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 being SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.13 and SEQ ID NO.29; 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 to the 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 related to 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 most rapidly increasing malignant tumors with the highest incidence and mortality rates, posing the greatest threat to public health and life. Over the past 50 years, many countries have reported a significant increase in both the incidence and mortality rates of lung cancer. Lung cancer ranks first in both incidence and mortality among all malignant tumors in men, and second in incidence and mortality among women. Chemotherapy is the primary treatment for lung cancer, requiring over 90% of lung cancer cases. While chemotherapy is highly effective for both early-stage and late-stage lung cancer, it can also significantly damage healthy cells, significantly weakening the patient's immune system and leading to complications.

[0004] As people gain a deeper understanding of the nature of tumor-driving gene mutations and personalized, precise immune responses, 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 various 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 effect has not yet been formed. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, the first purpose of the present invention is to provide an antigen peptide associated with lung cancer driver gene mutations. The antigen peptide associated with lung cancer driver gene mutations has a high affinity for MHC I molecules on DC cells and can effectively stimulate and induce the production of specific cytotoxic T lymphocytes (CTLs). It 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 antigenic peptide related to lung cancer driver gene mutation.

[0008] The first object of the present invention can be achieved by adopting the following technical solution: an antigen peptide associated with lung cancer driver gene mutations, where the lung cancer driver genes are 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 solution: an application of an antigen peptide related to a lung cancer driver gene mutation, wherein the antigen peptide related to a lung cancer driver gene mutation as described above is used to induce the production of specific cytotoxic T cell clones.

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

[0012] Alternatively, an application of an antigenic peptide associated with a lung cancer driver gene mutation, wherein the antigenic peptide associated with a lung cancer driver gene mutation as described above is used 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] Alternatively, an application of an antigenic peptide associated with a lung cancer driver gene mutation, wherein the antigenic peptide associated with a lung cancer driver gene mutation as described above is used to prepare a lung cancer risk intervention therapeutic agent.

[0014] This invention conducts comprehensive research based on specific types of cancer, specifically studying the pathogenesis of lung cancer. It eliminates lung cancer cells harboring mutations in EGFR, FAT4, KMT2C, KEAP1, KRAS, RB1, and TP53 lung cancer driver genes, inhibiting tumor cell growth and preventing the development of lung cancer. Immunological studies have confirmed that CD8-positive T lymphocytes (CTLs) function through cellular immunity by recognizing antigenic peptides bound to MHC-I molecules and becoming activated. Activated CTLs kill corresponding target cells, exerting an immune surveillance effect.

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

[0016] 1. The present invention's neoantigen peptides associated with lung cancer driver gene mutations are antigenic peptides expressed by tumor cells that can activate T cells. Through high-throughput gene sequencing and data analysis, numerous individualized somatic mutations in a patient's tumor are discovered. Antigenic peptides associated with driver gene mutations are screened as targets, chemically synthesized in vitro, and loaded onto antigen-presenting cells (APCs), thereby activating T cells to recognize multiple neoantigen peptides and, in turn, kill mutated tumor cells.

[0017] 2. The antigenic peptides screened by the present invention, which are related to lung cancer driver gene mutations, have a high affinity for MHC I molecules on DC cells and can effectively stimulate and induce the production of specific cytotoxic T lymphocytes (CTLs), inhibiting tumor cell growth, indicating that they have good potential as peptide vaccines and DC vaccines, and have good prospects for clinical translation and disease prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1-47 These are views of the results of the CTL IFN-γ release experiments specific to SEQ ID NO. 1-47;

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

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0021] The antigenic 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 procedures for establishing antigen peptide-specific CTL clones related to lung cancer driver gene mutations are 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 one week, 10 5 The cells were obtained by stimulating PBMCs treated with mitomycin C and loaded with antigenic peptides related to lung cancer driver gene mutations once and then screening by standard cytotoxicity assay.

[0026] T2 cells were loaded with 5uM antigen peptides related to lung cancer driver gene mutations as target cells, and the antigen peptide-specific cytotoxicity of CTLs related to 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 4 -2×10 5 / mL;

[0029] 2) Add target cells to a 96-well round-bottom cell culture plate, adding 100 μL of culture medium to each well. For the three effector cell natural release control wells, add only 100 μL of culture medium without adding target cells.

[0030] 3) Add 100 μL of CTL effector cells (CD8+ T cells) to each well, with a 10:1 effector cell to target cell ratio. For natural release wells, add only 100 μL of culture medium without effector cells. For maximum release wells, add 100 μL of 1% NP40. Perform each experiment in triplicate.

[0031] 4) Place in a carbon dioxide incubator at 37°C and 5% 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 enzyme-linked assay 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 (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 incubate at room temperature for 20 min;

[0033] 6) Measure the optical density (OD value) of each well on an enzyme-linked immunosorbent assay (ELISA) with a detection wavelength of 492 nm and a reference wavelength of 650 nm.

[0034] Using 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 of capture antibody and 12 mL of coat buff at 1:250. Coat 100 μL per well of a 96-well ELISA plate at 4°C overnight.

[0037] 2) Wash the plate three times. Prepare a wash buffer by adding 50 mL of the buffer to distilled water to make 1000 mL of wash buffer. Dilute the buffer 1:20 and wash the plate three times using an automatic plate washer.

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

[0039] 4) Wash the plate 3 times;

[0040] 5) Prepare the standard and dilute it in equal proportions to establish a concentration gradient: blank well, 4.7 pg / mL, 9.4 pg / mL, 18.8 pg / mL, 37.5 pg / mL, 75 pg / mL, 150 pg / mL, and 300 pg / mL. Add the sample to be tested and the replica well in sequence. If the concentration is too high, dilute it. Incubate at room temperature for 2 hours.

[0041] 6) Wash the plate 5 times;

[0042] 7) Prepare the detection antibody (secondary antibody) by adding 48 μL of detector antibody to 12 mL of assay dilution. Immediately after adding the sample, add 48 μL of SAV-HRP to the first solution. 100 μL per well should be incubated at room temperature for 1 hour.

[0043] 8) Wash the plate 7 times;

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

[0045] 10) Add stop solution and perform colorimetry on a microplate reader using 450 nm as the measurement wavelength and 620 nm as the reference wavelength to create a standard curve.

[0046] The results are as follows Figure 1-47 As shown, Figure 1-47 Sequence number 1 shows experimental data for IFN-γ release from antigenic peptides SEQ ID NOs. 1-47, sequence number 2 shows data from PBS phosphate buffer, and sequence number 3 shows data from a negative control peptide (the control peptide sequence is shown in SEQ ID NO. 48 as: AAAAAAAAA). The vertical axis of the graph represents absorbance. These experimental data demonstrate that the CTL epitopes established by this invention are extremely effective, with excellent agreement between the predicted and 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 for IFN-γ release experiments, with PBS phosphate buffer and negative control peptide (control peptide sequence as shown in SEQ ID NO.48: AAAAAAAAA) as controls. The experimental results are absorbance values. Figures 48-52 As shown, Figures 48-52 Sequence number 1 is the experimental data of combined antigen peptide IFN-γ release, Figures 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, and sequence number 4 is the data of the negative control peptide (the control peptide sequence is as shown in SEQ ID NO.48: AAAAAAAAA). 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, and 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 (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 thus achieving 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 chemical synthesis difficulty, 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] Those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all of these changes and modifications should fall within the scope of protection of the claims of the present invention. SEQUENCE LISTING <110> Shenzhen New Target Biotechnology Co., Ltd. <120> Antigen 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 mutations, It is characterized in that The sequences of the antigen peptide combination associated with lung cancer driver gene mutations are SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.13 and SEQ ID NO.

29.

2. Application of an antigen peptide combination associated with lung cancer driver gene mutations, It is 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 disease diagnosis and treatment.

3. Application of an antigen peptide combination associated with lung cancer driver gene mutations, It is 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. Application of an antigen peptide combination associated with lung cancer driver gene mutations, It is 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 mutations of EGFR, FAT4 and RB1 genes.

5. Application of an antigen peptide combination associated with lung cancer driver gene mutations, It is 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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