CN115850377A - Tumor neoantigen polypeptide based on NRAS gene Q61K mutation and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a tumor neoantigen polypeptide related to Q61K mutation on NRAS (tumor-driving gene-based oncogene) and application thereof. The technical problem to be solved by the invention is to provide a specific immunotherapy scheme aiming at the Q61K mutation of a tumor driving gene NRAS based on MHC-II molecules aiming at the defects in the prior art. The technical scheme for solving the technical problems is to provide a tumor neoantigen polypeptide based on NRAS gene Q61K mutation. The amino acid sequence of the antigen peptide is GKEEYSAMRDQYMRT. The antigen peptide can obviously activate human body-specific T cells aiming at NRAS mutation so as to increase the killing capability of the T cells to NRAS mutation tumor cells, can prepare medicines for preventing and treating NRAS mutation tumors, and is particularly used for preparing tumor cell vaccines.

Description

Tumor neoantigen polypeptide based on NRAS gene Q61K mutation and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a tumor neoantigen polypeptide related to Q61K mutation based on a tumor driver gene NRAS and application thereof.

Background

Human RAS (Rat sarcomas) tumor driver genes, including HRAS, KRAS and NRAS genes, are mutated in about 30% of malignancies. Activation of NRAS in the plasma membrane leads to protein phosphorylation and signal transduction, promoting cell growth, proliferation and differentiation. Unlike the G12, G13 and Q61 site mutations, which occur primarily in the activating mutations, the mutation at the Q61 position enhances GTP binding and thereby reduces GTP hydrolysis, which ultimately leads to overactivation of RAS, carcinogenic signaling and drug resistance. NRAS Q61K mutations occur mainly in various types of tumors such as melanoma, colorectal cancer, thyroid cancer, and lung cancer. However, because of the specificity of NRAS proteins themselves, it is very challenging to develop therapeutic approaches that target NRAS. Thus, despite over 30 years of intensive research, effective anti-RAS therapies have not entered the clinic.

Immunotherapy of tumors is considered as a new generation of tumor treatment following surgery, radiotherapy, chemotherapy and small molecule targeted therapy, which differs from the previous traditional treatment methods in that: the immunotherapy changes the treatment means from directly killing tumor cells by medicines to enhancing immune cells, treats tumors by improving the anti-tumor immunity of patients, and has the advantages of accurate killing, small side effect, lasting curative effect, high degree of individuation and the like compared with the traditional therapy. In addition, the immune system of the body has the characteristic of immunological memory, so that immunotherapy can help patients to form memory type immunity, and has remarkable advantages in preventing tumor recurrence and metastasis. Peptide-based vaccines present tumor mutant peptides to MHC molecules to induce the production of specific and long-term memory T cells to fight tumors. Tumor antigens are generally considered endogenous antigens that, in combination with MHC class I molecules, stimulate a CD8+ cytotoxic response. Also a small fraction of the larger polypeptides bind to MHC II molecules and specifically stimulate CD4+ T helper cells.

Given the high frequency of NRAS mutations in tumors, they have become potential targets for immunotherapy. It has been found that the mutated NRAS peptides can induce specific CD8+ and CD4+ T cell anti-RAS immune responses, and that the NRAS mutant peptides have enhanced immunogenicity. Therefore, a vaccine targeting the NRAS mutant peptide can be used as a specific immunotherapy approach for NRAS mutant tumor patients. Among the existing studies, research on NRAS has mainly focused on MHC class I molecules, for example, researchers found that in melanoma patients of HLA-base:Sub>A × 01 genotype, the use ofbase:Sub>A novel antigen against ras.q61k (ILDTAGKEEY, SEQ ID No. 31) can effectively activate T cells in patients and kill tumors (Peri et al, J Clin invest.2021oct 15), but only in patients containing HLA-base:Sub>A × 01 genotype. RAS neoantigens based on MHC-class II molecules have not been reported at present.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a specific immunotherapy scheme aiming at Q61K mutation on tumor driving gene NRAS based on MHC-II molecules aiming at the defects in the prior art.

The technical scheme for solving the technical problems is to provide a tumor neoantigen polypeptide based on NRAS gene Q61K mutation. The amino acid sequence of the antigen peptide is GKEEYSAMRDQYMRT (SEQ ID No. 17).

Or, the polypeptide is the same or similar in function obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of each polypeptide.

Wherein the same or similar function as described above means that the antigenic polypeptide is capable of activating T cells specific for tumors with NRAS Q61K mutation in patients HLA-typed HLA-DRB1 x 09.

The invention also provides the application of the tumor neoantigen polypeptide in preparing an immune activity regulator capable of inducing the generation of specific cytotoxic T cell clone.

The invention also provides application of the tumor neoantigen polypeptide in preparing a tumor risk intervention and/or treatment agent with NRAS high-frequency mutation. Wherein the NRAS high-frequency mutation is Q61K mutation.

Based on the scheme, the invention further provides the DC cell. The DC cell is obtained by the stimulation of the tumor neoantigen polypeptide. Further, the stimulation mode in the DC cells is that the DC cells and the tumor neoantigen polypeptide are incubated together.

Wherein the DC cell is a mature DC cell.

Wherein the DC cells are isolated DC cells of patients with Q61K mutation in NRAS.

Further, the DC cells are isolated DC cells of a patient who contains human leukocytes and has HLA-DRB1 × 09.

The invention also provides the application of the tumor neoantigen polypeptide and the DC cell in preparing an immunotherapy medicament for tumors accompanied with NRAS mutation.

Wherein, the tumor accompanied with NRAS mutation in the application is a tumor with NRAS undergoing Q61K mutation.

Wherein the tumor in the above application is melanoma, colorectal cancer, thyroid cancer and lung cancer.

Wherein, the tumor in the application is the tumor of a patient with human leukocyte HLA-DRB1 x 09.

In addition, the present invention also provides the above antibody against the tumor neoantigen polypeptide of claim 1.

Wherein, the antibody is a polyclonal antibody or a monoclonal antibody.

Further, the above-described antibody may also form a conjugate with a conjugate moiety. The coupling 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, and a biotin.

The invention also provides a gene encoding the tumor neoantigen polypeptide or the antibody.

Meanwhile, the invention also provides a vector loaded with the gene. Furthermore, the vector is an expression vector, and the expression vector can be selected from common vectors such as a plasmid vector, an adenovirus vector, a lentivirus vector or an adeno-associated virus vector. When an adenovirus vector is used, a replication-defective adenovirus vector is generally used.

The invention has the beneficial effects that: the antigenic peptide can obviously activate human body specific T cells aiming at NRAS mutation so as to increase the killing capacity of the T cells to NRAS mutation tumor cells, and can prepare medicaments for preventing and treating NRAS mutation tumors. After the DC cells of the NRAS mutant tumor patient stimulated by the antigenic peptide are returned back to the patient body, the DC cells of the NRAS mutant tumor patient can activate T cells specific to the NRAS mutant peptide so as to increase the killing capacity of the T cells to cancer cells with NRAS mutation. The antigenic peptide of the invention fills the gap of the individual antigenic peptide in the treatment of tumor patients with NRAS-Q61K somatic mutation and HLA-DRB1 x 09. Meanwhile, the antigen of the invention is convenient for large-scale synthesis, and can be used for the standardized and individualized immunotherapy of NRAS mutation tumor patients.

Drawings

FIG. 1 shows the results of ELISA experiments.

Detailed Description

The invention obtains a series of antigen peptides by using a biological information prediction method based on the amino acid sequence of NRAS gene mutation peptide and combined with human leukocyte antigen haplotype in MHC-II molecules. And further screening to obtain a new antigen peptide with the amino acid sequence of GKEEYSAMRDQYMRT, which is named as M9. The human leukocyte antigen typing corresponding to the new antigen peptide M9 is HLA-DRB1 x 09, namely, the antigen can enable a patient who has HLA-DRB1 x 09 typing and NRAS gene Q61K mutation to activate DC cells specific to NRAS mutation so as to increase the killing capacity of T cells to NRAS mutation tumor cells.

Those skilled in the art know that functionally identical or similar polypeptides obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the GKEEYSAMRDQYMRT antigen polypeptide are also within the scope of the present invention. Wherein said same or similar function means that the antigenic polypeptide activates T cells specific for tumors with NRAS Q61K mutation in patients HLA-typed HLA-DRB1 x 09.

In the present invention, 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 fragment" 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, and most preferably 1 to 3) amino acids, and addition of one or several (up to 40, usually up to 20, preferably up to 10, and more preferably up to 5) amino acids at the C-terminus and/or the N-terminus. For example, substitutions with amino acids having similar or similar properties in the polypeptide will generally not alter function. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein or polypeptide. The term also includes active fragments and active derivatives of the polypeptides.

The expression "peptide fragment obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of each peptide fragment" also includes, but is not limited to, polypeptides formed by substituting at most 10 (i.e., one or several), preferably at most 8, more preferably at most 5 (5, 4, 3, 2 or 1) amino acids with amino acids having similar or similar properties, i.e., conservative variant polypeptides. Further, these conservative variant polypeptides may be generated by making substitutions according to table 1.

TABLE 1 amino acid substitution Table

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；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)	Ile；Leu；Met；Phe；Ala	Leu

The polypeptide can be used as an active ingredient to prepare anti-tumor medicaments. In general, one skilled in the art can use the above polypeptides as antigen active ingredients to prepare vaccines for preventing and/or treating NRAS-mutated tumors. The vaccine takes the polypeptide as an antigen component and pharmaceutically acceptable auxiliary materials or auxiliary components.

In the preparation of vaccines, immunological adjuvants are often added to enhance the immune response of the body to the vaccine. Wherein 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.

Naturally, antibodies against the above-mentioned proteins can be easily obtained in the art based on the polypeptides described in the present invention. The antibody is a polyclonal antibody or a monoclonal antibody; preferably a monoclonal antibody. The above antibodies may also form conjugates with a conjugate moiety. Further, the coupling 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, and a biotin. Antibodies that specifically bind to the aforementioned proteins can be used, on the one hand, for the preparation of a medicament for the prophylaxis and/or treatment of tumors that are mutated in NRAS, and, on the other hand, for the immunodetection of tumors that are mutated in NRAS.

In addition, the present invention also encompasses a gene encoding the above protein. The coding gene of the protein can be used for expressing and preparing the polypeptide; on the other hand, the recombinant expression vector can be loaded in an expression vector in an operable way, and further can be prepared into vector vaccines or vector medicaments. Expression can be selected from commonly used vectors such as plasmid vectors, adenovirus vectors, lentiviral vectors, or adeno-associated virus vectors. When an adenovirus vector is used, a replication-defective adenovirus vector is generally used.

In some cases, the tumor neoantigen polypeptide of the present invention further includes an antigenic peptide having a length of not more than 40 amino acids, the amino acid sequence of which comprises the sequence set forth in SEQ ID No.17 and still having the same or similar function as the neoantigen peptide M9. Furthermore, the amino acid sequence of the tumor neoantigen polypeptide comprises the sequence of SEQ ID No.17, and the number of amino acids is not more than 30; it still has the same or similar function as the neoantigenic peptide M9.

Further, the tumor neoantigen polypeptide leaf of the present invention includes a polypeptide formed by connecting 1 to 20 amino acids at the nitrogen terminal of the sequence of SEQ ID No.17 and/or connecting no more than and no more than 1 to 20 amino acids at the carbon terminal of the sequence of SEQ ID No.17, which still has the same or similar function as the neoantigen peptide M9.

That is, according to the present invention, an amino acid sequence with a certain length can be added to either or both sides of the tumor neoantigen polypeptide M9 having the sequence shown in SEQ ID No.17, and still an antigen polypeptide that can activate T cells of a tumor having NRAS Q61K mutation, which is specific to a patient who is HLA-DRB1 x 09.

It is understood that the above amino acid sequences added to either or both sides of the tumor neoantigen polypeptide M9 may be derived from both sides of the corresponding site of GKEEYSAMRDQYMRT in NRAS protein.

The new antigenic peptide M9 can obviously activate human body specific T cells aiming at NRAS mutation, and increase the killing capacity of the T cells to NRAS mutation tumor cells. On the basis, the invention naturally also provides the application of the series of new antigenic peptides in the preparation of the immunotherapy drugs of NRAS mutated tumors. When the above neoantigen peptide is used for treating NRAS-mutated tumors, a tumor patient having NRAS-mutated tumor is inoculated with a vaccine obtained by stimulating dendritic cells with a neoantigen, and then activated and proliferated T lymphocytes to start to attack cancer cells targeting the neoantigen peptide as a recognition target. On the basis, the invention also provides a DC cell which is obtained by stimulating the tumor neoantigen polypeptide. Stimulation is typically by co-incubation of the DC cells with the tumor neoantigen polypeptide. The DC cells are usually ex vivo DC cells. Naturally, the DC cells described above are ex vivo DC cells of patients with Q61K mutation in NRAS in tumors. Generally, ex vivo DC cells are cultured to maturity before a neoantigen stimulation step is performed. Naturally, the DC cells described above are ex vivo DC cells of a patient who contains human leukocytes and is HLA-DRB1 x 09.

The invention also provides the application of the tumor neoantigen polypeptide and the DC cell in preparing an immunotherapy medicament for tumors accompanied with NRAS mutation. The tumor accompanied with NRAS mutation in the application is a tumor with NRAS having Q61K mutation. Tumors in which Q61K mutation has been caused by NRAS include various tumors such as melanoma, colorectal cancer, thyroid cancer, and lung cancer. Of course, this is mainly applicable to patients having the above-mentioned tumor, who have human leukocytes and have HLA-DRB1 x 09.

The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without inventive efforts based on the embodiments of the present invention, shall fall within the scope of protection of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

Example 1 neoantigen analysis and prediction

Peripheral blood and lung cancer tumor tissues of 1 lung cancer patient were collected by clinical sampling. Peripheral blood is subjected to DNA extraction and exome sequencing to obtain genome data, tumor tissues are simultaneously subjected to DNA and RNA extraction, and exome sequencing and transcriptome sequencing are respectively carried out to obtain the genome data and the transcriptome data of the tumor tissues.

And (3) carrying out comparison based on a reference genome, marking of a repeated sequence and the weight comparison of short insertion deletion on the original data of the high-throughput sequencing, and the like to finally obtain the comparison genome data and the comparison transcriptome data with qualified quality control. Based on these data, obtaining the HLA genotype of the patient using HLA genotyping software; obtaining data on changes in genome copy number in tumor tissue using copy number analysis software; obtaining the purity and the clone structure of the tumor tissue by using purity analysis software; obtaining somatic mutations in tumor tissue using somatic mutation analysis software; obtaining a mutant polypeptide sequence with changed amino acids by using mutation site translation software; data on gene expression levels were analyzed using gene expression quantification software.

After obtaining neoantigen-associated data, we analyzed whether the mutant polypeptide sequences could efficiently bind to HLA molecules using HLA-neoantigen binding prediction software (IC 50 values <500 nM). Based on the above, the mutant polypeptide with IC50 value of top 15 is selected as candidate tumor neoantigen (see Table 2) in combination with polypeptide expression.

TABLE 2 information on candidate tumor neoantigens

EXAMPLES results of antigenic peptides activating dendritic cells through MHC-II pathway

1. Peripheral blood PBMC collection and processing

The patient in the first example was mechanically harvested using a monocyte harvesting system to obtain monocytes in the peripheral blood of the patient. The patient contained HLA-DRB1 x 09.

a) Diluting the mononuclear cells by using normal saline 1;

b) After washing twice with physiological saline and once with AIM-V cell culture medium, the cells were counted, and a suitable amount of cells were frozen to leave specimens.

2. Dendritic Cell (DC) culture and co-incubation with neoantigens

a) Carrying out cell culture on the PBMC obtained in the step 1, and incubating in an incubator to allow the mononuclear cells to adhere to the wall;

b) Separating adherent cells for culturing, and adding recombinant human GM-CSF and recombinant human IL-4 into a culture medium to induce monocytes to differentiate into DC cells;

c) After culturing for 6 days, adding DC maturation promoting factors LPS and IFN-gamma into the cell culture medium to induce DC maturation;

d) After 7 days of culture with addition of maturation promoting factors, mature DC cells were harvested, the antigen peptides selected in example 1 were grouped according to HLA grouping, a total of 5 groups of DC cells were set, each group of HLA-grouped antigen peptides was added to each corresponding group of DC cells, and the cells were incubated for 4 to 6 hours, respectively, and then cryopreserved for use.

e) The five groups of incubated DC cells were returned to the patient for clinical treatment. Subcutaneous injections were made in the axillary and inguinal lymph node drainage areas. The feedback is respectively carried out at 1,2,4,6 and 8 weeks, the completion of the 5 injections is an immunization course, the curative effect is evaluated after the immunization course, the treatment is started from the next week if the curative effect is effective, and the treatment is stopped if the curative effect is ineffective. Cell mass 3 x 10 per reinfusion ⁸ And (4) cells.

After one treatment cycle, peripheral blood was drawn from the patient for efficacy assessment.

3. Enzyme-linked immunosorbent assay (ELISPOT method)

a) Separating the peripheral blood of the patient after the back transfusion treatment to obtain T cells, grouping according to the antigen peptides, and adding different antigen peptides respectively;

b) After incubation for 20 hours, cells were washed with deionized water;

c) Adding IFN-gamma antibody marked by biotin for incubation for 1 hour, and washing cells after incubation is finished;

d) Adding enzyme-labeled avidin, incubating for 1 hour, and washing the cells;

e) Adding a color development solution, incubating, stopping developing after the spots grow to the proper size, and counting the spots on the ELISPOT plate by using software.

4. Analysis of results

The activation effect of the selected 15 tumor neoantigen polypeptides on the patient's T cells was systematically analyzed by ELISPOT plate spot counting (see fig. 1, the arabic number at the bottom left of each field image is the number of spots in the wells in the experiment), and the polypeptide labeled M9 was found to have the best activation effect (mean of the number of spots in the wells in the tumor neoantigen group was 1050), while the mean of the number of spots in the control normal polypeptide was 143, which is the NRAS-derived Q61K-mutated polypeptide GKEEYSAMRDQYMRT. The experimental result shows that in NRAS-Q61K somatic mutation and HLA-DRB1 x 09.

In conclusion, the antigen peptide of the invention has verified the individuation effectiveness in human body experiments by using immune experiments, thereby making up the blank of the individuation antigen peptide in the treatment of tumor patients with NRAS-Q61K somatic mutation and HLA-DRB1 x 09; the antigen peptide can obviously activate the T cell of human body specificity aiming at the NRAS mutant peptide, increases the killing capability of the T cell to the cancer cell with NRAS mutation, and is beneficial to large-scale synthesis so as to be used in standardized individualized tumor immunotherapy.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims (18)

1. A tumor neoantigen polypeptide, characterized by: the amino acid sequence is GKEEYSAMRDQYMRT (SEQ ID No. 17);

or a polypeptide having the same or similar function obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the above polypeptide.

2. Use of a tumor neoantigen polypeptide of claim 1 in the preparation of a modulator of immune activity that induces the production of specific cytotoxic T cell clones.

3. Use of the tumor neoantigen polypeptide of claim 1 in the preparation of a tumor risk intervention and/or treatment agent with high-frequency mutation of NRAS.

4. The use of claim 3, wherein the NRAS high frequency mutation is a Q61K mutation.

DC cells, characterized in that they have been stimulated with a tumor neoantigen polypeptide according to claim 1.

6. The DC cell according to claim 6, wherein the stimulation is co-incubation of the DC cell with a tumor neoantigen polypeptide.

7. The DC cell according to claim 6, wherein the DC cell is a mature DC cell.

8. The DC cell according to claim 7, wherein the DC cell is an ex vivo DC cell of a patient having Q61K mutation in NRAS.

9. The DC cell according to claim 7, wherein the DC cell is an ex vivo DC cell of a patient who contains human leukocytes and is HLA-DRB1 x 09.

10. Use of a tumor neoantigen polypeptide of claim 1 and DC cells of any one of claims 5 to 9 in the manufacture of a medicament for immunotherapy of tumors associated with NRAS mutations.

11. The use according to claim 10, wherein said tumor associated with NRAS mutation is a tumor in which NRAS has Q61K mutation.

12. The use according to claim 11, wherein said tumor is melanoma, colorectal cancer, thyroid cancer, or lung cancer.

13. The use according to claim 11, wherein the tumor is a tumor of a patient having human leukocytes with HLA-DRB1 x 09.

14. An antibody against the tumor neoantigen polypeptide of claim 1.

15. The antibody of claim 14, wherein: is polyclonal antibody or monoclonal antibody.

16. The antibody of any one of claims 14 to 15, wherein: also forming a conjugate with a coupling moiety; the coupling 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, and a biotin.

17. A gene encoding the tumor neoantigen polypeptide of claim 1 or the antibody of any one of claims 14 to 16.

18. A vector carrying the gene of claim 17.

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