A new antigen lung cancer vaccine Fastkd1 and its application
By developing a novel lung cancer vaccine Fastkd1 containing specific lung cancer neoantigen peptides, combined with immune adjuvant, the problem of poor chemotherapy toxicity and immunotherapy effects was solved, and an efficient and low toxic anti-tumor immune response was achieved.
Patent Information
- Application Number
- CN202411988658.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-04
AI Technical Summary
Prior art In the treatment of advanced non-small cell lung cancer (NSCLC), the significant toxicity and side effects of chemotherapy are unbearable in many patients, and the immunotherapy is not effective, mainly due to the nonspecific infiltration of the tumor by T cells.
A novel lung cancer vaccine, Fastkd1, is developed to contain specific lung cancer neoantigen peptides, and combine the immune adjuvant Poly(I:C) and thymic peptide alpha1, to activate the immune system and enhance the specific immune response to lung cancer cells.
The vaccine significantly stimulates the body's anti-tumor immune response, can effectively identify and attack lung cancer cells, reduce toxicity and side effects, and provide new ideas and methods for immunotherapy of lung cancer.
Smart Images
Figure CN119751587A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a new antigen lung cancer vaccine Fastkd1 and application thereof, belonging to the technical field of new antigens. Background Art
[0002] Lung cancer is one of the most lethal malignancies worldwide, and its incidence has been trending toward younger patients in recent years. For patients with advanced non-small cell lung cancer (NSCLC) who do not have actionable driver gene mutations, chemotherapy-based combination therapy is the preferred first-line treatment option. However, due to the significant toxicity and side effects of chemotherapy, many patients cannot bear the burden of chemotherapy or have developed strong resistance to chemotherapy, especially in the elderly or patients with weak immune systems. Clinically, non-chemotherapy combination therapy strategies for patients with advanced NSCLC mainly include anti-angiogenic therapy and immunotherapy, such as bevacizumab (BEV) combined with immune checkpoint inhibitors (ICIs). However, the effects of these therapies are usually not as good as 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 specifically targeted at 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 are a type of tumor-specific antigen. They are immunogenic polypeptide substances produced by gene mutations during tumor development. These neoantigens 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 have high specificity and low toxicity and side effects, and are suitable 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 Fastkd1 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 Fastkd1 comprises the above-mentioned new lung cancer antigen.
[0008] In some embodiments, the neoantigen lung cancer vaccine Fastkd1 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 Fastkd1 in the preparation of a drug for treating lung cancer.
[0013] The beneficial effects of the present invention are:
[0014] The present invention proposes an immunogenic new lung cancer antigen peptide, which has unique structural and functional characteristics and can effectively activate the body's immune system. By utilizing this new lung cancer new antigen peptide, a new lung cancer vaccine LLCvac can be developed. This vaccine has a significant immune activation effect in vivo and can stimulate the body to produce a strong anti-tumor immune response. LLCvac vaccine can effectively identify and attack lung cancer cells by inducing the proliferation and activation of specific immune cells, thereby achieving the purpose of preventing and treating lung cancer. The development of this new vaccine provides new ideas and methods for the immunotherapy of lung cancer, and has important clinical application value and market potential. 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 results for the mouse lung cancer cell line LLC and C57BL / 6 mouse tails.
[0017] Figure 2 This is the detection data of the new lung cancer antigen Fastkd1. Figure 2 A shows the results of the lung cancer neoantigen Fastkd1 in the in vitro interferon (IFN)-γ ELISPOT test. Figure 2 B shows the performance of the new antigen lung cancer vaccine Fastkd1 in the evaluation of anti-tumor effects. 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 embodiment of the present invention aims to provide a novel lung cancer-associated antigen, whose specific amino acid sequence can be found in SEQ ID NO: 1. This novel antigen is screened out by comprehensive exome sequencing and transcriptome sequencing technology, thereby ensuring its potential application value in lung cancer diagnosis and treatment.
[0020] The embodiments of the present invention are intended to provide a novel antigen lung cancer vaccine, named Fastkd1, which contains a specific new lung cancer antigen.
[0021] In some specific embodiments, the neoantigen lung cancer vaccine Fastkd1 further includes an immune adjuvant component. In these embodiments, the immune adjuvant can be two components, Poly (I: C) and thymosin α1. Specifically, in some embodiments, the mass ratio of the three components, lung cancer neoantigen, Poly (I: C) and thymosin α1, can be set to 2: 0.5-1.5: 0.5-1.5. Such a ratio helps to enhance the immune effect of the vaccine, thereby more effectively stimulating the body's immune response to lung cancer.
[0022] The embodiments of the present invention are intended to provide an application of a novel antigen in the development of drugs for treating lung cancer. Specifically, the embodiments of the present invention relate to a specific neoantigen lung cancer vaccine Fastkd1. This neoantigen vaccine Fastkd1 can effectively identify and attack lung cancer cells by activating the human immune system, thereby showing potential clinical application value in the treatment of lung cancer.
[0023] In addition, the development process of the vaccine fully considers safety and effectiveness. Through precise screening and verification, the specificity and immunogenicity of the new antigen are ensured, thereby reducing the risk of autoimmune reactions or other adverse reactions that may be caused by the vaccine. At the same time, the production process of the vaccine has also undergone strict quality control to ensure the purity and stability of the vaccine, further ensuring the safety of its clinical application. In addition, the development of this new antigen vaccine also provides new ideas and possibilities for the personalized treatment of lung cancer, and is expected to bring good news to more lung cancer patients.
[0024] Example 1
[0025] Cell line culture and identification
[0026] 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 (provided 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 comprehensive validation process and have been carefully authenticated by short tandem repeat (STR) analysis within the past year to ensure the accuracy of their identity.
[0027] Before the experiment began, in order to ensure that the cell lines were free of mycoplasma contamination, the researchers used a PCR mycoplasma detection kit (MycoSEQ Mycoplasma Detection Kit, provided by Thermo Fisher Scientific) to test the cell lines. 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 rigorous testing and verification processes, researchers can use these cell lines for subsequent experimental research with greater confidence.
[0028] Neoantigen identification and immunogenicity validation
[0029] To develop a tumor neoantigen vaccine against lung cancer cell lines, we first performed whole exome and transcriptome sequencing of the mouse lung cancer cell line LLC and the tails of C57BL / 6 mice in order to identify potential neoantigen mutations in these cell lines. Figure 1A is shown. Through these sequencing data, we identified a total of 762 high-quality LLC somatic mutations that were found when compared with the C57BL / 6 genome. Among these mutations, 224 were found to be expressed at the RNA level through LLC transcriptome data analysis, with the specific criteria of variant allele frequency greater than or equal to 10%, read depth greater than or equal to 20, and corresponding gene transcript counts greater than or equal to 1 per million. 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 MHCI (LLC cells and C57BL / 6 mice: H-2Kb allele). The results showed that 60 mutations generated short peptides with high binding affinity to MHC (H-2Kb allele, median binding affinity percentile ranking of all MHC binding affinity prediction algorithms less than or equal to 2%), and these short peptides were considered to be potential tumor neoantigen mutations.
[0030] To verify the immunogenicity of these potential neoantigens, we selected a mutant long peptide, Fastkd1-W447S, from the top 20 potential neoantigen mutations, synthesized it, and evaluated its immunogenicity. Subsequently, we mixed this 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 spleen T cells were isolated for in vitro interferon (IFN)-γ ELISPOT detection. The results are shown in Figure 2. Figure 2 As shown in A.
[0031] from Figure 2 As can be seen in A, this synthetic mutant long peptide is able to induce a strong immune response in T cells under the stimulation of autologous mature dendritic cells (DCs) pulsed with the respective mutant long peptides. To further determine whether the mutant long peptide has immune cross-reactivity with its corresponding wild-type peptide, we formed a pool of mutant peptides and wild-type peptides separately and mixed them with Poly (I: C) to immunize and treat mice as described above. Then, the mutant peptide and the corresponding wild-type peptide were pulsed with autologous DCs for downstream ELISPOT analysis. As expected, this new antigenic peptide had no significant immunogenic cross-reaction compared with the wild type. These results indicate that this peptide can induce a strong targeted immune response in C57BL / 6 mice and can be recognized as a new antigen of LLC.
[0032] Through sequencing analysis, we determined that the amino acid sequence of the mutant long peptide Fastkd1-W447S is DLVVYLMRSIQSDLVCL (SEQ ID NO: 1). The amino acid sequence of the wild-type Fastkd1-WT is DLVVYLMRWIQSDLVCL (SEQ ID NO: 2). This sequence difference is exactly where the new antigen mutation we are concerned about is.
[0033] To evaluate the anti-tumor effect, we used an in vivo tumor model for experiments. In the experiment, we selected female C57BL / 6 mice aged 6-8 weeks as experimental subjects. By injecting LLC cells into these mice, we successfully established a tumor model for the experiment. Specifically, we successfully established a subcutaneous lung cancer model by subcutaneously injecting 2×10^6 LLC cells in the right armpit of the mouse. To monitor the growth of the tumor, we arranged an operator to measure the tumor in two perpendicular directions every three days using a caliper. By measuring the long diameter (A) and short diameter (B), we used the formula V=AB2 / 2 to calculate the volume of the tumor. When the tumor volume grew to 50-80mm3, we defined this time point as day 0 of the experiment. Subsequently, we randomly divided the mice into three groups, each containing 6 mice, and began to treat them.
[0034] In the experimental group, we used a neoantigen peptide containing the identified mutations - the neoantigen peptide vaccine (Fastkd1-W447S). Each mouse was vaccinated 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)]. Starting from day 0 of the experiment, we injected the mice subcutaneously once every four days for a total of three times. The mice in the control group were also treated during the same time period, but were injected with phosphate buffered saline (ctrl) and wild-type neoantigen peptide vaccine (Fastkd1-WT). The wild-type neoantigen peptide vaccine (Fastkd1-WT) group was also combined with a dual immune adjuvant [50μg Poly(I:C) + 50μg thymosin α-1 (ZadaxiN, Sciclone Pharmaceuticals, USA)]. The experimental results are shown in the following figure. Figure 2 As shown in B.
[0035] according to Figure 2 The results of B showed that the new antigen peptide vaccine (Fastkd1-W447S) exhibited significant anti-tumor effects.
[0036] 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 Fastkd1, characterized in that: Including the lung cancer neoantigen according to claim 1.
3. The new antigen lung cancer vaccine Fastkd1 according to claim 2, characterized in that: Also included are immune adjuvants.
4. The new antigen lung cancer vaccine Fastkd1 according to claim 3, characterized in that: The immune adjuvants are Poly(I:C) and thymosin α1.
5. The new antigen lung cancer vaccine Fastkd1 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 Fastkd1 according to any one of claims 2 to 5 in the preparation of a drug for treating lung cancer.