Mouse MC38 colorectal cancer neoantigen polypeptide and immunologic adjuvant composition and application thereof in tumor treatment

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a mouse MC38 colorectal cancer neoantigen polypeptide and immunologic adjuvant composition and application of the mouse MC38 colorectal cancer neoantigen polypeptide and immunologic adjuvant composition in tumor treatment, and amino acid sequences of the neoantigen polypeptide are shown as SEQ ID No.2, 13, 16 and 17. Four tumor neoantigen polypeptides really having immunogenicity are obtained through screening, the obtained tumor neoantigens are compatible with PolyI: C to prepare the vaccine preparation, and after mice are treated by the MC38 tumor vaccine preparation, the growth of tumors can be effectively inhibited; and the survival time of the mouse is prolonged, and the new antigen vaccine preparation generates very effective T cell reaction in the tumor-bearing mouse body.

Description

Technical Field

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

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

[0003] The concept of neoantigen vaccines was first proposed in the 1990s, but it was not until the advent of high-throughput sequencing technology and bioinformatics tools that this field began to gain momentum. Given that cancer cells often exhibit highly variable mutation rates, neoantigen vaccines hold promise as personalized treatments. Although still in the early stages of development, these vaccines have shown positive results in clinical trials across a range of cancer types. Neoantigen vaccines have been developed in a variety of formats, including peptide vaccines, RNA vaccines, DNA vaccines, and neoantigen-pulsed DC vaccines. These vaccines induce an immune response in cancer patients that specifically targets and eliminates cancer cells. A large number of studies have shown that neoantigen vaccines have great clinical potential.

[0004] Despite the success in immunotherapy of advanced solid tumors, neoantigen peptide vaccines have many advantages over other forms of cancer vaccines, as they can induce highly targeted immune responses and simplify the manufacturing process. However, these vaccines face challenges such as the complexity of neoantigen identification and selection, limited immunogenicity, short half-life, and potential immune tolerance, and more research is 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: (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.

[0007] (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.

[0008] (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.

[0009] 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

[0010] In view of the above technical problems, the present invention innovatively develops personalized tumor neoantigen vaccine preparations and verifies their functions, and provides mouse MC38 colorectal cancer neoantigen vaccine preparations and preparation methods thereof.

[0011] First, the study showed that the main sources of tumor neoantigens are mutations caused by non-synonymous mutations, selective splicing, and post-translational modifications. The mRNA sequencing and exon sequencing (WES) analysis of the MC38 tumor cell line were performed, and the binding affinity of each mutant peptide to the MHC molecule was predicted by computer; the binding affinity and stability of the mutant peptide to the HLA molecule were predicted; finally, the new antigen mutation sequence was scored using a combination of multiple methods. Then the top 24 mutant sequences were selected as candidate neoantigen peptides (see Table 1), and they were synthesized and purified by chemical synthesis. After immunizing mice, immunogenic peptides were screened as tumor neoantigen vaccines. The detailed information of the neoantigen sequence is shown in Table 3. Combined with the use of PolyI:C, it was prepared into a neoantigen vaccine preparation to improve the anti-tumor effect.

[0012] In view of the shortcomings of the prior art, according to the first aspect of the technical solution of the present invention, the present invention provides a mouse MC38 colorectal cancer neoantigen polypeptide, the amino acid sequence of the mouse MC38 colorectal cancer neoantigen polypeptide is shown in SEQ ID No.1-24.

[0013] In certain embodiments, the amino acid sequence of the mouse MC38 colorectal cancer neoantigen polypeptide is shown as SEQ ID No. 2, 13, 16, and 17.

[0014] According to the second aspect of the technical solution of the present invention, the present invention provides an immune adjuvant composition containing the above-mentioned mouse MC38 colorectal cancer neoantigen polypeptide.

[0015] In certain embodiments, the immunoadjuvant composition further comprises an adjuvant.

[0016] In certain embodiments, the adjuvant is PolyI:C.

[0017] In certain embodiments, the mass fraction ratio of mouse MC38 colorectal cancer neoantigen polypeptide to adjuvant in the immune adjuvant composition is 1:1.

[0018] In certain embodiments, the dosage of the mouse MC38 colorectal cancer neoantigen polypeptide and the adjuvant in the immune adjuvant composition is 50ug / peptide+50ugPoly I:C.

[0019] 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 immune adjuvant composition in the preparation of tumor neoantigen vaccine.

[0020] According to the fourth aspect of the technical solution of the present invention, the present invention provides a biomaterial, wherein the biomaterial is any one of the following: C1) a nucleic acid molecule encoding the above-mentioned mouse MC38 colorectal cancer neoantigen polypeptide; 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).

[0021] According to the fifth aspect of the technical solution of the present invention, the present invention provides the use of the above-mentioned biomaterial in the preparation of tumor neoantigen vaccines.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The 24 candidate tumor neoantigen peptides predicted by computer were combined with PolyI:C and subcutaneously immunized mice. After three immunizations, the immune response caused by the neoantigens was enhanced. The Elispot technology was used to screen and obtain four tumor neoantigen peptides that were truly immunogenic ( Figure 3A-3B ).

[0023] (2) The tumor neoantigen obtained by this method was combined with PolyI:C to prepare a vaccine preparation. After mice were treated with the MC38 tumor vaccine preparation, the growth of tumors was effectively inhibited ( Figure 4A ); prolong the survival of mice ( Figure 4B ).

[0024] (3) After tumor-bearing mice were treated with MC38 tumor neoantigen vaccine preparation, CD3 + T cells, CD4 + T cells, CD8 + The proportions of T cells and effector memory T cells (Tem) were significantly increased compared with the control group ( Figure 5 ). These data indicate that the MC38 tumor neoantigen vaccine formulation generated a very effective T cell response in tumor-bearing mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .Flowchart for identification of new tumor antigen epitopes in mouse tumor cell lines; Figure 2 .New antigen screening immunization flow chart; Figure 3A . Elispot validation of the immunogenicity of MC38 tumor neoantigen peptides; Figure 3B . Statistical analysis of the MC38 immunogenic peptide results verified by Elispot; Figure 4A . Schematic diagram of personalized tumor neoantigen vaccine for subcutaneous tumor model mice; Figure 4B . Tumor growth curve of MC38 model mice treated with MC38 tumor neoantigen vaccine; Figure 4C . Kaplan-Meier comparison of survival differences in MC38 model mice treated with tumor neoantigen vaccines; Figure 5 .Flow cytometry was used to evaluate the effect of tumor neoantigen vaccine therapy on mouse immune cells; data were expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant. Different significance levels were divided into: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1. Screening of candidate neoantigens of tumor cell lines The steps include: (1) Prediction of candidate neoantigenic peptides: (1.1): Perform mRNA sequencing and exome sequencing (WES) analysis on the MC38 tumor cell line; (1.2): The new antigen mutation sequences were scored using a combination of multiple methods, and the top 24 mutation sequences were selected as candidate new antigen peptides. The results are shown in Table 1.

[0028] Table 1. MC38 candidate neoantigen peptide sequences Peptide number Gene Wild-type peptide 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 Pig 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 Ryb VTVGNVTVI SEQ ID No.13: VTVRNVTVI missense_variant H-2-Db 1241 TP14 Dx17 SSQQFSGI SEQ ID No.14:SSLQFSGI missense_variant H-2-Kb 1247 TP15 Lrrc8 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 (2): Chemically synthesize and purify candidate neoantigen peptides.

[0029] (3): ELISPOT screening of new antigenic peptides with immunogenicity (3.1): Mix the chemically synthesized candidate neoantigen peptides with the adjuvant PolyI:C, with every 4 to 5 peptides forming a peptide pool (see Table 2 for details).

[0030] Table 2. Distribution of candidate new antigen peptide pool Peptide pool grouping Peptide number Peptide content ratio (50ug / 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 C57BL / 6 mice were immunized subcutaneously in the groin of mice, and booster immunization was performed on the 5th and 10th days. Seven days after the last immunization, the spleen of the mice was taken, and the spleen single cell suspension was prepared. The immunogenic peptides were screened using the Elispot technology. The number of IFN-γ spots produced was analyzed using the One-way ANOVA statistical method, and the positive peptides with statistical significance were used to screen the immunogenic peptides from the candidate new antigen peptides of MC38. The sequences of the screened new antigen peptides are shown in Table 3.

[0031] Table 3. MC38 immunogenic new antigen peptide sequences Peptide number Gene Wild-type peptide sequence Mutant polypeptide sequence result HLA-typing score TP02 Ugt1a6a YEGICNGVPM YEGICNGVLM missense_variant H-2-Db 453 TP13 Ryb 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 The results showed that the 24 candidate tumor neoantigen peptides predicted by computer were combined with PolyI:C and immunized mice subcutaneously. After three immunizations, the immune response caused by the neoantigens was enhanced. The Elispot technology was used to screen and obtain four tumor neoantigen peptides that were truly immunogenic (see Figure 3A and Figure 3B ).

[0032] Example 2: In vivo efficacy and pharmacodynamics testing of tumor neoantigen vaccine preparations (2.1) Preparation of tumor neoantigen vaccine preparations, including the following steps: (2.1.1): The four screened tumor neoantigen peptides were mixed together to form tumor neoantigens, and then mixed with the adjuvant Poly I:C in a 1:1 volume ratio to prepare a tumor neoantigen vaccine preparation. A mouse subcutaneous tumor model was established, and tumor-bearing mice were treated with tumor neoantigen vaccine preparations starting from the third day after tumor cells were inoculated subcutaneously in the axilla of the left upper limb of the mouse. The dose was 50ug / peptide + 50ugPoly I:C. Dosage method: In the early stage, the drug was administered once every 3 days for 4 times; in the later stage, the drug was administered once every 6 days for 3 times; the treatment cycle was 27 days. The tumor diameter was measured with a vernier caliper twice a week. The formula for calculating the tumor volume is: V = 0.5a×b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The tumor growth curve was used to evaluate the anti-tumor efficacy of the tumor neoantigen vaccine preparation. Based on the results of the efficacy, the tumor vaccine preparation with the best anti-tumor effect was screened. Each data point is indicated by the mean tumor volume ± standard error (SEM), n = 5.

[0033] (2.2) Pharmacodynamic testing, including the following steps: Step 6: At the end of the experiment, the spleen of the mouse was taken and prepared into a single cell suspension. Flow cytometry (FCM) was used to detect the immune cell components in the spleen of the mouse. The cells were resuspended in PBS staining buffer to a concentration of 1×10 6 cells / 100 μL, centrifuge at 300×g for 5 minutes at 4°C, remove the supernatant; resuspend the cells with 100 μL PBS staining buffer; add 2 μL of blocking antibody Fc Block to each well, 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.

[0034] Table 4. Antibodies for flow cytometry staining

[0035] After the antibody incubation is completed, centrifuge at 4°C, 300×g for 5 minutes, remove the supernatant, resuspend the cells with 1ml PBS buffer, centrifuge at 4°C, 300×g for 5 minutes, remove the supernatant, add 1ml fixation and permeabilization buffer to resuspend, fix and permeabilize at 4°C in the dark for 45 minutes, centrifuge at 4°C, 300×g for 5 minutes, remove the supernatant, repeat once, add intranuclear antibody, incubate at 4°C in the dark for 30 minutes. Centrifuge at 4°C, 300×g for 5 minutes, remove the supernatant, repeat once; finally, resuspend the cells with 500μL fixation buffer and detect on the flow cytometer. Detection was performed using Merck's Guava@ easyCyte flow cytometer. 5000 immune cells were collected. Flow data were analyzed using FlowJo V10, Prism GraphPad 8.0 and Excel to analyze CD3 in spleen immune cells. + T cells, CD3 + CD4 + T cells and CD3 + CD8 + Percentages of T cells, Treg cells, Central memory CD3 T cells, and EffectorMemory CD3 T cells. One-way ANOVA statistical method was used to analyze the differences in data among multiple groups. *p<0.05; **p<0.01; ***p<0.001.

[0036] Figure 4A , Figure 4B , Figure 4C , Figure 5 The results showed that the tumor neoantigen obtained by this method was combined with PolyI:C to prepare a vaccine preparation. After mice were treated with the MC38 tumor vaccine preparation, the growth of tumors was effectively inhibited ( Figure 4A ); prolong the survival of mice ( Figure 4B-4C ). After tumor-bearing mice were treated with MC38 tumor neoantigen vaccine preparation, CD3 + T cells, CD4 + T cells, CD8 + The proportions of T cells and effector memory T cells (Tem) were significantly increased compared with the control group ( Figure 5 ). These data indicate that the MC38 tumor neoantigen vaccine formulation generated a very effective T cell response in tumor-bearing mice.

[0037] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mouse MC38 colorectal cancer neoantigen polypeptide, characterized in that: The amino acid sequences of the mouse MC38 colorectal cancer neoantigen polypeptides are shown in SEQ ID No. 2, 13, 16, and 17.

2. An immune adjuvant composition containing the mouse MC38 colorectal cancer neoantigen polypeptide 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 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 and the adjuvant in the immune adjuvant composition is 50ug / peptide+50ugPoly I:C.

7. Use of the mouse MC38 colorectal cancer neoantigen polypeptide according to claim 1 or the immune adjuvant composition according to any one of claims 2 to 6 in the preparation of a tumor neoantigen vaccine.

8. 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 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).

9. Use of the biomaterial as claimed in claim 8 in the preparation of tumor neoantigen vaccines.

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