HLA-A1101 restricted antigen epitope peptide, targeted epitope peptide TCR and application thereof

Abstract

The present invention belongs to the technical field of T cell immunotherapy drugs, and specifically relates to an HLA-A1101 restricted antigen epitope peptide, a targeted epitope peptide TCR and its application. The epitope peptide is PIK3CA_H1047L 1046‑1054 The mutant polypeptide has an amino acid sequence as shown in SEQ ID NO: 56; it has a strong binding ability with MHC, and the binding ability is better than that of wild-type PIK3CA_H1047 1046‑1054 , is an ideal target for activating T cells; and based on the PIK3CA_H1047L 1046‑1054 The TCRs designed with mutant peptides all have strong specificity and can mediate T cells to secrete a large amount of IFN-γ and IL-2 cytokines to kill PIK3CA_H1047L 1046‑1054 Mutated tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of T cell immunotherapy drugs, and specifically relates to an HLA-A1101 restricted antigen epitope peptide, a targeted epitope peptide TCR and applications thereof. Background Art

[0002] With the progress of research in the biomedical field, the target selection of T cell immunotherapy targeting malignant solid tumors has continued to evolve, from the initial lineage antigens to viral antigens. With the emergence of high-throughput sequencing technology in recent years, targeting individual tumor mutations has become the focus of the new generation of T cell therapy.

[0003] T cell therapy currently mainly includes TCR-T and CAR-T treatment methods. At present, CAR-T technology treatment has played a significant role in the treatment of blood tumors such as leukemia and lymphoma, greatly improving the survival rate and quality of life of patients. However, for solid tumors, CAR-T treatment methods have limited specific targets, which greatly limits its application prospects. TCR-T technology is different from CAR-T cell technology. TCR is a characteristic marker on the surface of all T cells, which binds to CD3 with non-covalent bonds to form a TCR-CD3 complex. In peripheral blood, 90%-95% of T cells express TCR. T cells with genetically modified TCR can specifically recognize antigen molecules on the surface of tumor cells, and then produce immune responses against tumor cells.

[0004] At present, in human cancer, PIK3CA gene is one of the oncogenes in the field of oncology. The frequency of PIK3CA gene mutation is close to 44.5% in endometrial cancer, close to 30.7% in breast cancer, close to 23.6% in cervical cancer, close to 24.7% in colorectal cancer, close to 20.4% in bladder cancer, close to 13.6% in head and neck cancer, close to 13.0% in esophageal and gastric cancer, etc. In addition to the above cancers, PIK3CA gene mutation also occurs in glioma, non-small cell carcinoma, glioblastoma, bile duct cancer, melanoma, seminoma, hepatobiliary cancer, sarcoma, pancreatic cancer, pleural mesothelioma, prostate cancer, ovarian epithelial cell tumor, renal clear cell carcinoma, adrenocortical carcinoma, kidney, non-clear cell carcinoma and thyroid cancer (refer to Figure 1 ).

[0005] Currently, the wild-type PIK3CA has a weak ability to bind to MHC, and the formed antigen peptide-MHC complex (pMHC) is unstable and cannot form a good T cell killing site. Summary of the invention

[0006] In view of the above problems, the present invention has discovered an HLA-A1101-restricted antigen epitope peptide that can form a stable complex with HLA-A1101 and be presented on the cell surface. The epitope peptide is PIK3CA_H1047L 1046-1054 The mutant peptide has a strong binding ability with MHC and is an ideal target for activating T cells; and based on the PIK3CA_H1047L 1046-1054 The TCRs screened by mutant peptides all have strong specificity and can mediate T cells to secrete a large amount of IFN-γ and IL-2 cytokines to kill PIK3CA_H1047L 1046-1054 Tumor cells with mutated HLA-A1101 subtype.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides an HLA-A1101 restricted antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID NO:56.

[0009] Another aspect of the present invention provides a TCR targeting a polypeptide-HLA-A1101 complex, the polypeptide sequence of which is shown in SEQ ID NO: 56, and the TCR can be selected from the following four TCRs:

[0010] The first TCR:

[0011] The first TCR comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 encoded by the sequence shown in SEQ ID NO:1, an α chain CDR2 encoded by the sequence shown in SEQ ID NO:2, and an α chain CDR3 encoded by the sequence shown in SEQ ID NO:3; the β chain variable region comprises a β chain CDR1 encoded by the sequence shown in SEQ ID NO:4, a β chain CDR2 encoded by the sequence shown in SEQ ID NO:5, and a β chain CDR3 encoded by the sequence shown in SEQ ID NO:6.

[0012] The second TCR:

[0013] The second TCR comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 encoded by the sequence shown in SEQ ID NO:19, an α chain CDR2 encoded by the sequence shown in SEQ ID NO:20, and an α chain CDR3 encoded by the sequence shown in SEQ ID NO:21, and the β chain variable region comprises a β chain CDR1 encoded by the sequence shown in SEQ ID NO:22, a β chain CDR2 encoded by the sequence shown in SEQ ID NO:23, and a β chain CDR3 encoded by the sequence shown in SEQ ID NO:24.

[0014] The third type of TCR:

[0015] The third TCR comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 encoded by the sequence shown in SEQ ID NO:37, an α chain CDR2 encoded by the sequence shown in SEQ ID NO:38, and an α chain CDR3 encoded by the sequence shown in SEQ ID NO:39, and the β chain variable region comprises a β chain CDR1 encoded by the sequence shown in SEQ ID NO:40, a β chain CDR2 encoded by the sequence shown in SEQ ID NO:41, and a β chain CDR3 encoded by the sequence shown in SEQ ID NO:42.

[0016] The fourth TCR:

[0017] The fourth TCR comprises any one of the following three groups: (a) an α chain encoded by the sequence shown in SEQ ID NO:15 and a β chain encoded by the sequence shown in SEQ ID NO:16; (b) an α chain encoded by the sequence shown in SEQ ID NO:33 and a β chain encoded by the sequence shown in SEQ ID NO:34; (c) an α chain encoded by the sequence shown in SEQ ID NO:51 and a β chain encoded by the sequence shown in SEQ ID NO:52.

[0018] In another aspect, the present invention provides a polynucleotide encoding the above-mentioned TCR, and the polynucleotide can be selected from any one of the following polynucleotides:

[0019] The first polynucleotide, encoding the first TCR mentioned above, comprises the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0020] The second polynucleotide, encoding the second TCR, comprises the sequences shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0021] The third polynucleotide, encoding the third TCR, comprises the sequences shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42.

[0022] The fourth nucleotide comprises any one of the following three groups of nucleic acid sequences: (a) SEQ ID NO:15 and SEQ ID NO:16; (b) SEQ ID NO:33 and SEQ ID NO:34; (c) SEQ ID NO:51 and SEQ ID NO:52.

[0023] In another aspect, the present invention provides an expression vector comprising any of the polynucleotides described above.

[0024] In another aspect, the present invention provides an engineered cell comprising any one of the above expression vectors.

[0025] In another aspect, the present invention provides a pharmaceutical composition, characterized in that it comprises any of the above-mentioned TCRs or any of the above-mentioned polynucleotides or any of the above-mentioned expression vectors or any of the above-mentioned engineered cells.

[0026] In another aspect, the present invention provides a use of any of the above TCRs, or any of the above polynucleotides, or any of the above expression vectors, or any of the above engineered cells in the preparation of a drug for treating cancers that are positive for polypeptide-HLA-A1101 expression; the polypeptide sequence is shown in SEQ ID NO:56.

[0027] In another aspect, the present invention provides a use of any of the above TCRs, or any of the above polynucleotides, or any of the above expression vectors, or any of the above engineered cells in the preparation of a reagent or kit for detecting tumor cells that express positive polypeptide-HLA-A1101; the polypeptide sequence is shown in SEQ ID NO:56.

[0028] In another aspect, the present invention provides a use of a polypeptide comprising the above-mentioned HLA-A1101 restricted antigen epitope peptide in the preparation of a therapeutic tumor vaccine.

[0029] The beneficial effects of the present invention include at least:

[0030] (1) HLA-A1101 restricted antigen epitope peptide (mutant PIK3CA_H1047L) provided by the present invention 1046-1054 ) has a strong binding ability with MHC, and its binding ability is better than that of wild-type PIK3CA_H1047 1046-1054 , which is an ideal target for activating T cells.

[0031] (2) TCR provided by the present invention for PIK3CA_H1047L 1046-1054 The mutant peptides all have strong specificity and can mediate T cells to secrete a large amount of IFN-γ and IL-2 cytokines to kill PIK3CA_H1047L 1046-1054Mutated tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The frequency of PIK3CA gene mutations in different tumors;

[0033] Figure 2 This is the combination of PIK3CA mutant sequence and HLA-A*11:01 subtype;

[0034] Figure 3 PIK3CA_H1047L 1046-1054 The reactivity of specific T cells to the mutant sequence;

[0035] Figure 4 Detection of PIK3CA_H1047L by flow cytometry 1046-1054 Expansion of specific T cells;

[0036] Figure 5 Flow cytometry plots to verify the specific reactivity of PT1TCR, PT8TCR and PT10TCR to mutant antigens;

[0037] Figure 6 The bar graphs are used to verify the specific reactivity of PT1TCR, PT8TCR and PT10TCR to mutant antigens;

[0038] Figure 7 The expression of CD137 was analyzed for activation of PT1TCR, PT8TCR, and PT10TCR with different concentrations of antigen peptides;

[0039] Figure 8 ELISA was used to detect the secretion of IFN-γ by PT1TCR, PT8TCR and PT10TCR in response to antigen peptides of different concentrations;

[0040] Fig. 9 Flow cytometry detection of PT1TCR, PT8TCR and PT10TCR and expression of PIK3CA_H1047L 1046-1054 The expression of CD137 was analyzed after activation of different tumor cells co-cultured;

[0041] Fig.10 T1TCR, PT8TCR and PT10TCR in different tumor cells and expression of PIK3CA_H1047L 1046-1054 The expression of CD137 in the activation analysis of different tumor cells after co-culture is shown in the bar chart;

[0042] Fig.11 ELISA to detect PT1TCR, PT8TCR and PT10TCR and expression of PIK3CA_H1047L 1046-1054IFN-γ secretion of different tumor cells after co-culture;

[0043] Fig.12 ELISA to detect PT1TCR, PT8TCR and PT10TCR and expression of PIK3CA_H1047L 1046-1054 IL-2 secretion of different tumor cells after co-culture;

[0044] Fig.13 Expression of PIK3CA_H1047L for PT1TCR, PT8TCR, and PT10TCR killing 1046-1054 MDA-MB-231 cells;

[0045] Fig.14 Expression of PIK3CA_H1047L for PT1TCR, PT8TCR, and PT10TCR killing 1046-1054 The situation of SKOV3 cells. DETAILED DESCRIPTION

[0046] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0047] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0048] An embodiment of the present invention provides an HLA-A1101 restricted antigen epitope peptide, whose amino acid sequence is shown in SEQ ID NO:56.

[0049] It should be noted that this epitope peptide originates from one of the high-frequency mutation sites of the tumor driver gene PIK3CA, site H1047 (PIK3CA_H1047L 1046-1054 Mutant polypeptide), when H1047L, produces immunogenicity. 1046-1054 The mutant peptide has a strong binding ability with MHC, which is better than the wild-type PIK3CA_H1047 1046-1054As mentioned above, PIK3CA mutations often occur in many types of cancers and are a gene with high frequency mutations in the human population. Based on this, this epitope peptide is an ideal target for activating T cells.

[0050] Another embodiment of the present invention provides a TCR targeting the above-mentioned HLA-A1101 restricted antigen epitope peptide, the polypeptide sequence of which is shown in SEQ ID NO: 56, and the TCR can be selected from the following four TCRs:

[0051] The first TCR:

[0052] The first TCR comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 encoded by the sequence shown in SEQ ID NO:1, an α chain CDR2 encoded by the sequence shown in SEQ ID NO:2, and an α chain CDR3 encoded by the sequence shown in SEQ ID NO:3; the β chain variable region comprises a β chain CDR1 encoded by the sequence shown in SEQ ID NO:4, a β chain CDR2 encoded by the sequence shown in SEQ ID NO:5, and a β chain CDR3 encoded by the sequence shown in SEQ ID NO:6.

[0053] In some specific embodiments, in the above-mentioned first TCR, the α chain variable region further comprises: one or more of an α chain FR1 encoded by the sequence shown in SEQ ID NO:7, an α chain FR2 encoded by the sequence shown in SEQ ID NO:8, an α chain FR3 encoded by the sequence shown in SEQ ID NO:9, and an α chain FR4 encoded by the sequence shown in SEQ ID NO:10; the β chain variable region further comprises: one or more of a β chain FR1 encoded by the sequence shown in SEQ ID NO:11, a β chain FR2 encoded by the sequence shown in SEQ ID NO:12, a β chain FR3 encoded by the sequence shown in SEQ ID NO:13, and a β chain FR4 encoded by the sequence shown in SEQ ID NO:14.

[0054] It should be noted that, in the first TCR described above, the nucleic acid sequences and amino acid sequences corresponding to the CDR regions encoded by the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6 and the FR regions encoded by the sequences shown in SEQ ID NO: 7 to SEQ ID NO: 14 are shown in Table 1 below:

[0055] Table 1 CDR and FR region sequences of the first TCR mentioned above

[0056] area Nucleic acid sequence Amino acid sequence α-chain CDR1 SEQ ID NO:1 NSASDY α chain CDR2 SEQ ID NO:2 IRSNMDK α-chain CDR3 SEQ ID NO:3 AEGETGNQFY β-chain CDR1 SEQ ID NO:4 SGHRS β-chain CDR2 SEQ ID NO:5 YF β-chain CDR3 SEQ ID NO:6 ASSSGTGGFEQF α chain FR1 SEQ ID NO:7 GESVGLHLPTLSVQEGDNSIINCAYS α chain FR2 SEQ ID NO:8 FIWYKQESGKGPQFIID α chain FR3 SEQ ID NO:9 RQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFC α chain FR4 SEQ ID NO:10 FGTGTSLTVIP β-chain FR1 SEQ ID NO:11 KAGVTQTPRYLIKTRGQQVTLSCSPI β-chain FR2 SEQ ID NO:12 VSWYQQTPGQGLQFLFE β-chain FR3 SEQ ID NO:13 RNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLC β-chain FR4 SEQ ID NO:14 FGPGTRLTVL

[0057] The second TCR:

[0058] The second TCR comprises an α chain variable region and a β chain variable region; comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 encoded by the sequence shown in SEQ ID NO:19, an α chain CDR2 encoded by the sequence shown in SEQ ID NO:20, and an α chain CDR3 encoded by the sequence shown in SEQ ID NO:21, and the β chain variable region comprises a β chain CDR1 encoded by the sequence shown in SEQ ID NO:22, a β chain CDR2 encoded by the sequence shown in SEQ ID NO:23, and a β chain CDR3 encoded by the sequence shown in SEQ ID NO:24.

[0059] In some specific embodiments, in the above-mentioned second TCR, the α chain variable region further comprises: one or more of an α chain FR1 encoded by the sequence shown in SEQ ID NO:25, an α chain FR2 encoded by the sequence shown in SEQ ID NO:26, an α chain FR3 encoded by the sequence shown in SEQ ID NO:27, and an α chain FR4 encoded by the sequence shown in SEQ ID NO:28; the β chain variable region further comprises: one or more of a β chain FR1 encoded by the sequence shown in SEQ ID NO:29, a β chain FR2 encoded by the sequence shown in SEQ ID NO:30, a β chain FR3 encoded by the sequence shown in SEQ ID NO:31, and a β chain FR4 encoded by the sequence shown in SEQ ID NO:32.

[0060] It should be noted that, in the above-mentioned second TCR, the nucleic acid sequences and amino acid sequences corresponding to the CDR regions encoded by the sequences shown in SEQ ID NO: 19 to SEQ ID NO: 24 and the FR regions encoded by the sequences shown in SEQ ID NO: 25 to SEQ ID NO: 32 are shown in Table 2 below:

[0061] Table 2 CDR and FR region sequences of the second TCR mentioned above

[0062] area Nucleic acid sequence Amino acid sequence α-chain CDR1 SEQ ID NO:19 TRDTTYY α chain CDR2 SEQ ID NO:20 RNSFDEQ α-chain CDR3 SEQ ID NO:21 ALSEATGNQFY β-chain CDR1 SEQ ID NO:22 SQVTM β-chain CDR2 SEQ ID NO:23 ANQGSEA β-chain CDR3 SEQ ID NO:24 SVQTLTSSDEQY α chain FR1 SEQ ID NO:25 AQKVTQAQTEISVVEKEDVTLDCVYE α chain FR2 SEQ ID NO:26 LFWYKQPPSGELVFLIR α chain FR3 SEQ ID NO:27 EISGRYSWNFQKSTSSFNFTITASQVVDSAVYFC α chain FR4 SEQ ID NO:28 FGTGTSLTVIP β-chain FR1 SEQ ID NO:29 SAVISQKPSRDICQRGTSLTIQCQVD β-chain FR2 SEQ ID NO:30 MFWYRQQPGQSLTLIAT β-chain FR3 SEQ ID NO:31 TYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLC β-chain FR4 SEQ ID NO:32 FGPGTRLTVT

[0063] The third type of TCR:

[0064] The third TCR comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 encoded by the sequence shown in SEQ ID NO:37, an α chain CDR2 encoded by the sequence shown in SEQ ID NO:38, and an α chain CDR3 encoded by the sequence shown in SEQ ID NO:39, and the β chain variable region comprises a β chain CDR1 encoded by the sequence shown in SEQ ID NO:40, a β chain CDR2 encoded by the sequence shown in SEQ ID NO:41, and a β chain CDR3 encoded by the sequence shown in SEQ ID NO:42.

[0065] In some specific embodiments, in the above-mentioned third TCR, the α chain variable region further comprises: one or more of an α chain FR1 encoded by the sequence shown in SEQ ID NO:43, an α chain FR2 encoded by the sequence shown in SEQ ID NO:44, an α chain FR3 encoded by the sequence shown in SEQ ID NO:45, and an α chain FR4 encoded by the sequence shown in SEQ ID NO:46; the β chain variable region further comprises: one or more of a β chain FR1 encoded by the sequence shown in SEQ ID NO:47, a β chain FR2 encoded by the sequence shown in SEQ ID NO:48, a β chain FR3 encoded by the sequence shown in SEQ ID NO:49, and a β chain FR4 encoded by the sequence shown in SEQ ID NO:50.

[0066] It should be noted that, in the third TCR described above, the nucleic acid sequences and amino acid sequences corresponding to the CDR regions encoded by the sequences shown in SEQ ID NO:37-SEQ ID NO:42 and the FR regions encoded by the sequences shown in SEQ ID NO:43-SEQ ID NO:50 are shown in Table 3 below:

[0067] Table 3 CDR and FR region sequences of the third TCR mentioned above

[0068] area Nucleic acid sequence Amino acid sequence α-chain CDR1 SEQ ID NO:37 SSNFYA α chain CDR2 SEQ ID NO:38 MTLNGDE α-chain CDR3 SEQ ID NO:39 ASYSGAGSYQLT β-chain CDR1 SEQ ID NO:40 MNHNS β-chain CDR2 SEQ ID NO:41 SASEGT β-chain CDR3 SEQ ID NO:42 ASSEDFGGSSYNEQF α chain FR1 SEQ ID NO:43 ILNVEQSPQSLHVQEGDSTNFTCSFP α chain FR2 SEQ ID NO:44 LHWYRWETAKSPEALFV α chain FR3 SEQ ID NO:45 KKKGRISATLNTKEGYSYLYIKGSQPEDSATYLC α chain FR4 SEQ ID NO:46 FGKGTKLSVIP β-chain FR1 SEQ ID NO:47 NAGVTQTPKFQVLKTGQSMTLQCAQD β-chain FR2 SEQ ID NO:48 MYWYRQDPGMGLRLIYY β-chain FR3 SEQ ID NO:49 TDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFC β-chain FR4 SEQ ID NO:50 FGPGTRLTVL

[0069] The fourth TCR:

[0070] The fourth TCR comprises any one of the following three groups: (a) an α chain encoded by the sequence shown in SEQ ID NO:15 and a β chain encoded by the sequence shown in SEQ ID NO:16; (b) an α chain encoded by the sequence shown in SEQ ID NO:33 and a β chain encoded by the sequence shown in SEQ ID NO:34; (c) an α chain encoded by the sequence shown in SEQ ID NO:51 and a β chain encoded by the sequence shown in SEQ ID NO:52.

[0071] It should be noted that the fourth TCR belongs to the preferred sequence when the FR regions in the first TCR, the second TCR and the third TCR are all selected from the corresponding FRs.

[0072] It should also be noted that the above-mentioned TCR can only recognize tumor cells at the PIK3CA_H1047L site and perform tumor-specific killing, and has no recognition ability for normal human tissues expressing the wild type.

[0073] It should also be noted that in the above TCR, the CDR3 of the α chain and the β chain belong to the hypervariable region of the variable region. When the TCR recognizes the MHC-antigen peptide complex, the CDR3 directly binds to the antigen peptide, which directly affects the TCR's ability to recognize the PIK3CA_H1047L mutation. The hypervariable regions of the α chain and the β chain of the TCR also include the CDR1 and CDR2 hypervariable regions. The CDR1 and CDR2 hypervariable regions are relatively stable relative to the CDR3. When the TCR recognizes the MHC-antigen peptide complex, the CDR1 and CDR2 hypervariable regions recognize and bind to the sidewalls of the antigen binding groove of the MHC molecule.

[0074] It should also be noted that the FR (FR1, FR2, FR3, FR4) in the above three TCRs is a framework region, which is used to connect the CDR region and is relatively stable. In addition to the above sequences, the FR regions in the first, second and third TCRs can also be selected from sequences with ≥80% identity to the FR1, FR2, FR3 and FR4 sequences of the above three TCRs, such as ≥80%, 85%, 90% or 95%, and the source can be mouse or human (but other sources, such as rabbit, pig and other animal sources are not excluded). In certain specific implementations, the sequences of the FR regions of the above-mentioned α chain and β chain and the sequences of the CDR regions can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 to respectively constitute the α chain variable region and the β chain variable region of the TCR. For example, in the first TCR, the sequence encoding the α chain variable region is shown in SEQ ID NO:15, and the sequence encoding the β chain variable region is shown in SEQ ID NO:16; in the second TCR, the sequence encoding the α chain variable region is shown in SEQ ID NO:33, and the sequence encoding the β chain variable region is shown in SEQ ID NO:34; in the third TCR, the sequence encoding the α chain variable region is shown in SEQ ID NO:51, and the sequence encoding the β chain variable region is shown in SEQ ID NO:52.

[0075] It should also be noted that, in addition to the above-mentioned α chain variable region and β chain variable region, the α chain also includes an α chain constant region, and the β chain also includes a β chain constant region. The sources of the α chain constant region and the β chain constant region can be mouse, human or human-mouse chimera (but other sources, such as rabbit, pig and other animal sources are not excluded), which are almost no mutation regions. Specifically, in the first TCR, the α chain constant region can be selected as shown in SEQ ID NO:57, and the β chain constant region can be selected as shown in SEQ ID NO:58; in the second TCR, the α chain constant region can be selected as shown in SEQ ID NO:59, and the β chain constant region can be selected as shown in SEQ ID NO:60; in the third TCR, the α chain constant region can be selected as shown in SEQ ID NO:61, and the β chain constant region can be selected as shown in SEQ ID NO:62. In addition, in the above-mentioned TCR, regarding the sources of the variable region and constant region of the α chain or β chain, the α chain variable region or β chain variable region is from mouse or human (but other sources, such as rabbit, pig and other animal sources are not excluded), and the source of the α chain or β chain constant region may be the same as or different from the source of the α chain or β chain variable region.

[0076] In some specific embodiments, in the first TCR, the full-length sequence of the TCR α chain composed of the α chain variable region and the constant region may include the sequence shown in SEQ ID NO:17, and the full-length sequence of the TCR α chain composed of the β chain variable region and the constant region may include the sequence shown in SEQ ID NO:18; in the second TCR, the full-length sequence of the TCR α chain composed of the α chain variable region and the constant region may include the sequence shown in SEQ ID NO:35, and the full-length sequence of the TCR α chain composed of the β chain variable region and the constant region may include the sequence shown in SEQ ID NO:36; in the third TCR, the full-length sequence of the TCR α chain composed of the α chain variable region and the constant region may include the sequence shown in SEQ ID NO:53, and the full-length sequence of the TCR α chain composed of the β chain variable region and the constant region may include the sequence shown in SEQ ID NO:54.

[0077] Another embodiment of the present invention provides a polynucleotide encoding the above-mentioned TCR, and the polynucleotide can be selected from any one of the following polynucleotides:

[0078] The first polynucleotide, encoding the first TCR mentioned above, comprises the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0079] In some specific embodiments, the first polynucleotide mentioned above also contains one or more of the sequence shown in SEQ ID NO:8, the sequence shown in SEQ ID NO:9, the sequence shown in SEQ ID NO:10, the sequence shown in SEQ ID NO:11, the sequence shown in SEQ ID NO:12, the sequence shown in SEQ ID NO:13 and the sequence shown in SEQ ID NO:14.

[0080] The second polynucleotide, encoding the second TCR, comprises the sequences shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0081] In some specific embodiments, the second polynucleotide further comprises one or more of a sequence as shown in SEQ ID NO:25, a sequence as shown in SEQ ID NO:26, a sequence as shown in SEQ ID NO:27, a sequence as shown in SEQ ID NO:28, a sequence as shown in SEQ ID NO:29, a sequence as shown in SEQ ID NO:30, a sequence as shown in SEQ ID NO:31 and a sequence as shown in SEQ ID NO:32.

[0082] The third polynucleotide, encoding the third TCR, comprises the sequences shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42.

[0083] In some specific embodiments, the above-mentioned third polynucleotide also contains one or more of the sequence shown in SEQ ID NO:43, the sequence shown in SEQ ID NO:44, the sequence shown in SEQ ID NO:45, the sequence shown in SEQ ID NO:46, the sequence shown in SEQ ID NO:47, the sequence shown in SEQ ID NO:48, the sequence shown in SEQ ID NO:49 and the sequence shown in SEQ ID NO:50.

[0084] The fourth nucleotide comprises any one of the following three groups of nucleic acid sequences: (a) SEQ ID NO:15 and SEQ ID NO:16; (b) SEQ ID NO:33 and SEQ ID NO:34; (c) SEQ ID NO:51 and SEQ ID NO:52.

[0085] It should be noted that the above-mentioned polynucleotide may also contain other (in addition to the above-mentioned polynucleotide) nucleotides encoding any of the above-mentioned TCRs, such as sequences containing one or more codon-optimized sequences of SEQ ID NO:1-SEQ ID NO:6; for example, sequences containing one or more codon-optimized sequences of SEQ ID NO:19-SEQ ID NO:24; for example, sequences containing one or more codon-optimized sequences of SEQ ID NO:37-SEQ ID NO:42.

[0086] Another embodiment of the present invention provides an expression vector comprising any of the polynucleotides described above.

[0087] In some specific embodiments, the above-mentioned expression vector can be selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector and a plasmid.

[0088] In some specific embodiments, the lentiviral vector can be selected from the following group: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).

[0089] Yet another embodiment of the present invention provides an engineered cell, which comprises any of the above-mentioned expression vectors.

[0090] It should be noted that the above-mentioned engineered cells can be host cells, and the above-mentioned expression vector is introduced into the host cell to encode the TCR protein; it can also be T cells, and the above-mentioned expression vector (loaded with the target gene) is transfected into T cells to obtain TCR-T cells for PIK3CA_H1047L 1046-1054 Recognition and killing of mutant peptides.

[0091] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that it comprises any of the above-mentioned TCRs or any of the above-mentioned polynucleotides or any of the above-mentioned expression vectors or any of the above-mentioned engineered cells. Specifically, in the above-mentioned applications, the TCR can be any one of the above-mentioned four TCRs; the polynucleotide can be any one of the above-mentioned four polynucleotides.

[0092] It should be noted that the above-mentioned TCR or antigen binding fragment or polynucleotide or expression vector or engineered cell can be added with other drugs that can treat cancer to form a pharmaceutical composition to enhance the efficacy; among them, other drugs that can treat cancer can include chemotherapy drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immune preparations, etc. In addition, the above-mentioned TCR can also be combined with surgery for treatment. The specific situation can be based on the cancer situation. Medication or combined medication.

[0093] Yet another embodiment of the present invention provides a use of any of the above TCRs, or any of the above polynucleotides, or any of the above expression vectors, or any of the above engineered cells in the preparation of a drug for treating cancers that are positive for polypeptide-HLA-A1101 expression; the polypeptide sequence is shown in SEQ ID NO:56.

[0094] In some specific embodiments, the cancers used in the above applications include endometrial cancer, breast cancer, cervical cancer, colorectal cancer, bladder cancer, head and neck cancer, esophageal and gastric cancer, glioma, non-small cell carcinoma, glioblastoma, bile duct cancer, melanoma, seminoma, hepatobiliary cancer, sarcoma, pancreatic cancer, pleural mesothelioma, prostate cancer, ovarian epithelial cell tumors, renal clear cell carcinoma, adrenocortical carcinoma renal, non-clear cell carcinoma or thyroid cancer.

[0095] In some specific embodiments, the drugs used in the above applications include cell-based drugs, protein-based drugs, ADC drugs, or TCR and antigen combination drugs.

[0096] Yet another embodiment of the present invention provides a use of any of the above TCRs, or any of the above polynucleotides, or any of the above expression vectors, or any of the above engineered cells in the preparation of a reagent or kit for detecting tumor cells that express positive polypeptide-HLA-A1101; the polypeptide sequence is shown in SEQ ID NO:56.

[0097] It should be noted that, specifically, the kit involved can be divided into various small boxes, and then filled with various detection reagents. The detection reagents and detection kits can be used indirectly or directly in the expression of PIK3CA_H1047L 1046-1054 Various malignant tumors with mutations, such as pancreatic tumors, colorectal tumors, endometrial tumors, lung tumors, bile duct tumors, cervical tumors, etc.

[0098] Still another embodiment of the present invention provides a use of a polypeptide comprising the above-mentioned HLA-A1101 restricted antigen epitope peptide in the preparation of a therapeutic tumor vaccine.

[0099] It should be noted that therapeutic tumor vaccines are an emerging treatment method for treating cancer. It is an immunotherapy that aims to activate the patient's own immune system to recognize and attack tumor cells. Therapeutic tumor vaccines usually contain tumor-associated antigens or antigen fragments that can trigger the immune system's response to attack tumor cells. These vaccines work by guiding the immune system to produce antibodies or T cell responses to fight tumors. They can be used after cancer patients receive standard treatment (such as surgery, radiotherapy or chemotherapy) to reduce the risk of cancer recurrence or prolong the patient's survival. In the present invention, mutant antigen peptides are used to induce the immune system of tumor patients who are HLA-A1101 positive and have PIK3CA-H1047L mutations to produce T cell responses to fight tumors.

[0100] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0101] In the following examples, the gene amplification, sequencing and analysis methods involved were performed according to the following steps:

[0102] (1) RT-PCR: The downstream primers used were TCR gene constant region specific primers (Ca_RV1primer, Cb_RV1primer), and the upstream primers were sequences containing the outer linker and the initial 20 bp of the TCR signal peptide (AL primers, BL primers) (specifically, according to the contents and embodiments described in the literature "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A. A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun. 2016 Jun 10; 474(4): 709-714. doi: 10.1016 / j.bbrc.2016.05.015. Epub 2016 May 4. PMID: 27155153");

[0103] (2) Second round of PCR: The TCR PCR product obtained in (1) above was used as a template, the upstream primer was an outer linker primer (P2A-C primer), and the downstream primer was a specific primer for a segment of the TCR constant region upstream of the α chain constant region (Ca_RV2 primer), to obtain the second round of PCR product of the TCR α chain; the upstream primer was an outer linker primer (BES-AP primer), and the downstream primer was a specific primer for a segment of the TCR constant region upstream of the β chain constant region (Cb_RV2 primer), to obtain the second round of PCR product of the TCR β chain (specifically according to the literature "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A. A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun. 2016 Jun 2016). 10; 474(4):709-714.doi:10.1016 / j.bbrc.2016.05.015.Epub 2016May4.PMID:27155153)

[0104] (3) The amplified second-round PCR products containing the TCR α chain and β chain variable region genes were subjected to agarose gel electrophoresis to obtain the TCR α chain or β chain variable region target gene at the 500 bp-750 bp position;

[0105] (4) The target bands were subjected to first-generation sequencing (Beijing Qingke Company). After TCR sequence analysis, TCR clones with high frequencies were selected and named PT1, PT8, and PT10, respectively.

[0106] The amplification system used in the above-mentioned implementation steps involving gene amplification, sequencing and analysis methods is shown in Table 4 below (specifically implemented according to the contents recorded in the document "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A. A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun. 2016 Jun 10; 474 (4): 709-714. doi: 10.1016 / j.bbrc.2016.05.015. Epub 2016 May 4. PMID: 27155153").

[0107] Table 4 Amplification system

[0108]

[0109]

[0110] In the following examples, the nucleic acid sequences or amino acid sequences of the materials involved are shown in Table 5 below:

[0111] Table 5 Nucleic acid sequence or amino acid sequence of the materials involved in the examples

[0112] materials Nucleic acid sequence or amino acid sequence <![CDATA[PIK3CA_H1047 1046-1054 Wild-type peptide]]> SEQ ID NO:55 <![CDATA[PIK3CA_H1047L 1046-1054 Mutant Peptide]]> SEQ ID NO:56 PT1TCR α chain variable region (αV region) SEQ ID NO:15 PT1TCRβ chain variable region (βV region) SEQ ID NO:16 PT1TCRα chain full length sequence SEQ ID NO:17 PT1TCRβ chain full length sequence SEQ ID NO:18 PT10TCR α chain variable region (αV region) SEQ ID NO:33 PT10TCRβ chain variable region (βV region) SEQ ID NO:34 PT10TCRα chain full length sequence SEQ ID NO:35 PT10TCRβ chain full length sequence SEQ ID NO:36 PT8TCR α chain variable region (αV region) SEQ ID NO:51 PT8TCRβ chain variable region (βV region) SEQ ID NO:52 PT8TCRα chain full length sequence SEQ ID NO:53 PT8TCRβ chain full length sequence SEQ ID NO:54

[0113] In the above table, the PT1TCR α chain variable region refers to the fragment encoded by the genomic sequence of the α chain variable region composed of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 in the above-mentioned first TCR according to the arrangement order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the PT1TCR β chain variable region refers to the fragment encoded by the genomic sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 in the above-mentioned first TCR. The sequence shown in NO:14 is a fragment obtained by the genome encoding the β chain variable region arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the full-length sequence of the PT1TCR α chain is a structure in which the α chain variable region and the constant region of the PT1TCR are connected, and the full-length sequence of the PT1TCR β chain is a structure in which the β chain variable region and the constant region of the PT1TCR are connected.

[0114] The PT10TCR α chain variable region refers to the fragment encoded by the genomic sequence of the α chain variable region composed of the sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28 in the above-mentioned first TCR in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the PT1TCR β chain variable region refers to the fragment encoded by the genomic sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: The sequence shown in NO:32 is a fragment obtained by genome encoding the variable region of the β chain, which is composed of the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the full-length sequence of the PT10TCR α chain is a structure formed by connecting the variable region and constant region of the α chain of PT10TCR, and the full-length sequence of the PT10TCR β chain is a structure formed by connecting the variable region and constant region of the β chain of PT1TCR;

[0115] The PT8TCR α chain variable region refers to the fragment encoded by the genomic sequence of the α chain variable region composed of the sequences shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46 in the above-mentioned first TCR in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the PT8TCR β chain variable region refers to the fragment encoded by the genomic sequence of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID The sequence shown in NO:50 is a fragment obtained by the genome encoding the β chain variable region arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the PT1TCR α chain full-length sequence is a structure in which the α chain variable region and the constant region of PT1TCR are connected, and the PT8TCR β chain full-length sequence is a structure in which the β chain variable region and the constant region of PT8TCR are connected.

[0116] In the following examples, PIK3CA_H1047 1046-1054 Wild-type peptide and PIK3CA_H1047L 1046-1054 The mutant peptide was commissioned to be synthesized by Jinruis Biotechnology Co., Ltd.

[0117] 1. Screening of PIK3CA mutation sites

[0118] In the embodiment of the present invention, NetMHCV4.1 predicted that the H1047L mutation site of the PIK3CA gene may be immunogenic (a polypeptide sequence that can activate T cells to produce immune reactivity), and PIK3CA_H1047L was artificially synthesized. 1046-1054 Mutant polypeptide (SEQ ID NO: 56) and PIK3CA_H1047 1046-1054The wild-type polypeptide (SEQ ID NO: 55) was then cloned and the TAP1 gene of the K562 cell line was knocked out using CRISPR-Cas9 technology (reference "T cells discriminate between groups C1and C2 HLA-C, Malcolm JW Sim*t, Zachary Stotz, Jinghua Lu, Paul Brennan, Eric O Long, Peter D Sun*") (TAP1 gene (Transporter Associated with Antigen Processing 1, TAP1) encodes a protein called TAP1, which is a transporter that transports endogenously produced antigen fragments (usually peptide chains) from the cytoplasm to the endoplasmic reticulum and then binds to MHC-I molecules; cells lacking the TAP1 gene cannot present antigen fragments normally, so that the MHC-I molecules expressed on the cell membrane surface cannot exist stably) to construct a TAP1 gene knockout K562 cell model.

[0119] Using the TAP1 gene knockout K562 cell model, the different mutant peptides were further tested for their ability to bind to MHC with TAP1-KO-A11-K562 of the HLA-A*11:01 subtype. Figure 2 As shown, the results showed that HLA-A*11:01 molecules can bind to the mutant PIK3CA_H1047L 1046-1054 and wild-type PIK3CA_H1047 1046-1054 The complex is expressed on the cell membrane surface. 1046-1054 The binding affinity of PIK3CA was significantly increased compared with the wild type. The above results prove that the predicted PIK3CA mutant sequence can bind to the HLA-A*11:01 subtype and form a stable antigen peptide-MHC complex (pMHC), and compared with the wild type, the mutant sequence has better pMHC stability, suggesting that it may activate T cells and is a more ideal target.

[0120] 2. Verification of the immune response induced by T cells using PIK3CA mutant gene

[0121] (1) Peripheral blood of healthy volunteers with HLA-A*11:01 subtype was collected and separated CD8+T cells and monocytes; 800U / ml IL-4 and 800U / ml GM-CSF were added to the monocyte culture medium and cultured for 4 days to differentiate them into DC cells, and then the above-synthesized PIK3CA_H1047L was added to the DC cell culture medium. 1046-1054The mutant polypeptide (concentration of 10uM) was placed in an incubator and cultured for 16 hours; then, the DC cells loaded with the mutant polypeptide were CD8+T cells were mixed and cultured for 3 weeks. The T cell products after culture were tested for the content of specific T cells (IFN-γ secretion by T cells) by flow cytometry. Figure 3 As shown, the results showed that PIK3CA_H1047L 1046-1054 It is immunogenic and can induce, stimulate and expand specific T cells in vitro and secrete IFN-γ with anti-tumor ability.

[0122] (2) Using peptide-loaded dendritic cells to stimulate the induced T cells again, the secretion of IFN-γ by T cells was detected. The results were as follows: Figure 3 As shown, the results showed that PIK3CA_H1047L was successfully induced 1046-1054 Expansion of specific T cells.

[0123] (3) Take 1×10 7 The amplified T cells were centrifuged at 250 g for 10 min, the supernatant was discarded, and 100 ul of antibody staining solution (containing 1 ug / ml PIK3CA_H1047L 1046-1054 / HLA-A1101 tetramer, 1ug / mlAPC-CD3 antibody in PBS), incubate at room temperature for 30min; wash three times with 5ml PBS containing 0.5% BSA, centrifuge at 250g for 10min; resuspend the cells with 1ml PBS containing 0.5% BSA, and use flow cytometry to sort single specific T cells. The results are shown in Figure 4 shown.

[0124] (4) Total RNA was extracted from the sorted specific T cells, and then RT-PCR amplification was performed to obtain the TCR gene sequence. The sequence structure of TCR was analyzed by first-generation sequencing and comparison with the IMGT database, and the PT1 variable region was obtained, which consisted of an α chain variable region encoded by the sequence shown in SEQ ID NO: 15 (amino acid sequence of "GESVGLHLPTLSVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDIRSNMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAEGETGNQFYFGTGTSLTVIP") and a β chain variable region encoded by the sequence shown in SEQ ID NO: 16 (amino acid sequence of "KAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSSGTGGFEQFFGPGTRLTVL"); the PT10 variable region was obtained, which consisted of an α chain variable region encoded by the sequence shown in SEQ ID NO: 15 (amino acid sequence of "GESVGLHLPTLSVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDIRSNMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAEGETGNQFYFGTGTSLTVIP") and a β chain variable region encoded by the sequence shown in SEQ ID NO: 16 (amino acid sequence of "KAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSSGTGGFEQFFGPGTRLTVL"); NO:33 encoded by the α chain variable region (amino acid sequence is "AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEATGNQFYFGTGTSLTVIP") and the β chain variable region encoded by the sequence as shown in SEQ ID NO:34 (amino acid sequence is "SAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQTLTSSDEQYFGPGTRLTVT"); the PT8 variable region is composed of the sequence as shown in SEQ ID NO:34. It consists of the α chain variable region encoded by the sequence shown in NO:51 (amino acid sequence is "ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCASYSGAGSYQLTFGKGTKLSVIP") and the β chain variable region encoded by the sequence shown in SEQ ID NO:52 (amino acid sequence is "NAGVTQTPKFQVLKTGQSMTLQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASSEDFGGSSYNEQFFGPGTRLTVL").

[0125] 3. TCR Binding Specificity Verification

[0126] The PT1 α chain variable region (V region) nucleic acid sequence (as shown in SEQ ID NO: 15) and the PT1 α chain constant region (C region) nucleic acid sequence (SEQ ID NO: 57) are connected to obtain the PT1 α chain nucleic acid full-length sequence (as shown in SEQ ID NO: 17); the PT1 β chain variable region (V region) nucleic acid sequence (as shown in SEQ ID NO: 16) and the PT1 β chain constant region (C region) nucleic acid sequence (SEQ ID NO: 58) are connected to obtain the PT1 β chain nucleic acid full-length sequence (as shown in SEQ ID NO: 18);

[0127] The PT10 α chain variable region (V region) nucleic acid sequence (as shown in SEQ ID NO: 33) and the PT10 α chain constant region (C region) nucleic acid sequence (SEQ ID NO: 59) were connected to obtain the PT10 α chain nucleic acid full-length sequence (as shown in SEQ ID NO: 35); the PT10 β chain variable region (V region) nucleic acid sequence (as shown in SEQ ID NO: 34) and the PT10 β chain constant region (C region) nucleic acid sequence (SEQ ID NO: 60) were connected to obtain the PT10 β chain nucleic acid full-length sequence (as shown in SEQ ID NO: 36);

[0128] The PT8 α chain variable region (V region) nucleic acid sequence (as shown in SEQ ID NO: 51) and the PT8 α chain constant region (C region) nucleic acid sequence (SEQ ID NO: 61) were connected to obtain the PT8 α chain nucleic acid full-length sequence (as shown in SEQ ID NO: 53); the PT8 β chain variable region (V region) nucleic acid sequence (as shown in SEQ ID NO: 52) and the PT8 β chain constant region (C region) nucleic acid sequence (SEQ ID NO: 62) were connected to obtain the PT8 β chain nucleic acid full-length sequence (as shown in SEQ ID NO: 54);

[0129] Using the lentiviral expression plasmid pWPXL (purchased from Addegene) as the backbone vector, PT1, PT8 and PT10 lentiviral expression vectors, namely, lentiviral expression vectors pWPXL-PT1TCR, pWPXL-PT8TCR and pWPXL-PT10TCR, were constructed respectively; the pWPXL-PT1TCR, pWPXL-PT8TCR and pWPXL-PT10TCR lentiviral expression vectors were co-transfected with the lentiviral packaging plasmid pMD2.G and psPAX2 plasmid at a mass ratio of 1:0.5:1 into 293T cells, and the viral supernatant was collected two days later; the viral supernatant was used to infect CD8+T cells, the medium was changed 24 hours after infection, and after culturing for 2-3 days, CD8+T cells expressing PT1TCR, PT8TCR and PT10TCR were obtained respectively for downstream experiments.

[0130] CD8+T cells expressing PT1TCR, PT8TCR and PT10TCR were mixed with K562 cells expressing A1101 gene at a cell number ratio of 1:1, and loaded with a final concentration of 10uM wild type (WT) (as shown in SEQ ID NO:55), mutant (Mut) (as shown in SEQ ID NO:56) and blank control (DMSO) for co-culture, and the cells were recovered after 16 hours;

[0131] Then, the recovered cells were added with 50ul of cell staining solution containing APC-CD137 antibody and incubated at room temperature for 30min; then washed three times with PBS containing 0.5% BSA and centrifuged at 200g-250g for 10min; then resuspended the cells with PBS containing 0.5% BSA for downstream detection; CD8+TCR-T cell activation analysis was performed using flow cytometry to detect the expression of CD137 in CD8+T cell activation analysis.

[0132] The results of flow cytometry analysis of CD8+ T cells expressing PT1TCR, PT8TCR and PT10TCR are shown in Figure 2. Figure 5 As shown, and statistics are performed as Figure 6 As shown, the results show that PT1TCR, PT8TCR and PT10TCR will undergo specific activation after stimulation with K562 of the A1101 gene loaded with mutant (Mut) antigen peptide, and therefore they are PIK3CA_H1047L-specific TCRs.

[0133] 4. CD137 expression test and IFN-γ secretion level test

[0134] CD8+ T cells expressing PT1TCR, PT8TCR and PT10TCR were mixed with K562 cells expressing A1101 gene at a cell number ratio of 1:1 and loaded with gradient dilutions (10-5 μM, 10 -5 μM, 10 -4 μM, 10 -3 μM, 10 -2 μM, 10 -1 μM, 10 0 μM and 10 1 μM) of PIK3CA_H1047L 1046-1054 Mutant polypeptide (Mut) was co-cultured; cells and supernatant were recovered after 16 hours;

[0135] The above cells were added with 50ul of cell staining solution containing APC-CD137 antibody and incubated at room temperature for 30min; then washed three times with PBS containing 0.5% BSA and centrifuged at 200-250g for 10min; then resuspended with PBS containing 0.5% BSA for downstream detection; CD8+TCR-T cell activation analysis was performed using flow cytometry to detect the expression of CD137 in CD8+T cell activation analysis; the results are shown in Figure 7 As shown, PT1TCR, PT8TCR and PT10TCR will undergo specific activation after stimulation by K562 of the A1101 gene loaded with mutant (Mut) antigen peptides. As the concentration of the antigen peptides gradually decreases, the TCR-T cells can still recognize the mutant antigen peptides, so they are TCRs with higher affinity.

[0136] In addition, the supernatant was used for ELISA to detect IFN-γ cytokine secretion, specifically including: PT1-T cells, PT8-T cells and PT10-T cells prepared from T cells of human PBMC were respectively loaded with PIK3CA_H1047L 1046-1054 The mutant peptides were mixed with A1101-K562 cells at a ratio of 1:1 and 2 × 10 5 The number of cells / well was added to a 96-well plate, and each sample was repeated three times. The PMA / Ionomycin (ION) group was diluted into a working solution in advance by adding 1 μl of PMA / Ionomycin mixture (250×) to each 250 μl of cell culture medium, and used as a positive stimulation control. T cells (D1mock and D2mock) that were not infected with KT2, KT3, and KT8 lentiviruses were added in parallel as negative controls; after culturing at 37°C for 24 hours, the supernatant of the 96-well plate culture wells was taken and centrifuged at 500g for 5 minutes to remove the residual cells, and the supernatant was added to the ELISA plate coated with anti-IFN-γ antibody to detect the IFN-γ level in the supernatant. The results are as follows Figure 8 As shown, PT1TCR, PT8TCR and PT10TCR can secrete IFN-γ to kill tumor cells and exert effector function.

[0137] 5. Tumor cell endogenous mutant antigen recognition experiment

[0138] CD8+ T cells expressing PT1TCR, PT8TCR, and PT10TCR were compared with CD8+ T cells expressing PIK3CA_H1047L 1046-1054 Mutant polypeptide (SEQ ID NO: 56) and PIK3CA_H1047 1046-1054 K562 cells, MDA-MB-231 cells and SKOV3 cells containing wild-type peptide were respectively divided into 1:1 cell number (5x10 4 ) mixed co-culture; after 16 hours, the cells and supernatant were recovered;

[0139] The above cells were added with 50ul of cell stain containing APC-CD137 antibody and incubated at room temperature for 30min; then washed three times with PBS containing 0.5% BSA and centrifuged at 200g-250g for 10min; then resuspended with PBS containing 0.5% BSA for downstream detection; CD8+TCR-T cell activation analysis was performed using flow cytometry to detect the expression of CD137 in CD8+T cell activation analysis. The flow cytometry detection results are shown in Fig. 9 As shown, and the statistical graph is as follows Fig.10 As shown, the results show that PT1TCR, PT8TCR and PT10TCR are specifically activated after stimulation with K562 expressing the endogenous expression mutation PIK3CA_H1047L of the A1101 gene.

[0140] The supernatant was subjected to ELISA detection of IFN-γ and IL-2 cytokines, specifically including: PT1-T cells, PT8-T cells and PT10-T cells prepared by using T cells of human PBMC were respectively loaded with PIK3CA_H1047L 1046-1054 The mutant peptides were mixed with A1101-K562 cells at a ratio of 1:1, and 5 × 10 4 The number of cells / well was added to a 96-well plate, and each sample was repeated three times. The PMA / ionomycin (ION) group was diluted in advance into a working solution by adding 1 μl of PMA / ionomycin mixture (250×) to every 200 μl of cell culture medium, and used as a positive stimulation control. After culturing at 37°C for 24 hours, the supernatant of the 96-well plate culture wells was taken and centrifuged at 500g for 5 minutes to remove the residual cells. The supernatant was added to an ELISA plate coated with anti-IFN-γ antibody or IL-2 antibody, and the IFN-γ and IL-2 levels in the supernatant were detected. The results are shown in Fig.11 and Fig.12As shown, the results showed that PT1TCR, PT8TCR and PT10TCR secreted cytokines IFN-γ and IL-2 after stimulation with K562, MDA-MB-231 and SKOV3 expressing the endogenous mutation PIK3CA_H1047L of the A1101 gene.

[0141] Therefore, PT1TCR, PT8TCR and PT10TCR are high-affinity TCRs that can specifically recognize endogenous mutant antigens and secrete IFN-γ to kill tumor cells and exert effector function.

[0142] 6. Tumor Killing Experiment

[0143] CD8+ T cells expressing PT1TCR, PT8TCR, and PT10TCR were compared with CD8+ T cells expressing PIK3CA_H1047L 1046-1054 Mutant polypeptide (SEQ ID NO: 56) and PIK3CA_H1047 1046-1054 The wild-type peptides were mixed at (2.5:1, 5:1, 10:1, 20:1) and 2.5 × 10 4 , 5×10 4 , 2×10 5 and 4×10 5 The number of cells / well was added to a 96-well plate, and each sample was repeated three times; MDA-MB-231 and SKOV3 cell lines were previously modified cell lines (the mutant gene luciferase reporter gene (luciferase reporter gene refers to a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase; luciferase can catalyze the oxidation of luciferin to oxyluciferin, and during the oxidation process of luciferin, it will emit bioluminescence), PIK3CA_H1047L and HLA-A1101 genes were introduced into MDA-MB-231 and SKOV3 cell lines through a lentiviral system to stably express the above genes) to express the luciferase reporter gene;

[0144] After culturing at 37°C for 24 hours, the supernatant of the 96-well plate culture wells was discarded, and luciferase was added to detect the signal in the reaction system, thereby detecting the killing results of PT1TCR, PT8TCR and PT10TCR on MDAMB-231 cells and SKOV3. The killing results of MDAMB-231 cells are as follows: Fig.13 As shown, the killing results of SKOV3 are as follows Fig.14As shown, the results show that as the ratio of effector cells to target cells increases, PT1TCR, PT8TCR and PT10TCR all show higher specific killing ability. Therefore, PT1TCR, PT8TCR and PT10TCR have PIK3CA_H1047L mutation-specific killing ability.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A TCR targeting a polypeptide-HLA-A1101 complex, characterized in that: It comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 encoded by the sequence shown in SEQ ID NO:1, an α chain CDR2 encoded by the sequence shown in SEQ ID NO:2, and an α chain CDR3 encoded by the sequence shown in SEQ ID NO:3; the β chain variable region comprises a β chain CDR1 encoded by the sequence shown in SEQ ID NO:4, a β chain CDR2 encoded by the sequence shown in SEQ ID NO:5, and a β chain CDR3 encoded by the sequence shown in SEQ ID NO:6; the polypeptide sequence is shown in SEQ ID NO:

56.

2. The TCR according to claim 1, characterized in that The α chain variable region further comprises: one or more of an α chain FR1 encoded by the sequence shown in SEQ ID NO:7, an α chain FR2 encoded by the sequence shown in SEQ ID NO:8, an α chain FR3 encoded by the sequence shown in SEQ ID NO:9, and an α chain FR4 encoded by the sequence shown in SEQ ID NO:10; the β chain variable region further comprises: one or more of a β chain FR1 encoded by the sequence shown in SEQ ID NO:11, a β chain FR2 encoded by the sequence shown in SEQ ID NO:12, a β chain FR3 encoded by the sequence shown in SEQ ID NO:13, and a β chain FR4 encoded by the sequence shown in SEQ ID NO:

14.

3. A TCR targeting a polypeptide-HLA-A1101 complex, characterized in that The polypeptide sequence is shown in SEQ ID NO:56; TCR comprises an α chain encoded by the sequence shown in SEQ ID NO:15 and a β chain encoded by the sequence shown in SEQ ID NO:

16.

4. The polynucleotide encoding the TCR according to claim 1, characterized in that It comprises the sequences shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

5. The TCR polynucleotide according to claim 4, characterized in that It also includes one or more of the sequence shown in SEQ ID NO:8, the sequence shown in SEQ ID NO:9, the sequence shown in SEQ ID NO:10, the sequence shown in SEQ ID NO:11, the sequence shown in SEQ ID NO:12, the sequence shown in SEQ ID NO:13 and the sequence shown in SEQ ID NO:

14.

6. The polynucleotide encoding the TCR according to claim 1, characterized in that The polynucleotide comprises the sequences shown in SEQ ID NO:15 and SEQ ID NO:

16.

7. An expression vector, characterized in that Comprising the polynucleotide according to any one of claims 4 to 6.

8. An engineered cell, characterized in that Comprising the expression vector according to claim 7.

9. A pharmaceutical composition, characterized in that Comprising the TCR according to any one of claims 1 to 3, or the polynucleotide according to any one of claims 4 to 6, or the expression vector according to claim 7, or the engineered cell according to claim 8.

10. Use of the TCR according to any one of claims 1 to 3, the polynucleotide according to any one of claims 4 to 6, the expression vector according to claim 7, or the engineered cell according to claim 8 in the preparation of a medicament for treating cancer expressing positive polypeptide-HLA-A1101; the polypeptide sequence is shown in SEQ ID NO: 56; the cancer is endometrial cancer, breast cancer, cervical cancer, colorectal cancer, bladder cancer, head and neck cancer, esophageal and gastric cancer, glioma, non-small cell carcinoma, glioblastoma, bile duct cancer, melanoma, seminoma, hepatobiliary cancer, sarcoma, pancreatic cancer, pleural mesothelioma, prostate cancer, ovarian epithelial cell tumor, renal clear cell carcinoma, adrenocortical carcinoma, renal non-clear cell carcinoma or thyroid cancer.

11. Use of the TCR according to any one of claims 1 to 3, the polynucleotide according to any one of claims 4 to 6, the expression vector according to claim 7, or the engineered cell according to claim 8 in the preparation of a reagent or kit for detecting tumor cells positive for polypeptide-HLA-A1101 expression; the polypeptide sequence is shown in SEQ ID NO: 56.

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