New antigen lung cancer vaccine Calcoco1 and application thereof

Abstract

The invention discloses a new antigen lung cancer vaccine Calcoco1 and application thereof, the new antigen lung cancer vaccine Calcoco1 comprises a lung cancer new antigen, and the amino acid sequence of the lung cancer new antigen is shown as SEQ ID NO: 1. The neoantigen lung cancer vaccine can play a good anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to a new antigen lung cancer vaccine Calcocol and application thereof, belonging to the technical field of new antigens. Background Art

[0002] Lung cancer is the malignant tumor with the highest mortality rate in the world, and its incidence has been trending towards younger patients in recent years. For patients with advanced non-small cell lung cancer (NSCLC) without actionable driver gene mutations, chemotherapy-based combination therapy is the first-line treatment option. However, due to the significant toxicity and side effects of chemotherapy, a considerable number of patients cannot tolerate chemotherapy or have significant resistance to chemotherapy, especially in the elderly or immunocompromised groups. Clinically, non-chemotherapy combination treatment strategies for patients with advanced NSCLC mainly include anti-angiogenic therapy and immunotherapy, such as bevacizumab (BEV) plus immune checkpoint inhibitors (ICIs). However, the effects of these therapies are still inferior to chemotherapy-based combination therapy. One of the main reasons is that after anti-angiogenic and immunotherapy, most T cells infiltrating into tumor tissues are not specific to tumors. Therefore, how to increase the infiltration of tumor-specific lymphocytes without significantly increasing toxicity and side effects is a key strategy to improve the efficacy of chemotherapy-free treatment.

[0003] Tumor neoantigens, which are tumor-specific antigens, are immunogenic polypeptide substances produced by gene mutations during tumor development. They can bind to the major histocompatibility complex (MHC) and be displayed on the surface of tumor cells, thereby activating immune cells to enter the tumor and enhancing the effect of tumor immunotherapy. Therefore, therapeutic vaccines based on tumor neoantigens are highly specific and have low toxicity and side effects, and can be used for precise tumor immunotherapy. However, the immunosuppressive microenvironment (such as angiogenesis and high expression of PD-L1 in the tumor microenvironment) still significantly inhibits the anti-tumor activity of tumor-specific T cells induced by neoantigen lung cancer vaccines. Therefore, it is of great significance to design neoantigen-based lung cancer vaccines to achieve complete anti-tumor effects. Summary of the invention

[0004] The present invention provides a new antigen lung cancer vaccine Calcocol and application thereof, which can effectively solve the above problems.

[0005] The present invention is achieved in that:

[0006] A new lung cancer antigen, whose amino acid sequence is shown in SEQ ID NO:1.

[0007] A new antigen lung cancer vaccine Calcoco1, comprising the above-mentioned new lung cancer antigen.

[0008] In some embodiments, the neoantigen lung cancer vaccine Calcocol also includes an immune adjuvant.

[0009] In some embodiments, the immune adjuvant is Poly(I:C) and thymosin α1.

[0010] In some embodiments, the mass ratio of the lung cancer neoantigen, Poly (I:C) and thymosin α1 is 2:0.5-1.5:0.5-1.5.

[0011] A use of the new antigen in the preparation of a drug for treating lung cancer.

[0012] A use of the new antigen lung cancer vaccine Calcocol in the preparation of a drug for treating lung cancer.

[0013] The beneficial effects of the present invention are:

[0014] The present invention provides a novel immunogenic lung cancer neoantigen peptide, which can be used to prepare a neoantigen lung cancer vaccine (LLCvac), and which can stimulate a strong anti-tumor immune response in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 Whole exome and transcriptome sequencing maps were performed for the mouse lung cancer cell line LLC and the tail of C57BL / 6 mice.

[0017] Figure 2 These are the detection graphs of the lung cancer neoantigen Calcoco1, where 2A is the in vitro interferon (IFN)-γ ELISPOT detection graph of the lung cancer neoantigen Calcoco1, and 2B is the anti-tumor effect evaluation graph of the neoantigen lung cancer vaccine Fastkd1. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0019] The present invention discloses a newly discovered lung cancer-related antigen, whose amino acid sequence is specifically shown below, corresponding to SEQ ID NO: 1. This antigen has unique properties, making it an ideal candidate for developing a new lung cancer vaccine, and by utilizing the unique amino acid sequence of this new antigen, a strong anti-tumor immune response can be effectively stimulated in the human body.

[0020] The present invention discloses a novel lung cancer vaccine Calcocol, which contains a series of carefully selected lung cancer neoantigens. These neoantigens are extracted from lung cancer cells by advanced biotechnology, have high specificity and immunogenicity, and can effectively stimulate the body's immune response to fight lung cancer cells.

[0021] In certain specific embodiments, this new antigen lung cancer vaccine Calcocol is also specially added with immune adjuvants to further enhance the immune effect of the vaccine. The role of immune adjuvants is to activate and regulate the body's immune system, so that it produces a stronger immune response to the antigen components in the vaccine, thereby improving the protective effect of the vaccine.

[0022] In some specific embodiments, the immune adjuvants used include Poly(I:C) and thymosin α1. Poly(I:C) is a synthetic double-stranded RNA molecule that can simulate viral infection, activate the body's natural immune response, and enhance the immunogenicity of antigens. Thymosin α1 is a polypeptide extracted from the thymus that can regulate and enhance the body's cellular immune response, especially the immune response of T cells.

[0023] In certain specific embodiments, the mass ratio of lung cancer neoantigen, Poly (I: C) and thymosin α1 is optimized to 2: 0.5-1.5: 0.5-1.5. This ratio optimization is based on a large amount of experimental research and clinical trial data, aiming to ensure that the various components in the vaccine can work synergistically to achieve the best immune effect. Through this ratio optimization, the vaccine Calcoco1 can maximize its prevention and treatment effects on lung cancer while ensuring safety.

[0024] A use of the new antigen in the preparation of a drug for treating lung cancer.

[0025] A use of the new antigen lung cancer vaccine Calcocol in the preparation of a drug for treating lung cancer.

[0026] The application of the new antigen lung cancer vaccine Calcoco1 is not limited to direct use as a therapeutic drug, but can also be used as an adjuvant therapy in combination with existing lung cancer treatments such as surgery, radiotherapy and chemotherapy to improve the treatment effect and patient survival rate. By activating the body's immune system, especially the specific immune response to lung cancer cells, the Calcoco1 vaccine can more effectively eliminate cancer cells in the body and reduce the risk of recurrence and metastasis. In addition, due to its neoantigen-based characteristics, the vaccine is highly specific and safe, can reduce damage to normal tissues, and improve the quality of life of patients.

[0027] Example 1

[0028] The experimental method is as follows:

[0029] Cell line culture and identification

[0030] Mouse Lewis lung cancer cell lines (LLC) were provided by the Suzhou Beina Collection Center (BNCC) in China. These cell lines were cultured in DMEM medium (manufactured by Gibco brand) containing 10% fetal bovine serum (FBS). The culture conditions were set at a temperature of 37°C and a carbon dioxide concentration of 5%. These cell lines have undergone a rigorous validation process and have been carefully authenticated by short tandem repeat (STR) analysis over the past year to ensure the accuracy of their identity.

[0031] Before the experiment officially began, in order to ensure that there was no mycoplasma contamination in the cell line, the researchers used a PCR mycoplasma detection kit (MycoSEQ Mycoplasma Detection Kit, provided by Thermo Fisher Scientific) to test the cell line. This detection step is to ensure the accuracy and reliability of the experimental data and avoid experimental errors caused by mycoplasma contamination. Through this series of testing and verification processes, researchers can use these cell lines for subsequent experimental research with greater confidence.

[0032] Neoantigen identification and immunogenicity validation

[0033] To develop a tumor neoantigen vaccine specifically targeting lung cancer cell lines, we took the initial steps of conducting whole exome and transcriptome sequencing of the mouse lung cancer cell line LLC and the tails of C57BL / 6 mice. The goal of this process was to identify potential neoantigen mutations that might have unique expression patterns in lung cancer cell lines and thus be potential targets for vaccine development (see Figure 1 A). Through meticulous analysis, we successfully identified 762 high-quality LLC somatic mutations. These mutations passed strict screening criteria, including variant allele frequency ≥10%, read depth ≥20, and corresponding gene transcript number per million ≥1, and finally 224 mutations were confirmed to be expressed at the RNA level (see Figure 1 B). Based on these data, we further used eight different MHC binding affinity prediction algorithms (including NetMHCpan, NetMHC, NetMHCcons, PickPocket, MHCflurry, SMM, SMMPMBMC and MHCnuggetsI) to predict the binding affinity of these mutations to MHC I (especially the H-2Kb allele in LLC cells and C57BL / 6 mice). After comprehensive analysis, we found that there were 60 mutations that generated short peptides with high binding affinity to MHC (H-2Kb allele, median binding affinity percentile ranking of all MHC binding affinity prediction algorithms ≤2%), and these short peptides were considered potential tumor neoantigen mutations.

[0034] To verify the immunogenicity of these potential neoantigens, we carefully selected a mutant long peptide Calcoco1-P581L from the top 20 potential neoantigen mutations, synthesized it and evaluated its immunogenicity. Subsequently, we mixed the mutant long peptide with the immune adjuvant Poly(I:C) and immunized subcutaneously in the flank of C57BL / 6 mice on days 0, 4, and 8, respectively. On the 14th day after the initial immunization, the immunized mice were killed, and we isolated splenic T cells for in vitro interferon (IFN)-γ ELISPOT assay to evaluate the immune response of T cells. The results showed that this synthetic mutant long peptide was able to induce a strong immune response of T cells under the stimulation of autologous mature dendritic cells (DCs) pulsed with the respective mutant long peptides (see Figure 2 A).

[0035] To further determine whether the mutant long peptide has immunological cross-reactivity with its corresponding wild-type peptide, we pooled the mutant peptide and the corresponding wild-type peptide and mixed them with Poly(I:C) to immunize and treat mice as described above. The mutant peptide and the corresponding wild-type peptide were then pulsed with autologous DCs separately for downstream ELISPOT analysis. As expected, this neoantigenic peptide had no significant immunogenic cross-reactivity compared to the wild-type. These results indicate that this peptide is able to elicit a strong targeted immune response in C57BL / 6 mice and can be recognized as a neoantigen of the LLC cell line.

[0036] Through sequencing analysis, we successfully determined that the amino acid sequence of the mutant long peptide Calcoco-P581L1 is LVVINQPALIAPQFSGP (SEQ ID NO: 1). The amino acid sequence of the wild-type Calcoco1-WT is LVVINQPAPIAPQFSGP (SEQ ID NO: 2). This discovery provides important basic data for further research and development of new antigen vaccines against lung cancer.

[0037] In vivo tumor models and evaluation of antitumor effects

[0038] To conduct tumor experiments, the researchers selected female C57BL / 6 mice aged 6-8 weeks as experimental subjects. The experiment was implemented by injecting LLC cells into these mice. Specifically, the establishment of a subcutaneous lung cancer model was achieved by subcutaneously injecting 2×10^6 LLC cells under the right armpit of the mouse. To ensure the accuracy of the experiment, tumor monitoring was the responsibility of a dedicated operator. The operator used a caliper to measure the two dimensions of the tumor every three days to ensure the accuracy of the data.

[0039] The calculation formula of tumor volume is: V = AB2 / 2, where A represents the long diameter and B represents the short diameter. When the tumor volume grows to 50-80 mm3, this time point is defined as day 0 of the experiment. From this day on, mice are randomly divided into three groups, each containing 6 mice, in order to start receiving different treatment plans.

[0040] The mice in the experimental group were treated with a new antigen peptide vaccine (Calcoco1-P581L). Each mouse was injected with 100 micrograms / peptide, and this vaccine was combined with an optimized clinically available dual immune adjuvant [50μg Poly (I:C) + 50μg thymosin α-1 (ZadaxiN, Sciclone Pharmaceuticals, USA)]. From day 0, the vaccine was injected subcutaneously once every four days for a total of three injections.

[0041] The mice in the control group were also treated for the same period of time, but were injected with phosphate buffered saline (ctrl) and wild-type neoantigen peptide vaccine (Calcoco1-WT). The wild-type vaccine group was also combined with double immune adjuvants [50μg Poly(I:C) + 50μg thymosin α-1 (ZadaxiN, Sciclone Pharmaceuticals, USA)] to ensure the consistency of experimental conditions.

[0042] The experimental results are as follows Figure 2 As shown in B. Figure 2 As can be seen in B, the new antigen peptide vaccine (Calcoco1-P581L) showed significant anti-tumor effects. This discovery provides an important reference for future research and treatment.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A new lung cancer antigen, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A new antigen lung cancer vaccine Calcocol, characterized in that: Including the lung cancer neoantigen according to claim 1.

3. The new antigen lung cancer vaccine Calcocol according to claim 2, characterized in that: Also included are immune adjuvants.

4. The new antigen lung cancer vaccine Calcocol according to claim 3, characterized in that The immune adjuvants are Poly(I:C) and thymosin α1.

5. The new antigen lung cancer vaccine Calcocol according to claim 4, characterized in that: The mass ratio of the lung cancer neoantigen, Poly(I:C) and thymosin α1 is 2: 0.5-1.5：0.5-1.5。 6. Use of the new antigen according to claim 1 in the preparation of a drug for treating lung cancer.

7. Use of the new antigen lung cancer vaccine Calcocol according to any one of claims 2 to 5 in the preparation of a drug for treating lung cancer.

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