064] CDR2β-FQNEAQ (SEQ ID NO: 5); and

[0065] CDR3β-ASSKGTERYSPLH (SEQ ID NO:6).

[0066] In some embodiments (Y2383-11), the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, and the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:

[0067] CDR1α-DRGSQS (SEQ ID NO:7);

[0068] CDR2α-IYSNGD (SEQ ID NO:8); and

[0069] CDR3α-AVRGGGSNYKLT (SEQ ID NO: 9), and

[0070] The three complementarity determining regions (CDRs) of the TCRβ chain variable domain are:

[0071] CDR1β-SGHRS (SEQ ID NO:10);

[0072] CDR2β-YFSETQ (SEQ ID NO:11); and

[0073] CDR3β-ASSLASGGYEQY (SEQ ID NO: 12).

[0074] In some embodiments (Y24341-C1), the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, and the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:

[0075] CDR1α-VSGLRG (SEQ ID NO:20);

[0076] CDR2α-LYSAGEE (SEQ ID NO:21); and

[0077] CDR3α- CAVRPVSNSGGYQKVTF (SEQ ID NO: 22), and

[0078] The three complementarity determining regions (CDRs) of the TCRβ chain variable domain are:

[0079] CDR1β- SGHRS (SEQ ID NO:23);

[0080] CDR2β-YFSETQ (SEQ ID NO:24); and

[0081] CDR3β-CASSLGQGAYEQYF (SEQ ID NO:25).

[0082] In some embodiments (Y24342-C2), the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, and the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:

[0083] CDR1α-VSGLRG (SEQ ID NO:28);

[0084] CDR2α-LYSAGEE (SEQ ID NO:29); and

[0085] CDR3α- CAVRSFQKLVF (SEQ ID NO: 30), and

[0086] The three complementarity determining regions (CDRs) of the TCRβ chain variable domain are:

[0087] CDR1β- SGHRS (SEQ ID NO:31);

[0088] CDR2β-YFSETQ (SEQ ID NO:32); and

[0089] CDR3β-CASSLASGGYEQYF (SEQ ID NO:33).

[0090] In some embodiments (Y24342-C3), the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, and the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:

[0091] CDR1α-TRDTTYY (SEQ ID NO:36);

[0092] CDR2α-RNSFDEQN (SEQ ID NO:37); and

[0093] CDR3α- CALSERVRSGSRLTF (SEQ ID NO: 38), and

[0094] The three complementarity determining regions (CDRs) of the TCRβ chain variable domain are:

[0095] CDR1β-DFQATT (SEQ ID NO:39);

[0096] CDR2β-SNEGSKA (SEQ ID NO:40); and

[0097] CDR3β-CSARGAGFYNEQFF (SEQ ID NO:41).

[0098] In some embodiments (Y24351-C5), the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, and the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:

[0099] CDR1α-DRGSQS (SEQ ID NO:44);

[0100] CDR2α-IYSNGD (SEQ ID NO:45); and

[0101] CDR3α- CAVINTGTASKLTF (SEQ ID NO: 46), and

[0102] The three complementarity determining regions (CDRs) of the TCRβ chain variable domain are:

[0103] CDR1β-SNHLY (SEQ ID NO:47);

[0104] CDR2β-FYNNEI (SEQ ID NO:48); and

[0105] CDR3β-CASKWLSSYEQYF (SEQ ID NO:49).

[0106] In some embodiments (Y2358-21), the TCR comprises an α chain variable domain amino acid sequence as shown in SEQ ID NO: 13:

[0107] AQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLLTKPSAHMSDAAEYFCVVDVGNDMRFGAGTRLTVKPN;

[0108] The TCR comprises a β chain variable domain amino acid sequence as shown in SEQ ID NO: 14:

[0109] DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSKGTERYSPLHFGNGTRLTVTE;

[0110] In some embodiments (Y2383-11), the TCR comprises an α chain variable domain amino acid sequence as shown in SEQ ID NO: 15:

[0111] KEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGGGSNYKLTFGKGTLLTVNPN;

[0112] The TCR comprises a β chain variable domain amino acid sequence as shown in SEQ ID NO: 16:

[0113] KAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSLASGGYEQYFPGGTRLTVTE;

[0114] In some embodiments (Y24341-C1), the TCR comprises an α chain variable domain amino acid sequence as shown in SEQ ID NO: 26:

[0115] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVRPVSNSGGYQKVTFGTGTKLQVIP;

[0116] The TCR comprises a β chain variable domain amino acid sequence as shown in SEQ ID NO: 27:

[0117] KAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSLGQGAYEQYFGPGTRLTVT;

[0118] In some embodiments (Y24342-C2), the TCR comprises an α chain variable domain amino acid sequence as shown in SEQ ID NO: 34:

[0119] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVRSFQKLVFGGTRLLVSP;

[0120] The TCR comprises a β chain variable domain amino acid sequence as shown in SEQ ID NO: 35:

[0121] KAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSLASGGYEQYFGPGTRLTVT;

[0122] In some embodiments (Y24342-C3), the TCR comprises an α chain variable domain amino acid sequence as shown in SEQ ID NO:42:

[0123] AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSERVRSGSRLTFGEGTQLTVNP;

[0124] The TCR comprises a β chain variable domain amino acid sequence as shown in SEQ ID NO: 43:

[0125] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARGAGFYNEQFFGPGTRLTVL;

[0126] In some embodiments (Y24351-C5), the TCR comprises an α chain variable domain amino acid sequence as shown in SEQ ID NO:50:

[0127] KEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVINTGTASKLTFGGTRLQVTL;

[0128] The TCR comprises a β chain variable domain amino acid sequence as shown in SEQ ID NO: 51:

[0129] EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASKWLSSYEQYFGPGTRLTVT.

[0130] In some embodiments, the TCR is single-chain.

[0131] In some embodiments, the TCR is composed of an α chain variable region and a β chain variable region connected by a peptide linker sequence.

[0132] In some embodiments, cysteine ​​residues form an artificial disulfide bond between the α and β chain constant domains of the TCR.

[0133] In some embodiments, a conjugate is bound to the C- or N-terminus of the α chain and / or β chain of the TCR. Preferably, the conjugate is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these substances.

[0134] In some embodiments, the TCR is a murine TCR, a human-mouse chimeric TCR, or a humanized TCR.

[0135] In some embodiments, the vector includes an expression vector, i.e., a construct capable of being expressed in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages, and viral vectors.

[0136] In some embodiments, viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes virus vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Preferably, the vector can transfer the nucleic acid of the present invention into cells, such as T cells, such that the cells express a TCR specific for the PRAME antigen. The vector should be capable of sustained, high-level expression in T cells.

[0137] In some embodiments, the lentiviral vector may include: a lentiviral expression vector pLenti (Addgene).

[0138] In some embodiments, the host cell is a mammalian cell. For example, the host cell is a human cell. Although the host cell can be any cell type, can be derived from any type of tissue, and can be at 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.

[0139] In some embodiments, where host cells or a population of related cells are administered, the host cells can be allogeneic to the mammal or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0140] In some embodiments, the term "mammal" refers to any mammal, including but not limited to: mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. Preferably, the mammal is from the order Carnivora, including Felidae (cats) and Canidae (dogs). More preferably, the mammal is from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs), or from the order Perissodactyla, including Equine (horses). Most preferably, the mammal is from the order Primates, Apes, or Monkeys, or from the order Anthropoidea (humans and apes). Particularly preferred is a human.

[0141] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the examples is intended only to facilitate understanding of the method and central concept of the present invention. It should be noted that those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the claims of the present invention.

[0142] Example 1: Cloning of antigen short peptide-specific T cells

[0143] Peripheral blood lymphocytes (PBLs) from healthy volunteers with the HLA-A*02:01 genotype were stimulated with the synthetic peptide PRAME (VLDGLDVLL). These peptides were then annealed with biotinylated HLA-A*02:01 to generate pHLA haploids. These haploids were then combined with PE-labeled streptavidin (BD Biosciences) to form PE-labeled tetramers. These tetramers and anti-CD8-APC double-positive cells were then sorted using a BD Melody flow cytometer, with the positive cells sorted into 96-well plates, one cell per well.

[0144] Example 2: Construction of TCR genes and vectors for obtaining PRAME antigen short peptide-specific T cell clones

[0145] The cells obtained in Example 1 were lysed with 0.1% Triton-X (Sangon) and amplified using the Clontech SMARTRACE cDNA amplification kit with primers designed to target the C-terminal conserved region of the human TCR gene. The downstream primer for the conserved region of the TCR α chain C segment was tcagctggaccacagc (SEQ ID NO:18); the downstream primer for the conserved region of the TCR β chain C segment was aatcctttctcttgaccatggccatc (SEQ ID NO:19). The full-length TCR α and β chain genes were cloned into the lentiviral expression vector pCDH(SBI) using overlap PCR. Specifically, the full-length TCR α and β chain genes were ligated using overlap PCR to generate the TCRα-2A-TCRβ fragment. The lentiviral expression vector and TCRα-2A-TCRβ were digested and ligated to obtain the pCDH-TRA-2A-TRB plasmid, which was then sequenced and confirmed (IMGT) to obtain plasmids for two TCR clones: Y2358-21 and Y2383-11.

[0146] The sequencing results of the CDR1α, CDR2α, and CDR3α of the TCRα chain variable domain of Y2358-21 are SEQ ID NOs: 1-3, respectively; the sequencing results of the CDR1β, CDR2β, and CDR3β of the TCRβ chain variable domain are SEQ ID NOs: 4-6, respectively. The sequencing results of the TCRα chain variable domain are SEQ ID NO: 13, and the sequencing results of the TCRβ chain variable domain are SEQ ID NO: 14.

[0147] The sequencing results of the CDR1α, CDR2α, and CDR3α of the TCRα chain variable domain of Y2383-11 are SEQ ID NOs: 7-9, respectively; the sequencing results of the CDR1β, CDR2β, and CDR3β of the TCRβ chain variable domain are SEQ ID NOs: 10-12, respectively. The sequencing results of the TCRα chain variable domain are SEQ ID NO: 15, and the sequencing results of the TCRβ chain variable domain are SEQ ID NO: 16.

[0148] 293T cells were then used to package pseudoviruses. Specifically, the aforementioned plasmids were mixed with the VSVG plasmid, RRE plasmid, and Rev plasmid (purchased from Addgene) in a ratio of 20:7:13:5, and 20 μg was diluted into 0.5 mL of DMEM medium to form the DNA solution. 60 μL of polyetherimide (PEI 1 μg / μL) was added to 0.5 mL of DMEM, and the PEI / DMEM mixture was added to the prepared DNA solution. After incubation at room temperature for 20 minutes, the cells were added to 293T cells cultured in a 10 cm dish and mixed thoroughly. After 6 hours, the DMEM medium was replaced with fresh DMEM. After 72 hours, the supernatant containing the lentivirus was collected and used as the lentiviral supernatant for each TCR.

[0149] Example 3: Construction of a cell line overexpressing antigen short peptide-specific TCR

[0150] The NFAT-GFP element (Addgene) was synthesized using standard methods described in the Molecular Cloning Laboratory Manual and inserted into the expression vector pCDH(SBI). The fragment was sequenced and confirmed to be correct. Pseudoviruses were then packaged using 293T cells (Pnosai CL-0130). Jurkat cells were infected with pseudoviruses containing the NFAT-GFP element and TCR elements. By limiting dilution and monoclonal expansion, Jurkat-NFAT-GFP-TCR overexpressing cell lines were generated: Jurkat-NFAT-GFP-Y2358-21-TCR and Jurkat-NFAT-GFP-Y2383-11-TCR.

[0151] Example 4: TCR Binding Affinity Experiment for Mutant Peptides and Wild-Type Peptides or Tumor-Associated Antigen Peptides

[0152] (1) Construction of K562CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01

[0153] The HLA-A*02:01 element (IMGT / HLA Acc No: HLA00043) and CD80 (NP_005182.1) were synthesized and inserted into the expression vector pCDH(SBI). The fragments were sequenced and confirmed to be correct. Lentivirus was then packaged using 293T cells. Specifically, the plasmid containing the CD80-HLA-A*02:01 element was mixed with the VSVG plasmid, RRE plasmid, and Rev plasmid (purchased from Addgene) at a ratio of 20:7:13:5. 20 μg of the mixture was diluted into 0.5 mL of DMEM medium to prepare the DNA solution. 20 μL of polyetherimide (PEI 1 μg / μL) was added to 0.5 mL of DMEM. This PEI / DMEM mixture was then added to the prepared DNA solution. After incubation at room temperature for 20 minutes, the mixture was added to 293T cells cultured in a 10 cm dish and mixed thoroughly. After 6 hours, fresh DMEM medium was replaced. After 72 hours, the supernatant containing the lentivirus was collected to obtain the CD80-HLA-A*02:01 lentiviral supernatant. K562 or 293T cell lines were infected with pseudovirus containing the CD80-HLA-A*02:01 element. K562-CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01 overexpressing cell lines were obtained by limiting dilution and monoclonal expansion.

[0154] (2) Jurkat-NFAT-GFP-TCR overexpressing cell line co-cultured with K562-CD80-HLA-A*02:01

[0155] The cell lines (Jurkat-NFAT-GFP-Y2358-21-TCR and Jurkat-NFAT-GFP-Y2383-11-TCR) were co-incubated with K562-CD80-HLA-A*02:01 loaded with different concentrations of the target antigen peptide (PRAME-VLDGLDVLL). Specifically, K562 was incubated with different concentrations of the target antigen peptide in a 37°C incubator for 1 hour. After centrifugation, the cells were resuspended in culture medium. The Jurkat and K562 cells were counted separately, and 2×10 cells of Jurkat and peptide-loaded K562 were aspirated. 4 The cells were mixed and 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.

[0156] The results are as follows Figure 1The results showed that T cells expressing Y2358-21 and Y2383-11 had strong reactivity and specificity to the target antigen peptide PRAME (using AFP antigen peptide as a control), indicating that the relevant T cell receptor proteins are suitable for therapeutic use. In addition, based on similar experimental steps to the above steps, we also verified that T cells expressing other TCRs had strong reactivity and specificity to the antigen peptide PRAME (Y24341-C1, Y24342-C2, Y24342-C3, Y24351-C5) using similar methods and obtained similar results (see for details). Figure 1 ).

[0157] Example 5: Stability experiment of overexpressed TCR on cell membrane

[0158] TCR-T cells (Y23193 and Y23194) expressing TCRs were constructed using the TCR elements (TCR α-2A-TCR β fragments) of Y2358-21 and Y2383-11, respectively. The specific steps are as follows:

[0159] (1) Preparation of TCR lentivirus

[0160] Each TCR component and GFP (Addgene) were synthesized using standard methods described in the Molecular Cloning a Laboratory Manual (ISBN 978-1-936113-42-2; Chapter 3: Cloning and Transformation with Plasmid Vectors) and inserted into the expression vector pCDH(SBI). Sequencing confirmed the fragments. Pseudoviruses were then packaged using 293T cells (Pnosai CL-0130) according to the procedures described in Example 2.

[0161] (2) Construction of TCR-T cells expressing TCR

[0162] After thawing PBMCs, use an appropriate amount of X-VIVO 15 medium containing 100 IU / mL rhIL-2 to a density of 1 × 10^6 cells / mL. For every 2 × 10^6 cells, add 10 μL of MACS CD3 / CD28 T cell TransAct beads in X-VIVO 15 medium containing 100 IU / mL rhIL-2, mix gently, and culture in a cell culture incubator. After 24 hours, the cells were centrifuged and the supernatant was decanted 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 was added (infection at an MOI of 10) based on the total number of cells and the measured viral titer. A control group of cells was also set up without lentivirus. The culture medium of each cell was replenished to a final volume of 2 mL with X-VIVO 15 medium containing 100 IU / mL IL-2. Polybrene was added to a final concentration of 10 μg / mL. The cells were centrifuged at 2000 g for 60 minutes at 37°C and the infected cells were placed in a CO2 incubator for 24 hours. Fresh medium was replaced regularly and the cell density was adjusted. The cells were cultured until day 14.

[0163] 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.

[0164] Example 6: Specific IFN-γ and IL-2 secretion by TCR-overexpressing T cells against antigen-positive target cells

[0165] 1. Use TCR-expressing T cells (same as in Example 5) as effector cells, and culture PBMCs that have not been transduced with TCR in parallel as effector cell controls.

[0166] 2. K562-CD80-HLA-A*02:01 cells loaded with 10^-7 M of the PRAME (VLDGLDVLL) short peptide or an unrelated peptide were used as positive target cells (same as in Example 4); the E:T effector-target ratio (effector cell: target cell ratio) was 1:1. After 24 hours of incubation, the expression of IL-2 and / or IFN-γ in the cells was measured.

[0167] 3. K562-CD80-HLA-A*02:01 cells loaded with PRAME (VLDGLDVLL) peptide at concentrations ranging from 10 M to 10 M were used as positive target cells (same as in Example 4); the E:T effector-target ratio (effector cell: target cell ratio) was 1:1, and the expression of CD137 on the cells was detected after 24 hours of incubation.

[0168] The results showed that in the presence of positive target cells, the TCR-T overexpression group produced IL-2 and IFN-γ and upregulated CD137 expression, while the TCR-T overexpression group did not produce IFN-γ or IL-2 in response to negative target cells. Some results of IL-2 and INF-γ expression and secretion are shown in Figure 3 and Figure 4 Some of the results of CD137 upregulation are shown in Figure 5 The above data show that: the relevant T cells overexpressing TCR have specific activation efficacy on antigen-positive target cells, and the TCR protein obtained by the present invention is suitable for use for therapeutic purposes.

[0169] Example 7: Specific cytotoxicity of TCR-overexpressing T cells against antigen-positive tumor cells

[0170] 1. T cells expressing TCR (same as in Example 5) were used as effector cells, and PBMCs not transduced with TCR were expanded and cultured in parallel as effector cell controls (Mock).

[0171] 2. 293T-CD80-HLA-A*02:01 (same as in Example 5) loaded with 10^-9M~10^-6M PRAME (VLDGLDVLL) short peptide was used as the positive target cell (+); the E:T effector target (effector cell: target cell ratio) ratio was 10:1, and the target cell adhesion ability was detected in real time using an RTCA (real-time label-free cell analysis system that integrates a microelectronic cell sensor chip into the bottom of a cell detection plate and obtains biological information related to cell physiological functions, including cell growth, extension, morphological changes, death, and adhesion, through real-time dynamic electrode impedance detection). Specifically, the instrument collected the cell adhesion ability value (Cell Index) for each well every 15 minutes. In subsequent data processing, the data at the last time point before the addition of T cells was used as the normalized value, and the normalized cell adhesion ability value (Normalized Cell Index) at each time point in each group was calculated; the results are shown (see Figure 6 ): The TCR-T overexpression group only had significant killing activity against 293T-CD80-HLA-A*02:01 loaded with tumor-associated antigen peptides, and had no killing effect on 293T-CD80-HLA-A*02:01 loaded with irrelevant peptides, among which Y23193 had a better killing effect.

Claims

1. A T cell receptor targeting a PRAME peptide, wherein the sequence of the PRAME peptide is VLDGLDVLL, and the T cell receptor comprises a TCR α chain variable domain and a TCR β chain variable domain, in, The TCR α chain variable domain comprises: CDR1α with the sequence SSYSPS; CDR2α of YTSAATLV; and CDR3α with the sequence VVDVGNDMR, Wherein, the TCRβ chain variable domain comprises: The sequence is CDR1β of SEHNR; CDR2β of sequence FQNEAQ; and The CDR3β sequence is ASSKGTERYSPLH.

2. The T cell receptor according to claim 1, wherein The T cell receptor is soluble.

3. The T cell receptor according to claim 1, wherein An artificial disulfide bond is contained between the α chain constant region and the β chain constant region of the T cell receptor.

4. The T cell receptor according to claim 1, in, The sequence of the TCRα chain variable domain is: AQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCVVDVGNDMRFGAGTRLTVKPN, Wherein, the sequence of the TCRβ chain variable domain is: DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSKGTERYSPLHFGNGTRLTVTE.

5. A synthetic nucleic acid molecule encoding the T cell receptor according to claim 1. A vector comprising the synthetic nucleic acid molecule according to claim 5 . A host cell comprising the vector according to claim 6 .

8. A method for preparing a T cell receptor, comprising: (i) culturing the host cell according to claim 7 to express the T cell receptor according to claim 1; as well as (ii) isolating the T cell receptor.

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