Isolated TCR and use thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to an isolated TCR and a use thereof. The TCR comprises a TCR α chain and a TCR β chain; the TCR α chain comprises CDRα1, CDRα2 and CDRα3 in a TCR α chain variable region; the TCR β chain comprises CDRβ1, CDRβ2 and CDRβ3 in a TCR β chain variable region; the amino acid sequence of CDRα3 is as shown in SEQ ID NO. 2; and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO. 1. A TP53-R248Q-TCR-T cell prepared by stably expressing TP53-R248Q-TCR in a CD8+ T cell can specifically recognize and kill a tumor cell which expresses HLA-A*11:01 and a TP53-R248Q mutant antigen. A solid theoretical and experimental foundation is provided for developing TCR-T therapy targeting a TP53-R248Q tumor neoantigen.

Description

An isolated TCR and its use Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an isolated TCR and uses thereof. Background Art

[0002] p53 is a prominent tumor suppressor gene that regulates the cell cycle and prevents cell carcinogenesis. Known as the "guardian of the genome," it is also one of the most frequently mutated tumor suppressor genes in human cancers. The TP53-R248Q mutation, the second most common mutation in malignant tumors, is closely associated with disease recurrence, poor response to treatment, and drug resistance. Mutations in the p53 gene occur in 50% of cancer types, while the remaining cancer types frequently develop other mechanisms to inactivate p53 function. Missense mutations are generally divided into two categories. One type is DNA-contacting mutations, occurring in amino acids that contact DNA and prevent p53 from binding to DNA, such as the p53R273H and R248Q mutants. The other type is conformational mutations, occurring in amino acids that maintain structure and leading to protein unfolding, such as the p53R175H, Y220C, and R249S mutants. Researchers have found that acquired TP53R248Q mutations are associated with recurrence of disease during treatment, poor patient response to treatment, and resistance to multiple chemotherapy drugs. Studies have shown that the TP53-R248Q mutation not only leads to loss of tumor suppressor function, but also, as a gain-of-function mutation, can promote tumorigenesis in mouse models. The TP53-R248Q mutation also shows increased invasive behavior in cell lines and has been shown to lead to worse overall survival. Therefore, targeting the TP53-R248Q tumor neoantigen is of great significance for cell therapy of various tumors carrying this mutation.

[0003] In recent years, immune cell therapy has become a new approach to treating tumors, following surgical treatment, radiotherapy, chemotherapy, small molecule targeted therapy, or monoclonal antibody therapy. TCR-T cells use genetic engineering to transplant cloned TCRs that specifically recognize tumor antigens onto autologous CD8+ T cells, giving them the ability to specifically kill tumor cells. Compared to CAR-T cells, which can only recognize membrane antigens, TCR-T cells recognize antigen peptides presented by MHC, allowing them to target a wider range of antigens, including intracellular and membrane antigens. TCR-T cell therapy increases the number of T lymphocytes while enhancing their specificity for killing tumor cells, thereby achieving better tumor treatment outcomes. Recent clinical studies have demonstrated that TCR-T has a good therapeutic effect on cancer patients. However, due to the individual specificity of tumor-specific antigens and the richness of the TCR repertoire, obtaining TCR sequences that specifically recognize neoantigens in tumors is extremely challenging.

[0004] Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a separated TCR and use thereof to solve the problems in the prior art.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides an isolated TCR, wherein the TCR comprises a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises CDRα1, CDRα2 and CDRα3 in the variable region of the TCRα chain; the TCRβ chain comprises CDRβ1, CDRβ2 and CDRβ3 in the variable region of the TCRβ chain; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.2, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.3 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.4; and / or, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO.5 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO.6.

[0008] The present invention also provides a polynucleotide encoding the aforementioned TCR.

[0009] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide and a plasmid backbone.

[0010] The present invention provides a virus comprising the aforementioned polynucleotide.

[0011] The present invention provides a T cell comprising the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned virus.

[0012] The present invention also provides use of the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned T cell in preparing a tumor treatment product.

[0013] The present invention also provides a tumor treatment product, which comprises the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned T cell and a pharmaceutically acceptable excipient.

[0014] The present invention also provides a method for treating tumors, comprising administering the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus, or the aforementioned T cell to a tumor patient.

[0015] As described above, the isolated TCR and its use of the present invention have the following beneficial effects:

[0016] The present invention combines the distribution ratio of HLA subtypes and the mutation frequency of TP53 mutants, selects the high-frequency TP53-R248Q (Rank 2#) tumor-specific antigen (TSA) corresponding to the high-frequency HLA subtype (HLA-A*11:01), conducts TP53-R248Q tumor antigen-specific TCR screening in vitro, obtains the TCR sequence after single-cell TCR sequencing, and conducts comprehensive TP53-R248Q-TCR specificity and functional verification. The results of in vivo / in vitro experiments on TP53-R248Q-TCR-T cells prepared by stably expressing the TP53-R248Q-TCR of the present invention in CD8+T cells show that TP53-R248Q-TCR-T cells can specifically recognize and kill tumor cells expressing HLA-A*11:01 and TP53-R248Q mutant antigens, significantly increase the release of cytokine IFNγ, and ultimately achieve specific killing of target cells. The present invention can provide a solid theoretical and experimental basis for the development of TCR-T therapy targeting TP53-R248Q tumor-specific antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a schematic diagram showing the results of sorting and single-cell sequencing of TP53-R248Q-TCR-T cells of the present invention.

[0018] FIG2 is a schematic diagram showing the results of an in vitro binding experiment of TP53-R248Q-TCR-T cells of the present invention.

[0019] FIG3 is a schematic diagram showing the results of an in vitro activation experiment of TP53-R248Q-TCR-T cells of the present invention.

[0020] FIG4 is a schematic diagram showing the results of an in vitro killing experiment of TP53-R248Q-TCR-T cells of the present invention.

[0021] FIG5 is a schematic diagram showing the results of an in vivo killing experiment of TP53-R248Q-TCR-T cells of the present invention. DETAILED DESCRIPTION

[0022] The present invention provides an isolated TCR or fragment thereof, wherein the TCR comprises a TCRα chain and a TCRβ chain. The TCRα chain comprises a TCRα chain variable region and a TCRα chain constant region; the TCRβ chain comprises a TCRβ chain variable region and a TCRβ chain constant region; the TCRα chain comprises CDRα1, CDRα2, and CDRα3 in the TCRα chain variable region; the TCRβ chain comprises CDRβ1, CDRβ2, and CDRβ3 in the TCRβ chain variable region; the amino acid sequence of CDRα1 is shown in SEQ ID NO. 2, and the amino acid sequence of CDRβ3 is shown in SEQ ID NO. 1.

[0023] In some specific embodiments, the amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.3 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.4.

[0024] In some specific embodiments, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO.5 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO.6.

[0025] A CDR (complementarity determining region) generally refers to the region of an antibody or receptor that spatially complements an antigenic determinant. The variability in an antibody or receptor is typically not evenly distributed throughout the variable region. Both the α and β chain variable regions of a monoclonal antibody or receptor typically have three hypervariable regions (HVRs). These regions typically complement the antigenic determinant spatially, and are therefore also called complementarity determining regions (CDRs). The α chain variable region typically includes three CDRs: CDRα1, CDRα2, and CDRα3, while the β chain variable region also typically includes three CDRs: CDRβ1, CDRβ2, and CDRβ3.

[0026] In some specific embodiments, the α chain variable region and the β chain variable region may further include a framework region, and the framework region may be located between the complementarity determining regions or at both ends of the complementarity determining regions. In certain specific embodiments of the present invention, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence of a murine monoclonal antibody variable region, obtained by replacing, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0027] In some embodiments, the TCR α chain or TCR β chain further comprises a TCR α chain constant region or a TCR β chain constant region.

[0028] In some specific embodiments, the amino acid sequence of the TCR α chain variable region is shown as SEQ ID NO.14; and / or the amino acid sequence of the TCR β chain variable region is shown as SEQ ID NO.15.

[0029] In some specific embodiments, the amino acid sequence of the TCR α chain variable region can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.14; the amino acid sequence of the TCR β chain variable region can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.15.

[0030] In some specific embodiments, the amino acid sequence of the TCRα chain constant region is shown as SEQ ID No.11; the amino acid sequence of the TCRβ chain constant region is shown as SEQ ID No.10.

[0031] In some specific embodiments, the TCR α chain constant region may also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.11; and the TCR β chain constant region may also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.10.

[0032] In some specific embodiments, the amino acid sequence of the TCRα chain is shown as SEQ ID NO.8; the amino acid sequence of the TCRβ chain is shown as SEQ ID NO.7.

[0033] In some specific embodiments, in the case of having the aforementioned α and β chain variable region CDR sequences, the TCR α chain can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.8; the TCR β chain can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.7.

[0034] In some embodiments, the TCRα chain is selected from a human TCRα chain, a humanized TCRα chain, a chimeric TCRα chain, or a murine TCRα chain; and the TCRβ chain is selected from a human TCRβ chain, a humanized TCRβ chain, a chimeric TCRβ chain, or a murine TCRβ chain. "Chimeric TCRα chain" or "chimeric TCRβ" refers to a TCRα chain or TCRβ chain comprising sequences derived from more than one species, such as sequences derived from human and mouse.

[0035] In some embodiments, the TCR binds to the p53 antigen. Preferably, the TCR binds to the TP53-R248Q antigen.

[0036] The present invention also provides an antigen-binding protein, comprising the aforementioned TCR.

[0037] The present invention also provides a polynucleotide encoding the aforementioned TCR.

[0038] In some specific embodiments, the nucleotide sequence of the polynucleotide is shown as SEQ ID No.9.

[0039] In some specific embodiments, the nucleotide sequence of the polynucleotide is a polynucleotide having a sequence similarity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 99.5% to that of SEQ ID No. 9.

[0040] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide and a plasmid backbone.

[0041] In some specific embodiments, the plasmid backbone is a plasmid backbone of a virus, adenovirus or adeno-associated virus. More specifically, the plasmid backbone is selected from pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-T Any one or more of puroGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DEST, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP, and pHR-SFFV-Puro.

[0042] The present invention provides a virus comprising the aforementioned polynucleotide.

[0043] In some embodiments, the virus is selected from a lentivirus, an adenovirus, or an adeno-associated virus.

[0044] The present invention provides a T cell comprising the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned virus.

[0045] In some embodiments, the T cells are TCR-T cells.

[0046] In some embodiments, the T cells are selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed-type hypersensitivity T cells, naive T cells, or memory T cells. Helper T cells are T cells that assist humoral and cellular immunity; suppressor T cells are T cells that suppress cellular and humoral immunity; effector T cells are T cells that release lymphokines; cytotoxic T cells are T cells that kill target cells; delayed-type hypersensitivity T cells are T cells that participate in type IV hypersensitivity reactions and can act on helper and suppressor T cells to amplify the immune effect; naive T cells are T cells that have not yet differentiated; and memory T cells are T cells that have the ability to stimulate memory-specific antigens.

[0047] In some embodiments, the T cells are Jurkat T cells or CD8+ T cells.

[0048] The present invention also provides use of the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned T cell in preparing a tumor treatment product.

[0049] In some specific embodiments, the tumor is selected from one or more of adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

[0050] The present invention also provides a tumor treatment product, which comprises the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned T cell and a pharmaceutically acceptable excipient.

[0051] In some embodiments, the adjuvant includes various excipients and diluents, which are not necessary active ingredients and have no excessive toxicity after administration. The adjuvant includes sterile water or normal saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.) or adhesives. The adjuvant also includes other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol. When the adjuvant is used for an aqueous solution for injection, it is selected from normal saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannose or sugar alcohol isotonic solution. The aqueous solution for injection includes a solubilizing agent. The solubilizing agent is selected from alcohol (ethanol), polyol (propylene glycol or PEG) and / or nonionic surfactant (Tween 80 or HCO-50). In the tumor treatment products provided by the present invention, the aforementioned TCR, polynucleotide, nucleic acid construct, or T cell may be a single active ingredient, or may be combined with one or more other active ingredients useful for tumor treatment to form a combination preparation. The active ingredients may be other various drugs used for tumor treatment. The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient of the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned T cell and tumor treatment product depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the bifunctional compound as the active ingredient is 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0052] The present invention also provides a method for treating tumors, comprising administering an effective amount of the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus, or the aforementioned T cell to a tumor patient.

[0053] In some embodiments, the amount administered is 1-1000 mg / kg / day. Specifically, the amount administered is 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or 500 mg-1000 mg / kg / day.

[0054] In some embodiments, the subject of the method can be a mammal; preferably, the subject of the method is a human.

[0055] In some embodiments, the tumor is a tumor in which the wild-type TP53 gene has an R248Q mutation, wherein the specific nucleotide sequence or amino acid sequence information of the wild-type TP53 gene can be obtained from existing databases, such as NCBI or Ensembl.

[0056] In the present invention, a T cell receptor (TCR) is a molecule present on the surface of a T cell that is responsible for recognizing a peptide-MHC complex. In some embodiments, the TCR is a truncated or intact full-length TCR. In some embodiments, the TCR is a heterodimer composed of α and β chains. In some embodiments, the TCR may also be a single-chain TCR (scTCR).

[0057] In the present invention, the term "TCRα chain constant region" or "TCRβ chain constant region" includes the extracellular constant region, transmembrane region and intracellular constant region connected in sequence, wherein the extracellular constant region may include the hinge region of the TCRα chain and the TCRβ chain, and participate in the formation of disulfide bonds between the TCRα chain and the TCRβ chain; the transmembrane region also belongs to the constant region, and its functions include participating in the cell membrane anchoring of the TCRα chain and the TCRβ chain and the interaction with the CD3 subunit to form a TCR-CD3 complex; the possible functions of the intracellular constant region include participating in the conformational transition of the TCR-CD3 complex and signal transduction after TCR signal transduction.

[0058] In the present invention, the term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., TCR α-chain or β-chain (or gd chain and d chain) TCRs)) involved in the binding of an immunoglobulin superfamily binding protein (e.g., TCR) to an antigen. The variable domains of the a chain and b chain of a natural TCR (Va and nb, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three CDRs. The Va domain is encoded by two independent DNA segments, the variable gene segment and the joining gene segment (VJ); the nb domain is encoded by three independent DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). A single Va or nb domain may be sufficient to confer antigen binding specificity. In addition, a TCR that binds to a specific antigen can be separated from the antigen-binding TCR using the Va or nb domain to screen a library of complementary Va or nb domains, respectively.

[0059] In the present invention, the term "antigen" refers to a cell surface molecule or an intracellular molecule presented by an MHC molecule or an MHC-like molecule that can be bound by an antibody or a T cell receptor (TCR), including but not limited to polypeptide antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), or polysaccharide antigens (e.g., CA199, CA72-4, and CA125). The antigen can be a tumor antigen, such as a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0060] As used herein, the term "isolated" refers to a material that has been removed from its natural state or otherwise artificially manipulated, such as the α chains, β chains, T cell receptors, and nucleic acids described herein. An isolated material may be substantially or essentially free of components that normally accompany it in its natural state, or may be manipulated to be in an artificial state with components that normally accompany it in its natural state. An isolated material may be in natural, chemically synthesized, or recombinant form. An isolated material may also or alternatively be in an enriched, partially purified, or purified form.

[0061] In the present invention, the term "polynucleotide" also referred to as "nucleotide" or "nucleic acid" refers to a nucleic acid chain composed of deoxyribonucleic acid, ribonucleic acid, modified nucleic acid or base, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0062] In the present invention, the term "nucleic acid construct" also referred to as "vector" refers to a vector capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors.

[0063] In the present invention, the term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of these cells.

[0064] In the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleic acids at corresponding amino acid positions or nucleic acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleic acid at the corresponding position in the second sequence, then the molecules are identical at that position.

[0065] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percent identity between two nucleic acid sequences is determined using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0066] The percent identity between two amino acid or nucleic acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0067] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0068] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0069] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0070] The sequence information of the present invention is as follows:

[0071] SEQ ID No.1

[0072] SEQ ID No. 2

[0073] SEQ ID No. 3

[0074] SEQ ID No.4

[0075] SEQ ID No.5

[0076] SEQ ID No.6

[0077] SEQ ID No.7

[0078] SEQ ID No.8

[0079] SEQ ID No.9

[0080] SEQ ID No.10

[0081] SEQ ID No.11

[0082] SEQ ID No.12

[0083] SEQ ID No. 13

[0084] SEQ ID No. 14

[0085] SEQ ID No.15

[0086] Example 1 Sorting of TP53-R248Q-TCR-T cells and TCR sequencing

[0087] 1. Inducing healthy donor monocytes to differentiate into dendritic cells (DCs) in vitro

[0088] (1) PBMCs (or resuscitated frozen PBMCs) were obtained from healthy donors / patients by Lymphoprep density gradient centrifugation. The cells were resuspended in 10 ml of 1640 medium (Gbico) and centrifuged at 350 g for 5 min. The supernatant was removed and an appropriate amount of DC medium (Stem cell) was added to resuspend the PBMCs to a density of 5 × 106 cells / ml.

[0089] (2) Pipette 2 ml of the above PBMC suspension into a 6-well plate at a cell count of 1 × 107 cells per well. Incubate the 6-well plate in a 37°C, 5% CO2 incubator for 2 h. Gently swirl the 6-well plate until a thin layer of non-adherent cells is suspended in the center of the well. Gently tilt the plate and carefully aspirate the medium and any clumping of non-adherent cells without disturbing the adherent cells.

[0090] (3) Gently add 3 ml of DC medium (Stem cell) containing a final concentration of 1× DC Differentiation Supplement to each well along the side wall, and place the 6-well plate in a 37°C, 5% CO2 incubator for 3 days.

[0091] (4) On Day 5, without changing the culture medium, 30 μl of 100× DC Maturation Supplement (Stem Cell) was added to each well of the culture medium. The 6-well plate was placed in a 37°C, 5% CO2 incubator to induce maturation for 2 days.

[0092] (5) Use a pipette to blow and resuspend the cells at the bottom of the well plate to collect mature dendritic cells, i.e., mature DCs. Transfer the collected mature DCs to a 15 ml centrifuge tube and inspect the bottom of the well plate under a microscope to ensure that all mature DCs have been completely harvested.

[0093] 2. DC cells loaded with TP53-R248Q antigen peptide were co-stimulated with autologous CD8+ T cells and cultured

[0094] (1) Add 5 μg / ml of the target peptide TP53-R248Q to the above 15 ml centrifuge tube (Mature DC), seal the lid with Parafilm, place in a MACSmix rotator, and rotate in a cell culture incubator at 37°C, 5% CO2 for 4 hours.

[0095] (2) PBMCs (or frozen PBMCs) were obtained from healthy donors / patients by Lymphoprep density gradient centrifugation. The cells were resuspended in 10 ml of 1640 medium and centrifuged at 350 × g for 8 min. The supernatant was removed and CD8 was isolated from PBMCs using the EasySep™ Human CD8 Positive Selection Kit. + T cells.

[0096] (3) After the mature DCs were incubated with TP53-R248Q-peptide, the mature DCs loaded with TP53-R248Q-peptide were irradiated with a dose of 4000 rad (40 Gy) using an irradiator.

[0097] (4) After irradiation, mature DCs loaded with TP53-R248Q-peptide were stimulated in vitro at a DC:CD8+ T cell ratio of 1:2.5. 2 ml was added to each well of a 12-well plate, and the plate was then incubated overnight in a 37°C, 5% CO2 cell culture incubator.

[0098] (5) Subsequently, half of the medium was replaced every 2-3 days. On Day 14, irradiated mature DCs loaded with TP53-R248Q-peptide were prepared and added to the cells in the culture system described in (4) for a second round of in vitro stimulation.

[0099] (6) On Day 28, cells in the co-stimulation system were collected and FACS staining was started. First, 10 μl of HLA-A:1101-TP53-R248Q-Tetramer-PE was added, mixed and incubated at RT in the dark for 20 min. After the incubation, 5 μl of CD8a-APC was added, mixed and incubated at 4°C in the dark for 20 min. After the incubation, the cells were washed three times with FACS buffer, and the cells were resuspended in 400 μl of FACS buffer containing 1×DAPI working solution and transferred to FACS tubes. After setting up various single-color controls, unstained controls, and isotype controls, the CD8+ / Tetramer (%) ratio of each group was detected by flow cytometry.

[0100] 3. Isolation of TP53-R248Q-specific T cells and single-cell TCR sequencing

[0101] (1) CD8 from healthy donors + After two rounds of in vitro stimulation of T cells with autologous TP53-R248Q-peptide-loaded DCs, cells in the costimulation system were collected for flow cytometry analysis. The specific flow cytometry results are shown in Figure 1A. + / Tetramer + The T ratio was 0.14%, and the clustering was obvious. This indicated that TP53-R248Q-specific-TCR-T cells were specifically expanded in two rounds of in vitro stimulation. Then, the CD8 + / tetramer + T cells were specifically sorted and then sent to Jingneng Bio for 10×Genomics single-cell TCR+ transcriptome sequencing to obtain the TCR sequence and transcriptomic characteristics of CD8+ / tetramer+ T cells.

[0102] (2) After single-cell sequencing was completed, transcriptomic feature analysis was performed on the top four TCRs. The TCR sequencing results and frequency ranking results of the cell population obtained by analysis are shown in Figure 1B. Subsequently, the TCR sequence with the highest frequency was optimized. Specifically, the TCR-C region, i.e., the TCR constant region, was replaced with mTCR-C (wherein mTCR-C includes mTCRα-C and mTCRβ-C, the amino acid sequence of mTCRα-C is shown in SEQ ID No. 11, and the amino acid sequence of mTCRβ-C is shown in SEQ ID No. 10) to avoid TCR mispairing. The amino acid sequence of the TCRα chain in the final TCR was shown in SEQ ID NO. 8; the amino acid sequence of the TCRβ chain was shown in SEQ ID NO. 7; the amino acid sequences of CDRα1, CDRα2, and CDRα3 in the TCRα chain were shown in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 2, respectively; the amino acid sequences of CDRβ1, CDRβ2, and CDRβ3 in the TCRβ chain were shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively. NO.1; the TCR sequence was codon-optimized for human origin to increase the expression of TCR, and the nucleotide sequence of the DNA chain encoding the TCR was finally obtained as shown in SEQ ID No.12; P2A and Furin-cleavage were used to enable the simultaneous expression of the α and β chains of the TCR in a lentiviral expression vector, wherein the nucleotide sequence of the DNA chain encoding the protein expressed by the above-mentioned lentiviral expression vector is shown in SEQ ID No.13.

[0103] Example 2 In vitro binding experiment of TP53-R248Q-TCR-T cells

[0104] 1. TP53-R248Q-TCR-T cell preparation

[0105] (1) The optimized nucleotide sequence obtained in Example 1 was used to construct a lentiviral expression vector for TP53-R248Q-TC as shown in SEQ ID No. 13, and then the lentiviral packaging of TP53-R248Q-TCR was performed. The specific operation of viral packaging was as follows: 293T cells were digested and passaged, and the cell density was observed on the day of transfection. Transfection was performed when the cell density was 80-90% full. No culture medium change was required before transfection. Opti-MEM needed to be preheated in a 37-degree water bath, and Fugene-HD transfection reagent needed to be returned to room temperature before use and should be shaken before use. The complex components of each bottle of T75 for transfection were as follows: pHR-SFFV-TCR-Puro plasmid containing the target TCR: 10ug; pMD2.G (envelope plasmid): 5ug; pSPAX2 (core enzyme): 7.5ug. Make up to 1 mL of opti-MEM and mix thoroughly. Add 56 μL of Fugene-HD transfection reagent and pipette up and down 5-6 times. Let stand at room temperature for 15 minutes before adding to a T75 flask. Collect viral supernatants 48 and 72 hours after collection. Incubate at 4000 rpm for 5 minutes and filter through a 0.45 μm PES membrane before use. TP53-R248Q-TCR lentiviral fluids were collected 48 and 72 hours after viral packaging.

[0106] (2) Jurkat T cells were infected with TP53-R248Q-TCR lentiviral particles by adding 10 μg / ml polybrene and centrifuging at 800 × g for 90 min at 30°C. After centrifugation, the cells were placed in a cell culture incubator at 37°C, 5% CO2 for 24 h, and then replaced with fresh 1640 medium for culture.

[0107] (3) 20 μl of CD3 / 28 beads (Genscript) and 20 ng / ml of IL-2 were added to CD8+ T cells from healthy donors. After activating the T cells for 48 h, 10 μg / ml of polybrene was added for the first round of infection with the TP53-R248Q-TCR lentivirus. After centrifugation, the cells were cultured in a cell incubator at 37°C, 5% CO2 for 24 h. Subsequently, a second round of infection with the TP53-R248Q-TCR lentivirus was performed.

[0108] 2. FACS detection of TP53-R248Q-TCR-T cell binding specificity

[0109] The TP53-R248Q-TCR-T cells prepared above were stained with mTCRβC+ / HLA-A*11:01-TP53-R248Q-Tetramer. Flow cytometry results showed distinct cell populations of Tetramer+ / mTCRβC+Jurkat T (Figure 2A) and CD8+ / Tetramer+primary CD8+ T (Figure 2B). This result demonstrates that the TP53-R248Q-TCR screened and identified in this in vitro screening can be normally expressed in Jurkat T cell lines and primary CD8+ T cells, and can specifically bind to HLA-A:1101-TP53-R248Q-Tetramer.

[0110] Example 3 In vitro activation experiment of TP53-R248Q-TCR-T cells

[0111] The in vitro activation assay for TP53-R248Q-TCR-T cells, i.e., the IFNγ-Elispot assay, includes the following steps:

[0112] (1) Directly differentiating donor autologous monocytes into DC cells in vitro, loading the DCs with TP53-R248-WT or TP53-R248Q antigen peptides as in Example 1, and co-culturing the TP53-R248Q-TCR-T cells prepared in Example 2 with the DCs loaded with TP53-R248-WT or TP53-R248Q antigen peptides in vitro;

[0113] (2) After 24 h of in vitro co-culture, the cells were discarded and washed five times with 200 μl of DPBS to completely remove the cells. 100 μl of 1 μg / ml detection antibody 7-B6-1-biotin was added to the culture system and incubated at 37°C for 2 h. Subsequently, 100 μl of streptavidin-HRP (3420-2H, MABTECH) was added and incubated at room temperature for 1 h.

[0114] (3) After washing five times with 200 μl DPBS, 100 μl TMB substrate was added to each well until complete spots appeared in the wells. Sterile water was then added to interrupt the color development process.

[0115] The results are shown in Figure 3. DCs loaded with TP53-R248Q antigen peptide can specifically activate TP53-R248Q-TCR-T cells in vitro. After activation, the IFNγ expression level of TP53-R248Q-TCR-T cells was significantly increased (Figure 3A-C), while DCs loaded with TP53-R248-WT antigen peptide had no obvious activation effect on TP53-R248Q-TCR-T cells (Figure 3B-C). Therefore, the results indicate that the TP53-R248Q-TCR obtained in this screening and identification can specifically recognize the TP53-R248Q antigen peptide.

[0116] Example 4 In vitro killing experiment of TP53-R248Q-TCR-T cells

[0117] The in vitro killing assay of TP53-R248Q-TCR-T cells, also known as the Incucyte killing assay, includes the following steps:

[0118] (1) In vitro, TP53-R248Q-TCR-T effector cells and K562-A1101-TP53-R248Q target cells were constructed by lentiviral infection of T cells or K562-A1101 cells. Then, TP53-R248Q-TCR-T cells or CD8 + T cells and K562-A1101 control cells or K562-A1101-TP53-R248Q target cells were added to 96-well plates for co-culture;

[0119] (2) Dissolve by adding 100 μL DPBS Annexin V Dyes (Sartorius). Then Annexin V Dyes reagent was diluted in complete culture medium to a final dilution of 1:200.

[0120] (3) Place the 96-well plate containing cells into the Incucyte (Sartorius) live cell analyzer and monitor cell apoptosis using appropriate fluorescence channels. Specific parameters are as follows: A. Observation magnification: 20× objective lens; B. Channel selection: phase contrast Green|Red|Orange|NIRc; C. Scan type: 2-4 images per well; D. Scan interval: scan once every 30 minutes until the killing efficiency is completed after 48 hours of co-culture.

[0121] (4) After the test, the in vitro killing efficiency of TP53-R248Q-TCR-T was analyzed using the Incucyte live cell analysis system software (Sartorius).

[0122] The results are shown in Figure 4. This example uses the Incucyte high-throughput long-term dynamic imaging and analysis system to detect the killing of target cells by TP53-R248Q-TCR-T cells in real time. The results show that within 48 hours of co-culture, compared with negative control CD8+ T cells without TP53-R248Q-TCR, TCR-T cells with different effector-target ratios (1:1, 2:1, and 5:1) can more efficiently kill target cells that are HLA-A*11:01 positive and express the TP53-R248Q mutation (Figures 4A-D).

[0123] Example 5 In vivo killing experiment of TP53-R248Q-TCR-T cells

[0124] The in vivo killing experiment of TP53-R248Q-TCR-T cells, also known as the CDX animal experiment, includes the following steps:

[0125] (1) 6-8 week old female Jicui Yaokang NCG mice were selected and injected into the lateral tail vein with 3E6 HLA subtype-matched K562 cell line carrying TP53-R248Q mutant protein and expressing Luciferase reporter gene.

[0126] (2) Three days after transplantation of the K562-HLA-TP53-R248Q-luci cell line, 1E7 TCR-T cells were injected into the lateral tail vein, and control T lymphocytes without TCR gene transfection were used as negative controls.

[0127] (3) Every 7 days after tumor cell inoculation, in vivo imaging of small animals was performed, and the weight changes and survival of mice were detected to evaluate the in vivo killing effect of TCR-R248Q-TCR-T cells on target cells.

[0128] The results are shown in Figure 5. This example uses an in vivo CDX model to evaluate the effectiveness of TP53-R248Q-TCR-T cells in killing target cells. On day 0, 3E10 K562-HLA-A*11:01-R248Q-luci cells were injected into the lateral tail vein of immunodeficient NCG mice. Then, on day 3, 1E7 Mock T or TP53-R248Q-TCR-T cells were infused back. After the T cell infusion, 2500 IU of human IL-2 cytokine was injected intraperitoneally daily. Bioluminescence imaging was performed at 4 hours and 1-5 weeks after tumor cell inoculation (Figure 5A). In vivo imaging results showed that mice in the saline and mock T cell-infused groups showed significant tumor cell growth within 5 weeks after tumor cell infusion, while some mice (3 / 5) in the R248Q-TCR-T cell-infused group showed no significant tumor growth (Figure 5B). Furthermore, the total photon count and average photon intensity of luciferase in the R248Q-TCR-T group were significantly lower than those in the saline and mock T cell control groups (Figures 5C-D). CDX experiments demonstrated that HLA-A*11:01-restricted TP53-R248Q-TCR-T cells could efficiently and specifically kill K562-HLA-A*11:01-R248Q-luciferase target cells in vivo.

[0129] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. An isolated TCR, characterized in that The TCR comprises a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises CDRα1, CDRα2 and CDRα3 in the variable region of the TCRα chain; the TCRβ chain comprises CDRβ1, CDRβ2 and CDRβ3 in the variable region of the TCRβ chain; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.2, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.

1.

2. The TCR according to claim 1, characterized in that The amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.3 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.4; and / or, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO.5 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO.

6.

3. The TCR according to claim 1, characterized in that The amino acid sequence of the TCR α chain variable region is shown in SEQ ID NO.14; and / or, the amino acid sequence of the TCR β chain variable region is shown in SEQ ID NO.

15.

4. The TCR according to claim 1, characterized in that The amino acid sequence of the TCR α chain is shown in SEQ ID NO.8; and / or, the amino acid sequence of the TCR β chain is shown in SEQ ID NO.

7.

5. A polynucleotide, characterized in that The polynucleotide encodes the TCR described in any one of claims 1-4.

6. The polynucleotide according to claim 5, characterized in that The nucleotide sequence of the polynucleotide is shown in SEQ ID No.

9.

7. A nucleic acid construct, characterized in that The nucleic acid construct comprises the polynucleotide according to any one of claims 5 to 6 and a plasmid backbone.

8. The nucleic acid construct according to claim 7, characterized in that The plasmid backbone is a plasmid backbone of a lentivirus, an adenovirus or an adeno-associated virus; preferably, the plasmid backbone is selected from pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO .1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DEST, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP, pHR-SFFV-Puro, or any one or more thereof.

9. A virus, characterized in that The virus contains the polynucleotide according to any one of claims 5-6 or the nucleic acid construct according to any one of claims 7-8.

10. A T cell, characterized in that The T cell contains the TCR according to any one of claims 1 to 4, the polynucleotide according to any one of claims 5 to 6, the nucleic acid construct according to any one of claims 7 to 8, or the virus according to claim 9.

11. The T cell according to claim 10, characterized in that The T cell is a TCR-T cell; and / or, the T cell is selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed allergic reaction T cells, natural T cells or memory T cells; preferably, the T cell is a Jurkat T cell or a CD8+T cell.

12. Use of the TCR according to any one of claims 1 to 4, the polynucleotide according to any one of claims 5 to 6, the nucleic acid construct according to any one of claims 7 to 8, the virus according to claim 9 or the T cell according to any one of claims 10 to 11 in the preparation of a tumor treatment product.

13. The use according to claim 13, characterized in that The tumor is selected from one or more of adrenal cortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial carcinoma, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

14. A tumor treatment product, characterized in that: The tumor treatment product comprises the TCR as described in any one of claims 1-4, the polynucleotide as described in any one of claims 5-6, the nucleic acid construct as described in any one of claims 7-8, the virus as described in claim 9 or the T cell as described in any one of claims 10-11 and a pharmaceutically acceptable excipient.

15. A method for treating tumors, characterized in that: The method comprises administering to the subject an effective dose of a TCR as described in any one of claims 1-4, a polynucleotide as described in any one of claims 5-6, a nucleic acid construct as described in any one of claims 7-8, a virus as described in claim 9, a T cell as described in any one of claims 10-11, or a product as described in claim 14.

16. The method according to claim 15, characterized in that The method comprises one or more of the following features: 1) The amount of administration is 1-1000 mg / kg / day; 2) The subject is a mammal; preferably, the subject is a human; 3) The tumor is a tumor in which the wild-type TP53 gene undergoes R248Q mutation.

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