New antigen polypeptide and immune adjuvant composition of mouse MC38 colorectal cancer and its application in treating tumors

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a novel antigen polypeptide and an immune adjuvant composition of mouse MC38 colorectal cancer and their application in treating tumors. The amino acid sequences of the novel antigen polypeptides are shown in SEQ ID No.2, 13, 16, and 17. Four tumor neoantigen polypeptides with true immunogenicity are screened in the present invention. The obtained tumor neoantigens are formulated with PolyI:C into a vaccine preparation. After treatment with the MC38 tumor vaccine preparation, the growth of tumors can be effectively inhibited in mice; the survival period of mice is prolonged, and a very effective T cell response is generated in the tumor-bearing mice by the neoantigen vaccine preparation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a novel antigen polypeptide and an immune adjuvant composition of mouse MC38 colorectal cancer and their application in treating tumors. Background Art

[0002] Neoantigens are abnormal peptides generated by genetic and epigenetic alterations (point mutations, insertions / deletions, gene fusions / translocations, splicing variants, and post-translational modifications) in cancer cells, which are only expressed on cancer cells. They can be recognized by the human immune system as foreign substances and may trigger a strong immune response against tumors. The development of various neoantigen-targeted therapies has been widely studied to enhance the immune response against specific neoantigens expressed on the surface of cancer cells, thereby promoting recognition and subsequent elimination. Vaccination of patients with neoantigen vaccines can initiate and activate T cells, or enhance existing weak immune responses within lymph nodes. Compared with previous cancer vaccines, these innovative neoantigen vaccines have demonstrated feasibility, safety, and the ability to trigger vaccine-specific immune responses unprecedentedly.

[0003] The concept of neoantigen vaccines was initially proposed in the 1990s, but it was not until the emergence of high-throughput sequencing technologies and bioinformatics tools that this field began to gain momentum. Given that cancer cells often exhibit a highly variable mutation rate, neoantigen vaccines are promising as personalized therapies. Although still in the early stages of development, these vaccines have achieved positive results in clinical trials for a range of cancer types. Neoantigen vaccines have been developed in various forms, including peptide vaccines, RNA vaccines, DNA vaccines, and neoantigen-pulsed DC vaccines. These vaccines can induce immune responses in cancer patients, especially against and elimination of cancer cells. A large number of studies have shown that neoantigen vaccines have great clinical potential.

[0004] Despite certain achievements in the immunotherapy of advanced solid tumors, compared with other forms of cancer vaccines, neoantigen peptide vaccines have many advantages because they can trigger highly targeted immune responses and simplify the manufacturing process. However, these vaccines face challenges such as the complexity of neoantigen recognition and selection, limited immunogenicity, short half-life, and potential immune tolerance, and more research is still needed to address the existing limitations.

[0005] At present, the application of tumor neoantigen vaccines requires several key steps. The first step is the identification and characterization of tumor-specific neoantigens, which requires the use of advanced genome sequencing technology and bioinformatics tools to analyze tumor samples and identify unique gene mutations that produce neoantigens for each patient. Subsequently, the design of vaccines containing the identified neoantigens begins. The process involves selecting the most effective neoantigens and formulating the neoantigens in a way that enhances immunogenicity and stability. Neoantigens that can effectively stimulate autologous specific immune responses and kill tumor cells are screened out, and then advanced chemical synthesis technology is used to prepare the corresponding neoantigen vaccines for clinical application in cancer immunotherapy. The third step involves the use of in vitro models to evaluate safety, efficacy, and immunogenicity. This process requires the use of immunological assays to quantify the immune response of tumor neoantigen vaccines and evaluate the ability of tumor neoantigen vaccines to inhibit tumor growth and metastasis. Finally, clinical trials on humans are essential for testing the safety and efficacy of vaccines. Therefore, the formulation of neoantigen vaccines and their functional validation are extremely critical steps in application.

[0006] Personalized tumor neoantigen vaccines are a new direction of immunotherapy with great potential. However, there are still some problems to be solved in their screening, identification, and preparation:

[0007] (1) The steps for obtaining tumor neoantigens are rather complicated, involving multiple steps including sample sequencing, analysis, prediction, and in vitro verification, which is expensive and time-consuming.

[0008] (2) Tumors produce a large number of mutations, and only a very small proportion of mutation sites can induce autologous anti-tumor immune responses. Therefore, the accuracy of bioinformatics prediction is crucial to obtaining positive tumor neoantigens. However, the accuracy of prediction software in this field is generally low, and a large number of in vitro validation experiments are required for screening, making it extremely difficult to obtain positive sequences.

[0009] (3) Units engaged in the production of tumor neoantigen vaccines and basic research need to have multidisciplinary and multi-equipment platform conditions to meet the needs of subsequent research.

[0010] The present invention identifies and screens tumor-specific neoantigens, synthesizes neoantigen peptides, and prepares tumor neoantigen vaccines in combination with PolyI:C. Two main goals are achieved: first, to screen out tumor-specific neoantigens; second, to develop highly specific tumor neoantigen vaccine products. The vaccine composed of multiple neoantigen peptides and the immunomodulator PolyI:C is expected to improve the specificity and efficacy of neoantigen vaccine treatment, as well as reduce the risk of immune tolerance and side effects. Using the tumor neoantigen vaccine of mouse MC38 colorectal cancer as a model, combined with the early development and scientific research of tumor neoantigen vaccines, may be an important strategy to further enhance the efficacy of immunotherapy for solid tumors, and provide a reliable design and decision-making tool for preclinical tumor vaccines and their applications. Summary of the Invention

[0011] In view of the above technical problems, the present invention innovatively develops and verifies a personalized tumor neoantigen vaccine preparation and its functions. A neoantigen vaccine preparation for mouse MC38 colorectal cancer and a preparation method thereof are provided.

[0012] First, research shows that the main sources of tumor neoantigens include mutations generated by non-synonymous mutations, alternative splicing, post-translational modifications, etc. By performing mRNA sequencing and whole exome sequencing (WES) analysis on the MC38 tumor cell line, and then predicting the binding affinity of each mutant peptide to MHC molecules by computer; predicting the binding affinity and stability of mutant peptides to HLA molecules; finally, scoring the neoantigen mutant sequences using a combination of multiple methods. Then, the top 24 mutant sequences with the highest scores are selected as candidate neoantigen peptides (see Table 1) and synthesized and purified by chemical synthesis. After immunizing mice, the polypeptides with immunogenicity are screened out as tumor neoantigen vaccines, and the detailed information of the neoantigen sequences is shown in Table 3. By combining the use of PolyI:C, a neoantigen vaccine preparation is prepared to enhance the anti-tumor effect.

[0013] According to the first aspect of the technical solution of the present invention, in view of the deficiencies of the prior art, the present invention provides a neoantigen polypeptide for mouse MC38 colorectal cancer, and the amino acid sequence of the neoantigen polypeptide for mouse MC38 colorectal cancer is shown in SEQ ID No. 1-24.

[0014] In some embodiments, the amino acid sequence of the neoantigen polypeptide for mouse MC38 colorectal cancer is shown in SEQ ID No. 2, 13, 16, 17.

[0015] According to the second aspect of the technical solution of the present invention, the present invention provides an immunoadjuvant composition containing the above-mentioned neoantigen polypeptide for mouse MC38 colorectal cancer.

[0016] In some embodiments, the immunoadjuvant composition further contains an adjuvant.

[0017] In some embodiments, the adjuvant is PolyI:C.

[0018] In some embodiments, the mass fraction ratio of the neoantigen polypeptide for mouse MC38 colorectal cancer to the adjuvant in the immunoadjuvant composition is 1:1.

[0019] In some embodiments, the administration dose of the neoantigen polypeptide for mouse MC38 colorectal cancer and the adjuvant in the immunoadjuvant composition is 50 μg / peptide + 50 μg Poly I:C.

[0020] According to the third aspect of the technical solution of the present invention, the present invention provides the use of the above-mentioned mouse MC38 colorectal cancer neoantigen polypeptide or the above-mentioned immunoadjuvant composition in the preparation of a tumor neoantigen vaccine.

[0021] According to the fourth aspect of the technical solution of the present invention, the present invention provides a biological material, and the biological material is any one of the following:

[0022] C1) A nucleic acid molecule encoding the above-mentioned mouse MC38 colorectal cancer neoantigen polypeptide;

[0023] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0024] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0025] C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

[0026] C5) A recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

[0027] According to the fifth aspect of the technical solution of the present invention, the present invention provides the use of the above-mentioned biological material in the preparation of a tumor neoantigen vaccine.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] (1) Twenty-four candidate tumor neoantigen peptides predicted by computer are combined with PolyI:C and subcutaneously immunized into mice. After three immunizations, the immune response induced by the neoantigens can be enhanced, and four tumor neoantigen polypeptides with true immunogenicity are screened by the Elispot technique ( Figures 3A - 3B ).

[0030] (2) The tumor neoantigens obtained by this method are combined with PolyI:C to prepare a vaccine preparation. After treatment with the MC38 tumor vaccine preparation, the growth of tumors in mice can be effectively inhibited ( Figure 4A ); the survival period of mice can be prolonged ( Figure 4B ).

[0031] (3) After treatment with the MC38 tumor neoantigen vaccine preparation, the proportions of CD3 + T cells, CD4 + T cells, CD8 + T cells and effector memory T cells (Tem) in the spleen of tumor-bearing mice are all significantly increased compared with the control group ( Figure 5). These data indicate that the MC38 tumor neoantigen vaccine preparation generated a very effective T cell response in tumor-bearing mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 . Flow chart for the identification of tumor neoantigen epitopes of mouse tumor cell lines;

[0033] Figure 2 . Immunization flow chart for neoantigen screening;

[0034] Figure 3A . Elispot results showing the immunogenicity of MC38 tumor neoantigen polypeptides;

[0035] Figure 3B . Statistical analysis of the results of Elispot-verified immunogenic peptides of MC38;

[0036] Figure 4A . Schematic diagram of the treatment of subcutaneous tumor model mice with personalized tumor neoantigen vaccines;

[0037] Figure 4B . Tumor growth curve of MC38 model mice treated with MC38 tumor neoantigen vaccines;

[0038] Figure 4C . Kaplan-Meier comparison of survival differences in MC38 model mice treated with tumor neoantigen vaccines;

[0039] Figure 5 . Flow cytometry analysis was used to evaluate the effect of tumor neoantigen vaccine treatment on mouse immune cells; the data are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant. Different significance levels are divided into: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0041] Example 1. Screening of neoantigen candidates for tumor cell lines

[0042] The method includes the following steps:

[0043] (1) Prediction of candidate neoantigen peptides:

[0044] (1.1): Perform mRNA sequencing and whole exome sequencing (WES) analysis on the MC38 tumor cell line;

[0045] (1.2): Score the neoantigen mutant sequences using a combination of multiple methods and select the top 24 mutant sequences with the highest scores as candidate neoantigen peptides. The results are shown in Table 1.

[0046] Table 1. MC38 Candidate Neoantigen Peptide Sequences

[0047] Polypeptide number Gene Wild - type polypeptide sequence Mutant polypeptide sequence Result HLA - typing Score TP01 Ndufs6 AVLTFRRL SEQ ID No.1:AALTFRRL missense_variant H - 2 - Kb 119 TP02 Ugt1a6a YEGICNGVPM SEQ ID No.2:YEGICNGVLM missense_variant H - 2 - Db 453 TP03 Tnks2 SCYICHRQL SEQ ID No.3:SSYICHRQL missense_variant H - 2 - Kb 597 TP04 Pigu FWYFFAEM SEQ ID No.4:FLYFFAEM missense_variant H - 2 - Kb 707 TP05 Itch VAREWFFLL SEQ ID No.5:VASEWFFLL missense_variant&splice_region_variant H - 2 - Kb 756 TP06 Pik3ca SMLLSSEQL SEQ ID No.6:SMLLSSEPL missense_variant H - 2 - Db 837 TP07 Wdr11 FAIRMCPPL SEQ ID No.7:FAILMCPPL missense_variant H - 2 - Db 1058 TP08 Spg7 FNFEYAVER SEQ ID No.8:FNFEYAVEL missense_variant H - 2 - Kb 1074 TP09 Ankrd27 LIFKYVGTM SEQ ID No.9:LIFKYLGTM missense_variant H - 2 - Kb 1077 TP10 Tyk2 LVMEYVPL SEQ ID No.10:MVMEYVPL missense_variant H - 2 - Kb 1159 TP11 Sptbn1 SMVRDLML SEQ ID No.11:SMVRYLML missense_variant H - 2 - Kb 1186 TP12 Grik5 STMTFFQNS SEQ ID No.12:SSMTFFQNS missense_variant H - 2 - Kb 1219 TP13 Rybp VTVGNVTVI SEQ ID No.13:VTVRNVTVI missense_variant H - 2 - Db 1241 TP14 Ddx17 SSQQFSGI SEQ ID No.14:SSLQFSGI missense_variant H - 2 - Kb 1247 TP15 Lrrc8a LIDQYDPL SEQ ID No.15:LIYQYDPL missense_variant H-2-Kb 1292 TP16 Nmt1 YTLLNENYV SEQ ID No.16: YSLLNENYV missense_variant H-2-Db 1337 TP17 Aldh7a1 INYSTSLPL SEQ ID No.17: INYSSSLPL missense_variant H-2-Kb 1347 TP18 Pitpnm1 SSLLLADTL SEQ ID No.18: SSLLLSDTL missense_variant&splice_region_variant H-2-Db 1349 TP19 Xpo5 VSALFPFV SEQ ID No.19: VSALFSFV missense_variant H-2-Kb 1431 TP20 Dock8 TGLLFTEL SEQ ID No.20: IGLLFTEL missense_variant H-2-Kb 1445 TP21 Nadk YQVLNEVVI SEQ ID No.21: YQVLNEVMI missense_variant H-2-Db 1549 TP22 Zfp442 KAFGFQSGL SEQ ID No.22: KAFAFQSGL missense_variant H-2-Kb 1567 TP23 Ripk1 GVWYPPNL SEQ ID No.23: VVWYPPNL missense_variant H-2-Kb 1616 TP24 Elmod3 HSFESAGL SEQ ID No.24: LSFESAGL missense_variant H-2-Kb 1648

[0048] (2): Chemically synthesize and purify the candidate neoantigen peptides.

[0049] (3): Screen for immunogenic neoantigen peptides using ELISPOT

[0050] (3.1): Mix the chemically synthesized candidate neoantigen polypeptides with the adjuvant PolyI:C, with every 4 - 5 polypeptides as a peptide pool (see Table 2 for details).

[0051] Table 2. Distribution Table of Candidate Neoantigen Polypeptide Pools

[0052] Peptide pool grouping Peptide number Peptide content ratio (50 ug / peptide) Peptide pool 1 TP01, TP02, TP03, TP06, TP07 1:1:1:1:1 Peptide pool 2 TP09, TP10, TP11, TP12, TP13 1:1:1:1:1 Peptide pool 3 TP14, TP16, TP17, TP18, TP19 1:1:1:1:1 Peptide pool 4 TP21, TP22, TP23, TP24 1:1:1:1 Peptide pool 5 TP04, TP05, TP08, TP15, TP20 1:1:1:1:1

[0053] Immunize C57BL / 6 mice subcutaneously in the inguinal region. Perform a booster immunization on the 5th and 10th days respectively. Seven days after the last immunization, take the spleens of the mice, prepare single-cell suspensions of the spleens, and screen for immunogenic polypeptides using the Elispot technique. Analyze the number of IFN-γ spots using the One-way ANOVA statistical method. Those with statistical significance are regarded as positive polypeptides, and immunogenic polypeptides are respectively screened out from the candidate neoantigen polypeptides of MC38. The sequences of the screened neoantigen polypeptides are shown in Table 3.

[0054] Table 3. MC38 Immunogenic Neoantigen Polypeptide Sequences

[0055] Peptide number Gene Wild-type peptide sequence Mutant peptide sequence Result HLA-typing Score TP02 Ugt1a6a YEGICNGVPM YEGICNGVLM missense_variant H-2-Db 453 TP13 Rybp VTVGNVTVI VTVRNVTVI missense_variant H-2-Db 1241 TP16 Nmt1 YTLLNENYV YSLLNENYV missense_variant H-2-Db 1337 TP17 Aldh7a1 INYSTSLPL INYSSSLPL missense_variant H-2-Kb 1347

[0056] The results show that: 24 candidate tumor neoantigen peptides predicted by computer, in combination with PolyI:C, can enhance the immune response induced by neoantigens through subcutaneous immunization of mice. After 3 immunizations, 4 tumor neoantigen polypeptides with true immunogenicity are screened out using the Elispot technique (see Figure 3A and Figure 3B ).

[0057] Example 2. In Vivo Efficacy Test and Pharmacokinetics Test of Tumor Neoantigen Vaccine Preparation

[0058] (2.1) Preparation of tumor neoantigen vaccine preparation, including the following steps:

[0059] (2.1.1)Mix the 4 selected neoantigen peptides of tumors together to form a tumor neoantigen, and then formulate it with the adjuvant Poly I:C at a volume ratio of 1:1 to prepare a tumor neoantigen vaccine preparation. Establish a subcutaneous tumor model in mice. Starting from the 3rd day after inoculating tumor cells subcutaneously into the left axilla of the mice, treat the tumor-bearing mice with the tumor neoantigen vaccine preparation at a dose of 50 μg / peptide + 50 μg Poly I:C. Administration method: Administer once every 3 days in the early stage for 4 times; administer once every 6 days in the later stage for 3 times; the treatment cycle is 27 days. Measure the tumor diameter with a vernier caliper twice a week. The calculation formula for tumor volume is: V = 0.5a × b 2 , where a and b represent the major axis and minor axis of the tumor respectively. The antitumor efficacy of the tumor neoantigen vaccine preparation is evaluated by the tumor growth curve. According to the results of the drug efficacy, screen out the tumor vaccine preparation with the best antitumor effect. Each data point is marked with the mean tumor volume ± standard error of the mean (SEM), n = 5.

[0060] (2.2)Pharmacodynamic testing, including the following steps:

[0061] Step 6: At the end of the experiment, take the spleen of the mice, prepare a single-cell suspension, and use flow cytometry (FCM) to detect the immune cell components in the spleen of the mice. Resuspend the cells with PBS staining buffer to a concentration of 1 × 10 6 cells / 100 μL, centrifuge at 4°C and 300 × g for 5 minutes, and remove the supernatant; resuspend the cells with 100 μL of PBS staining buffer; add 2 μL of the blocking antibody Fc Block to each well and incubate at 4°C in the dark for 10 minutes. Then add the following surface antibodies (see Table 4) and incubate at 4°C in the dark for 30 minutes.

[0062] Table 4. Flow cytometry staining antibody table

[0063]

[0064] After antibody incubation is completed, centrifuge at 300×g for 5 minutes at 4°C, remove the supernatant, resuspend the cells with 1 ml of PBS buffer, centrifuge at 300×g for 5 minutes at 4°C, remove the supernatant, add 1 ml of fixation and permeabilization solution to resuspend, fix and permeabilize in the dark at 4°C for 45 minutes, centrifuge at 300×g for 5 minutes at 4°C, remove the supernatant, and repeat once. Add the antibody against the nucleus, incubate in the dark at 4°C for 30 minutes. Centrifuge at 300×g for 5 minutes at 4°C, remove the supernatant, and repeat once; finally, resuspend the cells with 500 μL of fixative and detect by flow cytometry on the machine. Use the Guava@ easyCyte flow cytometer from Merck to detect. Collect 5000 immune cells. Analyze the flow data using FlowJo V10, Prism GraphPad 8.0 and Excel, and analyze the percentages of CD3 + T cells, CD3 + CD4 + T cells and CD3 + CD8 + T cells, Treg cells, Central memory CD3 T cells, EffectorMemory CD3 T cells in spleen immune cells. Use the One-way ANOVA statistical method to statistically analyze the data differences among multiple groups. *p<0.05; **p<0.01; ***p<0.001.

[0065] Figure 4A , Figure 4B , Figure 4C , Figure 5 's results show that the novel tumor antigen obtained by this method is formulated into a vaccine preparation in combination with PolyI:C. After mice are treated with the MC38 tumor vaccine preparation, it can effectively inhibit the growth of tumors ( Figure 4A ); extend the survival period of mice ( Figure 4B - Figure 4C ). After tumor-bearing mice are treated with the MC38 novel tumor antigen vaccine preparation, the proportions of CD3 + T cells, CD4 + T cells, CD8 + T cells and effector memory T cells (Tem) are all significantly increased compared with the control group ( Figure 5 ). These data indicate that the MC38 novel tumor antigen vaccine preparation generates a very effective T cell response in tumor-bearing mice.

[0066] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle described in the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A mouse MC38 colorectal cancer neoantigen peptide group, characterized in that: The mouse MC38 colorectal cancer neoantigen polypeptide group consists of four polypeptides with amino acid sequences as shown in SEQ ID No. 2, 13, 16 and 17.

2. An immune adjuvant composition containing the mouse MC38 colorectal cancer neoantigen polypeptide group according to claim 1.

3. The immunoadjuvant composition according to claim 2, characterized in that: The immunoadjuvant composition further comprises an adjuvant.

4. The immune adjuvant composition according to claim 3, characterized in that The adjuvant is PolyI:C.

5. The immune adjuvant composition according to claim 3, characterized in that: The mass fraction ratio of the mouse MC38 colorectal cancer neoantigen polypeptide group to the adjuvant in the immune adjuvant composition is 1:

1.

6. The immune adjuvant composition according to claim 5, characterized in that The dosage of the mouse MC38 colorectal cancer neoantigen polypeptide group and the adjuvant in the immune adjuvant composition is 50ug / peptide+50ugPoly I:C.

7. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the mouse MC38 colorectal cancer neoantigen polypeptide group according to claim 1; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

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