Tumor antigen peptide and application thereof

Abstract

The invention relates to the field of biological medicines, in particular to a tumor antigen peptide and application thereof. The tumor antigen peptide provided by the invention comprises a peptide fragment with an amino acid sequence as shown in SEQ ID No.1. The tumor antigen peptide provided by the invention can be presented by the highest frequency HLA subtype (HLA-A * 11: 01) of Chinese population. In addition, through an in-vitro stimulation mode, it is verified that the tumor antigen peptide can effectively stimulate specific T cells, and a specific TCR element is successfully obtained. Therefore, a direct foundation is laid for further developing a TCR-T therapy, a therapeutic vaccine and other immunotherapies of the targeted human TP53R248Q.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a tumor antigen peptide and its application. Background Art

[0002] TP53 is one of the most frequently mutated tumor suppressor genes in human cancer 1 The TP53 gene is mutated in about 42% of cancer patients, involving more than half of cancer types, while the remaining cancer types frequently find other ways to promote the inactivation of P53 function. 2 . The main mutation types of P53 include missense mutations, truncating mutations, in-frame mutations, and splicing mutations (TP53 mutation database, http: / / www-p53.iarc.fr / ). About 80% of TP53 mutations are missense mutations, which result in single amino acid substitutions, such as R175H, R248Q, and R273H. Studies have shown that acquired TP53 R248Q mutations are associated with recurrence of the disease during treatment, poor treatment response in patients, and resistance to multiple chemotherapeutic drugs. 3 The study showed that the TP53-R248Q mutation not only leads to loss of tumor suppressor function, but also acts as a gain-of-function mutation that promotes tumorigenesis in mouse models. The TP53-R248Q mutation also showed increased invasive behavior in cell lines and has been shown to lead to worse overall survival. 4 Therefore, targeting TP53-R248Q is of great significance for the treatment of various tumors carrying this mutation.

[0003] In recent years, immune cell therapy technology has become a new method for tumor treatment after surgical treatment, radiotherapy, chemotherapy, small molecule targeted therapy or monoclonal antibody therapy. The representative one is adoptive T cell therapy, which includes tumor infiltrating T cell therapy (TIL), chimeric antigen T cell receptor therapy (CAR-T) and engineered T cell receptor therapy (TCR-T). Among them, TCR-T therapy has developed rapidly in recent years and is expected to bring new breakthroughs in tumor treatment. It is a cutting-edge immune cell therapy technology that uses genetic engineering technology to load cloned TCRs that specifically recognize tumor antigens onto autologous or allogeneic CD8+T cells, enabling them to acquire the ability to specifically kill tumor cells. 5. Compared with CAR-T cells that can only recognize membrane antigens, TCR-T cells recognize antigens that are peptides presented by MHC, and can therefore target a wider range of antigens, including intracellular and membrane antigens; TCR-T cell therapy increases the number of T lymphocytes while improving the specificity of T lymphocytes in killing tumor cells, thereby achieving better tumor treatment effects. Recent clinical studies have shown that TCR-T has a good therapeutic effect on tumor patients, but due to the individual specificity of tumor-specific antigens and the richness of the TCR library, it is extremely challenging to obtain TCR sequences that specifically recognize tumor neoantigens.

[0004] In addition to TCR-T, the development of therapeutic vaccines provides a new direction for tumor treatment. Therapeutic vaccines target tumor mutations, connect several tumor neoantigens in series in the form of mRNA, and inject them into the patient's body. Using the patient's own immune system, they stimulate T cells that specifically recognize tumor neoantigens to kill and eliminate tumor cells, thereby achieving the purpose of treatment. 6 The prerequisite for the application of both TCR-T therapy and therapeutic vaccines is to identify tumor neoantigens. Therefore, if a new immunotherapy targeting TP53 mutations is to be developed, it is crucial to identify tumor neoantigen peptides containing TP53 mutations.

[0005] References:

[0006] 1 Kastenhuber, ER, Lowe, SW Putting p53 in Context. Cell, 2017, 170(6): 1062-1078.

[0007] 2 Bykov, VJN, Eriksson, SE, Bianchi, J., Wiman, KGTargeting mutantp53 for efficient cancer therapy. NatRev Cancer, 2018, 18(2): 89-102.

[0008] 3Pan,M.,Jiang,C.,Tse,P.,Achacoso,N.,Alexeeff,S.,Solorzano,AV,Chung,E.,Hu,W.,Truong,TG,Arora,A.,Sundaresan,T.,Suga,JM,Thomas,S.,Habel,LATP53 Gain-of-Function and Non-Gain-of-FunctionMutations AreDifferentially Associated With Sidedness-Dependent Prognosis in MetastaticColorectal Cancer.

[0009] J Clin Oncol,2022,40(2):171-179.

[0010] 4Ng, JW, Lama, D., Lukman, S., Lane, DP, Verma, CS, Sim, AYR248Qmutation--Beyond p53-DNAbinding. Proteins, 2015, 83(12): 2240-2250.

[0011] 5Rosenberg, SA, Restifo, NPAdoptive cell transfer as personalized immunotherapy for human cancer.

[0012] Science, 2015, 348(6230): 62-68.

[0013] 6Saxena, M., van der Burg, SH, Melief, CJM, Bhardwaj, N. Therapeuticcancer vaccines. Nat Rev Cancer,

[0014] 2021,21(6):360-378. Summary of the invention

[0015] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a tumor antigen peptide and use thereof, so as to solve the problems in the prior art.

[0016] To achieve the above objectives and other related objectives, the present invention provides a tumor antigen peptide, wherein the tumor antigen peptide comprises one or more of the following features:

[0017] 1) The tumor antigen peptide comprises a peptide segment having an amino acid sequence as shown in SEQ ID No. 1;

[0018] 2) The tumor antigen peptide is a peptide segment that has at least 80% sequence similarity to the peptide segment described in 1) and has or partially has the activity of the tumor antigen peptide described in 1).

[0019] Preferably, the tumor antigen peptide can be recognized by an antibody that specifically binds to TP53 R248Q mutant tumor cells.

[0020] The present invention also provides a recombinant nucleic acid molecule, which encodes the aforementioned tumor antigen peptide.

[0021] The present invention also provides a recombinant expression vector, which comprises the aforementioned recombinant nucleic acid molecule.

[0022] The present invention also provides a cell, wherein the cell contains the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule or the aforementioned recombinant expression vector.

[0023] The present invention also provides the use of the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell in preparing any of the following products:

[0024] 1) HLA multimer products;

[0025] 2) TCR-T products;

[0026] 3) Tumor vaccine products;

[0027] 4)Tumor treatment products.

[0028] The present invention also provides a method for preparing isolated TCR-T cells, the preparation method comprising: co-culturing antigen presenting cells loaded with the aforementioned tumor antigen peptides with T cells, thereby obtaining the isolated TCR-T cells.

[0029] The present invention also provides a tumor vaccine, which comprises one or more of the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell.

[0030] The present invention also provides a pharmaceutical composition, which comprises the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell and a pharmaceutical auxiliary material or therapeutic adjuvant.

[0031] As described above, a tumor antigen peptide and its use of the present invention have the following beneficial effects:

[0032] The present invention utilizes the established K562 cell line that stably expresses HLA-A*11:01, introduces the tandem TP53 mutant candidate antigen peptide into the cell, and then immunoprecipitates the HLA-antigen peptide complex by HLA antibody, performs mass spectrometry analysis, identifies a short peptide containing the TP53 R248Q mutation site, and confirms that it can be presented by the highest frequency HLA subtype (HLA-A*11:01) in the Chinese population. In addition, the present invention verifies that it can effectively stimulate specific T cells and successfully obtain specific TCR elements by in vitro stimulation. This lays a direct foundation for the further development of immunotherapies such as TCR-T therapy and therapeutic vaccines targeting human TP53 R248Q. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown are the process and analysis results of the tumor antigen peptide mass spectrometry of the present invention.

[0034] Figure 2 Shown is a schematic diagram of the sorting and single-cell sequencing results of T cells induced by the tumor antigen peptide of the present invention.

[0035] Figure 3 A schematic diagram showing the results of an in vitro binding experiment of TP53-R248Q-TCR-T cells prepared using the tumor antigen peptides of the present invention.

[0036] Figure 4 A schematic diagram showing the results of an in vitro activation experiment of TP53-R248Q-TCR-T cells prepared using the tumor antigen peptides of the present invention.

[0037] Figure 5 A schematic diagram showing the results of an in vitro cytokine release experiment of TP53-R248Q-TCR-T cells prepared using the tumor antigen peptides of the present invention.

[0038] Figure 6 A schematic diagram showing the results of specific detection of TP53-R248Q-TCR-T cells prepared using the tumor antigen peptides of the present invention.

[0039] Figure 7 Schematic diagram showing the results of an in vitro tumor killing experiment of TP53-R248Q-TCR-T cells prepared using the tumor antigen peptides of the present invention. DETAILED DESCRIPTION

[0040] The present invention provides a tumor antigen peptide, which comprises a peptide segment with an amino acid sequence as shown in SEQ ID No.1.

[0041] In some specific embodiments, the tumor antigen peptide can be recognized by an antibody that specifically binds to TP53 R248Q mutant tumor cells.

[0042] In some specific embodiments, the tumor antigen peptide may also include a peptide segment having at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:1 and having or partially having the activity of the tumor antigen peptide shown in SEQ ID NO:1.

[0043] In some specific embodiments, the tumor antigen peptide can be a polypeptide obtained by substituting, deleting, adding or conservatively mutating one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids in the amino acid sequence shown in SEQ ID No.1, and the amino acid sequence of the polypeptide can have 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 with the amino acid sequence shown in SEQ ID NO.1.

[0044] In some specific embodiments, the tumor antigen peptide further comprises a tag peptide segment added at the N-terminus or C-terminus. More specifically, one or more tag peptide segments that can be removed by enzymatic hydrolysis in vivo or in vitro are added to both ends of the tumor antigen peptide, and after enzymatic hydrolysis and removal, a peptide segment with an amino acid sequence as shown in SEQ ID No. 1 can still be obtained.

[0045] In some specific embodiments, the tag peptide segment is selected from one or more of a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag.

[0046] In some specific embodiments, the tumor antigen peptide may be further subjected to one or more modifications based on the peptide segment comprising the amino acid sequence as shown in SEQ ID NO: 1. More specifically, the modification is selected from one or more of the following: coupling or fusion with an antibody, a carrier, a ligand, an albumin, an Fc fragment, phosphorylation modification, PEGylation modification, amidation modification, glycosylation modification, and biotinylation modification.

[0047] The present invention also provides a recombinant nucleic acid molecule, which encodes the aforementioned tumor antigen peptide.

[0048] In some embodiments, the recombinant nucleic acid molecule can be composed of ribonucleotides or deoxyribonucleotides.

[0049] The present invention also provides a recombinant expression vector, which comprises the aforementioned recombinant nucleic acid molecule.

[0050] In some embodiments, the recombinant expression vector further comprises an expression control element. More specifically, the expression control element is selected from one or more of the following: a promoter, a transposon, an enhancer, a polyA element, an LTR element, an ITR element, a WPRE element, and an SV40 element.

[0051] The present invention also provides a cell, wherein the cell contains the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule or the aforementioned recombinant expression vector.

[0052] In some embodiments, the cell is a cell expressing or preparing a tumor antigen peptide. Specifically, the cell expressing or preparing a tumor antigen peptide is selected from any one of animal cells (such as CHO, COS, N2A, human cervical cancer cells such as HELA or human embryonic kidney cells such as HEK293T), plant cells, bacterial cells (such as Escherichia coli, Streptomyces, Salmonella typhimurium), fungal cells (such as yeast), and insect cells (such as Sf9). More specifically, the cell is a human cell.

[0053] In some embodiments, the cell is a cell with tumor antigen peptides on its surface. Specifically, the cell with tumor antigen peptides on its surface is an antigen presenting cell. Preferably, the antigen presenting cell is selected from one or more of monocytes, dendritic cells, B cells, Langerhans cells or virus-infected target cells of tumor cells.

[0054] The present invention also provides the use of the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell in preparing any of the following products:

[0055] 1) HLA multimer products;

[0056] 2) TCR-T products;

[0057] 3) Tumor vaccine products;

[0058] 4)Tumor treatment products.

[0059] In some embodiments, the TCR-T product is an isolated TCR-T cell.

[0060] The present invention also provides a method for preparing isolated TCR-T cells, the preparation method comprising: co-culturing antigen presenting cells loaded with the aforementioned tumor antigen peptides with T cells, thereby obtaining the isolated TCR-T cells.

[0061] In some embodiments, the antigen presenting cells are monocytes or dendritic cells.

[0062] The present invention also provides a tumor vaccine, which comprises one or more of the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell.

[0063] In some embodiments, the tumor vaccine further comprises an immune adjuvant. More specifically, the immune adjuvant is selected from one or more of a radiotherapeutic agent, a chemotherapeutic agent or an immunotherapeutic agent.

[0064] In some embodiments, the tumor is selected from one or more of squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penis cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma, B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia or chronic myeloblastic leukemia.

[0065] The present invention also provides a pharmaceutical composition, comprising the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell and a pharmaceutically acceptable excipient. The pharmaceutical composition can be administered by a systemic route or a local route, selected from inner ear administration, ophthalmic administration, intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration and intralesional administration. The dosage form of the pharmaceutical composition is one or more of an injection, a tablet, a capsule, an aerosol, an eye drop or a nasal drop.

[0066] In some embodiments, the pharmaceutical composition further comprises a therapeutic adjuvant. More specifically, the therapeutic or therapeutic adjuvant is selected from one or more of a radiotherapeutic agent, a chemotherapeutic agent or an immunotherapeutic agent.

[0067] The adjuvant includes various excipients and diluents, which are not necessary active ingredients and have no excessive toxicity after application. The adjuvant includes sterile water or saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), 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. The adjuvant is selected from saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannoses or sugar alcohol isotonic solution when used for the aqueous solution of injection. The aqueous solution of 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 pharmaceutical composition provided by the present invention, the aforementioned tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell is a single effective ingredient, and can also be combined with one or more other active components useful for tumor treatment to form a combined preparation. The active component is other various drugs for treating tumors. 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 tumor antigen peptide, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned cell and the pharmaceutical composition depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the composition 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 more than 500 mg / kg / day.

[0068] The present invention also provides a method for treating or preventing tumors, comprising administering the aforementioned tumor vaccine or the aforementioned pharmaceutical composition to a subject.

[0069] In some specific embodiments, the method is: administering the aforementioned tumor vaccine or the aforementioned pharmaceutical composition to a subject intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesically, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by catheter, by lavage, or by infusion.

[0070] In some embodiments, the amount of administration is 1-1000 mg / kg / day. Specifically, the amount of administration 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.

[0071] In some embodiments, the subject of the method can be a mammal; preferably, the subject of the method is a human.

[0072] In this application, the term "peptide segment", "peptide" may also be "polypeptide", "protein" or "protein", all of which are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other operation, such as conjugation with a labeling component). Polypeptides can be isolated from natural sources, can be produced from eukaryotic or prokaryotic hosts by recombinant technology, and can be the product of synthetic methods.

[0073] In the present application, the term "conservative mutation" refers to a mutation (e.g., substitution, insertion and / or deletion of amino acids) that can normally maintain the function of a protein, such as a conservative substitution. Specifically, a "conservative substitution" generally exchanges an amino acid at one or more sites in a protein. This substitution can be conservative. As substitutions that are considered conservative substitutions, specifically, there can be cited substitutions of Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr. Conservative mutations include substitutions of Ile to Leu, Met, Val or Phe, substitutions of Leu to Ile, Met, Val or Phe, substitutions of Lys to Asn, Glu, Gln, His or Arg, substitutions of Met to Ile, Leu, Val or Phe, substitutions of Phe to Trp, Tyr, Met, Ile or Leu, substitutions of Ser to Thr or Ala, substitutions of Thr to Ser or Ala, substitutions of Trp to Phe or Tyr, substitutions of Tyr to His, Phe or Trp, and substitutions of Val to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations caused by individual differences, strains, species differences, etc. from which genes are derived.

[0074] In the present application, the term "sequence similarity", which may also be referred to as "sequence identity", generally refers to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotides or amino acid residues in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotides or amino acid residues by the total number of amino acid residues in the polynucleotides or polypeptides. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0075] In this application, the term "vaccine" generally refers to an immune preparation for preventing diseases that is made by artificially attenuating, inactivating or using genetic modification of pathogenic microorganisms (such as bacteria) and their metabolites.

[0076] In this application, the term "radiotherapeutic agent" includes the use of drugs that cause DNA damage.Radiotherapy has been widely used in cancer and disease treatment and includes what are commonly known as gamma rays, X-rays and / or the targeted delivery of radioisotopes to tumor cells.

[0077] In the present application, the term "chemotherapeutic agent" is a chemical compound that can be used to treat cancer. The categories of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators, anti-progestins, estrogen receptor downregulators, estrogen receptor antagonists, luteinizing hormone releasing hormone agonists, anti-androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents that can be used in the methods of treatment disclosed in the present application include cytostatic agents and / or cytotoxic agents.

[0078] In this application, the term "immunotherapeutic agent" includes "immunomodulators" and agents that promote or mediate antigen presentation that promotes cell-mediated immune responses. Among them, "immunomodulators" include immune checkpoint regulators, such as immune checkpoint protein receptors and their ligands that mediate T cell-mediated cytotoxicity inhibition, and are usually expressed by tumors or on anergic T cells in the tumor microenvironment, and allow tumors to escape immune attacks. Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands can overcome the immunosuppressive tumor environment to allow cytotoxic T cells to attack tumors. Examples of immune checkpoint proteins include, but are not limited to, PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT, and CD103.

[0079] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0080] 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 embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the 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.

[0081] When the embodiments give 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 can 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 grasp of the prior art by those skilled in the art and the record 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 can also be used to realize the present invention.

[0082] The sequence information used in this application is as follows:

[0083] SEQ ID No.1

[0084] SCMGGMNQR

[0085] Example 1 Screening and identification of tumor antigen peptides

[0086] 1. Antigenic peptide prediction

[0087] All the currently discovered P53 point mutations were sorted out from the human P53 mutation database. Combined with the distribution characteristics of HLA subtypes in the Chinese population, the top 10 HLA subtypes with the highest frequency were selected, and the online prediction tool NetMHCpan (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ) was used to predict antigenic peptides. The results with %ELRANK<2 were selected as candidate antigenic peptides.

[0088] 2. Mass spectrometry identification of candidate antigenic peptides

[0089] 1) Place the mutation site in the middle of the candidate antigen peptide selected above according to its amino acid sequence, and cut a short peptide of 25 amino acids. Connect multiple antigen peptides in series, and add G3S connecting peptides between the antigen peptides. Finally, clone it into a eukaryotic expression vector;

[0090] 2) Use K562 cells to stably transfer a single HLA gene to establish a cell line that stably expresses HLA (using HLA-A*11:01 as an example). The vector expressing the candidate antigen peptide constructed above is electroporated into cells expressing a single HLA subtype. After 48 hours, the cells are collected, washed with PBS, centrifuged and frozen for later use;

[0091] 3) After the cells collected above are lysed, immunoprecipitation is performed using HLA antibody (w6 / 32). The components pulled down by the antibody are analyzed by mass spectrometry. The mass spectrometry results are analyzed by protein sequence library search or de novo sequencing algorithm.

[0092] According to the above experimental process ( Figure 1 A) After the vector expressing the candidate TP53 mutant antigen peptide was electroporated into the HLA-A*11:01 stable transfectant, the mass spectrometry analysis results were obtained as follows Figure 1 As shown in B, the tumor antigen peptide sequence containing the TP53 R248Q mutation is finally obtained as shown in SEQ ID No. 1. The tumor antigen peptide used in the present application can be obtained by chemical synthesis based on the known specific amino acid sequence.

[0093] Example 2 Application of tumor antigen peptides in the development of therapeutic vaccines

[0094] 2.1 Tumor antigen peptides induce the generation of T cells that specifically bind to the TP53 R248Q mutation

[0095] 2.1.1 Inducing healthy donor monocytes to differentiate into dendritic cells (DC) in vitro

[0096] (1) Lymphoprep density gradient centrifugation was used to obtain PBMCs (or resuscitated frozen PBMCs) from the blood of healthy donors / patients. After resuspending in 10 ml of 1640 medium (Gbico), the supernatant was removed by centrifugation at 350 g for 5 min, and an appropriate amount of DC medium (Stem cell) was added to resuspend the PBMCs to 5 × 10 6 cells / ml density.

[0097] (2) Pipette 2 ml of the above PBMCs suspension into a 6-well plate. The number of cells is 1×10 7 cells per well. Place 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 clumping non-adherent cells without disturbing the adherent cells.

[0098] (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.

[0099] (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.

[0100] (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.

[0101] 2.1.2 DC cells loaded with TP53-R248Q antigen peptide were co-stimulated with autologous CD8+ T cells

[0102] (1) Add 5 μg / ml of the TP53-R248Q tumor antigen peptide with the amino acid sequence shown in SEQ ID No. 1 to the above 15 ml centrifuge tube (Mature DC). Seal the lid with Parafilm, place in a MACSmix rotator, and rotate and bind in a cell culture incubator at 37° C., 5% CO 2 for 4 hours.

[0103] (2) PBMCs (or resuscitated frozen PBMCs) were obtained from the blood of 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 the CD8+ T cells in the PBMCs were isolated using the EasySepTM HumanCD8 Positive Selection Kit.

[0104] (3) After the mature DCs were incubated with TP53-R248Q-peptide, the mature DCs loaded with TP53-R248Q tumor antigen peptide were irradiated with a dose of 4000 rad (40 Gy) using an irradiator.

[0105] (4) After irradiation of mature DCs loaded with TP53-R248Q-peptide, they were stimulated in vitro at a ratio of DC cells to CD8+ T cells of 1:2.5. 2 ml was added to each well of a 12-well plate, and then the 12-well plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated overnight.

[0106] (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 in the above (4) for a second round of in vitro stimulation.

[0107] (6) On Day 28, cells in the co-stimulation system were collected and FACS staining was then 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, 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 and the experimental group were detected by flow cytometry.

[0108] 2.1.3 Isolation of TP53-R248Q-specific T cells and single-cell TCR sequencing

[0109] (1) CD8+ T cells from healthy donors were stimulated by autologous TP53-R248Q-peptide-loaded DCs for two rounds in vitro Figure 2 A. Collect cells in the costimulation system for flow cytometry analysis. The specific flow cytometry results are as follows: Figure 2As shown in B, the proportion of CD8a+ / Tetramer+T in healthy donors is 0.14%, and the clustering is obvious. This shows that TP53-R248Q-specific-TCR-T cells have been specifically amplified in two rounds of in vitro stimulation. Subsequently, the CD8+ / tetramer+T cells in the co-culture system were specifically sorted using a flow cytometer, 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.

[0110] (2) After the single-cell sequencing is completed, the transcriptomic characteristics of the top four TCRs are analyzed, and the TCR sequencing results and frequency ranking results of the cell population obtained by the analysis are as follows: Figure 2 C. The TCR sequence with the highest frequency was then optimized; the TCR sequence was codon-optimized to increase the expression of TCR, and the final encoding TP53-R248Q-TCR was obtained; P2A and Furin-cleavage were used to allow the α and β chains of TCR to be expressed simultaneously in the lentiviral expression vector.

[0111] 2.1.4 Preparation of T cells expressing T cell receptors that specifically bind to TP53-R248Q mutation

[0112] (1) The optimized TP53-R248Q-TCR obtained above was used to construct a lentiviral expression vector ( Figure 3 AB), followed by TP53-R248Q-TCR lentiviral packaging. The specific operation of virus packaging is as follows: 293T cells are digested and passaged, and the cell density is observed on the day of transfection. Transfection can be performed when it is 80-90% full. No need to change the culture medium before transfection. Opti-MEM needs to be preheated in a 37-degree water bath, and Fugene-HD transfection reagent needs to be restored to room temperature before use. Shake well before use. The complex components of each bottle of T75 for transfection are as follows: pHR-SFFV-TCR-Puro plasmid containing the target TCR: 10ug; pMD2.G (envelope plasmid): 5ug; pSPAX2 (core enzyme): 7.5ug. After adding opti-MEM to 1mL, mix well; add 56ul Fugene-HD transfection reagent, blow normally 5-6 times, leave at room temperature for 15min, and then add it to the T75 bottle. Then, the virus supernatant was collected at 48h and 72h, and the supernatant was filtered through a 0.45um filter membrane (PES material) for use after 4000rpm for 5min. The TP53-R248Q-TCR lentiviral solution was collected at 48h and 72h after virus packaging.

[0113] (2) Jurkat T cells were infected with TP53-R248Q-TCR lentiviral particles, and 10 μg / ml polybrene was added, and the cells were centrifuged at 800×g, 90 min, and 30° C. After centrifugation, the cells were placed in a cell culture incubator at 37° C. and 5% CO2 for 24 h, and then fresh 1640 medium was replaced for culture.

[0114] (3) 20ul CD3 / 28 beads (Genscript) and 20ng / ml IL-2 were added to the CD8+T cells of healthy donors. After activating the T cells for 48h, 10μg / ml polybrene was added to perform the first round of TP53-R248Q-TCR lentivirus infection. After centrifugation, the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 24h. Subsequently, the second round of TP53-R248Q-TCR lentivirus infection was performed.

[0115] 2.2 Identification of binding specificity of T cells induced by tumor antigen peptides

[0116] 48 hours after the above-mentioned TP53-R248Q-TCR lentivirus infected Jurkat T cells or primary CD8+T cells, TP53-R248Q-TCR-Jurkat T cells or TP53-R248Q-TCR-T cells were aspirated, washed twice with FACS buffer, and then stained with HLA-A:1101-TP53-R248Q-Tetramer-PE and CD8a-APC antibodies.

[0117] The TP53-R248Q-TCR-T cells prepared above were stained with mTCRβC+ / HLA-A*11:01-TP53-R248Q-Tetramer. The flow cytometry results showed that Tetramer+ / mTCRβC+Jurkat T and CD8+ / Tetramer+primary CD8+T( Figure 3 C) The cell clustering is obvious. The results show that the TP53-R248Q-TCR screened and identified in vitro can be normally expressed in Jurkat T cell lines and primary CD8+T, and can specifically bind to HLA-A:1101-TP53-R248Q-Tetramer.

[0118] Example 3 Application of tumor antigen peptides in the preparation of TCR-T

[0119] 3.1 In vitro activation experiment of TP53-R248Q-TCR-T cells

[0120] The in vitro activation experiment of TP53-R248Q-TCR-T cells, i.e., the IFNγ-Elispot experiment, includes the following steps:

[0121] (1) Directly differentiating donor autologous monocytes into DC cells in vitro, loading DC with TP53-R248-WT or TP53-R248Q antigen peptide fragments as in Example 2, and co-culturing the TP53-R248Q-TCR-T cells prepared above with DC loaded with TP53-R248-WT or TP53-R248Q antigen peptide fragments in vitro;

[0122] (2) After 24 h of in vitro co-culture, the cells were discarded and then washed five times with 200 μl 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.

[0123] (3) After washing five times with 200 μl DPBS, add 100 μl TMB substrate to each well until complete spots appear in the wells, and then add sterile water to interrupt the color development process.

[0124] The results are as follows Figure 4 As shown in Figure 2, DCs loaded with TP53-R248Q antigen peptides can specifically activate TP53-R248Q-TCR-T cells in vitro, and the IFNγ expression level of TP53-R248Q-TCR-T cells after activation is significantly increased ( Figure 4 AC), while DC loaded with TP53-R248-WT antigen peptide had no significant activation effect on TP53-R248Q-TCR-T cells ( Figure 4 BC), so the results show that the TP53-R248Q-TCR obtained by this screening and identification can specifically recognize the TP53-R248Q antigen peptide.

[0125] 3.2 TP53-R248Q-TCR-T in vitro cytokine release assay

[0126] (1) In this example, K562 cells expressing the HLA-A*11:01 subtype of the donor were prepared by lentiviral infection and flow cytometry.

[0127] (2) After K562-A*11:01 control cells or cells loaded with TP53-R248Q mutant polypeptide were co-cultured with the donor's autologous TP53-R248Q-TCR-T for 24 h, flow cytometry detection of IFN-γ / TNF-α / IL-2 / GZMB was performed.

[0128] The results showed that TP53-R248Q-TCR-T specifically recognized the TP53-R248Q mutant peptide presented by HLA-A*11:01 and significantly secreted IFN-γ cytokine ( Figure 5 AD), while mTCRβ-negative CD8 + T cells did not secrete cytokines. The above results further showed that the TP53-R248Q-TCR sequence identified this time had excellent specificity, indirectly proving that the R248Q antigen peptide was immunogenic.

[0129] 3.3 Specific detection of R248Q mutant peptide

[0130] Through in vitro binding and activation experiments, this example verifies that TP53-R248Q-TCR-T can specifically recognize the TP53-R248Q mutant peptide presented by HLA-A*11:01, and TP53-R248Q-TCR-T can specifically secrete cytokines after recognition. Subsequently, in this example, the mutant amino acid Q position of the antigen peptide "SCMGGMNQR" was tested by in vitro activation experiments. The flow cytometry results showed that only the antigen peptide "SCMGGMNQR" peptide could specifically activate TP53-R248Q-TCR-T cells and upregulate the expression of CD137 and CD69 ( Figure 6 AB).

[0131] 3.4TP53-R248Q-TCR-T in vitro killing experiment

[0132] (1) As in the preparation of TP53-R248Q-TCR-T in Example 2, TP53-R248Q-TCR-T effector cells and K562-A1101-TP53-R248Q target cells were constructed in vitro by infecting T cells or K562-A1101 cells with lentivirus.

[0133] (2) K562 target cells or control cells were labeled with celltrace violet, incubated at room temperature and away from light for 15 min, and then washed by FACS at 1000 rpm for 5 min. After removing the supernatant, complete culture medium was added for resuspending and counting.

[0134] (3) TP53-R248Q-TCR-T cells and K562-A1101 control cells or K562-A1101-TP53-R248Q target cells were then added to a 96-well plate at an effector-target ratio of 5:1 for co-culture for 24 h.

[0135] (4) After collecting the cells in the 96-well plate, perform FACS washing at 1000 rpm for 5 min, remove the supernatant, add APC-Cy7-Live / dead dye to stain the dead cells, and use flow cytometry to detect the cell ratio of the culture system China CelltraceViolet+ / APC-Cy7+ after washing.

[0136] The above experimental results are as follows Figure 7 As shown in AB, TP53-R248Q-TCR-T can efficiently kill target cells in vitro, and its killing efficiency is significantly higher than that of primary CD8+T cells (P<0.0001). The results of the in vitro killing experiment show that the TP53-R248Q-TCR obtained by this screening and identification can specifically recognize and kill target cells expressing HLA-A1101-R248Q. This result also shows that the R248Q antigen peptide (SCMGGMNQR) identified in this study has strong immunogenicity and can specifically activate T cells. The R248Q-TCR-T identified in this study can specifically recognize and kill target cells.

[0137] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious 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 as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A tumor antigen peptide, characterized in that: The tumor antigen peptide comprises one or more of the following features: 1) The tumor antigen peptide comprises a peptide segment having an amino acid sequence as shown in SEQ ID No. 1; 2) The tumor antigen peptide is a peptide segment that has at least 80% sequence similarity to the peptide segment described in 1) and has or partially has the activity of the tumor antigen peptide described in 1).

2. The tumor antigen peptide according to claim 1, characterized in that The tumor antigen peptide can be recognized by an antibody that specifically binds to TP53R248Q mutant tumor cells.

3. A recombinant nucleic acid molecule, characterized in that The recombinant nucleic acid molecule encodes the tumor antigen peptide according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the recombinant nucleic acid molecule according to claim 3.

5. A cell, characterized in that The cell contains the tumor antigen peptide according to claim 1 or 2, the recombinant nucleic acid molecule according to claim 3, or the recombinant expression vector according to claim 4.

6. The cell according to claim 5, characterized in that The cell is a cell that expresses or produces a tumor antigen peptide; or, the cell is a cell that carries a tumor antigen peptide on its surface.

7. Use of the tumor antigen peptide according to claim 1 or 2, the recombinant nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, or the cell according to claim 5 or 6 in the preparation of any of the following products: 1) HLA multimer products; 2) TCR-T products; 3) Tumor vaccine products; 4)Tumor treatment products.

8. A method for preparing isolated TCR-T cells, characterized in that: The preparation method comprises: co-culturing antigen presenting cells loaded with the tumor antigen peptide as claimed in claim 1 or 2 with T cells, thereby obtaining the separated TCR-T cells.

9. A tumor vaccine, characterized in that: The tumor vaccine comprises one or more of the tumor antigen peptide according to claim 1 or 2, the recombinant nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, or the cell according to claim 5 or 6.

10. The tumor vaccine according to claim 9, characterized in that The tumor is selected from one or more of squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penis cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia or chronic myeloblastic leukemia.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the tumor antigen peptide according to claim 1 or 2, the recombinant nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, or the cell according to claim 5 or 6, and a pharmaceutical excipient or therapeutic adjuvant.

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