An antigen peptide combination related to esophageal cancer driver gene mutation and its application

Abstract

The invention discloses an antigen peptide combination associated with esophageal cancer driver gene mutations, wherein the esophageal cancer driver genes are MUC1, ERBB4 and PREX2; the sequences of the antigen peptide combination are SEQ ID NO.4, SEQ ID NO.9, SEQ ID NO.3 and SEQ ID NO.11; the application of the antigen peptide combination is to use the antigen peptide combination to induce the production of specific cytotoxic T cell clones; the antigen peptide combination has a high affinity with MHC I molecules on DC cells, can effectively stimulate and induce the production of specific cytotoxic T lymphocytes, can be used for immune clearance of tumor cells with esophageal cancer-related driver gene mutations, and has good therapeutic potential.

Description

[0001] This application is a divisional application of application number 202110598408.3, application date May 28, 2021, and invention name “An antigen peptide associated with esophageal cancer driver gene mutation and its application”. Technical Field

[0002] The present invention relates to an antigen peptide combination related to esophageal cancer driver gene mutation and application thereof, belonging to the technical field of biomedicine. Background Art

[0003] Esophageal cancer is a common digestive tract tumor, with about 300,000 people dying from it every year worldwide. Its incidence and mortality rates vary greatly among countries.

[0004] Conventional treatments for esophageal cancer are generally: surgical resection; radiotherapy, which can improve long-term survival rates but has many complications and unsatisfactory results; chemotherapy: which can sometimes improve efficacy, or relieve symptoms and prolong survival of esophageal cancer patients, but it is necessary to regularly check blood counts and liver and kidney function, and pay attention to drug reactions.

[0005] As people become more aware of the nature of tumor-driving gene mutations and personalized precise immune response reactions, personalized tumor-driving gene mutation-related peptide antigens have been proposed as a new concept for tumor-specific neoantigen immune cell therapy. Through the tumor's unique driver gene mutation-related antigens, the body's natural T cell immune defense system is induced to selectively destroy tumor gene mutation cells, thereby effectively preventing the occurrence of multiple cancers. This technical field has been rated as one of the top ten scientific and technological innovation research fields in the United States in 2019.

[0006] However, because the technology requires multidisciplinary, high-tech, and multi-platform collaborative research, the antigenic peptides related to esophageal cancer driver gene mutations have not been systematically discovered and a relatively complete peptide library has not been established, so a therapeutic agent with superior effects has not yet been formed. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide an antigen peptide combination related to esophageal cancer driver gene mutations. The antigen peptide combination related to esophageal cancer driver gene mutations has a high affinity for the MHCI molecules on DC cells, and can effectively stimulate and induce the production of specific cytotoxic T lymphocytes (CTLs), which can be used for immune clearance of tumor cells with esophageal cancer-related driver gene mutations, and has good therapeutic potential.

[0008] The second object of the present invention is to provide an application of the above-mentioned antigen peptide combination associated with esophageal cancer driver gene mutation.

[0009] The first object of the present invention can be achieved by adopting the following technical scheme: an antigen peptide combination associated with esophageal cancer driver gene mutations, wherein the esophageal cancer driver genes are at least two of EPCAM, MUC1, GPC3, LRP1B, ERBB4 and PREX2.

[0010] Among them, the sequence of the antigen peptide combination related to esophageal cancer driver gene mutation is at least two of SEQ ID NO.1-12.

[0011] The second purpose of the present invention can be achieved by adopting the following technical scheme: an application of an antigen peptide combination related to esophageal cancer driver gene mutation, using the antigen peptide combination related to esophageal cancer driver gene mutation as described above to induce the production of specific cytotoxic T cell clones.

[0012] Or, an application of an antigen peptide combination associated with esophageal cancer driver gene mutations, wherein the antigen peptide combination associated with esophageal cancer driver gene mutations as described above is used to prepare a human immune activity regulator that can induce the production of specific cytotoxic T cell clones.

[0013] Or, an application of an antigen peptide combination associated with esophageal cancer driver gene mutations, using the antigen peptide combination associated with esophageal cancer driver gene mutations as described above to prepare a cell culture fluid for preventing and intervening in mutations of at least two genes among EPCAM, MUC1, GPC3, LRP1B, ERBB4 and PREX2.

[0014] Or, an application of an antigen peptide combination associated with esophageal cancer driver gene mutations, using the antigen peptide combination associated with esophageal cancer driver gene mutations as described above to prepare an esophageal cancer risk intervention therapeutic agent.

[0015] The present invention conducts a comprehensive study based on a specific type of cancer, specifically studies the mechanism of esophageal cancer occurrence, and eliminates esophageal cancer cells with esophageal cancer driver gene mutations such as EPCAM, MUC1, GPC3, LRP1B, ERBB4, and PREX2, which can inhibit the growth of tumor cells and prevent the occurrence of esophageal cancer. Immunological studies have confirmed that the principle of CD8-positive T lymphocytes CTL in exerting cellular immunity is: CTL cells are activated by recognizing antigen peptides bound to MHC-I molecules, and the activated CTL can kill the corresponding target cells and exert immune surveillance.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The antigen peptide combination (neoantigen) associated with esophageal cancer driver gene mutations of the present invention refers to antigen peptides expressed by tumor cells that can activate T cells; through high-throughput gene sequencing and data analysis, the patient's tumor is found to have many individualized somatic mutations, and the antigen peptide combination associated with the driver gene mutation is screened out as a target, which is chemically synthesized in vitro and loaded onto antigen presenting cells (APCs), thereby activating T cells to recognize a variety of neoantigen peptides, thereby producing a therapeutic effect of killing mutated tumor cells;

[0018] 2. The antigen peptide combination related to esophageal cancer driver gene mutations screened by the present invention has a high affinity with the MHC I molecules on DC cells and can effectively stimulate and induce the production of specific cytotoxic T lymphocytes (CTLs) to inhibit the growth of tumor cells, indicating that it has good potential for peptide vaccines and DC vaccines, and has good prospects for clinical transformation and disease prevention. DETAILED DESCRIPTION

[0019] Below, in conjunction with specific implementation modes, the present invention is further described:

[0020] The antigen peptides related to esophageal cancer driver gene mutations used in the examples are shown in Table 1:

[0021] Table 1 Antigenic peptide sequences associated with esophageal cancer driver gene mutations

[0022]

[0023] The operation of establishing antigen peptide-specific CTL clones related to esophageal cancer driver gene mutations is as follows:

[0024] 10 from the same healthy donor 5 CD8 + T cells were loaded with 10 antigenic peptides associated with esophageal cancer driver gene mutations 4 After stimulating Mo-DCs twice with an interval of 1 week, 10 5 The cells were obtained by stimulating PBMCs treated with mitomycin C once with antigenic peptides associated with esophageal cancer driver gene mutations and then screening by standard cytotoxicity assay.

[0025] T2 cells were loaded with 5uM antigen peptides associated with esophageal cancer driver gene mutations as target cells, and the antigen peptide-specific cytotoxicity of CTLs associated with esophageal cancer driver gene mutations was confirmed by LDH release assay.

[0026] LDH lactate dehydrogenase cytotoxicity assay:

[0027] 1) Adjust the target cells to 5×10 cells / mL using RPMI-1640 medium containing 5% fetal calf serum.4 -2×10 5 / mL;

[0028] 2) Add target cells to a 96-well round-bottom cell culture plate, add 100 μL to each well. No target cells were added to the three effector cell natural release control wells, only 100 μL of culture medium was added;

[0029] 3) Add 100 μL of CTL effector cells (CD8+T cells) to each well, with the ratio of effector cells to target cells being 10:1; add only 100 μL of culture medium to the natural release wells without adding effector cells, and add 100 μL of 1vt% NP40 to the maximum release wells. Set up three replicate wells for each experiment;

[0030] 4) Place in a carbon dioxide incubator at 37°C and 5vt% CO2 for 4-6 hours;

[0031] 5) Centrifuge the culture plate at 200×g for 10 min; aspirate 150 μL of supernatant from each well and add it to another 96-well ELISA plate; add 20 μL of 0.4 mol / L lactic acid solution, 20 μL of 4 mmol / L 2-p-iodophenyl-3-p-nitrophenyltetrazole chloride, and 20 μL of reaction solution (containing 0.03 vt% BSA, 2.7 U / mL lipoamide dehydrogenase, 4.5 mmol / L hydrogenated coenzyme I (NAD+), and 1.2 vt% sucrose in PBS) to each well of the second plate, and place at room temperature for 20 min;

[0032] 6) Measure the optical density (OD value) of each well on an enzyme-linked detector with a detection wavelength of 492 nm and a reference wavelength of 650 nm.

[0033] By adopting the above in vitro induction method for establishing antigen peptide-specific CTL clones associated with esophageal cancer driver gene mutations, the present invention also established MHC-I restricted CTL clones, and confirmed their peptide-specific immune response effects through IFN-γ release experiments.

[0034] IFN-γ release assay:

[0035] 1) Coating antibody: dilute 44 μL capture antibody and 12 mL coat buff at 1:250, coat each well of a 96-well ELISA plate with 100 μL of the above solution at 4°C overnight;

[0036] 2) Wash the plate 3 times, prepare washing solution, add 50mL of distilled water to make 1000mL of wash buff, dilute 1:20, and wash the plate 3 times with an automatic plate washer;

[0037] 3) Block each well with 220 μL assay dilution and place at room temperature for 1 hour;

[0038] 4) Wash the plate 3 times;

[0039] 5) Prepare standard samples, dilute in equal proportions, and establish a concentration gradient, namely blank well, 4.7pg / mL, 9.4pg / mL, 18.8pg / mL, 37.5pg / mL, 75pg / mL, 150pg / mL, 300pg / mL. Add the sample to be tested and the secondary well in sequence. If the concentration is too high, dilute it. Leave at room temperature for 2h;

[0040] 6) Wash the plate 5 times;

[0041] 7) Prepare detection antibody (secondary antibody) 48μL detector antibody and add 12mL assay dilution to make one solution. When adding the sample, add 48μL SAV-HRP to one solution immediately. Place 100μL in each well at room temperature for 1 hour.

[0042] 8) Wash the plate 7 times;

[0043] 9) Add the colorimetric substrate streptavidin peroxidase and incubate in dark for 30 min;

[0044] 10) Add the stop solution and perform colorimetry on a microplate reader using 450 nm as the measurement wavelength and 620 nm as the reference wavelength to prepare a standard curve.

[0045] The results are shown in Table 2, which are the experimental data of IFN-γ release of antigen peptides of SEQ ID NO.1-12, with PBS phosphate buffer and negative control peptide (the control peptide sequence is shown in SEQ ID NO.13 as: AAAAAAAAA) as controls, and the experimental results are absorbance values. The above experimental data show that the CTL epitope established by the invention is extremely effective, and the predicted results are in very good agreement with the experimental results.

[0046] Table 2 Antigen peptide IFN-γ release test results (absorbance value)

[0047]

[0048]

[0049] Combination of at least two antigenic peptides:

[0050] Combination 1: SEQ ID NO. 3 and SEQ ID NO. 11;

[0051] Combination 2: SEQ ID NO.4, SEQ ID NO.9, SEQ ID NO.3 and SEQ ID NO.11;

[0052] Combination 3: SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.12;

[0053] The above three antigen peptide combinations were used to perform IFN-γ release experiments, with PBS phosphate buffer and negative control peptide (the control peptide sequence is shown in SEQ ID NO.13: AAAAAAAAA) as controls. The experimental results are absorbance values, and the results are shown in Table 3.

[0054] Table 3 IFN-γ release test results of antigen peptide combination (absorbance value)

[0055] combination Antigenic peptide PBS control Negative control Combination 1 570 29 34 Combination 2 585 31 40 Combination 3 590 29 35

[0056] It can be seen that by presenting the antigenic peptides related to the above-mentioned esophageal cancer driver gene mutations through dendritic cells and co-culturing with cytotoxic lymphocyte T cells, antigenic peptide-specific cytotoxic T lymphocytes can be induced and screened. Such antigen-specific cytotoxic T lymphocytes can be used for immune clearance of tumor cells with esophageal cancer-related driver gene mutations and prevent the occurrence of related diseases, especially tumor diseases.

[0057] At least two of the above antigenic peptides are loaded and re-infused into dendritic cells (DCs), which can be used as DC vaccines for disease prevention, stimulating the body to produce peptide-specific anti-cytotoxic T cells related to esophageal cancer driver gene mutations, thereby achieving the prevention of diseases related to esophageal cancer driver gene mutations.

[0058] The antigen peptide combination associated with esophageal cancer driver gene mutation of the present invention is short in length and easy to chemically synthesize. A high-purity product can be directly synthesized, which greatly reduces the application cost. At the same time, the effect is clear and has great application potential.

[0059] For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all of these changes and modifications should fall within the protection scope of the claims of the present invention. SEQUENCE LISTING <110> Shenzhen New Targeted Biotechnology Co., Ltd. <120> An antigen peptide combination related to esophageal cancer driver gene mutation and its application <130> 2021 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <400> 1 Leu Gln Lys Glu Ile Thr Thr Arg Tyr 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Lys Glu Ile Thr Thr Arg Tyr Gln Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Asp Thr Tyr His Pro Met Ser Glu Tyr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <400> 4 Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Gly Glu Phe Phe Thr Asp Val Ser Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Lys Ser Leu Gln Val Thr Arg Ile Phe 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <400> 7 Trp Thr Asp Ala Arg Ser Asp Ala Ile Tyr 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Met Ile Asp Pro Thr Lys Ala Arg Tyr 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <400> 9 Ala Pro Glu Arg Ser Pro Arg Gly Glu Leu 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <400> 10 Lys Met Pro Ile Lys Trp Met Ala Leu Glu 1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 Thr Val Asp Asn Val His Leu Glu Tyr 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Asn Thr Asp Val Pro Leu Glu Gly Tyr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <400> 13 Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5

Claims

1. An antigen peptide composition associated with esophageal cancer driver gene mutation, characterized in that: The antigen peptide composition related to esophageal cancer driver gene mutation is composed of antigen peptides with sequences of SEQ ID NO.4, SEQ ID NO.9, SEQ ID NO.3 and SEQID NO.

11.

2. An application of an antigen peptide composition associated with esophageal cancer driver gene mutation, characterized in that: The antigen peptide composition associated with esophageal cancer driver gene mutation as claimed in claim 1 is used to prepare a human immune activity regulator that can induce the production of specific cytotoxic T cell clones.

3. An application of an antigen peptide composition associated with esophageal cancer driver gene mutation, characterized in that: The antigen peptide composition associated with esophageal cancer driver gene mutation as claimed in claim 1 is used to prepare a cell culture fluid for preventing and intervening in mutations of MUC1, ERBB4 and PREX2 genes.

4. An application of an antigen peptide composition associated with esophageal cancer driver gene mutation, characterized in that: The antigen peptide composition associated with esophageal cancer driver gene mutation as described in claim 1 is used to prepare an esophageal cancer risk intervention therapeutic agent.

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