A specific t cell receptor for gp100 peptide and uses thereof

Abstract

The application belongs to the technical field of biological medicine and tumor immunotherapy, and particularly relates to a specific T cell receptor for GP100 peptide and application thereof. Experiments show that the T cell receptor provided by the application has high GP100 antigen peptide recognition specificity and immune response activity, can effectively activate effector T cells and induce the killing effect of the effector T cells on GP100 positive tumor cells, is suitable for prevention, diagnosis, auxiliary diagnosis or treatment of melanoma and other GP100 related tumors, and is particularly suitable for adoptive cell immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a specific T-cell receptor for GP100 peptide and its applications. Background Technology

[0002] Malignant tumors pose a serious threat to human life and health. Traditional treatments such as surgery, radiotherapy, and chemotherapy can control tumor progression to some extent, but they generally suffer from drawbacks such as high recurrence rates, significant toxic side effects, and limited efficacy for patients with advanced stages. In recent years, tumor immunotherapy has become an important development direction in the field of tumor treatment because it can mobilize the body's own immune system to recognize and eliminate tumor cells. Among them, adoptive cell therapy based on T cell receptors (TCRs) has shown broad application prospects in the treatment of solid tumors because it can recognize intracellular tumor antigen peptides presented by human leukocyte antigen (HLA) molecules, overcoming the limitation that chimeric antigen receptor T cells mainly recognize cell surface antigens.

[0003] GP100 (glycoprotein 100, also known as PMEL) is an important tumor-associated antigen gene, first discovered in melanoma. GP100 belongs to the melanoma differentiation antigen family and is abnormally highly expressed in various malignant tumors (especially melanoma), while in most normal tissues it is limited to melanocyte expression. GP100 protein products can be recognized by cytotoxic T lymphocytes (CD8+) and induce an immune response, thus it has long been considered an ideal target for immunotherapy. However, traditional GP100 antigens are mostly wild-type peptides, which are prone to cross-reaction with GP100 protein expressed in normal melanocytes, potentially leading to off-target toxicity and safety risks.

[0004] Currently, TCR-T products targeting other tumor antigens (such as MAGE-A4) (e.g., Afami-cel) have received regulatory approval for specific indications, demonstrating the clinical potential of TCR-T therapy. However, most TCR-T products targeting GP100 are still in the early stages of clinical research, and TCRs with high affinity and high specificity for GP100 antigen peptides have not yet been systematically developed and applied. Therefore, there is an urgent need to provide a TCR molecule that can specifically recognize GP100 antigen peptides (especially binding to common alleles such as HLA-A*02:01) and use it to construct TCR-T cells, with the aim of providing a safe and effective new personalized immunotherapy option for patients with GP100 antigen-positive tumors. Summary of the Invention

[0005] The technical problem that this invention aims to solve is that there are currently few treatment options for GP100 (melanoma antigen gene) and their effects are unsatisfactory. Patients need more and more effective products to meet their diverse clinical needs.

[0006] In a first aspect, the present invention relates to a specific T-cell receptor (TCR) targeting the GP100 peptide, the GP100 peptide having the sequence KTWGQYWQV, comprising a TCR α-chain variable region and a TCR β-chain variable region, wherein:

[0007] The variable region of the TCR α chain includes the following complementarity-determining regions (CDRs):

[0008] CDR1α, amino acid sequence is TRDTTYY;

[0009] CDR2α, with the amino acid sequence RNSFDEQN; and

[0010] CDR3α, amino acid sequence is ALSEADRGSTLGRLY;

[0011] The variable region of the TCR β chain includes the following complementarity-determining regions (CDRs):

[0012] CDR1β, amino acid sequence is KGHDR;

[0013] CDR2β, amino acid sequence is SFDVKD; and

[0014] CDR3β, amino acid sequence is ATSDLAGANRDTQY.

[0015] In some implementations, the T-cell receptor is soluble.

[0016] In some implementations, the T-cell receptor exists in a free form.

[0017] In some implementations, the T-cell receptor is further covalently or non-covalently linked to a detectable marker, therapeutic molecule.

[0018] In some implementations, the T-cell receptor is modified with polyethylene glycol (PEG) to improve its in vivo stability or pharmacokinetic properties.

[0019] In some implementations, engineered disulfide bonds are introduced into the α-chain constant region and β-chain constant region of the T-cell receptor.

[0020] In some implementations, the sequence of the variable region of the TCRα chain is as follows:

[0021] AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEADRGSTLGRLYFGRGTQLTVWP;

[0022] The sequence of the variable region of the TCRβ chain is:

[0023] DADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSDLAGANRDTQYFPGGTRLTVL.

[0024] In some implementations, the T-cell receptor is a fusion protein.

[0025] Secondly, the present invention provides a synthetic nucleic acid molecule that encodes the T-cell receptor of the first aspect of the present invention.

[0026] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecules synthesized by the present invention.

[0027] Fourthly, the present invention provides a host cell containing the recombinant vector of the present invention.

[0028] In some embodiments, the present invention also provides a host cell in which the synthetic nucleic acid molecules of the present invention are integrated into the chromosome of the host cell.

[0029] Fifthly, the present invention provides the use of the aforementioned T-cell receptor, or synthetic nucleic acid molecule, or recombinant vector or host cell, in the preparation of a drug for treating cancer.

[0030] Sixthly, the present invention provides a method for preparing a T-cell receptor, comprising:

[0031] (1) Expression of the aforementioned T cell receptor in the aforementioned host cells; and

[0032] (2) Isolate the aforementioned T cell receptor from the aforementioned host cells or their cultures.

[0033] In a seventh aspect, the present invention provides a composition comprising a fusion polypeptide comprising the aforementioned TCRα chain and / or TCRβ chain.

[0034] In some embodiments, cells are genetically modified by introducing isolated nucleic acid molecules encoding peptides, wherein the peptides contain at least one of the aforementioned TCRα and TCRβ chains.

[0035] In some implementations, the cells are immune cells.

[0036] In some implementation schemes, immune cells are selected from the group consisting of: antigen-presenting cells, B cells, dendritic cells, macrophages, Langerhans cells, T cells, NK cells, and NK T cells.

[0037] Eighthly, the present invention also relates to the use of the aforementioned T-cell receptor or synthetic nucleic acid molecule or recombinant vector or host cell in the preparation of a medicament for treating GP100-positive cancers.

[0038] In some embodiments, the GP100-positive cancer refers to cancer in which tumor cells present GP100-derived peptide-MHC complexes on their surface, including but not limited to:

[0039] Primary, recurrent, or metastatic melanomas expressing the GP100 protein (also known as PMEL, ME20, or HMB-45 antigen), such as cutaneous melanoma (including superficial, nodular, acral, mucosal, and other subtypes), uveal melanoma, and other rare melanoma subtypes; and other GP100-positive tumors, such as clear cell sarcoma.

[0040] In some implementations, the drug may be suitable for administration via any appropriate route, preferably parenteral (including subcutaneous, intramuscular, or preferably intravenous) route.

[0041] In a ninth aspect, the present invention also relates to a method for treating cancer in a patient, comprising administering to the patient the T-cell receptor of the present invention, a synthetic nucleic acid molecule, a recombinant vector, a host cell, or a drug.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) This invention successfully obtained T cell receptors that can specifically recognize GP100-A10 antigen by combining antigen peptide-HLA tetramer sorting with single-cell cloning technology, realizing precise screening of antigen-specific TCRs and improving the specificity and reliability of TCR sources.

[0044] (2) The TCRα chain and β chain obtained by this invention can be correctly paired and stably expressed in T cells, with good engineering modification performance and expression stability, and are suitable for the construction and large-scale preparation of TCR-T cells.

[0045] (3) The T cell receptor provided by the present invention has a highly specific recognition ability for the GP100-A10 antigen-HLA complex and does not react to unrelated antigens, thereby minimizing the potential off-target risk.

[0046] (4) The TCR-T cells constructed in this invention can effectively induce T cell activation and significantly secrete effector cytokines such as IL-2 and IFN-γ under antigen stimulation, while upregulating activation marker molecules, indicating that they have complete and effective immune functions.

[0047] (5) The TCR-T cells of the present invention can produce significant and antigen-dependent killing effects on antigen-positive tumor cells, while having no obvious killing activity on antigen-negative cells, demonstrating good tumor selectivity and safety, and have broad application prospects in tumor immunotherapy. Attached Figure Description

[0048] Figure 1 The figure shows the results of the binding affinity experiment of TCR to GP100 peptide.

[0049] Figure 2 This figure shows the results of experiments on the stability of TCR expression in the cell membrane.

[0050] Figure 3 Figure showing the experimental results of the effect of T cells overexpressing TCR on the specific IFN-γ secretion of antigen-positive target cells.

[0051] Figure 4 The figure shows the experimental results of the effect of T cells overexpressing TCR on the specific IL-2 secretion of antigen-positive target cells.

[0052] Figure 5 The figure shows the experimental results of the effect of T cells overexpressing TCR on CD137 expression.

[0053] Figure 6 This figure illustrates the experimental results of the specific killing activity of T cells overexpressing TCR against antigen-positive tumor cells. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0055] As used in this article, “CDR” is defined as the amino acid sequence of the complementarity-determining region of the TCR or TCR chain.

[0056] In the context of this invention, the following abbreviations for common nucleic acid bases are used: “A” for adenosine, “C” for cytidine, “G” for guanosine, “T” for thymidine, and “U” for uridine.

[0057] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting 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 to the maximum number of amino acids that can make up a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together 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).

[0058] As used in this article, "vector" can refer to a nucleic acid sequence containing an origin of replication. Vectors can be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA or RNA vectors. Vectors can be self-replicating extrachromosomal vectors or vectors integrated into the host genome.

[0059] The TCRs of the present invention may be non-naturally occurring and / or purified and / or engineered. Relative to the parental TCR, the TCRs of the present invention may have more than one mutation present in the α-chain variable region and / or the β-chain variable region. "Engineered TCR" and "mutated TCR" are used synonymously herein and generally refer to a TCR having one or more introduced mutations relative to the parental TCR, particularly in its α-chain variable region and / or β-chain variable region. These mutations can improve binding affinity against KTWGQYWQV (SEQ ID NO:35) complexed with HLA-A*02. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 mutations are present in the α-chain variable region, for example, 4 or 8 mutations, and / or 1, 2, 3, 4, or 5 mutations are present in the β-chain variable region, for example, 5 mutations.

[0060] In some implementations, the TCR includes a TCR α-chain variable region and a TCR β-chain variable region, and the three complementarity-determining regions (CDRs) of the TCR α-chain variable region are:

[0061] CDR1α-DSSSTY (SEQ ID NO:1);

[0062] CDR2α-IFSNMDM (SEQ ID NO:2); and

[0063] CDR3α-AENRDDKII (SEQ ID NO:3), and

[0064] The three complementarity-determining regions (CDRs) of the TCRβ chain variable region are:

[0065] CDR1β-SGHVS (SEQ ID NO:4);

[0066] CDR2β-FNYEAQ (SEQ ID NO:5); and

[0067] CDR3β-ASSLGGGGDIQY (SEQ ID NO:6).

[0068] In some implementations, the TCR includes a TCR α-chain variable region and a TCR β-chain variable region, and the three complementarity-determining regions (CDRs) of the TCR α-chain variable region are:

[0069] CDR1α-DSSSTY (SEQ ID NO:7);

[0070] CDR2α-IFSNMDM (SEQ ID NO:8); and

[0071] CDR3α-AESEAGNMLT (SEQ ID NO:9), and

[0072] The three complementarity-determining regions (CDRs) of the TCR β chain variable region are:

[0073] CDR1β-DFQATT (SEQ ID NO:10);

[0074] CDR2β-SNEGSKA (SEQ ID NO:11); and

[0075] CDR3β-SASQRGDEQF (SEQ ID NO: 12).

[0076] In some implementations, the TCR includes a TCR α-chain variable region and a TCR β-chain variable region, and the three complementarity-determining regions (CDRs) of the TCR α-chain variable region are:

[0077] CDR1α-TRDTTYY (SEQ ID NO:13);

[0078] CDR2α-RNSFDEQN (SEQ ID NO:14); and

[0079] CDR3α-ALSEADRGSTLGRLY (SEQ ID NO:15), and

[0080] The three complementarity-determining regions (CDRs) of the TCR β chain variable region are:

[0081] CDR1β-KGHDR (SEQ ID NO:16);

[0082] CDR2β-SFDVKD (SEQ ID NO:17); and

[0083] CDR3β-ATSDLAGANRDTQY (SEQ ID NO: 18).

[0084] In some implementations, the TCR includes a TCR α-chain variable region and a TCR β-chain variable region, and the three complementarity-determining regions (CDRs) of the TCR α-chain variable region are:

[0085] CDR1α-VSPFSN (SEQ ID NO:19);

[0086] CDR2α-MTFSENT (SEQ ID NO:20); and

[0087] CDR3α-VVSADLEYGNKLV (SEQ ID NO:21), and

[0088] The three complementarity-determining regions (CDRs) of the TCR β chain variable region are:

[0089] CDR1β-MNHNY (SEQ ID NO:22);

[0090] CDR2β-SVGAGI (SEQ ID NO:23); and

[0091] CDR3β-ASSYELAGPSTDTQY (SEQ ID NO:24).

[0092] In some embodiments (Y25054-X2Y3), the TCR includes the α-chain variable region amino acid sequence as shown in SEQ ID NO:25:

[0093] GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAENRDDKIIFGKGTRLHILP.

[0094] Furthermore, the TCR contains the β-chain variable region amino acid sequence as shown in SEQ ID NO:26:

[0095] GAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSLGGGGDIQYFGAGTRLSVL.

[0096] In some embodiments (Y25054-X3Y2), the TCR includes the α-chain variable region amino acid sequence as shown in SEQ ID NO:27:

[0097] GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESEAGNMLTFGGGTRLMVKP.

[0098] Furthermore, the TCR contains the β-chain variable region amino acid sequence as shown in SEQ ID NO:28:

[0099] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASQRGDEQFFGPGTRLTVL.

[0100] In some embodiments (Y25055-C3), the TCR includes the α-chain variable region amino acid sequence as shown in SEQ ID NO:29:

[0101] AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEADRGSTLGRLYFGRGTQLTVWP.

[0102] Furthermore, the TCR contains the β-chain variable region amino acid sequence as shown in SEQ ID NO:30:

[0103] DADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSDLAGANRDTQYFPGGTRLTVL.

[0104] In some embodiments (Y25054-X1Y1), the TCR includes the α-chain variable region amino acid sequence as shown in SEQ ID NO:31:

[0105] KNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSADLEYGNKLVFGAGTILRVKS.

[0106] Furthermore, the TCR contains the β-chain variable region amino acid sequence as shown in SEQ ID NO:32:

[0107] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYELAGPSTDTQYFPGGTRLTVL.

[0108] In some embodiments, the α-chain variable region of the TCR of the present invention may contain an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid residue sequences shown in sequences such as SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31. In some embodiments, the β-chain variable region of the TCR of the present invention may contain an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid residue sequences shown in sequences such as SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32.

[0109] In some implementations, the TCR is single-stranded.

[0110] In some implementations, the TCR is formed by linking the α-chain variable region and the β-chain variable region through a peptide linker sequence.

[0111] In some implementations, cysteine ​​residues form artificial disulfide bonds between the α and β chain constant regions of the TCR.

[0112] In some embodiments, the C- or N-terminus of the α-chain and / or β-chain of the TCR is bound with a conjugate, preferably a detectable marker, a therapeutic agent, a PK-modified moiety, or any combination of these substances.

[0113] In some embodiments, therapeutic agents that can bind to the TCR of the present invention include immunomodulators, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure toxicity at the desired site, the therapeutic agent can be contained within a liposome linked to the TCR, allowing for slow release. This prevents damaging effects during in vivo transport and ensures maximum efficacy of the therapeutic agent after the TCR binds to the relevant antigen-presenting cells.

[0114] In some implementations, the TCR is a mouse-derived TCR, a human-mouse chimeric TCR, or a humanized TCR.

[0115] In some implementations, 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.

[0116] In some embodiments, the viral vector includes, but is not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Preferably, the vector can transfer the nucleotides of the present invention into cells, such as T cells, causing the cells to express GP100 antigen-specific TCRs. The vector should be able to sustain high levels of expression in T cells.

[0117] In some implementations, the lentiviral vector may include: the lentiviral expression vector pLenti (addgene).

[0118] 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 originate from any type of tissue, and can be a cell at any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0119] In some embodiments, when host cells or related cell populations are administered, the host cells may be allogeneic or autologous to mammals. Preferably, the cells are autologous to mammals.

[0120] In some embodiments, mammal refers to any mammal, including but not limited to: rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits; preferably, the mammal is from the order Carnivora, including felines (cats) and canids (dogs). More preferably, the mammal is from the order Artiodactyla, including bovines (cattle) and suidae (pigs), or from the order Perissodactyla, including equines (horses); most preferably, the mammal is from the order Primates, apes, or monkeys, or from the suborder Anthropoidea (humans and apes). Particularly preferred is the mammal being human.

[0121] In some embodiments, the TCR, drug, recombinant vector, synthetic nucleic acid molecule and host cell of the present invention can be provided in a substantially pure form, for example at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% purity.

[0122] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0123] Example 1: Cloning antigen short peptide-specific T cells

[0124] Peripheral blood lymphocytes (PBLs) from healthy volunteers with the HLA-A*02:01 genotype were stimulated using the synthetic short peptide Genescript GP100-A10 (KTWGQYWQV, SEQ ID NO:35). The short peptide was then annealed with biotin-labeled HLA-A*02:01 to prepare pHLA haploids. These haploids were combined with PE-labeled streptavidin (BD Biosciences) to form PE-labeled tetramers. The tetramers and anti-CD8-APC double-positive cells were sorted using a BD Melody flow cytometer, with one positive cell per well in each 96-well plate.

[0125] Example 2: Construction of TCR gene and vector for antigen-specific T cell clones

[0126] Cells obtained in Example 1 were lysed using 0.1% Triton-X (Sangon Biotech), and amplified using the Clontech SMARTRACE cDNA amplification kit. Primers were designed for the conserved C-terminal region of the human TCR gene. The downstream primer for the conserved C-terminal region of the TCR α chain was tcagctggaccacagc (SEQ ID NO:33); the downstream primer for the conserved C-terminal region of the TCR β chain was tcagaaatcctttctcttgac (SEQ ID NO:34). The full-length TCR α and β chains were cloned into the lentiviral expression vector pCDH(SBI) using overlap PCR. Specifically, the full-length TCR α and β chains were ligated using overlap PCR to obtain the TCRα-2A-TCR fragment. The lentiviral expression vector was digested and ligated with TCRα-2A-TCRβ to obtain the pCDH-TRA-2A-TRB plasmid, which was then sequenced and confirmed (IMGT) to obtain plasmids of four TCR clones: Y25054-X2Y3, Y25054-X3Y2, Y25055-C3, and Y25054-X1Y1.

[0127] The sequencing results of CDR1α, CDR2α, and CDR3α of the TCRα chain variable region of Y25054-X2Y3 are SEQ ID NO:1-3, respectively; the sequencing results of CDR1β, CDR2β, and CDR3β of the TCRβ chain variable region are SEQ ID NO:4-6, respectively. The sequencing result of the TCRα chain variable region is SEQ ID NO:25, and the sequencing result of the TCRβ chain variable domain is SEQ ID NO:26.

[0128] The sequencing results of CDR1α, CDR2α, and CDR3α of the TCRα variable region of Y25054-X3Y2 are SEQ ID NO:7-9, respectively; the sequencing results of CDR1β, CDR2β, and CDR3β of the TCRβ variable region are SEQ ID NO:10-12, respectively. The sequencing result of the TCRα variable region is SEQ ID NO:27, and the sequencing result of the TCRβ variable region is SEQ ID NO:28.

[0129] The sequencing results of CDR1α, CDR2α, and CDR3α of the TCRα variable region of Y25055-C3 are SEQ ID NO:13-15, respectively; the sequencing results of CDR1β, CDR2β, and CDR3β of the TCRβ variable region are SEQ ID NO:16-18, respectively. The sequencing result of the TCRα variable region is SEQ ID NO:29, and the sequencing result of the TCRβ variable region is SEQ ID NO:30.

[0130] The sequencing results of CDR1α, CDR2α, and CDR3α of the TCRα variable region of Y25054-X1Y1 are SEQ ID NO:19-21, respectively; the sequencing results of CDR1β, CDR2β, and CDR3β of the TCRβ variable region are SEQ ID NO:22-24, respectively. The sequencing result of the TCRα variable region is SEQ ID NO:31, and the sequencing result of the TCRβ variable region is SEQ ID NO:32;

[0131] The pseudoviruses were then packaged using 293T cells. Specifically, the plasmids were mixed with VSVG, RRE, and Rev (Addgene) plasmids in a ratio of 4:5:4:10, and 20 μL of each mixture was diluted in 1.25 mL of DMEM medium to prepare the DNA solution. 20 μL of polyetherimide (PEI 1 µg / μL) was added to 1.25 mL of DMEM, and the PEI / DMEM mixture was added to the prepared DNA solution. After incubation at room temperature for 20 minutes, the mixture was added to 293T cells cultured in 15 cm dishes and mixed thoroughly. After 6 hours, the DMEM medium was replaced with fresh medium. After 72 hours, the supernatant containing the lentivirus was collected; this was the lentivirus supernatant for each TCR.

[0132] Example 3: Construction of a cell line overexpressing antigen short peptide-specific TCR

[0133] The NFAT-GFP element (Addgene) was synthesized and inserted into the expression vector pCDH(SBI) using the standard methods described in *Molecular Cloning Laboratory Manual*. The fragment was confirmed to be correct by sequencing. Then, pseudoviruses were packaged using 293T (Pronosai CL-0130). Jurkat cell lines were infected with pseudoviruses containing the NFAT-GFP element and pseudoviruses containing the TCR element. Through limiting dilution and single-clonal amplification, Jurkat-NFAT-GFP-TCR overexpressing cell lines were obtained, namely: Jurkat-NFAT-GFP-Y25054-X2Y3-TCR, Jurkat-NFAT-GFP-Y25054-X3Y2-TCR, Jurkat-NFAT-GFP-Y25055-C3-TCR, and Jurkat-NFAT-GFP-Y25054-X1Y1-TCR.

[0134] Example 4: Binding Affinity Experiment

[0135] (1) Construction of K562-CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01

[0136] 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 fragment was confirmed to be correct after sequencing. Lentiviral virus was then packaged using the 293T cell line. Specifically, the plasmid containing the CD80-HLA-A*02:01 element was mixed with VSVG plasmid, RRE plasmid, and Rev plasmid (purchased from Addgene) at a ratio of 4:5:4:10, and 20 μL was diluted in DMEM medium (1.25 mL) to prepare the DNA solution. 20 μL of polyetherimide (PEI 1 µg / µL) was added to DMEM (1.25 mL), and the PEI / DMEM mixture was added to the prepared DNA solution. After incubation at room temperature for 20 minutes, the mixture was added to 293T cells cultured in 15 cm plates and mixed thoroughly. Six hours later, the medium was replaced with fresh DMEM. After 72 hours, the supernatant containing the lentivirus was collected, which was the lentivirus supernatant containing CD80-HLA-A*02:01. K562 or 293T cell lines were infected with pseudoviruses containing the CD80-HLA-A*02:01 element. Through limiting dilution and monoclonal amplification, K562-CD80-HLA-A*02:01 overexpressing cell lines and 293T-CD80-HLA-A*02:01 were obtained.

[0137] (2) Jurkat-NFAT-GFP-TCR overexpressing cell lines were co-cultured with K562-CD80-HLA-A*02:01

[0138] Cell lines (Jurkat-NFAT-GFP-Y25054-X2Y3-TCR, Jurkat-NFAT-GFP-Y25054-X3Y2-TCR, Jurkat-NFAT-GFP-Y25055-C3-TCR, and Jurkat-NFAT-GFP-Y25054-X1Y1-TCR) were co-incubated with K562-CD80-HLA-A*02:01 cells loaded with different concentrations of the target antigen peptide GP100-A10 (KTWGQYWQV, SEQ ID NO:35). Specifically, K562 cells were incubated with different concentrations of the target antigen peptide at 37°C for 1 h. After centrifugation, the cells were resuspended in culture medium. Jurkat and K562 cells were counted separately, and 2 × 10⁶ cells each of Jurkat and peptide-loaded K562 cells were aspirated. 4 Cells were mixed and co-cultured in 96-well plates. After 24 hours of co-culture, flow cytometry was used to detect the activation levels of Jurkat cell reporter genes and CD69 cell activation levels.

[0139] The results are as follows Figure 1The results showed that TCR-T cells expressing Y25054-X2Y3, Y25054-X3Y2, Y25055-C3, and Y25054-X1Y1 exhibited strong reactivity and specificity to the target antigen peptide GP100-A10, but showed no response to the unrelated peptide (MAGE-A1). Therefore, they are well-suited for the use of related T cell receptor proteins for diagnostic and therapeutic purposes.

[0140] Example 5: Stability Experiment

[0141] TCR-T cells expressing TCR were constructed using TCR elements (TCR α-2A-TCR β fragments) from Y25054-X2Y3, Y25054-X3Y2, and Y25055-C3, respectively. The specific steps are as follows:

[0142] (1) Preparation of TCR lentivirus: Each TCR element and GFP (addgene) were synthesized and inserted into the expression vector pCDH (SBI) using the standard method described in Molecular Cloning Laboratory Manual. The fragments were confirmed to be correct after sequencing. Then, pseudoviruses were packaged using 293T (Pronosai CL-0130) according to the specific operation steps in Example 2.

[0143] (2) Construction of TCR-T cells expressing TCR: After thawing PBMCs, they were cultured in an appropriate amount of X-VIVO 15 medium containing 100 IU / mL rhIL-2 to a density of 1×10⁻⁶ cells / mL. 6 / mL. Every 2×10 6 Cells were added to 10 μL of MACS CD3 / CD28 T cellTransAct beads in X-VIVO15 medium containing 100 IU / mL rhIL-2, gently mixed, and cultured 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. Target lentivirus (with an MOI of 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 to a final volume of 2 mL of X-VIVO 15 medium containing 100 IU / mL IL-2, and polybrene was added to a final concentration of 10 μg / mL. The cells were plated and centrifuged at 37°C, 2000g for 60 minutes. The infected cells were then cultured in a CO2 incubator for 24 hours. The medium was changed periodically and the cell density was adjusted until day 14.

[0144] The expression rate of TCR-T was detected using GP100 Tetramer. The results showed (see...). Figure 2 Each TCR can be combined with GP100 Tetramer.

[0145] Example 6: Specific IFN-γ and IL-2 secretion assays of TCR-T cells overexpressing antigen-positive target cells

[0146] 1. T cells expressing TCR (same as in Example 5) were used as effector cells, and PBMCs that did not transduce TCR were expanded and cultured in parallel as control effector cells.

[0147] 2. Using a load of 10 -7 M's GP100 short peptide (KTWGQYWQV) or unrelated peptide K562-CD80-HLA-A*02:01 were used as positive target cells (same as in Example 4); the E:T effector-target ratio (ratio of effector cells to target cells) was 1:1, and the expression of IL-2 and / or IFN-γ in cells was detected after 24 h of incubation.

[0148] 3. Using a load of 10 -11 M to 10 -5 M. Different concentrations of GP100 short peptide (KTWGQYWQV) K562-CD80-HLA-A*02:01 were used as positive target cells (same as in Example 4); E:T effector-target ratio (ratio of effector cells to target cells) was 1:1, and the expression of CD137 in cells was detected after 24 h of incubation.

[0149] The results showed that in the presence of positive target cells, the TCR-T overexpression group produced IL-2 and IFN-γ, and CD137 expression was upregulated, while the TCR-T overexpression group did not produce IFN-γ or IL-2 in the presence of negative target cells. Partial results of IL-2 and IFN-γ expression and secretion are shown in [the table below]. Figure 3 and Figure 4 The results of CD137 upregulation are shown in the table below. Figure 5 The above data indicate that T cells overexpressing TCR have a specific activation effect on antigen-positive target cells, and the TCR protein obtained in this invention is suitable for use for diagnostic and therapeutic purposes.

[0150] Example 7: Specific killing activity of TCR-T cells overexpressing against antigen-positive tumor cells

[0151] 1. T cells expressing TCR (same as in Example 5) were used as effector cells, and PBMCs that were not transduced with TCR were expanded and cultured in parallel as a control (blank).

[0152] 2. Using a load of 10 -10 M to 10 -8M's GP100 (KTWGQYWQV) short peptide 293T-CD80-HLA-A*02:01 (same as Example 5) was used as a positive target cell (+); the E:T effector-target ratio (effector cell:target cell ratio) was 10:1. The adhesion ability of target cells was detected in real time using RTCA (Real-Time Label-Free Cell Analysis System). Specifically, the instrument collected cell adhesion ability values ​​(Cell Index) for each well every 15 minutes. In subsequent data processing, the data from the last time point before the addition of T cells was used as the normalized value to calculate the normalized cell adhesion ability value (Normalized Cell Index) for each group at each time point. The results show (see...) Figure 6 The TCR-T overexpression group showed significant killing activity only against 293T-CD80-HLA-A*02:01 loaded with tumor-associated antigen peptides, but no killing effect on 293T-CD80-HLA-A*02:01 without peptide loading. Among them, Y25055-C3 showed better killing effect.

[0153] In summary, the GP100-A10 specific T cell receptor obtained by this invention can be stably expressed in T cells and exhibits a high degree of specificity in recognizing the target antigen-HLA complex. It can effectively induce T cell activation, cytokine secretion, and tumor cell-specific killing effects, thus demonstrating that the TCR molecule of this invention has significant application value and unexpected technical effects in the fields of tumor immunotherapy and related diagnostics.

Claims

1. A specific T-cell receptor targeting GP100 peptide, characterized in that, The sequence of the GP100 peptide is KTWGQYWQV. The T cell receptor includes the TCR α chain variable region and the TCR β chain variable region. Wherein: the variable region of the TCR α chain includes: CDR1α, amino acid sequence is TRDTTYY; CDR2α, with the amino acid sequence RNSFDEQN; and CDR3α, amino acid sequence is ALSEADRGSTLGRLY; The variable region of the TCR β chain includes: CDR1β, amino acid sequence is KGHDR; CDR2β, amino acid sequence is SFDVKD; and CDR3β, amino acid sequence is ATSDLAGANRDTQY.

2. The T cell receptor according to claim 1, wherein, The T-cell receptor can specifically recognize the complex formed by the GP100 antigen peptide and human leukocyte antigen.

3. The T cell receptor according to claim 2, wherein, The human leukocyte antigen is HLA-A*02:

01.

4. The T-cell receptor according to any one of the preceding claims, wherein, The amino acid sequence of the α-chain variable region is as follows: AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEADRGSTLGRLYFGRGTQLTVWP; The amino acid sequence of the β-chain variable region is as follows: DADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSDLAGANRDTQYFPGGTRLTVL.

5. The T-cell receptor according to any one of claims 1 to 4, characterized in that, The T cell receptor includes engineered α-chain constant regions and / or β-chain constant regions.

6. A synthetic nucleic acid molecule, characterized in that, The synthesized nucleic acid molecule encodes the T cell receptor as described in any one of claims 1 to 5 or the TCR α chain and TCR β chain encoding the T cell receptor.

7. A recombinant vector, characterized in that, The recombinant vector comprises the synthetic nucleic acid molecule as described in claim 6.

8. A host cell, characterized in that, The host cell comprises the synthetic nucleic acid molecule of claim 6 or the recombinant vector of claim 7.

9. The use of the synthesized nucleic acid molecule of claim 6 or the recombinant vector of claim 7 in the preparation of a medicament for treating GP100-positive cancers, wherein, The GP100-positive cancers are primary, recurrent, or metastatic melanomas that express the GP100 protein.

10. An engineered T cell, characterized in that, The engineered T cells express the T cell receptor as described in any one of claims 1 to 5.

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