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

Abstract

The invention discloses an antigen peptide combination associated with liver cancer driver gene mutations, wherein the liver cancer driver genes are ERBB4, HNF1A, LRP1B, PREX2 and ZNF521; the sequences of the antigen peptide combination are SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.11, SEQ ID NO.14 and SEQ ID NO.15; application of the antigen peptide combination, using the antigen peptide combination to induce 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 generation of specific cytotoxic T lymphocytes, can be used for immune clearance of tumor cells with liver cancer-related driver gene mutations, and has good therapeutic potential.

Description

[0001] This application is a divisional application of application number 202110594226.9, application date May 28, 2021, and invention name “An antigen peptide associated with liver cancer driving gene mutation and its application”. Technical Field

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

[0003] Liver cancer is a malignant tumor of the liver, which can be divided into two categories: primary and secondary. Primary malignant liver tumors originate from the epithelial or mesenchymal tissue of the liver. The former is called primary liver cancer, which is a highly prevalent and extremely harmful malignant tumor in my country.

[0004] Treatment methods include surgery, hepatic artery ligation, hepatic artery chemoembolization, radiofrequency, cryosurgery, laser, microwave, chemotherapy and radiotherapy. Biological treatment and traditional Chinese medicine are also widely used in the treatment of liver cancer.

[0005] As people gain a better understanding of the nature of immune response, the concept of cellular immunity has been proposed. Through the unique mutations of tumors, the body's natural defense system is used to selectively destroy tumor cells, thereby effectively preventing the occurrence of various cancers.

[0006] 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.

[0007] However, due to the need for multidisciplinary, high-tech, and multi-platform collaborative research, the antigenic peptides associated with liver cancer driving gene mutations have not been systematically discovered and a relatively complete peptide library has not been established, so no therapeutic agents with superior effects have been formed. Summary of the invention

[0008] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide an antigen peptide combination associated with liver cancer driver gene mutations, which 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), which can be used for immune clearance of tumor cells with liver cancer-related driver gene mutations and has good therapeutic potential.

[0009] The second object of the present invention is to provide an application of the above-mentioned antigen peptide combination associated with liver cancer driver gene mutation.

[0010] The first object of the present invention can be achieved by adopting the following technical scheme: an antigen peptide combination related to liver cancer driver gene mutations, wherein the liver cancer driver genes are at least two of CDKN2A, ERBB4, HNF1A, NFE2L2, NTRK3, LRP1B, PREX2, ZFHX3 and ZNF521.

[0011] Among them, the sequence of the antigen peptide combination related to liver cancer driver gene mutation is at least two of SEQ ID NO.1-17.

[0012] 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 liver cancer driver gene mutations, using the antigen peptide combination related to liver cancer driver gene mutations as described above to induce the production of specific cytotoxic T cell clones.

[0013] Or, an application of an antigen peptide combination associated with liver cancer driver gene mutations, wherein the antigen peptide combination associated with liver 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.

[0014] Or, an application of an antigen peptide combination associated with liver cancer driver gene mutations, wherein the antigen peptide combination associated with liver cancer driver gene mutations as described above is used to prepare a cell culture fluid for preventing and intervening in mutations of at least two genes among CDKN2A, ERBB4, HNF1A, NFE2L2, NTRK3, LRP1B, PREX2, ZFHX3 and ZNF521.

[0015] Or, an application of an antigen peptide combination associated with liver cancer driver gene mutations, using the antigen peptide combination associated with liver cancer driver gene mutations as described above to prepare a liver cancer risk intervention therapeutic agent.

[0016] The present invention conducts a comprehensive study based on a specific type of cancer, specifically studies the mechanism of liver cancer occurrence, and eliminates liver cancer cells with mutations in CDKN2A, ERBB4, HNF1A, NFE2L2, NTRK3, LRP1B, PREX2, ZFHX3 and ZNF521 liver cancer driver genes, which can inhibit the growth of tumor cells and prevent the occurrence of liver 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 peptide combinations bound to MHC-I molecules, and the activated CTL can kill the corresponding target cells and play an immune surveillance role.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The antigen peptide combination (neoantigen) associated with liver 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 individual somatic mutations, and the antigen peptides associated with the 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;

[0019] 2. The antigen peptide combination related to liver cancer driving gene mutations screened by the present invention has a high affinity with 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 polypeptide vaccines and DC vaccines, and has good prospects for clinical transformation and disease prevention. DETAILED DESCRIPTION

[0020] Below, in conjunction with specific implementation modes, the present invention is further described:

[0021] The antigen peptides related to liver cancer driver gene mutations used in the examples are shown in Table 1:

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

[0023]

[0024] The operation of establishing antigen peptide-specific CTL clones related to liver 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 liver 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 liver cancer driver gene mutations, and then screened by standard cytotoxicity assay.

[0026] T2 cells were loaded with 5uM antigen peptides associated with liver cancer driver gene mutations as target cells, and the antigen peptide-specific cytotoxicity of CTLs associated with liver 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] Using the above in vitro induction method for establishing antigen peptide-specific CTL clones associated with liver 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 an ELISA reader using 450 nm as the measurement wavelength and 620 nm as the reference wavelength to prepare a standard curve.

[0046] The results are shown in Table 2, which are the experimental data of IFN-γ release of antigen peptides of SEQ ID NO.1-17, with PBS phosphate buffer and negative control peptide (the control peptide sequence is shown in SEQ ID NO.18 as: AAAAAAAAA) as controls, and the result data are absorbance values. The above experimental data show that the CTL epitope established by the present invention is extremely effective, and the predicted results are in very good agreement with the experimental results.

[0047] Table 2 Antigen peptide IFN-γ release test results (absorbance value)

[0048] Serial number Antigenic peptide PBS control Negative control SEQ ID NO.1 482 30 35 SEQ ID NO.2 469 28 39 SEQ ID NO.3 478 29 38 SEQ ID NO.4 168 29 37 SEQ ID NO.5 476 28 36 SEQ ID NO.6 182 29 35 SEQ ID NO.7 478 32 38 SEQ ID NO.8 485 32 37 SEQ ID NO.9 483 32 36 SEQ ID NO.10 469 32 35 SEQ ID NO.11 468 32 34 SEQ ID NO.12 467 32 35 SEQ ID NO.13 465 32 36 SEQ ID NO.14 471 30 37 SEQ ID NO.15 472 30 38 SEQ ID NO.16 480 33 41 SEQ ID NO.17 477 32 40

[0049] Combination of at least two antigenic peptides:

[0050] Combination 1: SEQ ID NO.2, SEQ ID NO.7, SEQ ID NO.15 and SEQ ID NO.17;

[0051] Combination 2: SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.11, SEQ ID NO.14 and SEQ ID NO.15;

[0052] Combination 3: SEQ ID NO. 8 and SEQ ID NO. 13;

[0053] Combination 4: SEQ ID NO.7, SEQ ID NO.14 and SEQ ID NO.15;

[0054] Combination 5: SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO.16;

[0055] The above five 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.18: AAAAAAAAA) as controls. The experimental results are absorbance values, and the results are shown in Table 3.

[0056] Table 3 IFN-γ release test results of antigen peptide combination (absorbance value)

[0057] combination Antigenic peptide PBS control Negative control Combination 1 609±12 31±1 41±2 Combination 2 615±13 31±2 41±3 Combination 3 608±14 30±2 43±3 Combination 4 615±15 32±1 42±2 Combination 5 623±14 30±1 42±3

[0058] It can be seen that by presenting at least two of the above liver 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 liver cancer-related driver gene mutations and prevention of the occurrence of related diseases, especially tumor diseases.

[0059] 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 liver cancer driver gene mutations, thereby achieving the prevention of diseases related to liver cancer driver gene mutations.

[0060] The antigen peptide combination associated with liver cancer driver gene mutations of the present invention is short in length and easy to chemically synthesize, and 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.

[0061] 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 liver cancer driver gene mutation and its application <130> 2021 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Ala Pro Asn Ser Tyr Gly Arg Arg Pro Ile 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <400> 2 Arg Leu Pro Val Asp Leu Ala Glu Glu Leu 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <400> 3 Ala Pro Glu Arg Ser Pro Arg Gly Glu Leu 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <400> 4 Lys Met Pro Ile Lys Trp Met Ala Leu Glu 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Gly Glu Asp Phe Thr Pro Pro Ile Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Ala Leu Tyr Ser His Lys Pro Glu Val 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Asn Thr Asp Phe Gly Asp Glu Phe Tyr 1 5 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <400> 8 Val Asn Thr Asp Phe Gly Asp Glu Phe Tyr 1 5 10 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <400> 9 Arg Glu Ala Glu Leu Leu Thr Asn Leu 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Gln Pro Arg Gln Ala Lys Gly Glu Leu 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <400> 11 Trp Thr Asp Ala Arg Ser Asp Ala Ile Tyr 1 5 10 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Met Ile Asp Pro Thr Lys Ala Arg Tyr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <400> 13 Thr Val Asp Asn Val His Leu Glu Tyr 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Asn Thr Asp Val Pro Leu Glu Gly Tyr 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <400> 15 Ser Glu Ala Asp Ile Gln Gln Leu Tyr 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Lys Glu Phe Pro Asn Gln Glu Ser Leu 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <400> 17 Ser Pro Asn Glu Asp Arg Ala Ala Leu 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <400> 18 Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5

Claims

1. An antigen peptide combination associated with liver cancer driver gene mutation, characterized in that: The sequences of the antigen peptide combination associated with liver cancer driver gene mutations are SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.11, SEQ ID NO.14 and SEQ ID NO.

15.

2. An application of an antigen peptide combination associated with liver cancer driver gene mutation, characterized in that: The antigen peptide combination associated with liver 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 liver cancer driver gene mutation, characterized in that: The antigen peptide combination associated with liver 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 liver cancer driver gene mutation, characterized in that: The antigen peptide combination associated with liver cancer driver gene mutation as claimed in claim 1 is used to prepare a cell culture fluid for preventing and intervening in mutations of ERBB4, HNF1A, LRP1B, PREX2 and ZFHX3 genes.

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

Images