CN114702569B - PD-L1 related vaccine and application thereof - Google Patents
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Abstract
The invention provides a PD-L1 related vaccine and application thereof, wherein the PD-L1 related vaccine comprises: an isolated polypeptide, an isolated nucleic acid, an antigen presenting cell, an immune cell, and/or an antibody, the isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1.3, 5,7, 8, 9, 11, 13, 14, 17, 18, 19, 21, 24, 25, 27, 28, 30 to 34 or a functional analogue thereof. The vaccine provided by the invention can take the PDL1 epitope as a target spot, improve the activity and the number of anti-tumor immunosuppressive cells in a tumor microenvironment, specifically remove negatively regulated cells such as stromal cells and immune cells in the tumor microenvironment and tumor cells expressing PDL1, has high safety, and has important significance in preventing and treating cancers.
Description
Technical Field
The invention relates to the field of biomedicine. In particular, the invention relates to PD-L1 related vaccines and uses thereof.
Background
The immune system contains many types of regulatory immune cells, which function to control the intensity of the immune response and maintain immune balance. Regulatory immune cells mainly include Regulatory T cells (Tregs), M2 macrophages (M2 macromacrophages), myeloid-derived suppressor cells (MDSCs) and different Dendritic Cell (DCs) subsets. The immunosuppressive process in which regulatory immune cells participate is a mechanism for controlling the scale and duration of a particular immune response. Immunosuppression differs from immune tolerance in that immunomodulation is an active immune activation. Research has shown that regulatory immune cells can express various immunosuppressive related proteins, such as IDO, PD-L1, TDO, arginase, CCL22, etc., and weaken the immune response effect, with the following three mechanisms: (1) Catabolic enzymes of amino acids, reducing the amino acids required for immune cell growth and function, such as tryptophan and arginine; (2) Inducing target cells and T cells to up-regulate immune checkpoint-related molecule expression, such as PD-1, PD-L2; (3) Chemokines are secreted, recruiting more subpopulations of regulatory immune cells to the area of inflammation.
Regulatory immune cells are considered to be of positive significance in the immune environment of healthy people, and can avoid damaging self cells by too strong immune responses. However, in the tumor immune environment, regulatory immune cells may suppress the body's immune killing response to malignant cells. Moreover, many types of cancer cells have been shown to adapt to the phenotype of regulatory immune cells, and like regulatory immune cells can express multiple immunosuppressive proteins that inhibit the activity of anti-tumor specific T cells in the tumor microenvironment. In order to balance the immunosuppression caused by regulatory immune cells, mechanisms for antagonizing immunosuppression are also formed in the immune system. In a tumor immune microenvironment, part of proinflammatory T cells can identify regulatory immune cells expressing immunosuppressive proteins in a targeted mode, the number of the regulatory immune cells is reduced, the tumor immunosuppressive effect is antagonized, proinflammatory cytokines are secreted at an inflammation part to assist in enhancing adaptive immune response, and the cells are defined as Anti-TIS cells.
The Anti-TIS (Tumor ImmunoSuppression) technology is an innovative immunotherapy scheme provided based on the action mechanism of Anti-TIS cells, and aims to activate human Anti-Tumor immunosuppressed cells, target regulatory immune cells and Tumor cells, break ImmunoSuppression within an acceptable toxicity range, activate immune response as much as possible, and generate Anti-Tumor therapy effect.
Disclosure of Invention
It should be noted that the present invention has been completed based on the following findings of the inventors:
HLA is the expression product of Major Histocompatibility Complex (MHC) of human, and is alloantigen with high polymorphism, and there are differences in HLA allelic type of people in different regions, for example, the allelic type with higher coverage in Europe and America is mainly HLA-A02:01, and the first two allelic types with highest coverage in China are HLA-A11:01 and HLA-A24:02.
Programmed cell death ligand 1 (PD-L1), also known as surface antigen cluster of differentiation 274 (CD274), is a transmembrane protein in the human body and is closely associated with the self-regulation of the immune system. Binding of programmed cell death receptor (PD-1) to PD-L1 on the surface of cancer cell or immunoregulatory cell can transmit immunosuppressive signal and reduce CD8 + Expansion and activation of T cells reduces the accumulation of antigen-specific T cells in lymph nodes, leading to immune escape of cancer cells.
Based on the above mechanism, the inventors selected the PD-L1 protein from many cancer-related genes to screen targets for anti-tumor immunosuppression. Further, the total length of the PD-L1 protein is 290 amino acids, and the total number of the obtained products is 8, 9, 10 and 11mer peptide segments 1126 according to 1 step displacement sliding shearing. The inventor analyzes all the peptide fragments and finds that only a few peptide fragments can be combined with HLA-A11:01 or HLA-A24:02 molecules to form an HLA complex and further combined with T cell receptors in organisms to stimulate the organisms to generate immune response, and most of the rest peptide fragments have no HLA affinity and immunogenicity, so that the research value of developing anti-tumor immunosuppressive vaccines does not exist. Thus, the inventors screened for immunologically functional epitopes from a wide variety of epitopes. The polypeptides can be used as vaccines or medicines, can take an epitope of the immune suppression protein PDL1 as a target spot, improve the activity and the number of Anti-TIS cells in a tumor microenvironment, and specifically eliminate negatively regulated cells such as stromal cells, immune cells and the like in the tumor microenvironment and tumor cells expressing PDL 1. In addition, since Anti-TIS cells occur naturally in the body, the body must have a mechanism to ensure immune homeostasis to control them, and thus the risk of triggering autoimmune-related adverse events is extremely low. Tests carried out at present show that mice inoculated with the polypeptide have no adverse reaction, and clinical research data also prove the safety of the method. Therefore, the compound has important significance in preventing and treating cancers.
In one aspect of the invention, the invention features an isolated polypeptide. According to an embodiment of the invention, the isolated polypeptide has the amino acid sequence as shown in SEQ ID NO: 1.3, 5,7, 8, 9, 11, 13, 14, 17, 18, 19, 21, 24, 25, 27, 28, 30 to 34 or a functional analogue thereof. The polypeptide has affinity to HLA-A11:01 and/or HLA-A24:02, can be naturally presented on the cell surface and combined with specific immune effector cells, specifically eliminates negatively regulated cells such as stromal cells and immune cells in a tumor microenvironment and tumor cells expressing PD-L1, has potential value as an immunotherapy target, and has important significance in preventing and treating cancers.
In another aspect of the invention, an isolated nucleic acid is provided. According to an embodiment of the invention, the isolated nucleic acid encodes the aforementioned polypeptide or a functional analog thereof.
In yet another aspect of the invention, an antigen presenting cell is provided. According to embodiments of the invention, the antigen presenting cells are used to present the polypeptides described above.
In yet another aspect of the invention, an immune cell is provided. According to an embodiment of the present invention, the binding target of the immune cell is the aforementioned isolated polypeptide or the aforementioned antigen presenting cell.
In yet another aspect of the invention, an antibody is provided. According to an embodiment of the invention, the antibody specifically recognizes the aforementioned isolated polypeptide.
In yet another aspect of the invention, a vaccine is provided. According to an embodiment of the invention, the vaccine comprises: the aforementioned isolated polypeptide, isolated nucleic acid, antigen presenting cell, immune cell and/or antibody.
In yet another aspect of the invention, a medicament is provided. According to an embodiment of the invention, the medicament comprises: the aforementioned isolated polypeptide, isolated nucleic acid, antigen presenting cell, immune cell and/or antibody.
In a further aspect of the invention, the invention proposes the use of the aforementioned isolated polypeptides, isolated nucleic acids, antigen presenting cells, immune cells and/or antibodies in the preparation of vaccines. According to an embodiment of the invention, the vaccine is for the prevention of cancer.
In a further aspect of the invention, the invention proposes the use of the aforementioned isolated polypeptide, isolated nucleic acid, antigen presenting cell, immune cell and/or antibody for the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment of cancer.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIGS. 1-4 show mass spectra of polypeptides according to embodiments of the present invention, wherein the upper half of the mass spectra is isolated and the lower half of the mass spectra is synthesis;
FIG. 5 shows an electron micrograph of polypeptides and in vitro immunogenic ELISPOTs according to embodiments of the invention;
FIGS. 6 and 7 show schematic diagrams of target cell analysis of immune cell specific killing presenting polypeptides according to embodiments of the invention, respectively;
FIGS. 8 and 9 show schematic diagrams of the analysis of the polypeptide vaccine for controlling the growth of the tumor of the mouse according to the embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
In one aspect of the invention, the invention features an isolated polypeptide. According to an embodiment of the invention, the isolated polypeptide has the amino acid sequence as shown in SEQ ID NO: 1.3, 5,7, 8, 9, 11, 13, 14, 17, 18, 19, 21, 24, 25, 27, 28, 30 to 34 or a functional analogue thereof. The polypeptide has affinity to HLA-A11:01 and/or HLA-A24:02, can be naturally presented on the cell surface and combined with specific immune effector cells, specifically eliminates negatively regulated cells such as stromal cells, immune cells and the like in a tumor microenvironment and tumor cells expressing PD-L1, has potential value of becoming an immunotherapy target, and has important significance in preventing and treating cancers.
Specifically, among the above polypeptides, SEQ ID NO: 1.3, 5,7, 8, 11, 14, 17, 24, 32, 33, 34 or a functional analogue thereof. SEQ ID NO: 3. 5, 8, 24, 32, 34 or functional analogues thereof, and has strong immunogenicity and capacity of stimulating the generation of specific T cells with high killing efficiency.
In another aspect of the invention, the invention features an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid encodes the aforementioned polypeptide or a complementary sequence thereof. As mentioned above, the polypeptide expressed by the nucleic acid provided by the above embodiments under appropriate conditions can be used for preventing or treating tumor, thereby exerting an immune killing effect. Furthermore, the polypeptide expressed by the nucleic acid provided by the embodiment of the invention under appropriate conditions can be used for preventing or treating tumors.
According to an embodiment of the invention, the isolated nucleic acid has the sequence as set forth in SEQ ID NO: 35. 37, 39, 41, 42, 43, 45, 47, 48, 51, 52, 53, 55, 58, 59, 61, 62, 64 to 68 or a functional analogue thereof. Specifically, in the above isolated nucleic acid, SEQ ID NO: 35. 37, 39, 41, 42, 45, 48, 51, 58, 66, 67, 68 or a functional analogue thereof, wherein the nucleotide sequence shown in any one of SEQ ID NOs: 37. 39, 42, 58, 66, 68 or a functional analogue thereof is more effective.
TABLE 1 Polypeptides and nucleic acid sequences
| Gene | Amino acid site | SEQ ID | Polypeptide sequence | SEQ ID | Nucleic acid sequences |
| PDL1 | 6-15 | NO:1 | VFIFMTYWHL | NO:35 | GTCTTTATATTCATGACCTACTGGCATTTG |
| PDL1 | 6-16 | NO:2 | VFIFMTYWHLL | NO:36 | GTCTTTATATTCATGACCTACTGGCATTTGCTG |
| PDL1 | 8-16 | NO:3 | IFMTYWHLL | NO:37 | ATATTCATGACCTACTGGCATTTGCTG |
| PDL1 | 8-17 | NO:4 | IFMTYWHLLN | NO:38 | ATATTCATGACCTACTGGCATTTGCTGAAC |
| PDL1 | 11-19 | NO:5 | TYWHLLNAF | NO:39 | ACCTACTGGCATTTGCTGAACGCATTT |
| PDL1 | 11-21 | NO:6 | TYWHLLNAFTV | NO:40 | ACCTACTGGCATTTGCTGAACGCATTTACTGTC |
| PDL1 | 15-25 | NO:7 | LLNAFTVTVPK | NO:41 | TTGCTGAACGCATTTACTGTCACGGTTCCCAAG |
| PDL1 | 17-25 | NO:8 | NAFTVTVPK | NO:42 | AACGCATTTACTGTCACGGTTCCCAAG |
| PDL1 | 32-41 | NO:9 | YGSNMTIECK | NO:43 | TATGGTAGCAATATGACAATTGAATGCAAA |
| PDL1 | 33-41 | NO:10 | GSNMTIECK | NO:44 | GGTAGCAATATGACAATTGAATGCAAA |
| PDL1 | 36-46 | NO:11 | MTIECKFPVEK | NO:45 | ATGACAATTGAATGCAAATTCCCAGTAGAAAAA |
| PDL1 | 37-46 | NO:12 | TIECKFPVEK | NO:46 | ACAATTGAATGCAAATTCCCAGTAGAAAAA |
| PDL1 | 79-89 | NO:13 | SSYRQRARLLK | NO:47 | AGTAGCTACAGACAGAGGGCCCGGCTGTTGAAG |
| PDL1 | 97-105 | NO:14 | AALQITDVK | NO:48 | GCTGCACTTCAGATCACAGATGTGAAA |
| PDL1 | 105-113 | NO:15 | KLQDAGVYR | NO:49 | AAATTGCAGGATGCAGGGGTGTACCGC |
| PDL1 | 110-118 | NO:16 | GVYRCMISY | NO:50 | GGGGTGTACCGCTGCATGATCAGCTAT |
| PDL1 | 116-124 | NO:17 | ISYGGADYK | NO:51 | ATCAGCTATGGTGGTGCCGACTACAAG |
| PDL1 | 116-125 | NO:18 | ISYGGADYKR | NO:52 | ATCAGCTATGGTGGTGCCGACTACAAGCGA |
| PDL1 | 117-126 | NO:19 | SYGGADYKRI | NO:53 | AGCTATGGTGGTGCCGACTACAAGCGAATT |
| PDL1 | 120-129 | NO:20 | GADYKRITVK | NO:54 | GGTGCCGACTACAAGCGAATTACTGTGAAA |
| PDL1 | 126-136 | NO:21 | ITVKVNAPYNK | NO:55 | ATTACTGTGAAAGTCAATGCCCCATACAACAAA |
| PDL1 | 127-136 | NO:22 | TVKVNAPYNK | NO:56 | ACTGTGAAAGTCAATGCCCCATACAACAAA |
| PDL1 | 129-136 | NO:23 | KVNAPYNK | NO:57 | AAAGTCAATGCCCCATACAACAAA |
| PDL1 | 133-141 | NO:24 | PYNKINQRI | NO:58 | CCATACAACAAAATCAACCAAAGAATT |
| PDL1 | 153-162 | NO:25 | LTCQAEGYPK | NO:59 | CTGACATGTCAGGCTGAGGGCTACCCCAAG |
| PDL1 | 159-167 | NO:26 | GYPKAEVIW | NO:60 | GGCTACCCCAAGGCCGAAGTCATCTGG |
| PDL1 | 168-178 | NO:27 | TSSDHQVLSGK | NO:61 | ACAAGCAGTGACCATCAAGTCCTGAGTGGTAAG |
| PDL1 | 169-178 | NO:28 | SSDHQVLSGK | NO:62 | AGCAGTGACCATCAAGTCCTGAGTGGTAAG |
| PDL1 | 181-189 | NO:29 | TTNSKREEK | NO:63 | ACCACCAATTCCAAGAGAGAGGAGAAG |
| PDL1 | 189-198 | NO:30 | KLFNVTSTLR | NO:64 | AAGCTTTTCAATGTGACCAGCACACTGAGA |
| PDL1 | 202-212 | NO:31 | TTNEIFYCTFR | NO:65 | ACAACTAATGAGATTTTCTACTGCACTTTTAGG |
| PDL1 | 252-260 | NO:32 | GVALTFIFR | NO:66 | GGTGTAGCACTGACATTCATCTTCCGT |
| PDL1 | 255-263 | NO:33 | LTFIFRLRK | NO:67 | CTGACATTCATCTTCCGTTTAAGAAAA |
| PDL1 | 273-281 | NO:34 | GIQDTNSKK | NO:68 | GGCATCCAAGATACAAACTCAAAGAAG |
It is to be noted that, with respect to the nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that any one or two of the complementary double strands are actually included. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the gene sequence in the present application includes a DNA form or an RNA form, and disclosure of one of them means that the other is also disclosed.
In yet another aspect of the invention, an antigen presenting cell is provided. According to an embodiment of the invention, the antigen presenting cell is used to present the isolated nucleic acid as described above. Therefore, the antigen presenting cell can deliver the polypeptide to the anti-tumor specific T cell, so as to activate the specific killing function of the T cell, and the antigen presenting cell can be effectively used for preventing or treating tumors.
According to an embodiment of the invention, the antigen presenting cell is a dendritic cell, a B cell or a monocyte-phagocytic cell, preferably a dendritic cell. Dendritic cells have extremely strong capacity of antigen endocytosis and processing, and can present antigens on the surfaces of cells. The inventors selected dendritic cells as antigen presenting cells that initiated, regulated and maintained an immune response to the polypeptide more strongly in vivo.
According to an embodiment of the invention, the antigen presenting cell expresses HLA-A11:01 and/or HLA-A24:02. The polypeptide has affinity to HLA-A11:01 and/or HLA-A24:02, can be naturally presented and combined with specific immune effector cells, specifically eliminates negatively regulated cells such as stromal cells, immune cells and the like in a tumor microenvironment and tumor cells expressing PD-L1, has potential value of becoming an immunotherapy target, and has important significance in preventing and treating cancers.
In yet another aspect of the invention, an immune cell is provided. According to an embodiment of the present invention, the binding target of the immune cell is the isolated polypeptide described above or the antigen presenting cell described above. This enables the immune killing function to be exhibited.
According to an embodiment of the invention, the immune cells are obtained by: contacting the antigen presenting cells with immune effector cells, or selecting and culturing from Tumor tissues (such as Tumor-infiltrating T cells, TILs), or transferring T Cell Receptor (TCR) gene capable of recognizing the isolated polypeptide into T cells (such as TCR-T). In particular, the cell with immune effector function is CD8 + T cells. By contacting an antigen-presenting cell presenting the aforementioned isolated peptide with a cell having an immune effector function, the antigen-presenting cell can activate an inactivated cell having an immune effector function to present an antigen-the aforementioned polypeptide, and further activate a cell having an immune effector function to produce a large number of immune effector cells having an action of specifically killing a target cell presenting the antigen-the aforementioned polypeptide. CD8 + The T cells have stronger capability of receiving the activation of antigen presenting cells, and the obtained CD8 + Specific killing of T cells presents antigen-the target cells of the isolated peptide are more potent.
In yet another aspect of the invention, an antibody is provided. According to an embodiment of the invention, the antibody specifically recognizes the isolated polypeptide as described above. Thus, the tumor antigen can be specifically recognized. According to the embodiment of the invention, the antibody can specifically recognize the antigen, and can be specifically combined with tumor cells or cells infected by virus (such as HBV, HCV, HIV, EBV and the like), so that the tumor cells or the cells infected by the virus are phagocytized by phagocytic cells, and the specific elimination of the tumor cells or the cells infected by the virus is realized.
In yet another aspect of the invention, a vaccine is provided. According to an embodiment of the invention, the vaccine comprises: isolated polypeptides, isolated nucleic acids, antigen presenting cells, immune cells and/or antibodies as described above. Therefore, when the vaccine enters the organism, specific T cells (such as CTL and TIL) can be specifically activated, negatively regulated cells such as stromal cells and immune cells in a tumor microenvironment and tumor cells expressing PD-L1 can be specifically eliminated, the safety is good, and the vaccine has important significance in the aspect of preventing cancers.
According to an embodiment of the invention, the vaccine further comprises an adjuvant. The invention does not strictly limit the type of the adjuvant, and can select the conventional adjuvant in the field, and particularly can flexibly select the adjuvant according to actual needs.
In yet another aspect of the invention, a medicament is provided. According to an embodiment of the invention, the medicament comprises: an isolated polypeptide, an isolated nucleic acid, an antigen presenting cell, an immune cell and/or an antibody as described above. Therefore, when entering the organism, the medicine provided by the embodiment of the invention can specifically activate specific T cells (such as CTL and TIL) to specifically remove negatively regulated cells such as stromal cells and immune cells in a tumor microenvironment and tumor cells expressing PD-L1, has good safety and has important significance in treating cancers.
According to an embodiment of the invention, the medicament further comprises a pharmaceutically acceptable excipient.
In a further aspect of the invention, the invention proposes the use of the aforementioned isolated polypeptides, isolated nucleic acids, antigen presenting cells, immune cells and/or antibodies in the preparation of vaccines. According to an embodiment of the invention, the vaccine is for the prevention of cancer. The vaccine enters into the organism, can specifically activate specific T cells (such as CTL and TIL), achieves the purpose of specifically removing negatively regulated cells such as stromal cells and immune cells in a tumor microenvironment and tumor cells expressing PD-L1, and has important significance in the aspect of preventing cancers.
Accordingly, the present invention provides a method of preventing cancer, comprising: administering to the subject the aforementioned isolated polypeptide, isolated nucleic acid, antigen presenting cell, immune cell, and/or antibody.
According to an embodiment of the invention, the subject of the vaccine expresses HLA-A11:01 and/or HLA-A24:02. As described above, the polypeptide has affinity to HLA-A11:01 and/or HLA-A24:02, can be naturally presented on the cell surface, can be combined with specific immune effector cells, can specifically eliminate negatively regulated cells such as stromal cells and immune cells in a tumor microenvironment and tumor cells expressing PD-L1, has high safety, and has important significance in preventing cancers.
In a further aspect of the invention, the invention proposes the use of the aforementioned isolated polypeptide, isolated nucleic acid, antigen presenting cell, immune cell and/or antibody for the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment of cancer. The medicine enters into organism, can specifically activate specific T cell (such as CTL, TIL), and specifically remove negatively regulated cells such as stromal cells, immunocytes and the like in tumor microenvironment and tumor cells expressing PD-L1, and has important significance in treating cancer.
According to an embodiment of the present invention, the cancer is breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia, thyroid cancer, lymphoma, bladder cancer, kidney cancer, uterine body cancer, ovarian cancer, gallbladder cancer, oral cancer, larynx cancer, bone cancer, testicular cancer or brain cancer.
According to an embodiment of the invention, the subject of the medicament expresses HLA-A11:01 and/or HLA-A24:02. As described above, the polypeptide has affinity to HLA-A11:01 and/or HLA-A24:02, can be naturally presented on the cell surface, can be combined with specific immune effector cells, can specifically eliminate negatively regulated cells such as stromal cells and immune cells in a tumor microenvironment and tumor cells expressing PD-L1, has high safety, and has important significance in treating cancers.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to techniques or conditions described in literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 immunogenicity prediction of Polypeptides
According to the invention, based on the selected HLA-A11:01 and HLA-A24:02 allelic typing, a biological information analysis process is utilized to cut PDL1 protein into polypeptide sequences with 8-11 mer amino acids in full length, the length of the protein coded by the PDL1 gene is 290 amino acids, the molecular weight is 33275 daltons, and 1126 cut polypeptide sequences. The 1126 polypeptides were subjected to a presentation prediction and an affinity prediction, respectively. A score of 0 to 1 is used to indicate a higher score indicating greater presentation, and a score greater than 0.1 indicates that the polypeptide is capable of presentation. Affinity score is expressed as an IC50 score, an IC50 of less than 500 indicates that the polypeptide has affinity, and an IC50 of less than 50 indicates that the polypeptide has high affinity. The screening condition is one of the following two conditions: (1) rendering a score value >0.5; (2) render score value >0.1 and IC50<500. The polypeptide sequences that met the conditions (table 2 and table 3) were subjected to the next step of T2 affinity validation.
TABLE 2 prediction scores for the presentation and affinity of the polypeptide sequences to HLA-A11:01 alleles
| SEQ ID | Gene | Amino acid site | Polypeptide sequence | HLA typing | Rendering scoring values | Affinity score |
| NO:7 | PDL1 | 15-25 | LLNAFTVTVPK | HLA-A11:01 | 0.2182 | 89.9590 |
| NO:8 | PDL1 | 17-25 | NAFTVTVPK | HLA-A11:01 | 0.7874 | 15.0000 |
| NO:9 | PDL1 | 32-41 | YGSNMTIECK | HLA-A11:01 | 0.1470 | 375.2523 |
| NO:10 | PDL1 | 33-41 | GSNMTIECK | HLA-A11:01 | 0.3712 | 34.6272 |
| NO:11 | PDL1 | 36-46 | MTIECKFPVEK | HLA-A11:01 | 0.5907 | 6.7074 |
| NO:12 | PDL1 | 37-46 | TIECKFPVEK | HLA-A11:01 | 0.1814 | 31.6022 |
| NO:13 | PDL1 | 79-89 | SSYRQRARLLK | HLA-A11:01 | 0.4621 | 38.1700 |
| NO:14 | PDL1 | 97-105 | AALQITDVK | HLA-A11:01 | 0.5077 | 90.5887 |
| NO:15 | PDL1 | 105-113 | KLQDAGVYR | HLA-A11:01 | 0.2106 | 73.2814 |
| NO:16 | PDL1 | 110-118 | GVYRCMISY | HLA-A11:01 | 0.2449 | 13.5977 |
| NO:17 | PDL1 | 116-124 | ISYGGADYK | HLA-A11:01 | 0.2952 | 8.1088 |
| NO:18 | PDL1 | 116-125 | ISYGGADYKR | HLA-A11:01 | 0.1865 | 71.6428 |
| NO:20 | PDL1 | 120-129 | GADYKRITVK | HLA-A11:01 | 0.1239 | 58.2933 |
| NO:21 | PDL1 | 126-136 | ITVKVNAPYNK | HLA-A11:01 | 0.3871 | 85.5169 |
| NO:22 | PDL1 | 127-136 | TVKVNAPYNK | HLA-A11:01 | 0.3067 | 23.8387 |
| NO:23 | PDL1 | 129-136 | KVNAPYNK | HLA-A11:01 | 0.1047 | 246.4766 |
| NO:25 | PDL1 | 153-162 | LTCQAEGYPK | HLA-A11:01 | 0.3360 | 15.1653 |
| NO:27 | PDL1 | 168-178 | TSSDHQVLSGK | HLA-A11:01 | 0.3165 | 37.5170 |
| NO:28 | PDL1 | 169-178 | SSDHQVLSGK | HLA-A11:01 | 0.4771 | 56.6550 |
| NO:29 | PDL1 | 181-189 | TTNSKREEK | HLA-A11:01 | 0.3763 | 36.1821 |
| NO:30 | PDL1 | 189-198 | KLFNVTSTLR | HLA-A11:01 | 0.2815 | 25.7653 |
| NO:31 | PDL1 | 202-212 | TTNEIFYCTFR | HLA-A11:01 | 0.1389 | 122.2114 |
| NO:32 | PDL1 | 252-260 | GVALTFIFR | HLA-A11:01 | 0.6331 | 18.2811 |
| NO:33 | PDL1 | 255-263 | LTFIFRLRK | HLA-A11:01 | 0.2900 | 6.6192 |
| NO:34 | PDL1 | 273-281 | GIQDTNSKK | HLA-A11:01 | 0.5256 | 24.7917 |
TABLE 3 predictive scores for the presentation and affinity of the polypeptide sequences to the HLA-A24:02 allele
| SEQ ID | Gene | Amino acid site | Polypeptide sequence | HLA typing | Rendering scoring values | Affinity score |
| NO:1 | PDL1 | 6-15 | VFIFMTYWHL | HLA-A24:02 | 0.3786 | 11.7606 |
| NO:2 | PDL1 | 6-16 | VFIFMTYWHLL | HLA-A24:02 | 0.1079 | 22.6598 |
| NO:3 | PDL1 | 8-16 | IFMTYWHLL | HLA-A24:02 | 0.8124 | 8.3178 |
| NO:4 | PDL1 | 8-17 | IFMTYWHLLN | HLA-A24:02 | 0.1026 | 69.4832 |
| NO:5 | PDL1 | 11-19 | TYWHLLNAF | HLA-A24:02 | 0.9231 | 7.5832 |
| NO:6 | PDL1 | 11-21 | TYWHLLNAFTV | HLA-A24:02 | 0.1202 | 28.6888 |
| NO:19 | PDL1 | 117-126 | SYGGADYKRI | HLA-A24:02 | 0.2008 | 155.6688 |
| NO:24 | PDL1 | 133-141 | PYNKINQRI | HLA-A24:02 | 0.8545 | 17.0088 |
| NO:26 | PDL1 | 159-167 | GYPKAEVIW | HLA-A24:02 | 0.1978 | 74.1338 |
Example 2 polypeptide T2 affinity validation
Take 2X 10 5 A500. Mu.L aliquot of T2 cells plated with human beta 2 IMDM serum-free medium of microglobulin (final concentration, 3. Mu.g/ml) was resuspended in 24-well plates, polypeptides from Table 1 (final concentration 100. Mu.M) were added, and the cells were incubated (37 ℃, 5% CO) 2 ) And cultured overnight. 2 multiple wells per group; t2 cells without added polypeptide were used as background control. Cells were harvested by centrifugation at 200g for 5 minutes. After the cells were washed twice with PBS, the cells were directly incubated with FITC monoclonal antibody against HLA-A11:01/HLA-A24:02 and maintained at 4 ℃ for 30 minutes. And then analyzed by flow cytometry. The Fluorescence Index (FI) was calculated using the following formula: FI = [ Mean Fluorescence Intensity (MFI) sample-MFI background ] / MFI background Wherein MFI background Represents the value without peptide. FI>1.5 shows that the peptide has high affinity for HLA-A11:01 or HLA-A24:02 molecules, 1.0<FI <1.5 shows that the peptide has moderate affinity for HLA-A11:01 or HLA-A24:02 molecules, and 0.5<FI <1.0 indicates that the peptide is HLA-A11:01 or HLA-A24:02 molecular low affinity.
The results are as followsThe following table shows that 12 polypeptides with high affinity for T2 affinity validation were selected for subsequent mass-spectrometric presentation validation, in vitro stimulation of CD8 amplification + T cells and ELISPOTs methods.
TABLE 4 detection of the affinity of the polypeptide sequences to HLA alleles
| Sample(s) | SEQ ID | Adding polypeptide concentration | Mean fluorescence intensity | FI | Conclusion |
| VFIFMTYWHL | NO:1 | 100μM | 690 | 3.57 | High affinity |
| VFIFMTYWHLL | NO:2 | 100μM | 11 | -0.93 | No affinity |
| IFMTYWHLL | NO:3 | 100μM | 970 | 5.42 | High affinity |
| IFMTYWHLLN | NO:4 | 100μM | 103 | -0.32 | No affinity |
| TYWHLLNAF | NO:5 | 100μM | 1000 | 5.62 | High affinity |
| TYWHLLNAFTV | NO:6 | 100μM | 131 | -0.13 | No affinity |
| LLNAFTVTVPK | NO:7 | 100μM | 531 | 2.51 | High affinity |
| NAFTVTVPK | NO:8 | 100μM | 540 | 2.57 | High affinity |
| YGSNMTIECK | NO:9 | 100μM | 236 | 0.56 | Low affinity |
| GSNMTIECK | NO:10 | 100μM | 102 | -0.32 | No affinity |
| MTIECKFPVEK | NO:11 | 100μM | 540 | 2.57 | High affinity |
| TIECKFPVEK | NO:12 | 100μM | 226 | 0.50 | No affinity |
| SSYRQRARLLK | NO:13 | 100μM | 299 | 0.98 | Low affinity |
| AALQITDVK | NO:14 | 100μM | 738 | 3.89 | High affinity |
| KLQDAGVYR | NO:15 | 100μM | 3 | -0.98 | No affinity |
| GVYRCMISY | NO:16 | 100μM | 31 | -0.80 | No affinity |
| ISYGGADYK | NO:17 | 100μM | 574 | 2.80 | High affinity |
| ISYGGADYKR | NO:18 | 100μM | 348 | 1.31 | Moderate affinity |
| SYGGADYKRI | NO:19 | 100μM | 341 | 1.26 | Moderate affinity |
| GADYKRITVK | NO:20 | 100μM | 138 | -0.09 | No affinity |
| ITVKVNAPYNK | NO:21 | 100μM | 376 | 1.49 | Moderate affinity |
| TVKVNAPYNK | NO:22 | 100μM | 39 | -0.74 | No affinity |
| KVNAPYNK | NO:23 | 100μM | 90 | -0.41 | No affinity for |
| PYNKINQRI | NO:24 | 100μM | 990 | 5.55 | High affinity |
| LTCQAEGYPK | NO:25 | 100μM | 304 | 1.01 | Moderate affinity |
| GYPKAEVIW | NO:26 | 100μM | 29 | -0.81 | No affinity |
| TSSDHQVLSGK | NO:27 | 100μM | 237 | 0.57 | Low affinity |
| SSDHQVLSGK | NO:28 | 100μM | 344 | 1.28 | Moderate affinity |
| TTNSKREEK | NO:29 | 100μM | 226 | 0.50 | No affinity for |
| KLFNVTSTLR | NO:30 | 100μM | 294 | 0.95 | Low affinity |
| TTNEIFYCTFR | NO:31 | 100μM | 373 | 1.47 | Moderate affinity |
| GVALTFIFR | NO:32 | 100μM | 745 | 3.93 | High affinity |
| LTFIFRLRK | NO:33 | 100μM | 681 | 3.51 | High affinity |
| GIQDTNSKK | NO:34 | 100μM | 852 | 4.64 | High affinity |
| Blank | 0μM | 151 | 0.00 | No affinity | |
| CMV | 100μM | 672 | 3.45 | High affinity |
Example 3 Mass Spectrometry experiments verify that the polypeptide is presented by HLA molecules on the surface of tumor cells
The invention enriches the polypeptide-MHC complex on the cell surface by the way of co-immunoprecipitation-mass spectrometry, and identifies whether the polypeptide is presented by MHC molecules on the surface of tumor cells. The specific method comprises the following steps:
1) Separation and purification of MHC-I restricted T cell epitope peptides: after binding of pan-MHC-IA/B/C antibody (clone No. w 6/32) to sepharose CL-4B beads having protein A molecules coupled to the surface thereof at 4 ℃ for 1 hour, the residual antibody content of the supernatant was examined using NanoDrop, and the pan-MHC-IA/B/C-bound sepharose was prepared at 4 ℃ until the antibody binding rate reached 90%. Respectively adding 40ml of RIPA lysate into SKMEL5 and HCT8 cell samples, incubating for 1 hour at 4 ℃, centrifuging for 30min at 12000rpm, adding sepharose CL-4B beads into supernatant for prehybridization, and incubating for 1 hour at 4 ℃; beads were removed by centrifugation, and the supernatant was added to pan-MHC-A/B/C-conjugated sepharose CL-4B beads and incubated overnight (16-18 hours) at 4 ℃. The beads were washed with 4 ℃ pre-chilled PBS and repeated three times; washing the beads with ultrapure water; centrifuging to remove washing liquid, eluting antibody-MHC-I protein complex on the surfaces of beads by using 0.1N acetic acid, dissociating the antibody-MHC-I protein complex under an acidic condition, further separating and purifying protein and polypeptide in an eluted product by using a 3kDa ultrafiltration tube or a C18 solid phase extraction column (25mg, waters), concentrating a purified product by using a freezing vacuum centrifuge, and storing the concentrated product on a mass spectrum machine at-20 ℃.
2) Mass spectrometric identification of MHC-I restricted T cell epitope peptides: the concentrated MHC-I restricted epitope peptide solution was analyzed by Q exact mass spectrometer (Thermo Fisher Scientific) connected to nanoflow HPLC (Thermo Fisher Scientific) on-line, using Rerosil-PurC 18-AQ1.9um packing manually packed in a 15cm long, 75um internal diameter separation column, and using a linear gradient of 2-30% buffer B (80% ACN/0.5% acetic acid) to elute the polypeptides at a flow rate of 250nl/min for a 90min elution time. The secondary mass spectrometry was fragmented using HCD and data acquisition selected the data-dependent "Top 20" method. The collection resolution of the MS atlas is 70,000, 200m/z, and the target value is 3E6 ions; ions 10 prior to ion intensity discharge are typically separated and accumulated using a maximum injection time of 120ms until the value of the automatic gain controller is shown as 1E5. The polypeptide matching option setting "disable", MS/MS resolution setting 17,500 (200 m/z).
3) Mass spectrometric data analysis of MHC-I restricted T cell epitope peptides: data analysis a mass spectrum was aligned with MaxQuant (version 1.3.10.15) to a library of human whole proteins (Uniprot, 86,749 proteins), tumor associated antigens, tumor specific mutant peptides and a list of profiles generated from a data set containing 247 common contaminants (keratin, bovine serum albumin and protease). Variable modification detection settings: n-terminal acetylation and methionine oxidation. Second polypeptide identification settings: enable; specific enzyme digestion setting: unspecific; the polypeptide identification FDR (false discovery rate) is set to 1 percent, and the protein identification FDR is not set; the sequence match length limit was set at 8-15aa, the maximum polypeptide mass was set at 1500Da, and the maximum charge state was set at 3. The initial allowable mass deviation of the precursor ion was set to 6ppm and the maximum fragment mass deviation was set to 20ppm. The "match between runs" setting is on. And outputting the identification result and storing the identification result in a "peptide. Txt" file, removing the polypeptides matched with the reverse library and the pollution library, and obtaining the identification result of the MHC-I restricted epitope in the rest.
The results showed that 11 of the 12 high affinity polypeptides in table 4 could identify HLA molecules expressed and presented on the cell surface in this experiment. The mass spectrum of the corresponding polypeptide is shown in figures 1 to 4.
EXAMPLE 4 in vitro stimulation of the Polypeptides to amplify CD8 + T cells
Taking PBMC cells of HLA-A11:01 subtype positive healthy volunteers, 2X 10 6 Separating PBMC cells by adherence method to monocyte (adherence 3 h), and separating CD8 by CD8 magnetic bead method to obtain CD8 + The T cell of (1). Adherent monocytes were induced to immature DC with GM-CSF (1000U/ml), IL-4 (1000U/ml), and then induced to mature DC polypeptide-specific with IFN-gamma (100U/ml), LPS (10 ng/ml), and the polypeptide sequences with high affinity of Table 4. Mature DC cells loaded with polypeptide were irradiated and matched with CD8 from the same volunteer + T cells are co-cultured, IL-21 is added, after 3 days, IL-2 and IL-7 are supplemented, then IL-2 and IL-7 are supplemented once on days 5 and 7, and the co-cultured cells are taken for counting on day 10, and subsequent ELISPOTs and LDH detection are carried out. The HLA-A24:02 subtype was also subjected to the above-mentioned operation. The counting results are as follows, thus showing that the polypeptides in tables 5 and 6 can stimulate the amplification of CD8 in vitro + T cells.
TABLE 5 HLA-A11:01 typing polypeptide count results after incubation
| Polypeptide sequence | HLA typing | Total number of cells before culture | Total number of cells after culture |
| LLNAFTVTVPK | HLA-A11:01 | 2.0×10^6 | 7.87×10^6 |
| NAFTVTVPK | HLA-A11:01 | 2.0×10^6 | 9.05×10^6 |
| MTIECKFPVEK | HLA-A11:01 | 2.0×10^6 | 8.28×10^6 |
| AALQITDVK | HLA-A11:01 | 2.0×10^6 | 8.32×10^6 |
| ISYGGADYK | HLA-A11:01 | 2.0×10^6 | 8.40×10^6 |
| GVALTFIFR | HLA-A11:01 | 2.0×10^6 | 8.30×10^6 |
| LTFIFRLRK | HLA-A11:01 | 2.0×10^6 | 9.87×10^6 |
| GIQDTNSKK | HLA-A11:01 | 2.0×10^6 | 9.82×10^6 |
TABLE 6 HLA-A24:02 typing polypeptide after incubation count results
| Polypeptide sequence | HLA typing | Total number of cells before culture | Total number of cells after culture |
| VFIFMTYWHL | HLA-A24:02 | 2.0×10^6 | 7.51×10^6 |
| IFMTYWHLL | HLA-A24:02 | 2.0×10^6 | 9.95×10^6 |
| TYWHLLNAF | HLA-A24:02 | 2.0×10^6 | 8.29×10^6 |
| PYNKINQRI | HLA-A24:02 | 2.0×10^6 | 9.87×10^6 |
Example 5 method of ELISPOTs to verify polypeptide activation of CD8 + T cell immune response
The cultured T cells of example 4 and T2 loaded with the experimental polypeptide or unrelated polypeptide (ALADGVQKV) were added to human IFN-gamma interferon ELISPOTs plates for culture, and after 20 hours, the spots generated in the ELISPOT experiment were counted according to the ELISPOTs assay (see kit instructions). The requirements for immunogenicity of the experimental polypeptides are as follows: the number of spots (polypeptide)/number of spots (irrelevant polypeptide) is more than or equal to 2, namely the number of spots caused by the experimental polypeptide exceeds the number of spots of the irrelevant polypeptide by two times or more.
ELISPOTs detection method principle: since T2 cells express HLA-A11:01/HLA-A24:02, T2 cells can present HLA-A11:01/HLA-A24:02 antigen peptides to T cells. CD8 + After the T cells specifically recognize the complex of HLA-A11:01/HLA-A24:02 and the polypeptide, the T cells can reactivate and secrete IFN-gamma interferon. And CD8 + IFN-gamma interferon secreted by activated T cells can be captured by antibodies on ELISPOTs plates, and the antibodies recognizing IFN-gamma can be developed by degrading substrates through enzymes coupled on the antibodies, and finallySpots are generated. The number of spots represents the number of cells activated to secrete IFN-gamma interferon. ELISPOTs results are shown in FIG. 5 and Table 7. The number of experimental group spots accumulated with 7 polypeptides is 2 times or more than that of irrelevant group spots, and the immunogenicity is stronger.
TABLE 7 polypeptide stimulation specificity CD8 + Secretion of IFN-gamma interferon from T cells
| SEQ ID | Polypeptide sequence | Number of polypeptide spots | Number of unrelated polypeptide spots | Multiple (experiment/irrelevant) |
| NO:1 | |
18±2.72 | 22±1.41 | 0.82 |
| NO:3 | IFMTYWHLL | 52±3.79 | 22±1.41 | 2.36 |
| NO:5 | TYWHLLNAF | 49±0.18 | 22±1.41 | 2.23 |
| NO:7 | |
14±0.61 | 22±1.41 | 0.64 |
| NO:8 | NAFTVTVPK | 65±4.32 | 22±1.41 | 2.95 |
| NO:11 | MTIECKFPVEK | 36±3.37 | 22±1.41 | 1.64 |
| NO:14 | AALQITDVK | 47±1.51 | 22±1.41 | 2.14 |
| NO:17 | |
15±3.90 | 22±1.41 | 0.68 |
| NO:24 | PYNKINQRI | 48±3.90 | 22±1.41 | 2.18 |
| NO:32 | GVALTFIFR | 64±4.77 | 22±1.41 | 2.91 |
| NO:33 | LTFIFRLRK | 38±3.86 | 22±1.41 | 1.73 |
| NO:34 | |
60±3.94 | 22±1.41 | 2.73 |
Example 6 LDH Release assay demonstrating CD8 + T cell polypeptide specific killing activity
The cells cultured in example 4 were co-cultured with T2 cells loaded with the experimental polypeptide or unrelated polypeptide or unloaded polypeptide, in which the maximum release holes, volume correction holes, medium control holes, spontaneous release holes, different effect-target ratios (ratio of the number of T cells to T2 cells) were set as controls, 3 multiple holes were set for each group, and after 4 hours, 50. Mu.l of the co-cultured cell supernatant was taken out and added to 50. Mu.l of LDH substrate mixture to allow the cell supernatant to catalyze the LDH substrate reaction, and finally the 490nm wavelength and 680nm reference wavelength were read, and the killing activity of the target cells against T2 was calculated according to the following formula based on the control holes.
The killing activity calculation formula is: killing efficiency (%) = (experimental wells-spontaneous release of effector cells-spontaneous release of target cells + media wells)/(maximum release of target cells-volume corrected wells-spontaneous release of target cells + media wells) × 100%.
The results are shown in table 8, fig. 6 and fig. 7, and the killing efficiency of the 7 immunogenic specific T cells in the corresponding 7 groups of T cells in E: T = 20.
TABLE 8T cell specific recognition and killing of target cells presenting experimental polypeptides
| E:T=1:1 | E:T=10:1 | E:T=20:1 | |
| T2 | -2.62% | 6.28% | 14.99% |
| T2+Irrelevant | 2.03% | 8.25% | 16.56% |
| T2+VFIFMTYWHL | 4.06% | 8.32% | 12.84% |
| T2 | 0.79% | 4.21% | 10.66% |
| T2+Irrelevant | 2.61% | 7.40% | 18.51% |
| T2+IFMTYWHLL | 6.73% | 34.30% | 77.67% |
| T2 | -2.49% | 4.89% | 10.70% |
| T2+Irrelevant | 0.27% | 12.50% | 15.22% |
| T2+TYWHLLNAF | 8.65% | 42.66% | 74.54% |
| T2 | -1.37% | 8.83% | 14.78% |
| T2+Irrelevant | -0.06% | 5.39% | 15.51% |
| T2+LLNAFTVTVPK | 3.38% | 9.66% | 11.98% |
| T2 | 2.06% | 5.28% | 12.47% |
| T2+Irrelevant | 4.66% | 10.02% | 13.33% |
| T2+NAFTVTVPK | 9.48% | 29.29% | 60.41% |
| T2 | -2.09% | 4.20% | 10.40% |
| T2+Irrelevant | -1.68% | 6.21% | 10.68% |
| T2+MTIECKFPVEK | 2.03% | 9.76% | 11.63% |
| T2 | 0.06% | 5.93% | 10.57% |
| T2+Irrelevant | 3.59% | 9.18% | 18.69% |
| T2+AALQITDVK | 8.79% | 31.20% | 58.98% |
| T2 | -0.61% | 4.65% | 10.98% |
| T2+Irrelevant | 0.71% | 6.78% | 15.13% |
| T2+ISYGGADYK | 3.17% | 8.69% | 14.22% |
| T2 | -4.90% | 6.22% | 10.47% |
| T2+Irrelevant | 2.45% | 12.48% | 18.35% |
| T2+PYNKINQRI | 9.96% | 39.50% | 57.71% |
| T2 | -0.76% | 6.78% | 9.27% |
| T2+Irrelevant | 1.88% | 8.76% | 15.89% |
| T2+GVALTFIFR | 7.78% | 22.31% | 52.78% |
| T2 | -0.53% | 5.40% | 10.43% |
| T2+Irrelevant | 1.62% | 6.88% | 12.74% |
| T2+LTFIFRLRK | 2.40% | 9.01% | 16.77% |
| T2 | -2.20% | 4.95% | 10.42% |
| T2+Irrelevant | 2.32% | 6.65% | 19.87% |
| T2+GIQDTNSKK | 7.97% | 32.55% | 59.97% |
Example 7 preparation of polypeptide vaccine and treatment protocol
1. Construction and packaging of recombinant lentiviruses expressing polypeptides
Synthesizing DNA sequences corresponding to the polypeptides, and respectively constructing corresponding lentiviral vectors pHBLV-Puro. The lentiviral plasmid was co-transfected with pSPAX2 and pMD2G helper plasmids into 293T cells and packaged as a polypeptide lentivirus.
2. Establishment of human cell line expressing polypeptide
Human melanoma cell line SKMEL5 was purchased from ATCC (accession number: HTB-70), and its HLA subtype was HLA-A11:01 positive. The human colorectal cancer cell line HCT8 was purchased from ATCC (accession number: CCL-244), and its HLA subtype was HLA-A24:02. The cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and streptomycin。37℃，5% CO 2 Culturing in an incubator. And transfecting the packed lentiviruses with SKMEL5 and HCT8 cell lines respectively, and continuously screening the surviving SKMEL5 and HCT8 cell lines by adopting Puromycin antibiotics (Puromycin), and finally establishing the SKMEL5 and HCT8 cell lines for expressing the polypeptide.
3. NOD/SCID mouse human immune reconstitution
Collecting 600-900 ml of anticoagulated peripheral blood of healthy volunteers. Ficoll Peripheral Blood Mononuclear Cells (PBMC) were isolated and the cells were collected for use. 300 NOD SCID mice with immunodiffusion excluded, 2X 10 PBMC per intraperitoneal injection 7 0.5ml, NOD SCID mice were human reconstituted immunologically. Mice successfully reconstituted by immunization after 4 weeks were selected and prepared for inoculation with models of human melanoma cell lines and human colorectal cancer cell lines.
4. Construction of human melanoma and human colorectal cancer tumor models
Established lines of human melanoma cell lines and human colorectal cancer cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and streptomycin. 37 ℃ and 5% CO 2 Culturing in an incubator. The tumor cells were collected, centrifuged at 200g/min, and washed 3 times with sterile physiological saline. Diluting properly, adding 10 microliters of 0.4% phloroglucinol blue into 40 microliters of cell suspension, staining and counting by microscopic examination to obtain the cell suspension with the concentration of 1 × 10 8 And selecting immune reconstituted NOD/SCID mice, and inoculating 100ml of tumor cell suspension per mouse subcutaneously. After inoculation, the inoculated part is observed day by day for infection and natural regression after tumor growth. After 7 days, the mouse subcutaneous tumor can be touched to be about rice grain-sized tumor, which indicates that the SKMEL5 or HCT8 subcutaneous tumor model NOD/SCID mouse model is successfully constructed.
5. Preparation of polypeptide vaccine
(1) 3 polypeptide sequences were selected in examples 3 and 5 that were equi-typed as HLA-A11:01 and had double positive results (positive presentation verification and positive immunogenicity verification): the preparation of polypeptide vaccine is carried out by NAFLTVPK (SEQ ID NO: 8), GVALTFIFR (SEQ ID NO: 32) and GIQDTNSKK (SEQ ID NO: 34).
SKMEL5 subcutaneous tumor model NOD/SCID mice immunized for 4 weeks were randomized into 6 groups: adjuvant group, adjuvant + irrelevant polypeptide group, adjuvant + NAFLTVPK polypeptide group, adjuvant + GVATFIFFR polypeptide group, adjuvant + GIQDTNSKK polypeptide group and adjuvant + polypeptide (polypeptide sequence is SEQ ID NO:8, 32 and 34) combination group, wherein each group comprises 8 individuals. The first immunization dose of the unrelated polypeptide group and the polypeptide combination was 100 ml/mouse. The above polypeptides were resuspended in PBS, mixed with 150 ml/Freund's complete adjuvant, adjusted to 300 ml/Freund's with PBS, and injected subcutaneously into the back at double spots. After 2 weeks, booster immunizations were performed 4 times using the same dose (complete Freund's adjuvant was used 1 time, and incomplete Freund's adjuvant was used thereafter). After the injection, the vital signs of the mice were observed, and the size of the tumor was measured with a vernier caliper every 3 to 4 days. Tumor volume was calculated as tumor volume =1/2 x length by width 2 . Meanwhile, the weight change of the mice is recorded. The results are shown in FIGS. 8A and B.
(2) 3 polypeptide sequences were selected in examples 3 and 5 that were equi-typed as HLA-A24:02 and had double positive results (positive presentation verification and positive immunogenicity verification): IFMTYWHLL (SEQ ID NO: 3), TYWHLLNAF (SEQ ID NO: 5) and PYNKINQRI (SEQ ID NO: 24) for the preparation of polypeptide vaccines.
HCT8 subcutaneous tumor model NOD/SCID mice immunized for 4 weeks were randomized into 6 groups: adjuvant group, adjuvant + irrelevant polypeptide group, adjuvant + IFMTYWHLL polypeptide group, adjuvant + TYWHLLNAF polypeptide group, adjuvant + PYNKINQRI polypeptide group and adjuvant + polypeptide (polypeptide sequence is SEQ ID NO:3, 5 and 24) combination group, each group has 8 pieces. The first immunization dose of the unrelated polypeptide group and the polypeptide combination was 100 ml/mouse. The above polypeptides were resuspended in PBS, mixed with 150 ml/Freund's complete adjuvant, adjusted to 300 ml/piece with PBS, and injected subcutaneously into the back at double spots. After 2 weeks, booster immunizations were performed with the same dose (1 st with complete Freund's adjuvant followed by 4 co-immunizations with incomplete Freund's adjuvant). After the injection, the vital signs of the mice were observed, and the size of the tumor was measured every 3 to 4 days with a vernier caliper. Tumor volume was calculated as tumor volume =1/2 x length by width 2 . Meanwhile, the weight change of the mice is recorded. The results are shown in FIGS. 9C and D.
The results show that the polypeptide vaccine group loaded by the adjuvant and the core polypeptide or the polypeptide combination can obviously slow down the growth of the tumor of the mouse and prolong the survival time of the mouse relative to the polypeptide vaccine group and the adjuvant group loaded by unrelated polypeptides.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Moreover, various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen Jinuoin Biotech, inc., dajinuoyin, wuhan, hua
<120> PD-L1 related vaccine and application thereof
<130> PDI220227
<160> 68
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
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Val Phe Ile Phe Met Thr Tyr Trp His Leu
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<213> Artificial Sequence
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Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu
1 5 10
<210> 3
<211> 9
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Ile Phe Met Thr Tyr Trp His Leu Leu
1 5
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Ile Phe Met Thr Tyr Trp His Leu Leu Asn
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Thr Tyr Trp His Leu Leu Asn Ala Phe
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Thr Tyr Trp His Leu Leu Asn Ala Phe Thr Val
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Leu Leu Asn Ala Phe Thr Val Thr Val Pro Lys
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Asn Ala Phe Thr Val Thr Val Pro Lys
1 5
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Tyr Gly Ser Asn Met Thr Ile Glu Cys Lys
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Gly Ser Asn Met Thr Ile Glu Cys Lys
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Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys
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Thr Ile Glu Cys Lys Phe Pro Val Glu Lys
1 5 10
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Ser Ser Tyr Arg Gln Arg Ala Arg Leu Leu Lys
1 5 10
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Ala Ala Leu Gln Ile Thr Asp Val Lys
1 5
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Lys Leu Gln Asp Ala Gly Val Tyr Arg
1 5
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Gly Val Tyr Arg Cys Met Ile Ser Tyr
1 5
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Ile Ser Tyr Gly Gly Ala Asp Tyr Lys
1 5
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Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg
1 5 10
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Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile
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Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys
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Ile Thr Val Lys Val Asn Ala Pro Tyr Asn Lys
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Thr Val Lys Val Asn Ala Pro Tyr Asn Lys
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Thr Val Lys Val Asn Ala Pro Tyr Asn Lys
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Pro Tyr Asn Lys Ile Asn Gln Arg Ile
1 5
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Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
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Gly Tyr Pro Lys Ala Glu Val Ile Trp
1 5
<210> 27
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<212> PRT
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Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
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Ser Ser Asp His Gln Val Leu Ser Gly Lys
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Thr Thr Asn Ser Lys Arg Glu Glu Lys
1 5
<210> 30
<211> 10
<212> PRT
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<220>
<223> 30
<400> 30
Lys Leu Phe Asn Val Thr Ser Thr Leu Arg
1 5 10
<210> 31
<211> 11
<212> PRT
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<223> 31
<400> 31
Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg
1 5 10
<210> 32
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<212> PRT
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<223> 32
<400> 32
Gly Val Ala Leu Thr Phe Ile Phe Arg
1 5
<210> 33
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<223> 33
<400> 33
Leu Thr Phe Ile Phe Arg Leu Arg Lys
1 5
<210> 34
<211> 9
<212> PRT
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<400> 34
Gly Ile Gln Asp Thr Asn Ser Lys Lys
1 5
<210> 35
<211> 30
<212> DNA
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<223> 35
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gtctttatat tcatgaccta ctggcatttg 30
<210> 36
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<212> DNA
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<220>
<223> 36
<400> 36
gtctttatat tcatgaccta ctggcatttg ctg 33
<210> 37
<211> 27
<212> DNA
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<223> 37
<400> 37
atattcatga cctactggca tttgctg 27
<210> 38
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 38
<400> 38
atattcatga cctactggca tttgctgaac 30
<210> 39
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 39
<400> 39
acctactggc atttgctgaa cgcattt 27
<210> 40
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 40
<400> 40
acctactggc atttgctgaa cgcatttact gtc 33
<210> 41
<211> 33
<212> DNA
<213> Artificial Sequence
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<223> 41
<400> 41
ttgctgaacg catttactgt cacggttccc aag 33
<210> 42
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 42
<400> 42
aacgcattta ctgtcacggt tcccaag 27
<210> 43
<211> 30
<212> DNA
<213> Artificial Sequence
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<223> 43
<400> 43
tatggtagca atatgacaat tgaatgcaaa 30
<210> 44
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 44
<400> 44
ggtagcaata tgacaattga atgcaaa 27
<210> 45
<211> 33
<212> DNA
<213> Artificial Sequence
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<400> 45
atgacaattg aatgcaaatt cccagtagaa aaa 33
<210> 46
<211> 30
<212> DNA
<213> Artificial Sequence
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<223> 46
<400> 46
acaattgaat gcaaattccc agtagaaaaa 30
<210> 47
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 47
<400> 47
agtagctaca gacagagggc ccggctgttg aag 33
<210> 48
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 48
<400> 48
gctgcacttc agatcacaga tgtgaaa 27
<210> 49
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 49
<400> 49
aaattgcagg atgcaggggt gtaccgc 27
<210> 50
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 50
<400> 50
ggggtgtacc gctgcatgat cagctat 27
<210> 51
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 51
<400> 51
atcagctatg gtggtgccga ctacaag 27
<210> 52
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 52
<400> 52
atcagctatg gtggtgccga ctacaagcga 30
<210> 53
<211> 30
<212> DNA
<213> Artificial Sequence
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<223> 53
<400> 53
agctatggtg gtgccgacta caagcgaatt 30
<210> 54
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 54
<400> 54
ggtgccgact acaagcgaat tactgtgaaa 30
<210> 55
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 55
<400> 55
attactgtga aagtcaatgc cccatacaac aaa 33
<210> 56
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 56
<400> 56
actgtgaaag tcaatgcccc atacaacaaa 30
<210> 57
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> 57
<400> 57
aaagtcaatg ccccatacaa caaa 24
<210> 58
<211> 27
<212> DNA
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<220>
<223> 58
<400> 58
ccatacaaca aaatcaacca aagaatt 27
<210> 59
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 59
<400> 59
ctgacatgtc aggctgaggg ctaccccaag 30
<210> 60
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 60
<400> 60
ggctacccca aggccgaagt catctgg 27
<210> 61
<211> 33
<212> DNA
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<220>
<223> 61
<400> 61
acaagcagtg accatcaagt cctgagtggt aag 33
<210> 62
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 62
<400> 62
agcagtgacc atcaagtcct gagtggtaag 30
<210> 63
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 63
<400> 63
accaccaatt ccaagagaga ggagaag 27
<210> 64
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 64
<400> 64
aagcttttca atgtgaccag cacactgaga 30
<210> 65
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 65
<400> 65
acaactaatg agattttcta ctgcactttt agg 33
<210> 66
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 66
<400> 66
ggtgtagcac tgacattcat cttccgt 27
<210> 67
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 67
<400> 67
ctgacattca tcttccgttt aagaaaa 27
<210> 68
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 68
<400> 68
ggcatccaag atacaaactc aaagaag 27
Claims (18)
1. An isolated polypeptide having an amino acid sequence set forth in SEQ ID NO: 3. 8, 24 or 32.
2. An isolated nucleic acid encoding the polypeptide of claim 1.
3. The isolated nucleic acid of claim 2, wherein the nucleotide sequence of the isolated nucleic acid is as set forth in SEQ ID NO: 37. 42, 58 or 66.
4. An isolated antigen presenting cell expressing HLA-A11:01 or HLA-A24:02,
wherein the antigen presenting cells expressing HLA-A11:01 are used for presenting an antigen with an amino acid sequence as shown in SEQ ID NO:8 or 32, antigen presenting cells expressing HLA-a24:02 for presenting an antigen having an amino acid sequence as set forth in SEQ ID NO:3 or 24.
5. The isolated antigen presenting cell of claim 4, wherein the isolated antigen presenting cell is a dendritic cell, a B cell, or a monocyte-phagocyte.
6. An isolated immune cell, wherein the binding target of the isolated immune cell is the isolated polypeptide of claim 1 or the isolated antigen presenting cell of claim 4 or 5.
7. The isolated immune cell of claim 6, wherein the isolated immune cell is obtained by:
contacting the isolated antigen presenting cell of claim 4 or 5 with a cell capable of an immune response.
8. The isolated immune cell of claim 7, wherein the immune effector-competent cell is CD8 + T cells.
9. A vaccine, comprising: an isolated polypeptide according to claim 1, an isolated nucleic acid according to claim 2 or 3, an isolated antigen presenting cell according to claim 4 or 5 and/or an isolated immune cell according to any one of claims 6 to 8.
10. The vaccine of claim 9, wherein the vaccine further comprises an adjuvant.
11. A medicament, comprising: an isolated polypeptide according to claim 1, an isolated nucleic acid according to claim 2 or 3, an isolated antigen presenting cell according to claim 4 or 5 and/or an isolated immune cell according to any one of claims 6 to 8.
12. The medicament of claim 11, further comprising a pharmaceutically acceptable excipient.
13. Use of the isolated polypeptide of claim 1, the isolated nucleic acid of claim 2 or 3, the isolated antigen presenting cell of claim 4 or 5, and/or the isolated immune cell of any one of claims 6 to 8 in the preparation of a vaccine for the prevention of cancer in which tumor cells express PD-L1.
14. Use according to claim 13, wherein the subject of the vaccine expresses HLA-a11:01 and/or HLA-a24:02.
15. Use of the isolated polypeptide of claim 1, the isolated nucleic acid of claim 2 or 3, the isolated antigen presenting cell of claim 4 or 5, and/or the isolated immune cell of any one of claims 6 to 8 in the manufacture of a medicament for the treatment of cancer in which tumor cells express PD-L1.
16. Use according to claim 15, wherein the subject of the medicament expresses HLA-a11:01 and/or HLA-a24:02.
17. The use according to any one of claims 13 to 16, wherein the cancer is breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia, thyroid cancer, lymphoma, bladder cancer, kidney cancer, uterine body cancer, ovarian cancer, gallbladder cancer, oral cancer, laryngeal cancer, bone cancer, testicular cancer, or brain cancer.
18. The use according to any one of claims 13 to 16, wherein the cancer is melanoma.
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| PCT/CN2022/128039 WO2023226295A1 (en) | 2022-05-25 | 2022-10-27 | Pd-l1-related vaccine and use thereof |
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| CN113061192B (en) * | 2021-04-12 | 2023-08-22 | 佰思巢(上海)生物科技有限公司 | PDL1 fusion protein with high affinity to PD-1 receptor and application thereof as T cell inhibitor |
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