Tumor neoantigen polypeptide based on nras gene q61k mutation and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a tumor neoantigen polypeptide based on Q61K mutation of tumor driver gene NRAS and application thereof. The present application aims to solve the technical problem of the prior art, and provides a specific immunotherapy scheme for Q61K mutation of tumor driver gene NRAS based on MHC-II molecules. The technical scheme for solving the above technical problem is to provide a tumor neoantigen polypeptide based on Q61K mutation of NRAS gene. The amino acid sequence of the antigen peptide is GKEEYSAMRDQYMRT. The antigen peptide of the present application can significantly activate human specific T cells against NRAS mutation, so as to increase the killing ability of T cells to NRAS mutant tumor cells, and can be used for preparing a drug for preventing and treating NRAS mutant tumor, and particularly for preparing a tumor cell vaccine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a tumor neoantigen polypeptide related to Q61K mutation of tumor driver gene NRAS and application thereof. BACKGROUND

[0002] Human RAS (Rat SArcoma) tumor driver genes include HRAS, KRAS and NRAS genes, and mutations can occur in about 30% of malignant tumors. Activation of NRAS in the plasma membrane can lead to protein phosphorylation and signal transduction, promoting cell growth, proliferation and differentiation. Its activating mutations mainly occur at G12, G13 and Q61 sites. Unlike G12 and G13 site mutations, Q61 position mutations can enhance GTP binding, thereby reducing GTP hydrolysis, and finally leading to overactivation of RAS, generation of pro-cancer signals and drug resistance. NRAS Q61K mutations mainly occur in melanoma, colorectal cancer, thyroid cancer and lung cancer and other types of tumors. However, due to the particularity of the NRAS protein itself, it is very challenging to develop treatment methods targeting NRAS. Therefore, despite more than 30 years of in-depth research, effective anti-RAS therapy has not yet entered the clinic.

[0003] Immunotherapy of tumors is considered to be a new generation of tumor treatment method after surgery, radiotherapy, chemotherapy and small molecule targeted therapy. The difference between immunotherapy and previous traditional treatment methods is that the treatment method is changed from direct killing of tumor cells by drugs to enhancement of immune cells, and the tumor is treated by improving the patient's own anti-tumor immune ability. Compared with traditional treatment, it has the advantages of precise killing, small side effects, long-term efficacy, high degree of individualization, etc. In addition, the immune system of the body has the characteristics of immune memory, so immunotherapy can help patients form memory-type immunity, which has a significant advantage in preventing tumor recurrence and metastasis. Peptide-based vaccines provide tumor mutant peptides to MHC molecules to induce specific and long-term memory T cells to fight tumors. Tumor antigens are usually considered to be endogenous antigens, which are combined with MHC class I molecules to stimulate CD8+ cytotoxic responses. A small part of larger polypeptides are combined with MHC II molecules to specifically stimulate CD4+ T helper cells.

[0004] In view of the high frequency of NRAS mutations in tumors, it has become a potential target for immunotherapy. Studies have found that mutant NRAS peptides can induce specific CD8+ and CD4+ T cell anti-RAS immune responses, and NRAS mutant peptides have enhanced immunogenicity. Therefore, vaccines targeting NRAS mutant peptides can be used as specific immunotherapy for patients with NRAS mutant tumors. In existing studies, research on NRAS has mainly focused on MHC-I molecules, for example, researchers found that in melanoma patients with HLA-A*01:01 genotype, using a neoantigen (ILDTAGKEEY, SEQ ID No. 31) against RAS.Q61K can effectively activate T cells in patients and kill tumors (Peri et. al, J Clin Invest. 2021 Oct 15), but it is only for patients with HLA-A*01:01 genotype. There is no report on RAS neoantigens based on MHC-II molecules. SUMMARY

[0005] The technical problem solved by the present application is to provide a specific immunotherapy targeting the Q61K mutation of the tumor driver gene NRAS based on MHC-II molecules to overcome the shortcomings of the prior art.

[0006] The technical solution of the present application to solve the above technical problem is to provide a tumor neoantigen polypeptide based on the Q61K mutation of the NRAS gene. The amino acid sequence of the antigen peptide is GKEEYSAMRDQYMRT (SEQ ID No. 17).

[0007] Or, a polypeptide with the same or similar function obtained by substituting, deleting and / or adding at least one amino acid in the amino acid sequence of each of the above polypeptides.

[0008] Wherein, the same or similar function mentioned above refers to the ability of the antigen polypeptide to activate specific T cells against tumors with NRAS Q61K mutation in patients with HLA genotype HLA-DRB1*09:01 and accompanying Q61K mutation of NRAS.

[0009] The present application also provides the use of the above-mentioned tumor neoantigen polypeptide in the preparation of an immune activity modulator that can induce the production of specific cytotoxic T cell clones.

[0010] The present application also provides the use of the above-mentioned tumor neoantigen polypeptide in the preparation of a risk intervention and / or therapeutic agent for tumors with high frequency of NRAS mutations. Wherein, the high frequency of NRAS mutations mentioned above is Q61K mutation.

[0011] Based on the above-mentioned scheme, the present application further provides a DC cell. The DC cell is obtained by stimulating the above-mentioned tumor neoantigen polypeptide. Further, the stimulation mode in the above-mentioned DC cell is that the DC cell is co-incubated with the tumor neoantigen polypeptide.

[0012] In the above-mentioned application, the DC cell is a mature DC cell.

[0013] In the above-mentioned application, the DC cell is an ex vivo DC cell of a patient with NRAS Q61K mutation.

[0014] Further, the above-mentioned DC cell is an ex vivo DC cell of a patient with human leukocyte HLA type HLA-DRB1*09:01.

[0015] The present application also provides the above-mentioned tumor neoantigen polypeptide and the above-mentioned DC cell for use in the preparation of an immunotherapeutic drug for a tumor with NRAS mutation.

[0016] In the above-mentioned use, the tumor with NRAS mutation is a tumor with NRAS Q61K mutation.

[0017] In the above-mentioned use, the tumor is melanoma, colorectal cancer, thyroid cancer and lung cancer.

[0018] In the above-mentioned use, the tumor is a tumor of a patient with human leukocyte HLA type HLA-DRB1*09:01.

[0019] In addition, the present application also provides an antibody against the above-mentioned tumor neoantigen polypeptide of claim 1.

[0020] In the above-mentioned antibody, the antibody is a polyclonal antibody or a monoclonal antibody.

[0021] Further, the above-mentioned antibody can form a conjugate with a conjugated moiety. The conjugated moiety is at least one selected from the group consisting of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein or biotin.

[0022] The present application also provides a gene encoding the above-mentioned tumor neoantigen polypeptide or the above-mentioned antibody.

[0023] Meanwhile, the present application also provides a vector loaded with the above-mentioned gene. Further, the vector is an expression vector, which can be selected from a commonly used vector such as a plasmid vector, an adenovirus vector, a lentivirus vector or an adeno-associated virus vector. When using an adenovirus vector, a replication-defective adenovirus vector is generally used.

[0024] The beneficial effects of this invention are as follows: The antigenic peptide of this invention can significantly activate human T cells specifically targeting NRAS mutations, thereby increasing the killing ability of T cells against NRAS-mutant tumor cells, and enabling the preparation of drugs for the prevention and treatment of NRAS-mutant tumors. Furthermore, DC cells from NRAS-mutant tumor patients stimulated by the antigenic peptide of this invention, when reinfused into the patient, can activate T cells specifically targeting the NRAS mutation peptide, thereby increasing the killing ability of T cells against cancer cells with NRAS mutations. The antigenic peptide of this invention fills the gap in the treatment of tumor patients with both NRAS-Q61K somatic mutations and the HLA-DRB1*09:01 genotype using personalized antigenic peptides. Simultaneously, the antigen of this invention is easy to synthesize on a large scale and can be used in standardized and personalized immunotherapy for NRAS-mutant tumor patients. Attached Figure Description

[0025] Figure 1 This is the result of an enzyme-linked immunosorbent assay (ELISA). Detailed Implementation

[0026] This invention, based on the amino acid sequence of the NRAS gene mutant peptide and combined with the human leukocyte antigen haplotype in MHC class II molecules, uses bioinformatics prediction methods to obtain a series of antigenic peptides. Further screening yielded a neoantigen peptide with the amino acid sequence GKEEYSAMRDQYMRT, named M9. This neoantigen peptide M9 corresponds to the HLA-DRB1*09:01 human leukocyte antigen typing, meaning it can activate specific dendritic cells (DCs) targeting NRAS mutations in patients with the HLA-DRB1*09:01 typing and NRAS gene Q61K mutation, thereby increasing the killing ability of T cells against NRAS-mutant tumor cells.

[0027] Those skilled in the art will understand that peptides with the same or similar functions obtained by substituting and / or deleting and / or adding at least one amino acid in the amino acid sequence of the GKEEYSAMRDQYMRT antigen peptide are also within the scope of protection of this invention. The same or similar function refers to the antigen peptide's ability to activate T cells specifically targeting tumors with NRAS Q61K mutations in patients with HLA-DRB1*09:01 HLA typing and accompanying NRAS Q61K mutations.

[0028] In the present application, the expression "a protein having the same or similar function as the above-mentioned protein, which is obtained by substituting and / or deleting and / or adding at least one amino acid in the amino acid sequence of each peptide segment" includes, but is not limited to, deletion, insertion and / or substitution of several (usually 1 to 20, preferably 1 to 10, more preferably 1 to 5, most preferably 1 to 3) amino acids, and addition of one or several (up to 40, usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and / or N-terminus. For example, when a substitution is made with an amino acid having similar or similar properties in the polypeptide, the function is usually not changed. For another example, addition of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein or polypeptide. The term also includes active fragments and active derivatives of the polypeptide.

[0029] The expression "a peptide segment obtained by substituting and / or deleting and / or adding at least one amino acid in the amino acid sequence of each peptide segment" also includes, but is not limited to, polypeptides in which up to 10 (i.e. one or several), preferably up to 8, more preferably up to 5 (5, 4, 3, 2 or 1) amino acids are replaced by amino acids having similar or similar properties, i.e. conservative variant polypeptides. Further, these conservative variant polypeptides can be replaced according to Table 1.

[0030] Table 1 Table of amino acid substitutions

[0031] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0032] The above-mentioned polypeptide can be used as an active ingredient to prepare an anti-tumor drug. Generally, a person skilled in the art can use the above-mentioned polypeptide as an antigen active ingredient to prepare a vaccine for preventing and / or treating tumors with NRAS mutations. The vaccine uses the above-mentioned polypeptide as an antigen component, as well as pharmaceutically acceptable adjuvants or auxiliary components.

[0033] When preparing a vaccine, an immunological adjuvant is often added to enhance the immune response of the body to the vaccine. Among them, the immunological adjuvant is Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant, microbial adjuvant, etc.

[0034] Naturally, on the basis of the polypeptide described in the present application, the antibody against the above-mentioned protein can be easily obtained in the art. The above-mentioned antibody is a polyclonal antibody or a monoclonal antibody; preferably a monoclonal antibody. The above-mentioned antibody can also form a conjugate with a conjugated moiety. Further, the conjugated moiety is one or more selected from the group consisting of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein or biotin. The antibody that can specifically bind to the aforementioned protein can be used for the preparation of a vaccine for preventing and / or treating tumors with NRAS mutations, and for the immunological detection of tumors with NRAS mutations.

[0035] In addition, the present application also comprises the coding gene of the above-mentioned protein. The coding gene of the above-mentioned protein can be used for expressing the above-mentioned polypeptide, and can also be operatively loaded in an expression vector, and then can be prepared into a vector vaccine or a vector drug. The expression can be selected from a plasmid vector, an adenovirus vector, a lentivirus vector or an adeno-associated virus vector, etc. When the adenovirus vector is used, a replication-defective adenovirus vector is generally used.

[0036] In some cases, the tumor neoantigen polypeptide of the present application also comprises an antigenic peptide with a length of not more than 40 amino acids, the amino acid sequence of which comprises the sequence of SEQ ID No. 17, and still has the same or similar function as the neoantigen peptide M9. Further, the tumor neoantigen polypeptide with the amino acid sequence comprising the sequence of SEQ ID No. 17 has a length of not more than 30 amino acids, and still has the same or similar function as the neoantigen peptide M9.

[0037] Further, the tumor neoantigen polypeptide of the present application comprises a polypeptide formed by connecting 1-20 amino acids to the nitrogen end of the sequence of SEQ ID No. 17 and / or connecting not more than 1-20 amino acids to the carbon end of the sequence of SEQ ID No. 17, and still has the same or similar function as the neoantigen peptide M9.

[0038] That is, the present application can add an amino acid sequence with a certain length to either side or both sides of the tumor neoantigen polypeptide M9 of the sequence of SEQ ID No. 17, and still obtain an antigenic peptide capable of activating T cells specific to tumors with NRAS Q61K mutation in patients with HLA typing of HLA-DRB1*09:01 and accompanied by NRAS Q61K mutation.

[0039] It can be understood that the above-mentioned amino acid sequence added to either side or both sides of the tumor neoantigen polypeptide M9 can be derived from the positions on both sides of GKEEYSAMRDQYMRT in the NRAS protein.

[0040] Since the novel antigenic peptide M9 of the present application can significantly activate human T cells specific to NRAS mutation, the killing ability of T cells to NRAS mutant tumor cells is increased. On this basis, the present application naturally also provides the use of the above-mentioned series of novel antigenic peptides in the preparation of immunotherapeutic drugs for NRAS mutant tumors. Since the above-mentioned novel antigenic peptides are used to treat NRAS mutant tumors, at present, the dendritic cells are stimulated by using novel antigens as vaccines, and after the vaccines are inoculated into patients with NRAS mutant tumors, T lymphocytes are activated and proliferated, and begin to attack cancer cells with novel antigenic peptides as recognition targets. On this basis, the present application also provides a DC cell, which is obtained by stimulating the above-mentioned tumor novel antigenic peptide. The stimulation mode is generally co-incubation of DC cells and tumor novel antigenic peptides. The DC cells are generally ex vivo DC cells. Naturally, the above-mentioned DC cells are ex vivo DC cells of patients with Q61K mutation of NRAS in tumors. Generally speaking, the ex vivo DC cells need to be cultured to maturity before the stimulation step of novel antigens. Naturally, the above-mentioned DC cells are ex vivo DC cells of patients with HLA typing of HLA-DRB1*09:01 of human leukocytes.

[0041] The present application also provides the use of the above-mentioned tumor novel antigenic peptide and the above-mentioned DC cell in the preparation of immunotherapeutic drugs for tumors with NRAS mutation. The tumor with NRAS mutation in the above-mentioned use is a tumor with Q61K mutation of NRAS. The tumor with Q61K mutation of NRAS includes melanoma, colorectal cancer, thyroid cancer and lung cancer and the like. Of course, it is mainly suitable for patients with the above-mentioned tumors with HLA typing of HLA-DRB1*09:01 of human leukocytes.

[0042] The present application will be further described below in conjunction with the drawings and specific embodiments, but is not limited to the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0043] Example 1 Novel antigen analysis and prediction

[0044] The peripheral blood and lung cancer tumor tissue of 1 lung cancer patient were collected by clinical sampling. DNA extraction was performed on the peripheral blood, and genomic data was obtained by exome sequencing. DNA and RNA extraction was performed on the tumor tissue at the same time, and exome sequencing and transcriptome sequencing were performed respectively to obtain genomic data and transcriptome data of the tumor tissue.

[0045] The raw data of high-throughput sequencing is subjected to reference genome-based alignment, repeat sequence labeling, and short insertion-deletion re-alignment, and finally the qualified alignment genome data and alignment transcriptome data are obtained. Based on these data, the HLA genotype of the patient is obtained using HLA genotype typing software; the genomic copy number change data in the tumor tissue is obtained using copy number analysis software; the purity and clonal structure of the tumor tissue are obtained using purity analysis software; the somatic mutation in the tumor tissue is obtained using somatic mutation analysis software; the mutated polypeptide sequence with amino acid change is obtained using mutation site translation software; and the gene expression level data is analyzed using gene expression quantification software.

[0046] After obtaining the neoantigen-related data, we use HLA-neoantigen binding prediction software to analyze whether the mutated polypeptide sequence can effectively bind to the HLA molecule (IC50 value < 500 nM). And on this basis, combined with polypeptide expression and other conditions, the top 15 mutated polypeptides with IC50 value are selected as the candidate tumor neoantigens (see Table 2).

[0047] Table 2 Information of candidate tumor neoantigens

[0048]

[0049] Example Two Results of Antigen Peptide Activating Dendritic Cells through MHC-II Pathway

[0050] 1. Collection and processing of peripheral blood PBMC

[0051] The patient in Example One was subjected to apheresis using a mononuclear cell collection system to obtain mononuclear cells in the patient's peripheral blood. The patient contains HLA-DRB1*09:01 typing.

[0052] a) Dilute the mononuclear cells with physiological saline 1:1, add human lymphocyte separation medium, and centrifuge, carefully aspirate the white membrane layer where the mononuclear cells are located to another clean centrifuge tube;

[0053] b) After washing with physiological saline twice and AIM-V cell culture medium once, count the cells, and take an appropriate amount of cell cryopreservation sample.

[0054] 2. Dendritic cell (DC) culture and co-incubation with neoantigens

[0055] a) Culture the PBMC obtained in step 1 in an incubator to allow the mononuclear cells to adhere;

[0056] b) Separate the adherent cells for culture, and add recombinant human GM-CSF and recombinant human IL-4 to the culture medium to induce mononuclear cells to differentiate into DC cells;

[0057] c) After 6 days of culture, DC maturation factors LPS, IFN-gamma are added to the cell culture medium to induce DC maturation;

[0058] d) After 7 days of culture with maturation factors, the mature DC cells are harvested, the antigen peptides selected in Example 1 are grouped according to HLA, and a total of 5 groups of DC cells are set up, the HLA-grouped antigen peptides are added to the corresponding groups of DC cells, and after 4-6 hours of incubation, the cells are stored for later use.

[0059] e) The five groups of incubated DC cells are returned to the patient for clinical treatment. Subcutaneous injection is performed in the axillary and inguinal lymph node drainage area. The return is performed at weeks 1, 2, 4, 6, and 8, and the completion of the 5 injections constitutes one course of immunotherapy. After one course of immunotherapy, the efficacy is evaluated, and if effective, the treatment is continued the following week, and if ineffective, the treatment is terminated. The amount of cells returned each time is 3*10 8 cells.

[0060] After one treatment cycle, the patient's peripheral blood is drawn for effect identification.

[0061] 3. Enzyme-linked immunosorbent assay (ELISPOT method)

[0062] a) T cells are isolated from the peripheral blood of the patient treated as described above, and are grouped according to antigen peptides, and different antigen peptides are added respectively;

[0063] b) After 20 hours of incubation, the cells are washed with deionized water;

[0064] c) Biotin-labeled IFN-gamma antibody is added and incubated for 1 hour, and after incubation, the cells are washed again;

[0065] d) Enzyme-labeled avidin is added and incubated for 1 hour, and after incubation, the cells are washed again;

[0066] e) Color developing solution is added and incubated, and after the spots grow to an appropriate size, the color development is terminated and ELISPOT plate spot counting is performed using software.

[0067] 4. Result analysis

[0068] The activation effect of the selected 15 tumor neoantigen polypeptides on the patient's T cells is systematically analyzed by ELISPOT plate spot counting (see Table 1). Figure 1The polypeptide M9 has the best activation effect (the average number of spots in the experimental small holes is 1050), and the average number of spots of the control normal polypeptide is 143, and the polypeptide is a polypeptide GKEEYSAMRDQYMRT derived from the Q61K mutation of NRAS. The experimental results show that in the tumor patients with NRAS-Q61K somatic mutation and HLA-DRB1*09:01 genotype, the M9 polypeptide GKEEYSAMRDQYMRT can significantly activate human specific T cells against NRAS mutant peptides, so as to increase the killing ability of T cells to cancer cells with NRAS mutation.

[0069] In summary, the antigenic peptide of the present application has been verified by immunological experiments in human experiments, so as to make up for the blank of individualized antigenic peptides in the treatment of tumor patients with NRAS-Q61K somatic mutation and HLA-DRB1*09:01 genotype; the antigenic peptide of the present application can significantly activate human specific T cells against NRAS mutant peptides, increase the killing ability of T cells to cancer cells with NRAS mutation, and is beneficial to large-scale synthesis for use in standardized individualized tumor immunotherapy.

[0070] The above only describes the preferred embodiments of the present application, and does not limit the embodiments and protection scope of the present application. It should be realized by those skilled in the art that any equivalent replacement and obvious changes made according to the content of the present application should be included in the protection scope of the present application.

Claims

1. A tumor neoantigen polypeptide, characterized in that: Its amino acid sequence is GKEEYSAMRDQYMRT (SEQ ID No. 17).

2. The use of the tumor neoantigen polypeptide of claim 1 in the preparation of an immunotherapeutic drug for tumors with Q61K mutations associated with NRAS; wherein the tumor is a tumor in a lung cancer patient with human leukocyte HLA typing HLA-DRB1*09:01; wherein the tumor is lung cancer.

3. A gene encoding the tumor neoantigen polypeptide of claim 1.

4. A vector loaded with the gene of claim 3.

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