CN117736310A

CN117736310A - HLA-A1101 restrictive epitope peptide, targeting epitope peptide TCR and application thereof

Info

Publication number: CN117736310A
Application number: CN202311731253.1A
Authority: CN
Inventors: 申美莹; 金艾顺; 韩晓建; 刘胜春; 郝亚楠
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2023-12-15
Filing date: 2023-12-15
Publication date: 2024-03-22

Abstract

The invention belongs to the technical field of T cell immunotherapy medicaments, and in particular relates to an HLA-A1101 restrictive epitope peptide,Targeting epitope peptide TCR and uses thereof. The epitope peptide is PIK3CA_H21047L _1046‑1054 The amino acid sequence of the mutant polypeptide is shown as SEQ ID NO. 56; it has strong binding ability to MHC, and the binding ability is better than that of wild PIK3CA_H21047 _1046‑1054 Is an ideal target for activating T cells; and based on the PIK3CA_H21047L _1046‑1054 The TCR designed by the mutant polypeptide has stronger specificity, and can mediate T cells to secrete a large amount of IFN-gamma and IL-2 cytokines to kill PIK3CA_H21047L _1046‑1054 A mutated tumor cell.

Description

HLA-A1101 restrictive epitope peptide, targeting epitope peptide TCR and application thereof

Technical Field

The invention belongs to the technical field of T cell immunotherapy medicaments, and particularly relates to HLA-A1101 restricted epitope peptide, a targeting epitope peptide TCR and application thereof.

Background

With the progress of research in the biomedical field, target selection of T cell immunotherapy targeting malignant solid tumors is evolving continuously, from initial lineage antigens to viral antigens, and with the recent advent of high throughput sequencing technology, targeted tumor individual mutations are called new generation T cell therapy focused targets.

T cell therapy currently includes mainly TCR-T and CAR-T therapeutic approaches. The existing CAR-T technology has remarkable effect in treating hematoma such as leukemia, lymphoma and the like, and greatly improves the survival rate and the survival quality of patients, but aiming at solid tumors, the CAR-T treatment means has limited application prospect due to limited specific targets at present. TCR-T technology differs from CAR-T cell technology in that TCRs are characteristic markers on the surface of all T cells, binding to CD3 non-covalently to form a TCR-CD3 complex. In peripheral blood, 90% -95% of T cells express TCR, and the T cells of the genetically modified TCR can specifically identify antigen molecules on the surface of tumor cells so as to generate immune response to the tumor cells.

Currently, in human cancers, the PIK3CA gene is one of oncogenes in the oncology field, and the gene mutation frequency of PIK3CA is approximately 44.5% in endometrial cancer, approximately 30.7% in breast cancer, approximately 23.6% in cervical cancer, approximately 24.7% in colorectal cancer, approximately 20.4% in bladder cancer, approximately 13.6% in head and neck cancer, approximately 13.0% in esophageal and gastric cancer, and the like, and in addition to the above cancers, the gene mutation of PIK3CA also occurs in gliomas, non-small cell cancers, glioblastomas, cholangiocarcinomas, melanomas, seminomas, hepatobiliary carcinomas, sarcomas, pancreatic cancers, pleural mesothelioma, prostate cancer, ovarian epithelial cell tumors, renal clear cell carcinoma, adrenocortical carcinoma kidney, non-clear cell carcinoma, thyroid cancer, and the like (see fig. 1).

At present, the binding capacity of wild PIK3CA and MHC is weak, the formed antigen peptide-MHC complex (pMHC) is unstable, and a better T cell killing site cannot be formed.

Disclosure of Invention

In view of the above problems, the present invention has found that a stable complex can be formed with HLA-A1101 and a HLA-A 1101-restricted epitope peptide is presented on the surface of a cell, which is PIK3CA_H21047L _1046-1054 The mutant polypeptide has strong binding capacity with MHC, and is an ideal target for activating T cells; and based on the PIK3CA_H21047L _1046-1054 The TCR screened by the mutant polypeptide has stronger specificity, and can mediate T cells to secrete a large amount of IFN-gamma and IL-2 cytokines to kill PIK3CA_H21047L _1046-1054 Tumor cells of the mutated HLA-A1101 subtype.

In order to achieve the above purpose, the present invention may adopt the following technical scheme:

in one aspect, the invention provides a HLA-A1101 restrictive epitope peptide, and the amino acid sequence of the HLA-A1101 restrictive epitope peptide is shown as SEQ ID NO. 56.

In another aspect, the invention provides a TCR targeting a polypeptide-HLA-A 1101 complex, the polypeptide sequence is as shown in SEQ ID NO:56, the TCR being selected from four TCR:

first TCR:

the first TCR comprises an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 1, an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 2 and an alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 3, and the beta chain variable region comprises a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 4, a beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 5 and a beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 6.

Second TCR:

the second TCR comprises an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 19, an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 20 and an alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 21, and the beta chain variable region comprises a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 22, a beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 23 and a beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 24.

Third TCR:

the third TCR comprises an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 37, an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 38 and an alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 39, and the beta chain variable region comprises a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 40, a beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 41 and a beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 42.

Fourth TCR:

the fourth TCR comprises any one of the following three groups: (a) An alpha chain encoded by the sequence shown in SEQ ID NO. 15 and a beta chain encoded by the sequence shown in SEQ ID NO. 16; (b) An alpha chain encoded by the sequence shown in SEQ ID NO. 33 and a beta chain encoded by the sequence shown in SEQ ID NO. 34; (c) An alpha chain encoded by the sequence shown in SEQ ID NO. 51 and a beta chain encoded by the sequence shown in SEQ ID NO. 52.

In a further aspect the invention provides a polynucleotide encoding a TCR as described above, the polynucleotide being selected from any one of:

the first polynucleotide, encoding the first TCR, 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.

A second polynucleotide, which codes for the second TCR, and comprises sequences shown as 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.

And a third polynucleotide encoding the third TCR, comprising the sequences shown as 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.

The fourth nucleotide comprises any one of the following three sets 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.

In a further aspect the invention provides an expression vector comprising a polynucleotide as defined in any one of the preceding.

In yet another aspect, the invention provides an engineered cell comprising an expression vector as described in any one of the above.

In a further aspect the invention provides a pharmaceutical composition comprising a TCR as defined in any one of the preceding claims or a polynucleotide as defined in any one of the preceding claims or an expression vector as defined in any one of the preceding claims or an engineered cell as defined in any one of the preceding claims.

In a further aspect, the invention provides the use of a TCR as claimed in any one of the preceding claims or a polynucleotide as claimed in any one of the preceding claims or an expression vector as claimed in any one of the preceding claims or an engineered cell as claimed in any one of the preceding claims in the manufacture of a medicament for the treatment of a cancer positive for expression of polypeptide-HLA-A 1101; the polypeptide sequence is shown as SEQ ID NO. 56.

In a further aspect, the invention provides the use of a TCR as defined in any one of the preceding claims or a polynucleotide as defined in any one of the preceding claims or an expression vector as defined in any one of the preceding claims or an engineered cell as defined in any one of the preceding claims in the preparation of a tumour cell reagent or kit for detecting positive expression of polypeptide-HLA-A 1101; the polypeptide sequence is shown as SEQ ID NO. 56.

In a further aspect, the invention provides the use of a polypeptide comprising a HLA-A1101 restricted epitope peptide as described above in the preparation of a therapeutic tumour vaccine.

The beneficial effects of the invention at least comprise:

(1) The HLA-A1101 restriction epitope peptide (mutant PIK3CA_H21047L) _1046-1054 ) Has strong binding capacity to MHCIs superior to wild PIK3CA_H21047 _1046-1054 Is an ideal target for activating T cells.

(2) The invention provides a TCR pair PIK3CA_H21047L _1046-1054 The mutant polypeptide has stronger specificity and can mediate T cells to secrete a large amount of IFN-gamma and IL-2 cytokines to kill PIK3CA_H21047L _1046-1054 A mutated tumor cell.

Drawings

FIG. 1 shows the frequency of gene mutation of PIK3CA in different tumors;

FIG. 2 shows the binding of the PIK3CA mutant sequence to subtype HLA-A 11:01;

FIG. 3 is a diagram of PIK3CA_H21047L _1046-1054 The reactivity of the specific T cells to the mutated sequence;

FIG. 4 shows a flow cytometry detection of PIK3CA_H21047L _1046-1054 The expansion of specific T cells;

FIG. 5 is a flow cytometry plot of the specific reactivity of PT1TCR, PT8TCR and PT10TCR to mutant antigens;

FIG. 6 is a histogram of validation of the specific reactivity of PT1TCR, PT8TCR and PT10TCR to mutant antigens;

FIG. 7 shows the expression of CD137 by PT1TCR, PT8TCR and PT10TCR on antigen peptide activation assays at various concentrations;

FIG. 8 shows ELISA for the detection of IFN-gamma secretion by PT1TCR, PT8TCR and PT10TCR for antigen peptides of different concentrations;

FIG. 9 is a schematic representation of flow cytometry detection of PT1TCR, PT8TCR and PT10TCR and expression of PIK3CA_H2 1047L _1046-1054 Is activated and analyzed for CD137 expression after co-culture of different tumor cells;

FIG. 10 shows T1TCR, PT8TCR and PT10TCR and expression of PIK3CA_H21047L in different tumor cells _1046-1054 Histogram of CD137 expression profile of activation analysis after co-culture of different tumor cells;

FIG. 11 is a schematic illustration of ELISA for detection of PT1TCR, PT8TCR and PT10TCR and expression of PIK3CA_H21047L _1046-1054 The secretion of IFN-gamma after co-culture of different tumor cells;

FIG. 12 ELISA for detection of PT1TCR, PT8TCR and PT10TCR and expression of PIK3CA_H1047L _1046-1054 The secretion of IL-2 after co-culture of different tumor cells;

FIG. 13 shows killing expression of PIK3CA_H277L by PT1TCR, PT8TCR and PT10TCR _1046-1054 MDA-MB-231 cell condition;

FIG. 14 shows killing expression of PIK3CA_H277L by PT1TCR, PT8TCR and PT10TCR _1046-1054 SKOV3 cell case.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like 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 be present or may be added. As used herein, "/" may be interpreted as "and" or "as appropriate.

The embodiment of the invention provides an HLA-A1101 restrictive antigen epitope peptide, and the amino acid sequence of the HLA-A1101 restrictive antigen epitope peptide is shown as SEQ ID NO. 56.

The epitope peptide is derived from one of high-frequency mutation sites H1047 site (PIK3CA_H21047L of tumor driving gene PIK3CA _1046-1054 Mutant polypeptides) that develop immunogenicity when H1047L. The PIK3CA_H21047L _1046-1054 The mutant polypeptide has strong binding capacity with MHC, and the binding capacity is superior to that of wild PIK3CA_H21047 _1046-1054 The method comprises the steps of carrying out a first treatment on the surface of the As described above, PIK3CA mutation often occurs in various types of cancers, a gene which is a high-frequency mutation in a population, based on which the epitope peptide activates T cells preferablyA target point.

Another embodiment of the invention provides a TCR targeting the HLA-A1101 restricted epitope peptide described above, the polypeptide sequence is shown in SEQ ID NO:56, which TCR can be selected from four TCRs:

first TCR:

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

In the first TCR, the sequence of SEQ ID NO. 1-SEQ ID NO. 6 encodes a CDR region and the sequence of SEQ ID NO. 7-SEQ ID NO. 14 encodes a nucleic acid sequence and an amino acid sequence corresponding to the FR region as shown in Table 1 below:

TABLE 1 CDR and FR region sequence cases for the first TCR described above

Region(s)	Nucleic acid sequences	Amino acidsSequence(s)
			Alpha chain CDR1	SEQ ID NO:1	NSASDY
Alpha chain CDR2	SEQ ID NO:2	IRSNMDK
			Alpha chain CDR3	SEQ ID NO:3	AEGETGNQFY
Beta chain CDR1	SEQ ID NO:4	SGHRS
			Beta chain CDR2	SEQ ID NO:5	YFSETQ
Beta chain CDR3	SEQ ID NO:6	ASSSGTGGFEQF
			Alpha chain FR1	SEQ ID NO:7	GESVGLHLPTLSVQEGDNSIINCAYS
Alpha chain FR2	SEQ ID NO:8	FIWYKQESGKGPQFIID
			Alpha chain FR3	SEQ ID NO:9	RQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFC
Alpha chain FR4	SEQ ID NO:10	FGTGTSLTVIP
			Beta chain FR1	SEQ ID NO:11	KAGVTQTPRYLIKTRGQQVTLSCSPI
Beta chain FR2	SEQ ID NO:12	VSWYQQTPGQGLQFLFE
			Beta chain FR3	SEQ ID NO:13	RNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLC
Beta chain FR4	SEQ ID NO:14	FGPGTRLTVL

Second TCR:

the second TCR comprises an alpha chain variable region and a beta chain variable region; comprising an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 19, an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 20 and an alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 21, and the beta chain variable region comprises a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 22, a beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 23 and a beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 24.

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

In the second TCR, the sequence shown in SEQ ID NO. 19-SEQ ID NO. 24 encodes a CDR region and the sequence shown in SEQ ID NO. 25-SEQ ID NO. 32 encodes a nucleic acid sequence and an amino acid sequence corresponding to the FR region as shown in Table 2 below.

TABLE 2 CDR and FR region sequence cases for the second TCR described above

Region(s)	Nucleic acid sequences	Amino acid sequence
			Alpha chain CDR1	SEQ ID NO:19	TRDTTYY
Alpha chain CDR2	SEQ ID NO:20	RNSFDEQN
			Alpha chain CDR3	SEQ ID NO:21	ALSEATGNQFY
Beta chain CDR1	SEQ ID NO:22	SQVTM
			Beta chain CDR2	SEQ ID NO:23	ANQGSEA
Beta chain CDR3	SEQ ID NO:24	SVQTLTSSDEQY
			Alpha chain FR1	SEQ ID NO:25	AQKVTQAQTEISVVEKEDVTLDCVYE
Alpha chain FR2	SEQ ID NO:26	LFWYKQPPSGELVFLIR
			Alpha chain FR3	SEQ ID NO:27	EISGRYSWNFQKSTSSFNFTITASQVVDSAVYFC
Alpha chain FR4	SEQ ID NO:28	FGTGTSLTVIP
			Beta chain FR1	SEQ ID NO:29	SAVISQKPSRDICQRGTSLTIQCQVD
Beta chain FR2	SEQ ID NO:30	MFWYRQQPGQSLTLIAT
			Beta chain FR3	SEQ ID NO:31	TYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLC
Beta chain FR4	SEQ ID NO:32	FGPGTRLTVT

Third TCR:

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

In the third TCR, the sequence shown in SEQ ID NO. 37-SEQ ID NO. 42 encodes a CDR region and the sequence shown in SEQ ID NO. 43-SEQ ID NO. 50 encodes a nucleic acid sequence and an amino acid sequence corresponding to the FR region as shown in Table 3 below.

TABLE 3 CDR and FR region sequence cases for the third TCR described above

Region(s)	Nucleic acid sequences	Amino acid sequence
			Alpha chain CDR1	SEQ ID NO:37	SSNFYA
Alpha chain CDR2	SEQ ID NO:38	MTLNGDE
			Alpha chain CDR3	SEQ ID NO:39	ASYSGAGSYQLT
Beta chain CDR1	SEQ ID NO:40	MNHNS
			Beta chain CDR2	SEQ ID NO:41	SASEGT
Beta chain CDR3	SEQ ID NO:42	ASSEDFGGSSYNEQF
			Alpha chain FR1	SEQ ID NO:43	ILNVEQSPQSLHVQEGDSTNFTCSFP
Alpha chain FR2	SEQ ID NO:44	LHWYRWETAKSPEALFV
			Alpha chain FR3	SEQ ID NO:45	KKKGRISATLNTKEGYSYLYIKGSQPEDSATYLC
Alpha chain FR4	SEQ ID NO:46	FGKGTKLSVIP
			Beta chain FR1	SEQ ID NO:47	NAGVTQTPKFQVLKTGQSMTLQCAQD
Beta chain FR2	SEQ ID NO:48	MYWYRQDPGMGLRLIYY
			Beta chain FR3	SEQ ID NO:49	TDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFC
Beta chain FR4	SEQ ID NO:50	FGPGTRLTVL

Fourth TCR:

The FR regions of the fourth TCR belonging to the first, second and third TCRs are selected from the preferred sequences for the corresponding FRs.

It should be noted that the TCR described above can recognize only tumor cells at pik3ca—h1047l site, and perform tumor-specific killing, and does not recognize wild-type human normal tissues.

In the TCR, the CDR3 of the α chain and the β chain belongs to the hypervariable region of the variable region, and when the TCR recognizes the MHC-antigen peptide complex, the CDR3 directly binds to the antigen peptide, and directly affects the recognition ability of the TCR to pik3ca—h1047l mutation. In the hypervariable regions of the α and β chains of the TCR, two hypervariable regions of CDR1 and CDR2 are also included, the CDR1 and CDR2 hypervariable regions being relatively stable with respect to CDR3, and when the TCR recognizes MHC-antigen peptide complexes, the CDR1 and CDR2 two hypervariable regions recognize and bind to the side walls of the MHC molecule antigen binding groove.

The FRs (FR 1, FR2, FR3 and FR 4) in the three TCRs are framework regions for connecting with the CDR regions and are relatively stable. The FR regions in the first, second and third TCRs may be selected from sequences other than those described above, and may be selected from sequences having greater than or equal to 80% identity to the FR1, FR2, FR3 and FR4 sequences of the three TCRs, such as greater than or equal to 80%, 85%, 90% or 95%, from murine sources or from human sources (although other sources such as animal sources such as rabbit sources, porcine sources, etc. are not excluded). In certain embodiments, the sequences of the FR regions and CDR regions of the α and β chains described above can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 to form, respectively, the α and β chain variable regions of a TCR, e.g., in a first TCR, the sequence encoding the α chain variable region is shown as SEQ ID NO. 15 and the sequence encoding the β chain variable region is shown as SEQ ID NO. 16; in the second TCR, the sequence encoding the variable region of the alpha chain is shown in SEQ ID NO. 33 and the sequence encoding the variable region of the beta chain is shown in SEQ ID NO. 34; in the third TCR, the sequence encoding the variable region of the alpha chain is shown in SEQ ID NO. 51 and the sequence encoding the variable region of the beta chain is shown in SEQ ID NO. 52.

The TCR may include an α chain constant region and a β chain constant region in addition to the α chain variable region and the β chain variable region described above, and the source of the α chain constant region and the β chain constant region may be a murine source, a human source or a human-mouse chimeric (but other sources such as animal sources such as rabbit sources and pig sources are not excluded), or a region in which mutation hardly occurs. Specifically, in the first TCR, the alpha chain constant region may be selected from the sequence shown in SEQ ID NO. 57 and the beta chain constant region may be selected from the sequence shown in SEQ ID NO. 58; in the second TCR, the alpha chain constant region can be selected to have the sequence shown in SEQ ID NO. 59 and the beta chain constant region can be selected to have the sequence shown in SEQ ID NO. 60; in the third TCR, the alpha chain constant region can be selected as set forth in SEQ ID NO. 61 and the beta chain constant region can be selected as set forth in SEQ ID NO. 62. In the TCR described above, the source of the variable and constant regions of the α chain or β chain may be the same or different from that of the variable regions of the α chain or β chain, and the variable regions of the α chain or β chain may be derived from murine sources or human sources (although other sources such as animal sources including rabbit sources and pig sources are not excluded).

In some embodiments, in the first TCR, the full-length TCR α chain sequence comprising the α chain variable region and the constant region can comprise a sequence as set forth in SEQ ID No. 17, and the full-length TCR α chain sequence comprising the β chain variable region and the constant region can comprise a sequence as set forth in SEQ ID No. 18; in the second TCR, the full-length TCR alpha chain sequence comprising the alpha chain variable region and the constant region may comprise the sequence shown in SEQ ID NO. 35 and the full-length TCR alpha chain sequence comprising the beta chain variable region and the constant region may comprise the sequence shown in SEQ ID NO. 36; in a third TCR, the full length TCR alpha chain sequence comprising the alpha chain variable and constant regions may comprise the sequence shown in SEQ ID NO:53 and the full length TCR alpha chain sequence comprising the beta chain variable and constant regions may comprise the sequence shown in SEQ ID NO: 54.

In yet another embodiment, the invention provides a polynucleotide encoding a TCR as described above, wherein the polynucleotide is selected from any one of the following polynucleotides:

In some embodiments, the first polynucleotide further comprises 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.

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

In some embodiments, the third polynucleotide further comprises 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.

A fourth nucleotide comprising any one of the following three sets 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.

It should be noted that the polynucleotide may also comprise other (other than the polynucleotide) any nucleotide encoding any one of the TCRs described above, such as a sequence comprising one or more of SEQ ID NOs 1 to 6 or a sequence codon optimized therewith; for example, one or more of SEQ ID NO. 19-SEQ ID NO. 24; and further, for example, comprises one or more of SEQ ID NO. 37-SEQ ID NO. 42.

Yet another embodiment of the present invention provides an expression vector comprising a polynucleotide as described in any one of the above.

In some embodiments, the expression vector may be 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.

In some embodiments, the lentiviral vector may be selected from the group consisting of: 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).

In yet another embodiment of the invention, an engineered cell is provided comprising an expression vector as described in any of the above.

The engineering cell may be a host cell, and the expression vector may be introduced into the host cell to encode a TCR protein; t cells may be obtained by transfecting T cells with the above expression vector (loaded with the target gene) to obtain TCR-T cells for PIK3CA_H21047L _1046-1054 Recognition and killing of mutant polypeptides.

In yet another embodiment, the invention provides a pharmaceutical composition comprising a TCR as described in any one of the above or a polynucleotide as described in any one of the above or an expression vector as described in any one of the above or an engineered cell as described in any one of the above. Specifically, in several of the above applications, the TCR may select any one of the four TCRs described above; polynucleotides any one of the four polynucleotides described above is selected.

It should be noted that the above TCR or antigen-binding fragment or polynucleotide or expression vector or engineering cell may be added with other drugs capable of treating cancer to form a pharmaceutical composition for enhancing therapeutic effect; wherein, other medicines for treating cancer can include chemotherapy drugs such as alkylating agent, antimetabolite, antitumor antibiotic, plant anticancer drug, hormone, immune preparation, etc.; in addition, the TCR described above may also be used in combination with surgery. The specific conditions can be used according to the cancer conditions or combined.

In a further embodiment, the invention provides the use of a TCR as defined in any one of the preceding claims or a polynucleotide as defined in any one of the preceding claims or an expression vector as defined in any one of the preceding claims or an engineered cell as defined in any one of the preceding claims in the manufacture of a medicament for the treatment of a cancer positive for expression of polypeptide-HLA-A 1101; the polypeptide sequence is shown as SEQ ID NO. 56.

In some embodiments, the cancer in the above applications comprises endometrial cancer, breast cancer, cervical cancer, colorectal cancer, bladder cancer, head and neck cancer, esophageal gastric cancer, glioma, non-small cell cancer, glioblastoma, cholangiocarcinoma, melanoma, seminoma, hepatobiliary carcinoma, sarcoma, pancreatic cancer, pleural mesothelioma, prostate cancer, ovarian epithelial cell tumor, renal clear cell carcinoma, renal adrenocortical carcinoma, non-clear cell carcinoma, or thyroid cancer.

In some embodiments, the agent in the above application comprises a cell-based agent, a protein-based agent, an ADC agent, or a combination of TCR and antigen.

In a further embodiment, the invention provides the use of a TCR as defined in any one of the preceding claims or a polynucleotide as defined in any one of the preceding claims or an expression vector as defined in any one of the preceding claims or an engineered cell as defined in any one of the preceding claims in the preparation of a tumour cell reagent or kit for detecting positive expression of polypeptide-HLA-A 1101; the polypeptide sequence is shown as SEQ ID NO. 56.

It should be noted that, in particular, the kit may be divided into various small boxes, and then various detection reagents are contained therein. The detection reagent and the detection kit can be indirectly or directly applied to the expression of PIK3CA_H21047L _1046-1054 Mutated various malignant tumors, such as including pancreatic swellingTumor, colorectal malignancy, endometrial malignancy, lung malignancy, bile duct malignancy cancer, cervical malignancy, and the like.

In a further embodiment, the invention provides the use of a polypeptide comprising the HLA-A1101 restricted epitope peptide described above in the preparation of a therapeutic tumor vaccine.

It is noted that therapeutic tumor vaccines are an emerging therapeutic approach for the treatment of cancer. It is an immunotherapy aimed at activating the patient's own immune system to recognize and attack tumor cells. Therapeutic tumor vaccines typically comprise tumor-associated antigens or antigen fragments that trigger a response of the immune system that attacks tumor cells. These vaccines work by directing the immune system to produce antibodies or T cell responses against the tumor. They may be used after a cancer patient has received standard treatment (such as surgery, radiation or chemotherapy) to reduce the risk of cancer recurrence or to extend the patient's survival. In the present invention, mutant antigenic peptides are used to induce T cell responses in the immune system of tumor cells positive for HLA-A1101 and having PIK3CA-H1047L mutation to combat tumors.

For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.

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

(1) RT-PCR: the downstream primer used was a TCR gene constant region specific primer (Ca-RV 1primer, cb-RV 1 primer) and the upstream primer was an initial 20bp sequence (AL primer, BLprimer) containing an outer adaptor and TCR signal peptide (in particular according to the examples described in the documents Hamana H, shitaoka K, kishi H, ozawa T, muraguchi A. Anovel, rapidandefficientmethodofcloning functional antigen-specific T-cell receptors from single human andmouse T-cells. Biochemim Biophys Res Commun.2016Jun10;474 (4): 709-714.Doi:10.1016/j. Bbrc.2016.05.015.Epub 2016May 4.PMID:27155153);

(2) Second round PCR: taking the PCR product of the TCR obtained in the step (1) as a template, wherein an upstream primer is an outer joint primer (P2A-Cprimer), and a downstream primer is a specific primer (Ca_RV2 primer) of a section of TCR constant region upstream of an alpha chain constant region to obtain a second round PCR product of the TCR alpha chain; the upstream primer is an outer adapter primer (BES-AP primer), the downstream primer is a specific primer (Cb_RV2 primer) of a TCR constant region at the upstream of the beta chain constant region, and the second round PCR product of the TCR beta chain is obtained (specifically, the PCR product is implemented according to the contents of documents HamanaH, shitaoka K, kishi H, ozawa T, muraguchi A. Anovil, rapid and efficientmethod ofcloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochemim Biophys Res Commun.2016Jun10;474 (4): 709-714.doi:10.1016/j.bbrc.2016.05.015.Epub 2016May 4.PMID:27155153);

(3) Performing agarose gel electrophoresis on the amplified second round PCR amplification product containing TCR alpha chain and beta chain variable region genes to obtain TCR alpha chain or beta chain variable region target genes at 500bp-750bp positions;

(4) The target band was subjected to a first generation sequencing (Beijing Optimaceae) and TCR sequence analysis, TCR clones were selected in which high frequency of occurrence were designated PT1, PT8 and PT10, respectively.

The amplification systems used in the above-described procedures involving the implementation of the methods of gene amplification, sequencing and analysis are shown in Table 4 below (specifically, the procedures described in the documents Hamana H, shitaoka K, kishi H, ozawaT, muraguchiA.Anovel, rapid andefficientmethod ofcloning functional antigen-specific T-cell receptors from single human andmouse T-cells. Biochemim Biophys Res Commun.2016Jun 10;474 (4): 709-714.doi:10.1016/j.bbrc.2016.05.015.Epub 2016May 4.PMID:27155153).

TABLE 4 amplification System

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

table 5 nucleic acid sequences or amino acid sequences of the materials involved in the examples

Material	Nucleic acid sequence or amino acid sequence
		PIK3CA_H1047 _1046-1054 Wild-type polypeptides	SEQ ID NO:55
PIK3CA_H1047L _1046-1054 Mutant polypeptides	SEQ ID NO:56
		PT1TCR alpha chain variable region (alpha V region)	SEQ ID NO:15
PT1TCR beta chain variable region (beta V region)	SEQ ID NO:16
		PT1TCR alpha chain full-length sequence	SEQ ID NO:17
PT1TCR beta chain full-length sequence	SEQ ID NO:18
		PT10TCR alpha chain variable region (alpha V region)	SEQ ID NO:33
PT10TCR beta chain variable region (beta V region)	SEQ ID NO:34
		PT10TCR alpha chain full-length sequence	SEQ ID NO:35
PT10TCR beta chain full-length sequence	SEQ ID NO:36
		PT8TCR alpha chain variable region (alpha V region)	SEQ ID NO:51
PT8TCR beta chain variable region (beta V region)	SEQ ID NO:52
		PT8TCR alpha chain full-length sequence	SEQ ID NO:53
PT8TCR beta chain full-length sequence	SEQ ID NO:54

In the above table, the PT1TCR alpha chain variable region refers to a fragment obtained by encoding the alpha chain variable region genome of the first TCR, wherein the fragment is formed by sequences shown by 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 according to the arrangement sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the PT1TCR beta chain variable region refers to a fragment obtained by encoding a beta chain variable region genome of the first TCR, wherein the fragment is formed by sequences shown by 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 according to the arrangement sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the full-length PT1TCR α chain sequence is a structure in which the variable and constant regions of the α chain of the PT1TCR are linked, and the full-length PT1TCR β chain sequence is a structure in which the variable and constant regions of the β chain of the PT1TCR are linked.

The PT10TCR alpha chain variable region refers to a fragment obtained by encoding the alpha chain variable region genome of the first TCR, wherein the fragment is formed by the sequences shown by 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 according to the arrangement sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the PT1TCR beta chain variable region refers to a fragment obtained by encoding a beta chain variable region genome of the first TCR, wherein the fragment is formed by sequences shown by 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. 32 according to the arrangement sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the full-length sequence of the PT10TCR alpha chain is a structure formed by connecting an alpha chain variable region and a constant region of the PT10TCR, and the full-length sequence of the PT10TCR beta chain is a structure formed by connecting a beta chain variable region and a constant region of the PT1 TCR;

The PT8TCR alpha chain variable region refers to a fragment obtained by encoding the alpha chain variable region genome of the first TCR, wherein the fragment is formed by the sequences shown by 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 according to the arrangement sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the PT8TCR beta chain variable region refers to a fragment obtained by encoding a beta chain variable region genome consisting of sequences shown by 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 NO:50 in the arrangement order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the above first TCR; the full-length PT1TCR α chain sequence is a structure in which the variable and constant regions of the α chain of the PT1TCR are linked, and the full-length PT8TCR β chain sequence is a structure in which the variable and constant regions of the β chain of the PT8TCR are linked.

In the following examples, PIK3CA_H21047 _1046-1054 Wild-type polypeptide and PIK3CA_H21047L _1046-1054 Mutant polypeptides were synthesized by Jin Ruisi Biotechnology Inc.

1. PIK3CA mutation site screening

In the embodiment of the invention, the NetMHCV4.1 predicts that the H1047L mutation site of the PIK3CA gene may have immunogenicity (polypeptide sequence capable of activating T cells to generate immunoreactivity), and the PIK3CA_H21047L is artificially synthesized _1046-1054 Mutant polypeptides (SEQ ID N)O: 56) and PIK3CA_H21047) _1046-1054 Wild-type polypeptide (SEQ ID NO: 55); the TAP1 gene of the K562 cell line (TAP 1 gene (TransporterAssociatedwithAntigen Processing, TAP 1) was then knocked out using CRISPR-Cas9 technology (ref. T cells discriminate between groups C and C2 HLA-C, malcolm J W Sim. T., zachary Stotz, jinghua Lu, paul Brennan, eric O Long, peter D Sun. Times.), which encodes a protein called TAP1, which is a transporter, delivers endogenously produced antigen fragments (usually peptide chains) from the cytoplasm to the endoplasmic reticulum, and then binds to MHC-I type molecules, and the deleted cells fail to normally present antigen fragments such that MHC-I molecules expressed to the cell membrane surface do not exist stably), to construct a TAP1 gene knocked-out K562 cell model.

The binding capacity of peptides to MHC was further tested using TAP1 knock-out K562 cell model with different mutant peptide fragments and TAP1-KO-A11-K562 of subtype HLA-A 11:01. The results are shown in FIG. 2, and show that HLA-A 11:01 molecules can be combined with mutant PIK3CA_H21047L _1046-1054 Wild-type pik3ca_h1047 _1046-1054 The complex is formed and expressed on the surface of the cell membrane. Wherein the mutant PIK3CA_H21047L _1046-1054 The binding force of (C) is obviously increased compared with that of a wild type. The above results demonstrate that the predicted PIK3CA mutant sequences can bind to HLA-A x 11:01 subtype and form stable antigen peptide-MHC complex (pMHC), and that the mutated sequences have better pMHC stability compared to the wild type, suggesting that it is possible to activate T cells and be a more desirable target.

2. PIK3CA mutant gene-induced T cell generation immune response verification

(1) Isolation of peripheral blood from healthy human volunteers pooled with subtype HLA-A 11:01Cd8+ T cells and monocytes; adding 800U/ml IL-4 and 800U/ml GM-CSF into monocyte culture solution, culturing for 4 days to differentiate into DC cells, and adding the above synthesized PIK3CA_H21047L into DC cell culture solution _1046-1054 Mutant polypeptide (10 uM concentration) was placed in incubator for 16 hours of stationary culture; along with itAfter that, the DC cells loaded with the above mutant polypeptide are combined with +.>Cd8+ T cells were mixed and cultured for 3 weeks; the content of specific T cells (IFN-. Gamma.secretion from T cells) of the cultured T cell product was detected by flow cytometry, and the results are shown in FIG. 3, which shows that PIK3CA_H21047L _1046-1054 Has immunogenicity, can induce and stimulate specific T cells to be amplified in vitro, and secrete IFN-gamma with anti-tumor capability.

(2) The induced T cells are stimulated again by the dendritic cells loaded with the polypeptide, the secretion of IFN-gamma by the T cells is detected, the result is shown in figure 3, and the PIK3CA_H21047L is successfully induced _1046-1054 Is a specific T cell expansion.

(3) Taking 1×10 ⁷ 250g for 10min, removing supernatant, adding 100ul of antibody dye solution (containing 1ug/ml PIK3CA_H21047L) _1046-1054 1ug/ml PBS of APC-CD3 antibody,/HLA-A 1101 tetramer), 30min incubation at room temperature; washing three times with 5ml PBS containing 0.5% BSA, and centrifuging at 250g for 10min; after resuspension of the cells with 1ml of PBS containing 0.5% BSA, single specific T cells were sorted using a flow cell sorting apparatus, and the results are shown in FIG. 4.

(4) Extracting total RNA from the sorted specific T cells, performing RT-PCR amplification to obtain a TCR gene sequence, and analyzing the sequence structure of the TCR by means of primary sequencing and IMGT database comparison to obtain a PT1 variable region which consists of an alpha chain variable region (the amino acid sequence is GESVGLHLPTLSVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDIRSNMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAEGETGNQFYFGTGTSLTVIP) coded by a sequence shown as SEQ ID NO. 15 and a beta chain variable region (the amino acid sequence is KAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSSGTGGFEQFFGPGTRLTVL) coded by a sequence shown as SEQ ID NO. 16; the PT10 variable region is composed of an alpha chain variable region (the amino acid sequence is 'AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEATGNQFYFGTGTSLTVIP') coded by a sequence shown as SEQ ID NO. 33 and a beta chain variable region (the amino acid sequence is 'SAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVQTLTSSDEQYFGPGTRLTVT') coded by a sequence shown as SEQ ID NO. 34; the PT8 variable region is composed of an alpha chain variable region encoded by a sequence shown as SEQ ID NO. 51 (the amino acid sequence is "ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCASYSGAGSYQLTFGKGTKLSVIP") and a beta chain variable region encoded by a sequence shown as SEQ ID NO. 52 (the amino acid sequence is "NAGVTQTPKFQVLKTGQSMTLQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASSEDFGGSSYNEQFFGPGTRLTVL").

3. TCR binding specificity validation

Connecting a PT1 alpha chain variable region (V region) nucleic acid sequence (shown as SEQ ID NO: 15) with a PT1 alpha chain constant region (C region) nucleic acid sequence (SEQ ID NO: 57) to obtain a PT1 alpha chain nucleic acid full-length sequence (shown as SEQ ID NO: 17); connecting a PT1 beta chain variable region (V region) nucleic acid sequence (shown as SEQ ID NO: 16) with a PT1 beta chain constant region (C region) nucleic acid sequence (SEQ ID NO: 58) to obtain a PT1 beta chain nucleic acid full-length sequence (shown as SEQ ID NO: 18);

connecting a PT10 alpha chain variable region (V region) nucleic acid sequence (shown as SEQ ID NO: 33) with a PT10 alpha chain constant region (C region) nucleic acid sequence (SEQ ID NO: 59) to obtain a PT10 alpha chain nucleic acid full-length sequence (shown as SEQ ID NO: 35); connecting a PT10 beta chain variable region (V region) nucleic acid sequence (shown as SEQ ID NO: 34) with a PT10 beta chain constant region (C region) nucleic acid sequence (SEQ ID NO: 60) to obtain a PT10 beta chain nucleic acid full-length sequence (shown as SEQ ID NO: 36);

connecting a PT8 alpha chain variable region (V region) nucleic acid sequence (shown as SEQ ID NO: 51) with a PT8 alpha chain constant region (C region) nucleic acid sequence (SEQ ID NO: 61) to obtain a PT8 alpha chain nucleic acid full-length sequence (shown as SEQ ID NO: 53); connecting a PT8 beta chain variable region (V region) nucleic acid sequence (shown as SEQ ID NO: 52) with a PT8 beta chain constant region (C region) nucleic acid sequence (SEQ ID NO: 62) to obtain a PT8 beta chain nucleic acid full-length sequence (shown as SEQ ID NO: 54);

The slow virus expression plasmids pWPXL (purchased from Addegene company) are taken as skeleton vectors to respectively construct PT1, PT8 and PT10 slow virus expression vectors, namely slow virus expression vectors pWPXL-PT1TCR, pWPXL-PT8TCR and pWPXL-PT10TCR; the pWPXL-PT1TCR, pWPXL-PT8TCR and pWPXL-PT10TCR lentiviral expression vectors are respectively transfected with lentiviral packaging plasmids pMD2.G and psPAX2 plasmid according to the mass ratio of 1:0.5:1 to prepare 293T cells, and virus supernatants are collected after two days; the virus supernatant was used to infect cd8+ T cells, and 24 hours after infection, after 2-3 days of culture, cd8+ T cells expressing PT1TCR, PT8TCR and PT10TCR were obtained, respectively, for downstream experiments.

Cd8+ T cells expressing PT1TCR, PT8TCR and PT10TCR were mixed with K562 cells expressing a1101 gene in a cell number ratio of 1:1, and were loaded with 10uM Wild Type (WT) (as shown in SEQ ID No. 55), mutant (Mut) (as shown in SEQ ID No. 56) and blank (DMSO) at final concentrations, respectively, and were co-cultured, and cells were recovered after 16 hours;

then adding 50ul of cell dye solution containing APC-CD137 antibody into the recovered cells respectively, and incubating for 30min at room temperature; then washing three times with PBS containing 0.5% BSA, centrifuging for 10min at 200g-250 g; cells were then resuspended with PBS containing 0.5% bsa for downstream detection; the expression of CD137 is detected by using flow cytometry to perform CD8+ TCR-T cell activation analysis.

The results of cd8+ T cell flow assays for PT1TCR, PT8TCR and PT10TCR are shown in figure 5 and statistics are shown in figure 6, showing that PT1TCR, PT8TCR and PT10TCR are specifically activated following stimulation of K562 of the a1101 gene loaded with mutant (Mut) antigenic peptide and are thus pik3ca_h1047l specific TCRs.

4. Expression test of CD137 and IFN-gamma secretion level test

Cd8+ T cells expressing PT1TCR, PT8TCR and PT10TCR were mixed with K562 cells expressing a1101 gene in a cell number ratio of 1:1, loaded with gradient dilutions (10 ^-5 μM、10 ^-5 μM、10 ^-4 μM、10 ^-3 μM、10 ^-2 μM、10 ^-1 μM、10 ⁰ mu.M and 10 ¹ μM) pik3ca_h1047l _1046-1054 Co-culturing mutant polypeptides (Mut); cells and supernatant were recovered after 16 hours;

adding 50ul of cell dye solution containing APC-CD137 antibody into the cells, and incubating for 30min at room temperature; then washing three times with PBS containing 0.5% BSA, centrifuging 200-250g for 10min; cells were then resuspended with PBS containing 0.5% bsa for downstream detection; performing CD8+ TCR-T cell activation analysis by adopting a flow cytometry, and detecting the expression condition of CD137 by the CD8+ T cell activation analysis; as shown in FIG. 7, the PT1TCR, PT8TCR and PT10TCR were activated specifically after the stimulation of K562 of A1101 gene loaded with mutant (Mut) antigen peptide, and the TCR-T cell was still able to recognize the mutant antigen peptide as the concentration of the antigen peptide was gradually decreased, so that it was a TCR with higher affinity.

In addition, the supernatant was subjected to ELISA for IFN-. Gamma.cytokine secretion assay, specifically comprising: PT1-T cells, PT8-T cells and PT10-T cells prepared by using T cells of human PBMC are respectively loaded with PIK3CA_H21047L _1046-1054 A1101-K562 cells of mutant polypeptide were 1:1 mixed and 2X 10 in 100. Mu.l volume ⁵ Cell number/well was added to 96-well plates and three replicates were made for each sample, with the PMA/Ionomycin (ION) group diluted in advance to working fluid with 1 μl PMA/ionomycin mix (250×) per 250 μl cell culture medium and used as positive stimulation control. T cells (D1 mock and D2 mock) of lentiviruses that were not infected with KT2, KT3 and KT8, respectively, were added in parallel as negative controls; after 24h incubation at 37 ℃, the supernatant from the 96-well plate incubation well was removed by centrifugation at 500g for 5min, the supernatant was added to ELISA plates coated with anti-IFN-gamma antibodies, and IFN-gamma levels in the supernatant were detected, as shown in FIG. 8, and PT1TCR, PT8TCR and PT10TCR were able to secrete IFN-gamma that kills tumor cells to exert effector functions.

5. Tumor cell endogenous mutation antigen recognition experiment

CD8+ T cells expressing PT1TCR, PT8TCR and PT10TCR and PIK3CA_H21047L _1046-1054 Mutant polypeptides (SEQ ID NO: 56) and PIK3CA_H21047 _1046-1054 K562 cells, MDA-MB-231 and SKOV3 cells of the wild-type polypeptide were expressed in 1:1 cell number (5X 10 ⁴ ) Mixing and co-culturing; cells and supernatant were recovered after 16 hours;

adding 50ul of cell dye solution containing APC-CD137 antibody into the cells, and incubating for 30min at room temperature; then washing three times with PBS containing 0.5% BSA, centrifuging for 10min at 200g-250 g; cells were then resuspended with PBS containing 0.5% bsa for downstream detection; the CD8+ TCR-T cell activation analysis is carried out by adopting the flow cytometry, the CD137 expression condition is detected by the CD8+ T cell activation analysis, the flow cytometry detection result is shown in figure 9, and the statistical chart is shown in figure 10, and the results show that the PT1TCR, the PT8TCR and the PT10TCR can be specifically activated after being stimulated by K562 of the endogenous expression mutation PIK3CA_H21047L of the expressed A1101 gene.

ELISA detection of IFN-gamma and IL-2 cytokines is carried out on the supernatant, and specifically comprises the following steps: PT1-T cells, PT8-T cells and PT10-T cells prepared by using T cells of human PBMC are respectively loaded with PIK3CA_H21047L _1046-1054 A1101-K562 cells of mutant polypeptide were 1:1 mixed and 5X10 in 100. Mu.l volume ⁴ Cell number/well was added to 96-well plates and three replicates were made for each sample, with the PMA/Ionomycin (ION) group diluted in advance to working fluid with 1 μl PMA/ionomycin mix (250×) per 200 μl cell culture medium and used as positive stimulation control. After 24H incubation at 37℃the supernatant from the 96-well plate culture wells was removed and centrifuged at 500g for 5min to remove residual cells, the supernatant was added to ELISA plates coated with anti-IFN-gamma or IL-2 antibodies, and the levels of IFN-gamma and IL-2 in the supernatant were detected, as shown in FIGS. 11 and 12, and the results showed that PT1TCR, PT8TCR and PT10TCR secreted cytokines IFN-gamma and IL-2 upon stimulation with K562, MDA-MB-231 and SKOV3 expressing the endogenous mutation PIK3CA_H1047L of the A1101 gene.

Thus, PT1TCR, PT8TCR and PT10TCR are relatively high affinity TCRs capable of specifically recognizing endogenous mutant antigens and secreting killer tumor cells to exert effector functions IFN- γ.

6. Tumor killing experiment

CD8+ T cells expressing PT1TCR, PT8TCR and PT10TCR and PIK3CA_H21047L _1046-1054 Mutant polypeptides (SEQ ID NO: 56) and PIK3CA_H21047 _1046-1054 Wild-type polypeptides were mixed (2.5:1, 5:1, 10:1, 20:1) and 2.5X10 by 100. Mu.l volume, respectively ⁴ 、5×10 ⁴ 、2×10 ⁵ And 4X 10 ⁵ Cell number/well was added to 96-well plates and three replicates were made for each sample; MDA-MB-231 and SKOV3 cell lines are cell lines which are modified in advance (mutant genes are used for detecting firefly luciferase (firefly luciferase) activity by taking luciferin as a substrate through a lentiviral system, the luciferin can catalyze the oxidation of the firefly luciferase into oxyuciferin, bioluminescence (bioluminescence) can be emitted in the oxidation process of the firefly luciferase), PIK3CA_H21047L and HLA-A1101 genes are introduced into the MDA-MB-231 and SKOV3 cell lines, and the expression of the luciferase reporter genes is stably expressed;

after incubation for 24h at 37 ℃, the supernatant of the 96-well plate culture well was discarded, luciferase was added to develop the low matters, and signals in the reaction system were detected, thereby detecting the killing results of PT1TCR, PT8TCR and PT10TCR on MDAMB-231 cells and SKOV3, wherein the killing results of MDAMB-231 cells are shown in fig. 13, the killing results of SKOV3 are shown in fig. 14, and the results show that PT1TCR, PT8TCR and PT10TCR all show higher specific killing ability with increasing proportion of effector cell target cells. Thus PT1TCR, PT8TCR and PT10TCR have pik3ca_h1047l mutation-specific killing ability.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims

1. The HLA-A1101 restrictive antigen epitope peptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 56.

2. A TCR targeting polypeptide-HLA-A 1101 complex, comprising an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 1, an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 2 and an alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 3, and the beta chain variable region comprises a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 4, a beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 5 and a beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 6; the polypeptide sequence is shown as SEQ ID NO. 56.

3. A TCR as claimed in claim 2 wherein the α chain variable region further comprises: one or more of an alpha chain FR1 encoded by a sequence shown in SEQ ID NO. 7, an alpha chain FR2 encoded by a sequence shown in SEQ ID NO. 8, an alpha chain FR3 encoded by a sequence shown in SEQ ID NO. 9 and an alpha chain FR4 encoded by a sequence shown in SEQ ID NO. 10; the beta chain variable region further comprises: one or more of a beta-chain FR1 encoded by a sequence shown in SEQ ID NO. 11, a beta-chain FR2 encoded by a sequence shown in SEQ ID NO. 12, a beta-chain FR3 encoded by a sequence shown in SEQ ID NO. 13 and a beta-chain FR4 encoded by a sequence shown in SEQ ID NO. 14.

4. A TCR targeting polypeptide-HLA-A 1101 complex, comprising an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 19, an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 20 and an alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 21, and the beta chain variable region comprises a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 22, a beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 23 and a beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 24; the polypeptide sequence is shown as SEQ ID NO. 56.

5. A TCR as claimed in claim 4 wherein the α chain variable region further comprises: one or more of an alpha chain FR1 encoded by a sequence shown in SEQ ID NO. 25, an alpha chain FR2 encoded by a sequence shown in SEQ ID NO. 26, an alpha chain FR3 encoded by a sequence shown in SEQ ID NO. 27 and an alpha chain FR4 encoded by a sequence shown in SEQ ID NO. 28; the beta chain variable region further comprises: one or more of the beta-chain FR1 encoded by the sequence shown in SEQ ID NO. 29, the beta-chain FR2 encoded by the sequence shown in SEQ ID NO. 30, the beta-chain FR3 encoded by the sequence shown in SEQ ID NO. 31 and the beta-chain FR4 encoded by the sequence shown in SEQ ID NO. 32.

6. A TCR targeting polypeptide-HLA-A 1101 complex, comprising an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 37, an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 38 and an alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 39, and the beta chain variable region comprises a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 40, a beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 41 and a beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 42; the polypeptide sequence is shown as SEQ ID NO. 56.

7. A TCR as claimed in claim 6 wherein the α chain variable region further comprises: one or more of an alpha chain FR1 encoded by a sequence shown in SEQ ID NO. 43, an alpha chain FR2 encoded by a sequence shown in SEQ ID NO. 44, an alpha chain FR3 encoded by a sequence shown in SEQ ID NO. 45 and an alpha chain FR4 encoded by a sequence shown in SEQ ID NO. 46; the beta chain variable region further comprises: one or more of a beta-chain FR1 encoded by a sequence shown in SEQ ID NO:47, a beta-chain FR2 encoded by a sequence shown in SEQ ID NO:48, a beta-chain FR3 encoded by a sequence shown in SEQ ID NO:49 and a beta-chain FR4 encoded by a sequence shown in SEQ ID NO: 50.

8. A TCR targeting polypeptide-HLA-A 1101 complex, wherein the polypeptide sequence is as shown in SEQ ID No. 56; TCR (thyristor controlled reactor)

Comprising any one of the following three groups: (a) An alpha chain encoded by the sequence shown in SEQ ID NO. 15 and a beta chain encoded by the sequence shown in SEQ ID NO. 16; (b) An alpha chain encoded by the sequence shown in SEQ ID NO. 33 and a beta chain encoded by the sequence shown in SEQ ID NO. 34; (c) An alpha chain encoded by the sequence shown in SEQ ID NO. 51 and a beta chain encoded by the sequence shown in SEQ ID NO. 52.

9. A polynucleotide encoding a TCR as claimed in claim 2 comprising sequences as set out 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.

10. The composition of claim 10 further comprising 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.

11. A TCR polynucleotide encoding a polypeptide according to claim 4 comprising sequences as set out 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.

12. The polynucleotide of claim 12, further comprising one or more of the sequence shown in SEQ ID No. 25, the sequence shown in SEQ ID No. 26, the sequence shown in SEQ ID No. 27, the sequence shown in SEQ ID No. 28, the sequence shown in SEQ ID No. 29, the sequence shown in SEQ ID No. 30, the sequence shown in SEQ ID No. 31 and the sequence shown in SEQ ID No. 32.

13. A polynucleotide encoding a TCR as claimed in claim 8, wherein the polynucleotide comprises any one of the following three sets 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.

14. An expression vector comprising the polynucleotide of any one of claims 9 to 13.

15. An engineered cell comprising the expression vector of claim 14.

16. A pharmaceutical composition comprising a TCR according to any one of claims 2 to 8 or a polynucleotide according to any one of claims 9 to 13 or an expression vector according to claim 14 or an engineered cell according to claim 15.

17. Use of a TCR according to any one of claims 2 to 8 or a polynucleotide according to any one of claims 9 to 13 or an expression vector according to claim 14 or an engineered cell according to claim 15 in the manufacture of a medicament for the treatment of a cancer positive for expression of polypeptide-HLA-A 1101; the polypeptide sequence is shown as SEQ ID NO. 56.

18. The use according to claim 17, wherein the cancer comprises endometrial, breast, cervical, colorectal, bladder, head and neck, esophageal gastric, glioma, non-small cell, glioblastoma, cholangiocarcinoma, melanoma, seminoma, hepatobiliary carcinoma, sarcoma, pancreatic, pleural mesothelioma, prostate, ovarian epithelial cell tumor, renal clear cell carcinoma, renal adrenocortical carcinoma, non-clear cell carcinoma or thyroid carcinoma.

19. Use of a TCR according to any one of claims 2 to 8 or a polynucleotide according to any one of claims 9 to 13 or an expression vector according to claim 14 or an engineered cell according to claim 15 in the preparation of a tumour cell reagent or kit for detecting positive expression of polypeptide-HLA-A 1101; the polypeptide sequence is shown as SEQ ID NO. 56.

20. Use of a polypeptide comprising an HLA-A 1101-restricted epitope peptide of claim 1 in the preparation of a therapeutic tumor vaccine.