Tumor neoantigen immune peptides and their applications

Abstract

The present invention discloses a tumor neoantigen immune polypeptide and its application, the tumor neoantigen immune polypeptide comprises a first antigen, a second antigen, a third antigen, a fourth antigen, a fifth antigen, a sixth antigen, a seventh antigen, an eighth antigen, a ninth antigen, and a tenth antigen, wherein the amino acid sequences of the first antigen, the second antigen, the third antigen, the fourth antigen, the fifth antigen, the sixth antigen, the seventh antigen, the eighth antigen, the ninth antigen, and the tenth antigen are respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10. The present invention has developed a tumor neoantigen immune polypeptide for treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a tumor neoantigen immune polypeptide and applications thereof. Background Art

[0002] After chemotherapy and molecular targeted therapy, tumor treatment has entered the era of tumor immunotherapy. The basic idea of ​​tumor immunotherapy is to activate the body's own immune system by relieving the immunosuppression of the tumor microenvironment, improving the antigen presentation function of dendritic cells and promoting the production of protective T cells, so as to achieve the purpose of identifying and killing tumor cells.

[0003] In recent years, personalized immunotherapy drugs based on tumor neoantigen immune peptides have been applied in related fields. Tumor neoantigens are abnormal proteins produced by cancer cells through "non-synonymous mutations". They only exist in cancer cells, not in normal cells, and therefore have high immunogenicity. Researchers extract and analyze blood samples from tumor patients, find tumor-specific genes, synthesize tumor-specific antigens, prepare personalized immunotherapy drugs based on tumor neoantigen immune peptides, and then re-infuse them into patients to stimulate the patient's own specific immune response to tumor cells, so as to achieve the therapeutic goal of clearing tumors without killing surrounding normal cells. Tumor neoantigen immune peptides can also induce immune memory cells, produce long-term immune effects, and prevent tumor metastasis and recurrence. Therefore, the development of new tumor neoantigen immune peptides for the treatment of tumors is a technical problem that technicians in this field need to solve. Summary of the invention

[0004] The present invention provides a tumor neoantigen immune polypeptide and its application, and develops a tumor neoantigen immune polypeptide for treating tumors.

[0005] One aspect of the present invention provides a tumor neoantigen immune polypeptide, including a first antigen, a second antigen, a third antigen, a fourth antigen, a fifth antigen, a sixth antigen, a seventh antigen, an eighth antigen, a ninth antigen, and a tenth antigen, wherein the amino acid sequence of the first antigen is shown in SEQ ID NO.1, the amino acid sequence of the second antigen is shown in SEQ ID NO.2, the amino acid sequence of the third antigen is shown in SEQ ID NO.3, the amino acid sequence of the fourth antigen is shown in SEQ ID NO.4, the amino acid sequence of the fifth antigen is shown in SEQ ID NO.5, the amino acid sequence of the sixth antigen is shown in SEQ ID NO.6, the amino acid sequence of the seventh antigen is shown in SEQ ID NO.7, the amino acid sequence of the eighth antigen is shown in SEQ ID NO.8, the amino acid sequence of the ninth antigen is shown in SEQ ID NO.9, and the amino acid sequence of the tenth antigen is shown in SEQ ID NO.10.

[0006] Another aspect of the present invention provides the use of the above-mentioned tumor neoantigen immune polypeptide in the preparation of immunotherapeutic drugs for treating tumors.

[0007] Optionally, the tumor is lung cancer.

[0008] The present invention uses ctDNA whole exome sequencing technology to perform gene detection on peripheral blood samples of lung cancer patients, screen out tumor neoantigens with gene mutations in cancer cells of lung cancer patients, and then artificially synthesize tumor neoantigen immune polypeptides. The present invention uses the highly sensitive cell function detection technology enzyme-linked immunosorbent assay (ELISpot) to detect the level of tumor neoantigen immune polypeptides stimulating PBMCs to secrete cytokines such as IFN-γ, and verifies the ability of the tumor neoantigen immune polypeptides to activate immune cells of lung cancer patients. Based on the tumor neoantigen immune polypeptides, immune preparations based on tumor neoantigen immune polypeptides can be prepared, and the immune preparations can be used as immunotherapy drugs for lung cancer. Clinical trials have shown that after a lung cancer patient used the immune preparations prepared by the tumor neoantigen immune polypeptides provided by the present invention for 6 times, his lung tumor was reduced from 15x10mm to 5.2x3.1mm; another lung cancer patient used the immune preparations prepared by the tumor neoantigen immune polypeptides provided by the present invention for a total of 7 times, and his tumor marker carcinoembryonic antigen (CEA) dropped from 6.06 ng / mL before using the immune preparations to the normal range of 3.97ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 A spot diagram showing that the tumor neoantigen immune polypeptide provided by the present invention stimulates human PBMCs to secrete IFN-γ cytokine in an ELISpot experiment; Figure 2 This is a chest CT comparison image of a lung cancer patient before and after using the immune preparation prepared with the tumor neoantigen immune polypeptide provided by the present invention; Figure 3 This is a graph showing changes in carcinoembryonic antigen levels in another lung cancer patient after using the immune preparation prepared with the tumor neoantigen immune polypeptide provided by the present invention. DETAILED DESCRIPTION

[0010] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0011] Human leukocyte antigen (HLA) is encoded by the HLA gene complex and is the expression product of the human major histocompatibility complex (MHC). HLA is a highly polymorphic alloantigen, and its allele typing varies among people in different regions. 11:01 has a high frequency of the "A" allele.

[0012] In view of this, the present invention uses ctDNA whole exome sequencing technology to perform gene testing on peripheral blood samples of lung cancer patients, screens out tumor neoantigens with gene mutations in cancer cells of lung cancer patients, and then artificially synthesizes tumor neoantigen immune peptides to obtain peptides that can bind to HLA-A. 11:01 tumor new antigen immune peptide, which can achieve the effect of targeting HLA-A 11:01 Personalized immunotherapy for lung cancer patients.

[0013] The present invention utilizes circulating tumor DNA (ctDNA) sequencing technology to screen out tumor neoantigen immune polypeptides through whole exome sequencing detection, and the tumor neoantigen immune polypeptides include a first antigen, a second antigen, a third antigen, a fourth antigen, a fifth antigen, a sixth antigen, a seventh antigen, an eighth antigen, a ninth antigen, and a tenth antigen, and the amino acid sequences of the first antigen, the second antigen, the third antigen, the fourth antigen, the fifth antigen, the sixth antigen, the seventh antigen, the eighth antigen, the ninth antigen, and the tenth antigen are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. Specifically, as shown in Table 1: Table 1

[0014] The tumor neoantigen immune peptide corresponds to the gene mutation of cancer cells in lung cancer patients, but there is no amino acid sequence of these mutations in normal cells. Combining the tumor neoantigen immune peptide, using it to prepare immunotherapy drugs, including off-the-shelf tumor neoantigen immune preparations and personalized tumor neoantigen immune preparations, can be used for immunotherapy of lung cancer patients, which can improve the efficacy of traditional cancer treatment and combined treatment.

[0015] The tumor neoantigen immune polypeptide provided by the present invention optimizes the combination of amino acid sequences as shown in SEQ ID NO.1~10 for the first time, and uses these 10 amino acid sequences simultaneously when preparing immunotherapy drugs, instead of using any one of them alone. Breaking through the limitation of single tumor gene mutation, the tumor neoantigen immune polypeptide finally achieves the killing effect of lung cancer cells. The tumor neoantigen immune polypeptide is different from a single neoantigen immune polypeptide drug, and it cannot be understood as a simple combination of a single drug.

[0016] The tumor new antigen immune peptide is HLA-A 11:01 has immune affinity and can be loaded onto cells expressing HLA-A 11:01 Antigen presenting cells.

[0017] Among them, the first antigen, the fourth antigen, the fifth antigen, the seventh antigen, and the eighth antigen are tumor-specific antigens (TSAs), and the second antigen, the third antigen, the sixth antigen, the ninth antigen, and the tenth antigen are tumor-associated antigens (TAAs).

[0018] Preferably, the tumor-specific antigen is tumor-specific antigen class I (TSA-I) or tumor-specific antigen class II (TSA-II).

[0019] Tumor-specific antigens are a type of antigen that is expressed in tumor cells and is almost not expressed in normal cells. The expression of these antigens in tumor cells usually originates from abnormal events such as gene mutations and chromosomal rearrangements in tumor cells, which makes the expression of these antigens in normal cells very limited or even completely absent.

[0020] The tumor-specific antigens in the present invention are potential ideal immunotherapy targets.

[0021] Preferably, the tumor-associated antigens are human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR) and prostate specific antigen (PSA).

[0022] Tumor-associated antigens are a class of molecules that exist on the surface of tumor cells or are produced in tumor cells. They can be recognized by the immune system as abnormal or abnormally expressed substances. These antigens can induce the immune system to produce immune responses against tumor cells, including T cell and B cell responses, thereby participating in anti-tumor immune responses.

[0023] The tumor-associated antigens in the present invention also exist in normal tissues, but their expression levels in tumor cells are significantly increased. The tumor-associated antigens in the present invention participate in processes such as cell proliferation, differentiation and survival, and their abnormal expression is related to the formation and development of tumors.

[0024] The present invention is verified by different embodiments below.

[0025] Example 1: ELISpot experiment verifies that tumor neoantigen immune peptides stimulate human PBMCs to secrete IFN-γ cytokine 1.1 Materials and Reagents: (1) tumor neoantigen peptides as shown in Table 1; (2) 96-well ELISpot plate (PVDF membrane or nylon membrane); (3) IFN-γ capture antibody (monoclonal antibody against IFN-γ); (4) Biotinylated secondary antibody (capture antibody against IFN-γ); (5) Enzyme-labeled substrates (such as TMB, BCIP / NBT); (6) PBMCs (mononuclear cells isolated from peripheral blood); (7) Culture medium (RPMI-1640, 10% FBS, antibiotics, etc.); (8) Antibody washing buffer (PBS or TBS buffer containing Tween-20); (9) Enzyme-labeled secondary antibody (such as horseradish peroxidase-labeled anti-biotin antibody); (10) Positive group (such as PHA or anti-CD3 antibody, which is known to activate T cells).

[0026] 1.2 Experimental steps: Step 1: Prepare the ELISpot plate.

[0027] (1) Wash the 96-well plate with PBS (or other appropriate buffer) to remove impurities; (2) Add IFN-γ capture antibody at 1 μg / mL and incubate overnight at 4°C; (3) The next day, wash the plate three times with PBS to remove unbound antibodies.

[0028] Step 2: PBMCs preparation.

[0029] (1) Isolate PBMCs from peripheral blood using the Ficoll-Paque separation method; (2) Count PBMCs by flow cytometry or cell counting plate; (3) Resuspend PBMCs in appropriate culture medium (e.g., RPMI-1640, 10% FBS).

[0030] Step 3: Peptide antigen stimulation.

[0031] (1) Add tumor neoantigen peptides at a concentration of 10-20 μg / mL to each well; (2) Add positive control (such as PHA or anti-CD3 antibody).

[0032] Step 4: PBMCs are added to the plate.

[0033] (1) Add 100 μL of PBMCs suspension to each well; (2) Set up negative control wells (no antigen) together with positive control wells (with PHA or anti-CD3) to ensure the reliability of experimental controls; (3) Incubate at 37°C, 5% CO2 for 18-24 hours.

[0034] Step 5: Wash.

[0035] (1) After incubation, wash the plate three times with PBS (or TBS) to remove unbound cells and cytokines; (2) Each time you wash, gently shake the plate and let it sit for a while before changing the solution.

[0036] Step 6: Add biotin-labeled secondary antibody.

[0037] (1) Add biotin-labeled secondary antibody (against IFN-γ) at a concentration of 1-2 μg / mL to each well; (2) Incubate at 37°C for 1-2 hours.

[0038] Step 7: Wash again.

[0039] Wash the plate three times with PBS (or TBS) to remove unbound secondary antibody.

[0040] Step 8: Add enzyme-labeled substrate.

[0041] (1) Add enzyme-labeled substrate (such as TMB or BCIP / NBT) to each well and incubate for 10-20 minutes until spots are visible. (2) Pay attention to the progress of the reaction and ensure that the color change is moderate. Too long a time may result in an overly strong signal.

[0042] Step 9: Stop the reaction.

[0043] The reaction was stopped with PBS, the plates were washed and patted dry.

[0044] Step 10: Count the spots.

[0045] (1) Count the number of spots in each well using an ELISpot plate reader or microscope. Each spot represents a cell that secretes IFN-γ. (2) The number of IFN-γ-secreting cells can be used to evaluate the immune response stimulated by tumor neoantigen peptides.

[0046] Step 11: Result analysis.

[0047] (1) Background signal: The number of spots in the negative control well should be close to zero.

[0048] (2) Positive signal: Positive control wells (such as PHA or anti-CD3 antibody stimulation) should show obvious spots.

[0049] (3) Experimental group: The number of spots displayed in the experimental group stimulated with tumor neoantigen peptides can be used to evaluate the immune response of antigen-specific T cells.

[0050] The experimental results are as follows Figure 1 As shown by Figure 1 From the spot scanning diagram marked by the boxes, it can be seen that the number of spots of IFN-γ produced by human PBMCs stimulated by the 10 antigens of the present invention are 162, 209, 127, 134, 136, 72, 84, 120, 172 and 104, respectively, which are significantly different from those in the background group, indicating that these 10 antigens can stimulate the immune T cells and NK cells in human PBMCs to secrete IFN-γ cytokines.

[0051] Example 2: Preparation of immune preparations using tumor neoantigen immune polypeptides (neoantigen-loaded DC vaccines) The steps of the method for preparing a new antigen-loaded DC vaccine using the tumor new antigen immune polypeptide provided by the present invention are as follows: 1. Use solid phase peptide synthesis (SPPS) to synthesize the tumor neoantigen immune polypeptide provided by the present invention. By introducing D-amino acids or cyclization, the stability of the polypeptide is enhanced to prevent its rapid degradation in the human body; at the same time, through mutation design, the affinity of the immune polypeptide to the MHC molecule is enhanced to improve its presentation efficiency.

[0052] 2. PBMCs were isolated from the peripheral blood of each lung cancer patient using the Ficoll-Paque stratification method.

[0053] 3. Isolate monocytes from PBMCs by adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), induce them to differentiate into DC cells, and culture them for 5-7 days.

[0054] 4. The synthesized tumor neoantigen immune peptide is loaded onto DC cells through a cell membrane permeabilizing agent (such as LPS or a synthetic carrier molecule).

[0055] 5. Add maturation factors such as TNF-α, IL-1β, IL-6 and CD40L to activate DC cells.

[0056] 6. Use flow cytometry (FACS) to detect DC cell maturation markers (such as CD80, CD86, HLA-DR) and antigen presentation function to ensure that DC cells can effectively activate specific T cells in lung cancer patients.

[0057] 7. Cultivate mature neoantigen-loaded DC cells to expand the cell number and ensure the vaccine dose required for each lung cancer patient.

[0058] 8. Ensure that the quantity and functional quality of DC cells meet the standards through cell counting and cell viability testing.

[0059] 9. Perform aseptic operations on the completed neoantigen-loaded DC vaccine, package it into the final dosage form and refrigerate it to ensure that the vaccine remains active during the immunization process.

[0060] In this example, the specific parameters of the neoantigen-loaded DC vaccine prepared by tumor neoantigen immune polypeptides are shown in Table 2.

[0061] Table 2

[0062] Example 3: Therapeutic effect of immune preparations prepared using tumor neoantigen immune polypeptides Taking two lung cancer patients receiving immunotherapy as examples, the therapeutic effect of the immune preparation prepared with the tumor neoantigen immune polypeptide provided in Example 2 on lung cancer is illustrated.

[0063] The experimental design is shown in Table 3: Table 3

[0064] The immune preparation prepared by the tumor neoantigen immune polypeptide provided in Example 2 was used to treat LC 1 lung cancer patients. After 6 injections of the immune preparation, the imaging test results showed that the lung tumor was reduced from 15x10mm to 5.2x3.1mm. Figure 2 shown.

[0065] LC 2 lung cancer patients received a total of 7 injections of immune preparations, such as Figure 3 As shown, the tumor marker carcinoembryonic antigen (CEA) decreased from 6.06 ng / mL before vaccination to 3.97 ng / mL in the normal range. It can be seen that the immune preparation prepared using the tumor neoantigen immune polypeptide provided by the present invention has an effective therapeutic effect on lung cancer.

[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A tumor neoantigen immune polypeptide, characterized in that: It includes a first antigen, a second antigen, a third antigen, a fourth antigen, a fifth antigen, a sixth antigen, a seventh antigen, an eighth antigen, a ninth antigen and a tenth antigen. The amino acid sequence of the first antigen is shown in SEQ ID NO.1, the amino acid sequence of the second antigen is shown in SEQ ID NO.2, the amino acid sequence of the third antigen is shown in SEQ ID NO.3, the amino acid sequence of the fourth antigen is shown in SEQ ID NO.4, the amino acid sequence of the fifth antigen is shown in SEQ ID NO.5, the amino acid sequence of the sixth antigen is shown in SEQ ID NO.6, the amino acid sequence of the seventh antigen is shown in SEQ ID NO.7, the amino acid sequence of the eighth antigen is shown in SEQ ID NO.8, the amino acid sequence of the ninth antigen is shown in SEQ ID NO.9, and the amino acid sequence of the tenth antigen is shown in SEQ ID NO.

10.

2. The tumor neoantigen immune polypeptide according to claim 1, characterized in that: The first antigen, the fourth antigen, the fifth antigen, the seventh antigen, and the eighth antigen are tumor-specific antigens, and the second antigen, the third antigen, the sixth antigen, the ninth antigen, and the tenth antigen are tumor-associated antigens.

3. Use of the tumor neoantigen immune polypeptide according to claim 1 in the preparation of immunotherapeutic drugs for treating tumors.

4. The use according to claim 3, characterized in that: The tumor is lung cancer.

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