Antigenic peptide associated with lung cancer driver gene mutation and its application

Abstract

An antigen peptide associated with a lung cancer driver gene mutation, the sequences of the antigen peptide are SEQ ID NO.17 and SEQ ID NO.21; application of the antigen peptide, wherein the antigen peptide is used to induce the production of specific cytotoxic T cell clones; the antigen peptide has a high affinity with MHC I molecules on DC cells, and can effectively stimulate and induce the production of specific cytotoxic T lymphocytes, and can be used for immune clearance of tumor cells with lung cancer-related driver gene mutations, and has good therapeutic potential.

Description

[0001] This application is a divisional application of application number 202110594210.8, application date May 28, 2021, and invention name “An antigen peptide associated with lung cancer driver gene mutation and its application”. Technical Field

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

[0003] Lung cancer is one of the malignant tumors with the fastest growing incidence and mortality rate, and it poses the greatest threat to people's health and life. In the past 50 years, many countries have reported that the incidence and mortality of lung cancer have increased significantly. The incidence and mortality of lung cancer in men rank first among all malignant tumors, and the incidence and mortality of lung cancer in women rank second. Chemotherapy is the main treatment for lung cancer, and more than 90% of lung cancers require chemotherapy. The efficacy of chemotherapy for lung cancer is relatively certain whether in the early or late stages, but chemotherapy also causes great damage to healthy cells, greatly reduces the patient's immunity, and easily leads to complications.

[0004] As people become more aware of the nature of tumor-driving gene mutations and personalized precise immune response, 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.

[0005] However, because the technology requires multidisciplinary, high-tech, and multi-platform collaborative research, the antigenic peptides related to lung 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

[0006] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide an antigen peptide associated with a lung cancer driver gene mutation, which has a high affinity for the MHC I molecules on DC cells and can effectively stimulate and induce the production of specific cytotoxic T lymphocytes (CTLs), and can be used for immune clearance of tumor cells with lung cancer-related driver gene mutations, and has good therapeutic potential.

[0007] The second object of the present invention is to provide an application of the above-mentioned antigen peptide associated with lung cancer driver gene mutation.

[0008] The first object of the present invention can be achieved by adopting the following technical scheme: an antigen peptide associated with lung cancer driver gene mutation, the lung cancer driver gene is at least two of EGFR, FAT4, KMT2C, KEAP1, KRAS, RB1 and TP53.

[0009] Among them, the sequences of the antigenic peptides associated with lung cancer driver gene mutations are at least two of SEQ No. 1-47.

[0010] The second object of the present invention can be achieved by adopting the following technical scheme: an application of an antigen peptide associated with a lung cancer driver gene mutation, wherein the antigen peptide associated with a lung cancer driver gene mutation as described above is used to induce the production of specific cytotoxic T cell clones.

[0011] Or, an application of an antigen peptide associated with a lung cancer driver gene mutation, using the antigen peptide associated with a lung cancer driver gene mutation as described above to prepare a human immune activity regulator that can induce the production of specific cytotoxic T cell clones.

[0012] Or, an application of an antigen peptide associated with lung cancer driver gene mutation, using the antigen peptide associated with lung cancer driver gene mutation as described above to prepare a cell culture fluid for preventing and intervening in mutations of at least two genes among EGFR, FAT4, KMT2C, KEA P1, KRAS, RB1 and TP53.

[0013] Or, an application of an antigenic peptide associated with a lung cancer driver gene mutation, using the antigenic peptide associated with a lung cancer driver gene mutation as described above to prepare a lung cancer risk intervention therapeutic agent.

[0014] The present invention conducts a comprehensive study based on a specific type of cancer, specifically studies the pathogenesis of lung cancer, and eliminates lung cancer cells with mutations in EGFR, FAT4, KMT2C, KEAP1, KRAS, RB1 and TP53 lung cancer driver genes, which can inhibit the growth of tumor cells and prevent the occurrence of lung cancer. Immunological studies have confirmed that the principle of CD8 positive T lymphocytes CTL to exert cellular immunity is that CTL cells are activated by recognizing antigen peptides bound to MHC-I molecules, and the activated CTL can kill the corresponding target cells and play an immune surveillance role.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The antigen peptides (neoantigen) associated with lung cancer driver gene mutations in the present invention refer to antigen peptides expressed by tumor cells that can activate T cells; through high-throughput gene sequencing and data analysis, numerous individual somatic mutations of the patient's tumor are discovered, and antigen peptides associated with driver gene mutations are screened out as targets, which are 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;

[0017] 2. The antigenic peptides related to lung cancer driver gene mutations screened by the present invention have 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) and inhibit the growth of tumor cells, indicating that they have good potential for polypeptide vaccines and DC vaccines, and have good prospects for clinical transformation and disease prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1-47 They are respectively the results of the CTL IFN-γ release experiment of SEQ ID NO.1-47;

[0019] Figure 48-52 These are the results of antigen peptide combination-specific CTL IFN-γ release experiment. DETAILED DESCRIPTION

[0020] Below, the present invention is further described in conjunction with the accompanying drawings and specific embodiments:

[0021] The antigen peptides associated with lung cancer driver gene mutations used in the examples are shown in Table 1:

[0022] Table 1 Antigenic peptide sequences associated with lung cancer driver gene mutations

[0023]

[0024] The operation of establishing antigen peptide-specific CTL clones related to lung cancer driver gene mutations is as follows:

[0025] 10 from the same healthy donor 5 CD8 + T cells were loaded with 10 antigenic peptides associated with lung cancer driver gene mutations 4 After stimulating Mo-DCs twice with an interval of 1 week, 10 5 The PBMCs were treated with mitomycin C and loaded with antigenic peptides associated with lung cancer driver gene mutations, and then screened by standard cytotoxicity assay.

[0026] T2 cells were loaded with 5uM antigen peptides associated with lung cancer driver gene mutations as target cells, and the antigen peptide-specific cytotoxicity of CTLs associated with lung cancer driver gene mutations was confirmed by LDH release assay.

[0027] LDH lactate dehydrogenase cytotoxicity assay:

[0028] 1) Adjust the target cells to 5×10 cells / mL using RPMI-1640 medium containing 5% fetal calf serum. 4 -2×10 5 / mL;

[0029] 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;

[0030] 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 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;

[0031] 4) Place in a carbon dioxide incubator at 37°C and 5vt% CO2 for 4-6 hours;

[0032] 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-chloronitrophenyltetrazole, and 20 μL of reaction solution (PBS containing 0.03 vt% BSA, 2.7 U / mL lipoamide dehydrogenase, 4.5 mmol / L hydrogenated coenzyme I (NAD+), and 1.2 vt% sucrose) to each well of the second plate in sequence, and place at room temperature for 20 min;

[0033] 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.

[0034] By adopting the above in vitro induction method for establishing antigen peptide-specific CTL clones associated with lung 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.

[0035] IFN-γ release assay:

[0036] 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;

[0037] 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;

[0038] 3) Block each well with 220 μL assay dilution and place at room temperature for 1 hour;

[0039] 4) Wash the plate 3 times;

[0040] 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 2 hours;

[0041] 6) Wash the plate 5 times;

[0042] 7) Prepare detection antibody (secondary antibody) by adding 48μL detector antibody to 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 1h.

[0043] 8) Wash the plate 7 times;

[0044] 9) Add the colorimetric substrate streptavidin peroxidase and incubate in dark for 30 min;

[0045] 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.

[0046] The results are as follows Figure 1-47 As shown, Figure 1-47 In the figure, sequence number 1 is the experimental data of antigen peptide IFN-γ release of SEQ ID NO.1-47, sequence number 2 is the data of PBS phosphate buffer, sequence number 3 is the data of negative control peptide (the control peptide sequence is shown as SEQ ID NO.48: AAAAAAAAA), and the vertical axis is the absorbance value. The above experimental data show that the CTL epitope established by the present invention is extremely effective, and the predicted results are in good agreement with the experimental results.

[0047] Combination of at least two antigenic peptides:

[0048] Combination 1: SEQ ID NO.5, SEQ ID NO.17, SEQ ID NO.25 and SEQ ID NO.38; 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; Combination 3: SEQ ID NO.17 and SEQ ID NO.21; Combination 4: SEQ ID NO.5, SEQ ID NO.9 and SEQ ID NO.24; Combination 5: SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.13 and SEQ ID NO.29;

[0049] The above five antigen peptide combinations were used to perform IFN-γ release experiments, using PBS phosphate buffer and negative control peptide (the control peptide sequence is shown in SEQ ID NO.48 as: AAAAAAAAA) as controls. The experimental results are absorbance values. Figure 48-52 As shown, Figure 48-52 Sequence No. 1 is the experimental data of combined antigen peptide IFN-γ release, Figure 48-52 Sequence number 2 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, sequence number 3 is the data of PBS phosphate buffer, sequence number 4 is the data of negative control peptide (the control peptide sequence is as shown in SEQ ID NO.48: AAAAAAAAA), and the vertical axis in the figure is the absorbance value.

[0050] It can be seen that by presenting at least two of the above-mentioned lung cancer driver gene mutation-related antigenic peptides through dendritic cells and co-culturing with cytotoxic lymphocyte T cells, antigen 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 lung cancer-related driver gene mutations to prevent the occurrence of related diseases, especially tumor diseases. At least two of the above-mentioned antigenic peptides are loaded and re-infused with dendritic cells (dendritic cells, DC), which can be used as DC vaccines for disease prevention, stimulating the body to produce polypeptide-specific anti-cytotoxic T cells related to lung cancer driver gene mutations, and then achieving the prevention of diseases related to lung cancer driver gene mutations. The antigenic peptides related to lung cancer driver gene mutations of the present invention are short in length, have low difficulty in chemical synthesis, can be directly synthesized to obtain high-purity products, and the application cost is greatly reduced. At the same time, the effect is clear and has good application potential.

[0051] 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> Antigenic peptide associated with lung cancer driver gene mutation and its application <130> 2021 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Arg Pro Ala Gly Ser Val Gln Asn Pro Val 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <400> 2 His Ser Thr Ala Val Gly Asn Pro Glu Tyr 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <400> 3 Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <400> 4 Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <400> 5 Tyr Thr His Gln Ser Asp Val Trp Ser Tyr 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <400> 6 Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <400> 7 Thr Ile Asp Glu Val Lys Gly Thr Ile Tyr 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <400> 8 Val Pro Ile Glu Thr Arg Arg Tyr Ala Leu 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <400> 9 Val Ile Asp Arg Glu Glu Gln Ser Phe Tyr 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <400> 10 Ser Pro Met Pro Leu Gly Ala Ser Ser Leu 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <400> 11 Ser Leu Pro Phe Ser Gly Lys His Ser Leu 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <400> 12 Leu Leu Asp Tyr Glu Val Gln Gln Tyr Tyr 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <400> 13 Ser Leu Asp Pro Asn Asn Asn Tyr Ile Tyr 1 5 10 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Lys Glu Leu Asp Pro Pro Lys Thr Tyr 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <400> 15 Lys Leu Trp Asp Glu Leu Ser Leu Val 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Ser Pro Ser Asn His Val Ser Ser Leu 1 5 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <400> 17 Lys Tyr Gln Asp Ala Pro Ala Ala Gln Phe 1 5 10 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <400> 18 Lys Val Met Glu Arg Leu Ile Glu Phe 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <400> 19 Val Ser Ile Glu Gly Ile His Pro Lys 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <400> 20 Phe Met Ala His Lys Val Val Leu Ala 1 5 <210> twenty one <211> 10 <212> PRT <213> Artificial Sequence <400> twenty one Gly Val Ile Asp Gly His Ile Tyr Ala Val 1 5 10 <210> twenty two <211> 9 <212> PRT <213> Artificial Sequence <400> twenty two Val Ile Asp Gly His Ile Tyr Ala Val 1 5 <210> twenty three <211> 10 <212> PRT <213> Artificial Sequence <400> twenty three Ser Gln His Gly Asn Arg Thr Phe Ser Tyr 1 5 10 <210> twenty four <211> 9 <212> PRT <213> Artificial Sequence <400> twenty four Lys Ser Phe Glu Asp Ile His His Tyr 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <400> 25 Phe Val Asp Glu Tyr Asp Pro Thr Ile 1 5 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <400> 26 Lys Ser Phe Glu Asp Ile His His Tyr Arg 1 5 10 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <400> 27 Lys Gln Ala Gln Asp Leu Ala Arg Ser Tyr 1 5 10 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <400> 28 Ala Thr Ser Ala Phe Gln Thr Gln Lys 1 5 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <400> 29 Glu Glu Tyr Asp Ser Ile Ile Val Phe Tyr 1 5 10 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <400> 30 Thr Glu Ile Asn Ser Ala Leu Val Leu 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <400> 31 Lys Tyr Asp Val Leu Phe Ala Leu Phe 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <400> 32 Leu Pro His Ala Val Gln Glu Thr Phe 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <400> 33 Val Tyr Phe Lys Asn Phe Ile Pro Phe 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <400> 34 Gln Pro Ser Glu Asn Leu Ile Ser Tyr 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <400> 35 Ala Glu Thr Gln Ala Thr Ser Ala Phe 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <400> 36 Arg Glu Leu Asn Glu Ala Leu Glu Leu 1 5 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <400> 37 Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe 1 5 10 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <400> 38 Lys Thr Tyr Gln Gly Ser Tyr Gly Phe 1 5 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <400> 39 Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe 1 5 10 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <400> 40 Ser Gln Lys Thr Tyr Gln Gly Ser Tyr 1 5 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <400> 41 Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys 1 5 10 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <400> 42 Gly Ser Asp Cys Thr Thr Ile His Tyr 1 5 <210> 43 <211> 9 <212> PRT <213> Artificial Sequence <400> 43 Arg Pro Ile Leu Thr Ile Ile Thr Leu 1 5 <210> 44 <211> 10 <212> PRT <213> Artificial Sequence <400> 44 Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu 1 5 10 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <400> 45 Thr Pro Arg Pro Pro Gly Pro Gly Leu 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <400> 46 Phe Ala Ile Asn Asn Thr Lys Ser Phe 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <400> 47 Ala Ala Pro Ala Pro Ala Pro Ser Trp 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <400> 48 Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5

Claims

1. An antigen peptide combination associated with lung cancer driver gene mutation, characterized in that: The sequences of the antigen peptide combination associated with lung cancer driver gene mutations are SEQ ID NO.17 and SEQ ID NO.

21.

2. An application of an antigen peptide combination associated with lung cancer driver gene mutation, characterized in that: The antigen peptide combination associated with lung cancer driver gene mutation as claimed in claim 1 is used to induce the production of specific cytotoxic T cell clones; the application is not for the purpose of diagnosis and treatment of the disease.

3. An application of an antigen peptide combination associated with lung cancer driver gene mutation, characterized in that: The antigen peptide combination associated with lung 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.

4. An application of an antigen peptide combination associated with lung cancer driver gene mutation, characterized in that: The antigen peptide combination associated with lung cancer driver gene mutation as claimed in claim 1 is used to prepare a cell culture fluid for preventing and intervening in KEAP1 gene mutation.

5. An application of an antigen peptide combination associated with lung cancer driver gene mutation, characterized in that: The antigen peptide combination associated with lung cancer driver gene mutation as claimed in claim 1 is used to prepare a lung cancer risk intervention therapeutic agent.

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