CN113173986B

CN113173986B - Antigenic peptide related to lung cancer driver gene mutation and application thereof

Info

Publication number: CN113173986B
Application number: CN202110594210.8A
Authority: CN
Inventors: 郝冰娜; 李许峰; 蔡睿; 赵环; 罗夫辛克纳吉; 张积仁
Original assignee: Shenzhen New Target Biotechnology Co ltd
Current assignee: Shanghai Baoyiren Biomedical Co ltd
Priority date: 2021-03-24
Filing date: 2021-05-28
Publication date: 2022-01-07
Anticipated expiration: 2041-05-28
Also published as: CN113896781A; CN113861283A; CN113667002A; CN113667002B; CN113667001B; CN113667001A; CN113173986A

Abstract

An antigenic peptide associated with a mutation in a lung cancer driver gene selected from at least two of EGFR, FAT4, KMT2C, KEAP1, KRAS, RB1 and TP 53; the sequences of the antigenic peptides are at least two of SEQ No. 1-47; the use of an antigenic peptide for inducing the production of specific cytotoxic T cell clones; 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 lung cancer related driver gene mutation, and has good treatment potential.

Description

Antigenic peptide related to lung cancer driver gene mutation and application thereof

Technical Field

The invention relates to an antigenic peptide related to lung cancer driver gene mutation and application thereof, belonging to the technical field of biological medicines.

Background

Lung cancer is one of the most rapidly growing malignancies that threaten human health and life. In many countries, the incidence and mortality of lung cancer have been reported to be significantly higher in recent 50 years, with lung cancer incidence and mortality in men accounting for the first of all malignancies, in women accounting for the second, and mortality accounting for the second. Chemotherapy is the primary treatment for lung cancer, and over 90% of lung cancers require chemotherapy. The curative effect of chemotherapy on lung cancer is more certain in early stage or late stage, but chemotherapy treatment also has great damage to healthy cells, so that the immunity of a patient is greatly reduced, and complications are easily caused.

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 unique tumor-driving gene mutation-associated antigen, so that various cancers are effectively prevented from happening, and the technical field is evaluated as ten technical innovation research fields in 2019 of the United states.

However, because the technology needs multidisciplinary, high-technology and multi-platform cooperative research, the antigen peptide related to the lung 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 lung cancer driver gene mutation, which 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 the immune clearance of tumor cells related to lung cancer driver gene mutation, and has good therapeutic potential.

The second purpose of the invention is to provide an application of the above antigen peptide related to the lung cancer driver 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 lung cancer driver gene selected from at least two of EGFR, FAT4, KMT2C, KEAP1, KRAS, RB1 and TP 53.

Wherein, the sequences of the antigenic peptides related to the lung cancer driving gene mutation are at least two of SEQ No. 1-47.

The second purpose of the invention can be achieved by adopting the following technical scheme: the application of the antigenic peptide related to the lung cancer driver gene mutation is to use the antigenic peptide related to the lung cancer driver gene mutation for inducing and generating specific cytotoxic T cell clone.

Or, the application of the antigen peptide related to the lung cancer driving gene mutation, the antigen peptide related to the lung cancer driving gene mutation is used for preparing the human body immunity activity regulator capable of inducing the generation of specific cytotoxic T cell clone.

Or, the application of the antigenic peptide related to the lung cancer driver gene mutation is to use the antigenic peptide related to the lung cancer driver gene mutation in preparing a cell culture solution for preventing and interfering at least two gene mutations in EGFR, FAT4, KMT2C, KEA P1, KRAS, RB1 and TP 53.

Or, the application of the antigenic peptide related to the lung cancer driver gene mutation, and the antigenic peptide related to the lung cancer driver gene mutation is used for preparing a lung cancer risk intervention therapeutic agent.

The invention carries out comprehensive research from the starting point of specific types of cancers, specifically researches the occurrence mechanism of the lung cancer, realizes the elimination of lung cancer cells with EGFR, FAT4, KMT2C, KEAP1, KRAS, RB1 and TP53 lung cancer driving gene mutation, can inhibit the growth of tumor cells and prevent the occurrence of the lung 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 invention relates to an antigenic peptide (neoantigen) related to lung cancer driver gene mutation, which is an antigenic peptide expressed by tumor cells and capable of activating 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 lung 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.

Drawings

FIGS. 1-47 are graphs showing the results of the CTL IFN- γ release assay specific to SEQ ID Nos. 1-47, respectively;

FIGS. 48 to 52 are views showing the results of experiments on the release of antigen peptide combination-specific CTL IFN-. gamma.s, respectively.

Detailed Description

The invention will be further described with reference to the accompanying drawings and the detailed description below:

antigenic peptides associated with mutations in the lung cancer driver genes used in the examples are shown in table 1:

TABLE 1 antigenic peptide sequences associated with mutations in the lung cancer driver genes

The establishment of antigenic peptide specific CTL clone related to lung cancer driver gene mutation is as follows:

10 of the same healthy donor⁵An individual CD8⁺T cell loading antigen peptide 10 related to lung cancer driving gene mutation ⁴2 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 lung cancer driver 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 lung cancer driver gene mutation as target cells, and the antigen peptide-specific cytotoxicity of CTL associated with the lung 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 (0.03 vt% BSA, 2.7U/mL lipoamide dehydrogenase, 4.5mmol/L hydrogenated coenzyme I (NAD +), 1.2 vt% sucrose in 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 lung cancer driver gene mutation by in vitro induction is adopted, the MHC-I restrictive CTL clone is also established, and the polypeptide specific immune response effect 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.

As shown in FIGS. 1 to 47, the data of IFN-. gamma.release experiments for the antigen peptides of SEQ ID Nos. 1 to 47 are shown as sequence No.1, the data of PBS phosphate buffer solution is shown as sequence No.2, the data of negative control peptide (AAAAAAAAA is shown as sequence No.48 for control peptide) is shown as sequence No.3, and the absorbance values are shown as the vertical axis in the figure. 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.

Combining at least two antigenic peptides:

combination 1: SEQ ID NO.5, SEQ ID NO.17, SEQ ID NO.25 and SEQ ID NO. 38; and (3) combination 2: SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.13, SEQ ID NO.19, SEQ ID NO.25, SEQ ID NO.36, SEQ ID NO.38 and SEQ ID NO. 43; and (3) combination: SEQ ID NO.17 and SEQ ID NO. 21; and (4) combination: SEQ ID NO.5, SEQ ID NO.9 and SEQ ID NO. 24; and (3) combination 5: SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.13 and SEQ ID NO. 29;

the IFN-gamma release experiment is carried out on the five antigen peptide combinations, PBS phosphate buffer solution and negative control peptide (the sequence of the control peptide is shown as SEQ ID NO. 48: AAAAAAAAA) are used as a control, the experiment result is an absorbance value, the result is shown as figures 48-52, the sequence number 1 in the figures 48-52 is the combined antigen peptide IFN-gamma release experiment data respectively, the sequence number 2 in the figures 48-52 is the data of SEQ ID NO.17, SEQ ID NO.43, SEQ ID NO.21, SEQ ID NO.5 and SEQ ID NO.6 respectively, the sequence number 3 is the data of PBS phosphate buffer solution, the sequence number 4 is the data of the negative control peptide (the sequence of the control peptide is shown as SEQ ID NO. 48: AAAAAAAAA), and the longitudinal axis in the figures is the absorbance value.

Therefore, at least two of the antigen peptides related to the lung cancer driver gene mutation are subjected to dendritic cell presentation and cytotoxic lymphocyte T cell co-culture, so that the antigen peptide specific cytotoxic T lymphocyte can be obtained through induction screening. The antigen specific cytotoxic T lymphocyte can be used for immune elimination of tumor cells with driver gene mutation related to lung cancer 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, stimulate organisms to generate polypeptide specific anti-cytotoxic T cells related to lung cancer driver gene mutation, and further realize the prevention of diseases related to the lung cancer driver gene mutation. The antigen peptide related to the lung cancer driver 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 lung cancer driver gene mutation and application thereof

<130> 2021

<160> 48

<170> PatentIn version 3.5

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Claims

1. The antigenic peptide combination related to the lung cancer driver gene mutation is characterized in that the sequences of the antigenic peptide combination related to the lung cancer driver gene mutation are SEQ ID NO.5, SEQ ID NO.17, SEQ ID NO.25 and SEQ ID NO. 38.

2. Use of the combination of antigenic peptides associated with a mutation in a driver gene for inducing the production of a specific cytotoxic T cell clone according to claim 1.

3. Use of the combination of antigenic peptides related to a mutation in the driver gene of lung cancer in the preparation of a modulator of human immune activity inducing the generation of specific cytotoxic T cell clones, according to claim 1.

4. Use of the combination of antigenic peptides associated with a mutation in the driver gene of lung cancer according to claim 1 for the preparation of a cell culture broth for the prevention and intervention of mutations in the EGFR, KEAP1, KRAS and TP53 genes.

5. Use of the combination of antigenic peptides associated with a mutation in the driver gene of lung cancer according to claim 1 for the preparation of an agent for intervention in the risk of lung cancer.