CN113307859A - Antigenic peptide related to breast cancer driver gene mutation and application thereof - Google Patents
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Abstract
The invention discloses an antigenic peptide related to breast cancer driver gene mutation, wherein the breast cancer driver gene is at least two of ABCB11, ESR1, GATA3, PALB2, SNX4, SLC24A4, HAUS3 and KMT 2C; the sequences of the antigenic peptides are at least two of SEQ No. 1-18; the application of the antigenic peptide is to use the antigenic peptide for inducing and generating specific cytotoxic T cell clone; the antigen peptide has high affinity with MHC I molecules on DC cells, can effectively stimulate and induce to generate specific cytotoxic T lymphocytes, can be used for immune elimination of tumor cells with breast cancer related driving gene mutation, and has good treatment potential.
Description
Technical Field
The invention relates to an antigenic peptide related to breast cancer driver gene mutation and application thereof, belonging to the technical field of biological medicines.
Background
Breast cancer has become a major public health problem in the current society. The etiology of breast cancer is not completely clear, and researches show that a certain regularity exists in the pathogenesis of breast cancer, and women with high risk factors of breast cancer are susceptible to breast cancer. The high risk factors refer to various risk factors related to the onset of breast cancer.
The treatment of breast cancer mainly adopts various means such as operation, radiotherapy, chemotherapy, endocrine therapy, biological targeting therapy, traditional Chinese medicine adjuvant therapy and the like. Surgery plays an important role in the diagnosis, staging and overall treatment of breast cancer. Radiotherapy is the method of destroying the growth and reproduction of cancer cells by radioactive rays to control and eliminate cancer cells. Surgery and radiotherapy are both local treatments. Chemotherapy is a treatment method that uses anticancer drugs to inhibit cancer cell division and destroy cancer cells, and is called chemotherapy for short. Endocrine therapy is to regulate the endocrine function in the body by using drugs or a method of removing endocrine glands, and reduce the secretion of endocrine hormones, thereby achieving the purpose of treating breast cancer. However, chemotherapy and radiotherapy can improve the long-term survival rate, but the complications are many and the curative effect is unsatisfactory.
With the understanding of the nature of tumor-driving gene mutation and personalized precise immune response, a personalized and customized tumor-driving gene mutation-associated peptide antigen is provided as a new concept of tumor-specific new antigen immune cell therapy, and natural T cell immune defense systems of human bodies are induced to selectively destroy tumor-driving gene mutation cells through the tumor-specific driving gene mutation-associated antigen, so that various cancers are effectively prevented from happening, and the technical field has been evaluated as ten technical innovation research fields in the United states in 2019.
However, because the technology needs multidisciplinary, high-technology and multi-platform cooperative research, the antigen peptide related to breast cancer driving gene mutation cannot be systematically found and a relatively complete peptide library is established, and therefore, a therapeutic agent with excellent effect cannot be formed.
Disclosure of Invention
In order to overcome the defects of the prior art, the first objective of the present invention is to provide an antigenic peptide related to breast cancer driver gene mutation, wherein the antigenic peptide related to breast cancer driver gene mutation has high affinity with MHC I molecules on DC cells, can effectively stimulate and induce the generation of specific Cytotoxic T Lymphocytes (CTLs), can be used for immune clearance of tumor cells with breast cancer driver gene mutation, and has good therapeutic potential.
The second purpose of the invention is to provide an application of the above antigenic peptide related to breast cancer driving gene mutation.
The first purpose of the invention can be achieved by adopting the following technical scheme: an antigenic peptide associated with a mutation in a breast cancer driver gene selected from at least two of ABCB11, ESR1, GATA3, PALB2, SNX4, SLC24A4, HAUS3 and KMT 2C.
Wherein, the sequences of the antigenic peptides related to the breast cancer driving gene mutation are at least two of SEQ No. 1-18.
The second purpose of the invention can be achieved by adopting the following technical scheme: the application of the antigenic peptide related to the breast cancer driving gene mutation is to use the antigenic peptide related to the breast cancer driving gene mutation for inducing and generating specific cytotoxic T cell clone.
Or, the application of the antigenic peptide related to the breast cancer driving gene mutation, the antigenic peptide related to the breast cancer driving gene mutation is used for preparing the human body immunological activity regulator capable of inducing the generation of specific cytotoxic T cell clone.
Or, the application of the antigenic peptide related to the breast cancer driving gene mutation is to use the antigenic peptide related to the breast cancer driving gene mutation in the preparation of cell culture solution for preventing and interfering the mutation of at least two genes of ABCB11, ESR1, GATA3, PALB2, SNX4, SLC24A4, HAUS3 and KMT 2C.
Or, the application of the antigenic peptide related to the breast cancer driving gene mutation, and the antigenic peptide related to the breast cancer driving gene mutation is used for preparing a breast cancer risk intervention therapeutic agent.
The invention carries out comprehensive research from the starting point of specific cancers, specifically researches the generation mechanism of the breast cancer, realizes the elimination of the breast cancer cells with ABCB11, ESR1, GATA3, PALB2, SNX4, SLC24A4, HAUS3 and KMT2C breast cancer driver gene mutation, can inhibit the growth of the tumor cells and prevent the generation of the breast cancer. The immunological research proves that the principle that CD8 positive T lymphocyte CTL plays cellular immunity is as follows: CTL cells are activated by recognizing antigen peptides bound to MHC-I molecules, and the activated CTL can kill corresponding target cells to exert an immune surveillance effect.
Compared with the prior art, the invention has the beneficial effects that:
1. the antigenic peptide (neoantigen) related to the breast cancer driver gene mutation refers to an antigenic peptide which is expressed by tumor cells and can activate T cells; through high-throughput gene sequencing and data analysis, individual somatic cell mutations of the tumor of a patient are found, antigen peptides related to driving gene mutations are screened out to serve as targets, and the targets are subjected to in vitro chemical synthesis and loaded on antigen-presenting cells (APC), so that T cells are activated to identify various new antigen peptides, and the curative effect of killing the mutated tumor cells is further generated;
2. the antigen peptide related to the breast cancer driver gene mutation, which is obtained by screening, has high affinity with MHC I molecules on DC cells, can effectively stimulate and induce the generation of specific Cytotoxic T Lymphocytes (CTLs) and inhibit the growth of tumor cells, so that the antigen peptide has the potential of good polypeptide vaccines and DC vaccines and has good clinical transformation and disease prevention prospects.
Detailed Description
The invention will be further described with reference to specific embodiments:
the antigenic peptides used in the examples in association with mutations in the breast cancer driver gene are shown in table 1:
TABLE 1 antigenic peptide sequences associated with mutations in the breast cancer driver genes
The establishment of antigenic peptide-specific CTL clones associated with breast cancer driver mutations was performed as follows:
10 of the same healthy donor5An individual CD8+T cell activation by loading 10 antigenic peptides associated with breast cancer driver gene mutations42 times of stimulation of Mo-DCs at intervals of 1 week and then 10 times of self-body5The mitomycin C treated PBMC loaded with antigen peptide related to breast cancer driving gene mutation is obtained by standard cytotoxic test screening after being stimulated for 1 time.
T2 cells were loaded with 5uM of the antigen peptide associated with the breast cancer driver gene mutation as target cells, and the antigen peptide-specific cytotoxicity of CTL associated with the breast cancer driver gene mutation was confirmed by LDH release assay.
Detection of LDH lactate dehydrogenase cytotoxicity:
1) the target cells were adjusted to 5X 10 with RPMI-1640 medium containing 5 vt% calf serum4-2×105/mL；
2) Target cells were added to 96-well round bottom cell culture plates at 100. mu.L per well. 3 effector cells are naturally released into control wells, no target cells are added, and only 100 mu L of culture solution is added;
3) add 100. mu.L of CTL effector cells (CD8+ T cells) to each well, with a ratio of effector cells to target cells of 10: 1; the natural release hole was filled with 100. mu.L of culture medium without effector cells, and 100. mu.L of 1 vt% NP40 was added to the maximum release hole. Each experiment is provided with three multiple wells;
4) placing at 37 ℃ for 5 vt% CO2Culturing for 4-6h in a carbon dioxide incubator;
5) centrifuging the culture plate for 200 Xg 10 min; sucking out 150 microliter of supernatant from each hole, and correspondingly adding the supernatant into another 96-hole enzyme-linked assay plate; to each well of the second plate were added 20. mu.L of a 0.4mol/L lactic acid solution, 20. mu.L of 4 mmol/L2-P-iodophenyl-3-P-chloronitrophenyltetrazole, and 20. mu.L of a reaction solution (containing 0.03 vt% BSA, 2.7U/mL lipoamide dehydrogenase, 4.5mmol/L hydrogenated coenzyme I (NAD +), 1.2 vt% sucrose, PBS) in this order, and the mixture was left at room temperature for 20 min;
6) the optical density (OD value) of each well was measured on an enzyme-linked detector at a detection wavelength of 492nm and a reference wavelength of 650 nm.
The method for establishing the antigen peptide specific CTL clone related to the breast cancer driver gene mutation by adopting the in vitro induction also establishes MHC-I restricted CTL clone, and the polypeptide specific immune response effect of the MHC-I restricted CTL clone is verified by IFN-gamma release experiments.
IFN- γ release assay:
1) coating antibody, taking 44 uL capture antibody and 12mL coat buff to dilute by 1:250 times, coating the liquid on a 96-well enzyme label plate by 100 uL per well, and standing overnight at 4 ℃;
2) washing the plate for 3 times, preparing washing liquor, adding 50mL of distilled water to prepare 1000mLwash buff, diluting by 1:20 times, and washing the plate for 3 times by using an automatic plate washing machine;
3) blocking 220 mu L of assay dilution in each hole and placing for 1h at room temperature;
4) washing the plate for 3 times;
5) prepare standard, dilute by equal ratio, and establish concentration gradient as blank well, 4.7pg/mL, 9.4pg/mL, 18.8pg/mL, 37.5pg/mL, 75pg/mL, 150pg/mL, 300pg/mL, respectively. And sequentially adding a sample to be detected, the auxiliary hole and the sample with overhigh concentration to be diluted. Standing at room temperature for 2 h;
6) washing the plate for 5 times;
7) preparing a detection antibody (secondary antibody) 48 mu L of detector antibody, adding 12mL of assay solution to prepare a solution, adding 48 mu L of SAV-HRP into the solution when adding the sample, and standing 100 mu L of each well at room temperature for 1 h;
8) washing the plate for 7 times;
9) adding chromogenic substrate streptomycin avidin peroxidase, keeping out of the sun for 30 min;
10) adding the stop solution, and carrying out colorimetric preparation on a standard curve by using 450nm as a measurement wavelength and 620nm as a reference wavelength on an enzyme-linked immunosorbent assay.
The results are shown in Table 2, and the results are absorbance values using PBS phosphate buffer and negative control peptide (control peptide sequence: AAAAAAAAA shown in SEQ ID NO. 19) as controls for IFN-. gamma.release test data for the antigen peptides of SEQ ID NO.1-18, respectively. The experimental data show that the CTL epitope established by the invention is extremely effective, and the predicted result and the experimental result are very good in conformity.
TABLE 1 antigenic peptide IFN-. gamma.Release test results (absorbance values)
| Serial number | Antigenic peptides | PBS control | Negative control |
| SEQ No.1 | 485 | 32 | 36 |
| SEQ No.2 | 479 | 32 | 37 |
| SEQ No.3 | 469 | 30 | 38 |
| SEQ No.4 | 472 | 31 | 39 |
| SEQ No.5 | 473 | 34 | 40 |
| SEQ No.6 | 475 | 33 | 39 |
| SEQ No.7 | 478 | 32 | 38 |
| SEQ No.8 | 485 | 32 | 37 |
| SEQ No.9 | 483 | 32 | 36 |
| SEQ No.10 | 469 | 32 | 35 |
| SEQ No.11 | 468 | 32 | 34 |
| SEQ No.12 | 467 | 32 | 35 |
| SEQ No.13 | 465 | 32 | 36 |
| SEQ No.14 | 471 | 30 | 37 |
| SEQ No.15 | 472 | 30 | 38 |
| SEQ No.16 | 479 | 30 | 39 |
| SEQ No.17 | 475 | 30 | 40 |
| SEQ No.18 | 476 | 30 | 41 |
Combining at least two antigenic peptides:
combination 1: SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.13 and SEQ ID NO. 17;
and (3) combination 2: SEQ ID NO.10 and SEQ ID NO. 17;
and (3) combination: SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO. 17;
IFN-gamma release experiments were performed with the above three antigen peptide combinations, with PBS phosphate buffer and negative control peptide (control peptide sequence: AAAAAAAAA shown in SEQ ID NO. 19) as controls, and the experimental results were absorbance values, and the results are shown in Table 3.
TABLE 3 IFN-. gamma.Release test results (absorbance values) for antigen peptide combinations
| Combination of | Antigenic peptides | PBS control | Negative control |
| Combination 1 | 623±18 | 28±2 | 39±3 |
| Combination 2 | 635±35 | 29±1 | 38±3 |
| Combination 3 | 610±14 | 32±1 | 39±5 |
Therefore, at least two of the antigen peptides related to the breast cancer driver gene mutation are subjected to dendritic cell presentation and are co-cultured with cytotoxic T lymphocytes, so that the antigen peptide specific cytotoxic T lymphocytes can be obtained through induction screening. The antigen specific cytotoxic T lymphocyte can be used for immune elimination of tumor cells with breast cancer related driving gene mutation, and prevention of related diseases, especially tumor diseases.
At least two of the antigen peptides and Dendritic Cells (DC) are loaded and returned for transfusion, and the antigen peptides can be used as DC vaccines for disease prevention and can stimulate organisms to generate polypeptide specific anti-cytotoxic T cells related to breast cancer driver gene mutation, so that prevention of diseases related to the breast cancer driver gene mutation is realized.
The antigen peptide related to breast cancer driving gene mutation has the advantages of short length, small chemical synthesis difficulty, capability of directly synthesizing to obtain a high-purity product, greatly reduced application cost, definite effect and good application potential.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
SEQUENCE LISTING
<110> Shenzhen City New Targeted Biotech Limited
<120> antigenic peptide related to breast cancer driver gene mutation and application thereof
<130> 2021
<160> 19
<170> PatentIn version 3.5
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Claims (6)
1. An antigenic peptide associated with a mutation in a breast cancer driver gene, wherein said breast cancer driver gene is at least two of ABCB11, ESR1, GATA3, PALB2, SNX4, SLC24a4, HAUS3 and KMT 2C.
2. The antigenic peptides associated with breast cancer driver mutations of claim 1, wherein said antigenic peptides associated with breast cancer driver mutations have at least two of the sequences of SEQ No. 1-18.
3. Use of the antigenic peptide associated with a mutation in a breast cancer driver gene of claim 1 for inducing the generation of a specific cytotoxic T cell clone.
4. Use of the antigenic peptide associated with a mutation in a breast cancer driver gene of claim 1 for the preparation of a modulator of human immune activity capable of inducing the production of specific cytotoxic T cell clones.
5. Use of the antigenic peptide related to breast cancer driver gene mutation of claim 1 in the preparation of a cell culture solution for the prevention and intervention of mutation of at least two genes selected from ABC B11, ESR1, GATA3, PALB2, SNX4, SLC24a4, HAUS3 and KMT 2C.
6. Use of the antigenic peptide related to breast cancer driver gene mutation according to claim 1 for the preparation of a breast cancer risk intervention therapeutic agent.
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| US20200157633A1 (en) * | 2017-04-01 | 2020-05-21 | The Broad Institute, Inc. | Methods and compositions for detecting and modulating an immunotherapy resistance gene signature in cancer |
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| US20200157633A1 (en) * | 2017-04-01 | 2020-05-21 | The Broad Institute, Inc. | Methods and compositions for detecting and modulating an immunotherapy resistance gene signature in cancer |
| CN112142837A (en) * | 2019-06-28 | 2020-12-29 | 天津亨佳生物科技发展有限公司 | New antigenic peptide composition and application thereof in tumor immunotherapy drugs |
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| CN113307859B (en) | 2022-03-01 |
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