Tumor neoantigen polypeptide and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to tumor neoantigen polypeptide based on EGFR gene mutation and application of the tumor neoantigen polypeptide. According to the effective method for predicting and screening the tumor neoantigen by using the tumor neoantigen prediction platform, the specific mutation of tumor tissues is obtained, and the neoantigen polypeptide with high immunogenicity, high affinity and high activation effect is more accurately predicted; the screened new antigen polypeptide makes up the blank of individualized antigen peptide in treatment of lung cancer NSCLC patients with EGFR-L858R somatic mutation, can be synthesized in a large scale, is used for subsequent standardized and individualized immunotherapy of EGFR-L858R mutation tumor patients, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a tumor neoantigen polypeptide based on EGFR gene mutation and an application thereof. Background Art

[0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world. It includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), of which 85% is NSCLC, and histological types include adenocarcinoma, squamous cell carcinoma and large cell carcinoma (MILLERM, HANNAN. Advances insystemic therapy for non-small cell lung cancer [J]. BMJ, 2021, 375: n2363.). Most patients are already in the middle and late stages when diagnosed and have lost the opportunity for surgical treatment. The main treatment methods are chemotherapy, molecular targeted therapy and immunotherapy (CHEN PX, LIU YH, WEN YK, et al. Non-small cell lung cancer in China [J]. Cancer Commun, 2022, 42 (10): 937-970). However, traditional chemotherapy has a wide range of cytotoxic effects, and patients are prone to serious adverse reactions such as nausea, vomiting, diarrhea, mucositis, ototoxicity, and nephrotoxicity (ZRAIK IM, HEβ-BUSCHY. Management of chemotherapy side effects and their long-term sequelae [J]. Urologe A, 2021, 60 (7): 862-871). The average overall survival (OS) of patients is 8-10 months, and the 5-year survival rate is only 15%. Although targeted therapy can prolong the PFS of patients with NSCLC who are positive for driver gene mutations and achieve a high disease control rate, there is no significant improvement in OS, and almost all patients will eventually experience resistance-mediated disease progression. Immunotherapy has a good prospect, but not all patients can obtain survival benefits from a single immunosuppressant. Therefore, it is of great significance to find relevant therapeutic targets and therapeutic drugs for NSCLC.

[0003] Epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family, also known as human epidermal growth factor receptor 1 (HER1). It is a transmembrane protein receptor responsible for regulating epidermal growth in the body. It has tyrosine kinase (TK) activity, can promote endothelial cell division, increase vascular permeability, and play an active role in regulating angiogenesis (Melincovici CS, AB, S, et al. Vascular endothelialgrowth fact or (VEGF)-key factor in normal and pathological angiogenesis [J]. Rom J Morphol Embryol, 2018, 59 (2): 455-467). EGFR is located on the short arm of human chromosome 7, including 28 exons, and is a member of the ErbB receptor TK family. EGFR gene mutation is an important driver mutation that leads to tumorigenesis (Zhou Wensheng, Zhang Wei, Han Baohui. Research and progress of EGFR gene exon 20 insertion mutation in non-small cell lung cancer [J]. Chinese Journal of Lung Cancer, 2020, 23 (2): 118-126). The percentage of this mutation is between 15% and 40%. The incidence in Caucasian non-small-cell lung cancer (NSCLC) patients is about 10%. In Asian NSCLC patients, the number of patients carrying this gene mutation accounts for 30%-50%. Among them, exons 18 to 21 of the intracellular tyrosine kinase structure region are the main areas where mutations occur, including three different types of deletion mutations, insertion mutations and point mutations. Deletion mutations in exon 19 (45%) and point mutations in exon 21 (40%-45%) account for about 85% to 90% of all known EGFR mutations (Paez JG, PasiA. Lee JC, et al. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy [J]. Science, 2015, 9 (5): 242-250.). The L858R point mutation in exon 21 accounts for 41% of all EGFR mutations and is one of the most common oncogenic mutations. Mutation of the 858th codon causes the L858R mutation, leading to further conversion of TG. Ultimately, the 858th codon in the EGFR protein is converted from the original amino acid to arginine. After conversion to this structure, it also promotes the sensitivity of cells to EGFR-TKI treatment. Previous studies have shown that the phosphorylation levels of downstream signaling pathways of EGFR gene exon 19 deletion and exon 21 L858R mutation are different, and exon 21 L858R mutation is more likely to occur simultaneously with other rare mutations (Hata A, Yoshioka H, ​​Fujita S, et al. Complex mutations in the epidermal growth factor receptor gene in non-small cell lung cancer [J]. J Thorac Oncol, 2010, 5 (10): 1524-1528.). EGFR overexpression may lead to cell proliferation, promote angiogenesis, tumor invasion and metastasis, and inhibit cell apoptosis, playing an important role in the development and progression of NSCLC. Chemoradiotherapy and EGFR tyrosine kinase inhibitors (EGFR-TKI) are the first-line treatment options for advanced EGFR L858R-positive NSCLC. However, in clinical practice, patients with EGFR L858R have a worse response to TKI than patients with EGFR 19Del. Some patients have poor first-line response to TKI, and patients who are effectively treated will inevitably develop drug resistance, and the efficacy of treatment after drug resistance is limited. New treatment models are urgently needed to improve the prognosis of these patients.

[0004] Although immunotherapy represented by immune checkpoint inhibitors (ICI) has become the standard first-line treatment for NSCLC, a study by Borghaei H et al. (Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer [J]. N Engl J Med. 2015; 373(17): 1627-1639.) showed that in clinical trials for NSCLC, patients with EGFR mutations benefited less from ICIs than patients with KARS, BRAF, and MET mutations. With the development of science and technology, it has been discovered that T cells recognize antigens by recognizing a sequence of 8-10 amino acids in the antigen molecule. This sequence determines the antigen specificity and is called the antigen epitope, which is a special chemical group in the antigen molecule that determines the antigen specificity. The peptide will be captured and phagocytosed by antigen presenting cells APCs (mainly dendritic cells DC, followed by macrophages), and then processed and presented by intracellular lysosomes or proteasomes, providing recognition (MHC) and co-stimulatory signals for the activation of corresponding T lymphocytes. Finally, with the help of the killing effect of CD8+T cells (such as the release of perforin, etc.), the tumor cells in the lesion site are lysed and cleared. Finding T cell mutation sites unique to the mutant genes of tumor patients and then designing and screening effective tumor neoantigen peptides has become a hot research direction. Tumor neoantigen peptides are considered to be the next generation of treatment after surgery, radiotherapy, chemotherapy and small molecule targeted therapy. Tumor neoantigens are combined with major histocompatibility complex (MHC) molecules and can be specifically recognized by cytotoxic T cell receptors (TCR) on the surface of tumor cells, thereby activating the immune response of T cells. Compared with traditional treatment methods, immunotherapy focuses on killing tumor cells by activating and enhancing the patient's own immune system, so it has advantages such as precise treatment, few side effects and lasting effects.

[0005] Earlier studies by Carreno BM et al. (Carreno BM, Magrini V, Becker-Hapak M, et al. A dendritic cell vaccine increases the breadth and diversity of melanomaneoantigen-specific T cells [J]. Science. 2015 May 15; 348 (6236): 803-8) showed that the strategy of using personalized neoantigen vaccines for cancer derived from mutant genes has been successful in both mouse models and clinical settings. The antigenic potential of top recurrent somatic mutations was evaluated by calculating the binding affinity between mutant epitopes and MHC class I, and it was found that most recurrent mutations were predicted to be antigenic. Developing a vaccine for personalized neoantigens for NSCLC patients with lung cancer hotspot mutations is a promising treatment for advanced lung cancer, but there are currently no reports of effective tumor neoantigens for the EGFR gene L858R mutation. Summary of the invention

[0006] Based on the inadequacy of the existing technology for the treatment of EGFR gene L858R mutant lung cancer, the present invention screened out a new antigen polypeptide, which can specifically activate killer T cells against EGFR gene L858R mutant tumor cells, thereby providing a therapeutic effect for this type of lung cancer. Based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides a tumor neoantigen polypeptide, the amino acid sequence of which is SEQID No.1, and the neoantigen polypeptide is a neoantigen polypeptide with an antigenic recognition region determined based on the EGFR gene L858R mutation.

[0008] Furthermore, the tumor is lung cancer.

[0009] Furthermore, the lung cancer is lung cancer accompanied by a high-frequency mutation of EGFR.

[0010] Furthermore, the high-frequency EGFR mutation is L858R mutation.

[0011] Furthermore, the lung cancer is lung cancer suffered by patients whose HLA typing of human leukocytes is HLA-A*02:06.

[0012] Furthermore, the new antigen polypeptide may also be a polypeptide with the same or similar function obtained by replacing and / or deleting and / or adding at least one amino acid in the amino acid sequence of the above polypeptide.

[0013] Furthermore, the polypeptide with the same or similar function refers to a tumor antigen polypeptide that can activate patients with HLA typing of HLA-A*02:06 and accompanied by EGFR L858R mutation to produce T cells specific to tumors with EGFR L858R mutation.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the tumor neoantigen polypeptide described in the first aspect.

[0015] In a third aspect, the present invention provides a gene expression vector, comprising a nucleotide encoding a tumor neoantigen polypeptide as described in the first aspect.

[0016] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect, wherein the host cell is transformed or transfected with the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect.

[0017] Furthermore, the host cells include bacteria, fungi and / or animal cells.

[0018] In a fifth aspect, the present invention provides a pharmaceutical composition, comprising the new antigen polypeptide described in the first aspect, and optional pharmaceutically acceptable excipients.

[0019] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0020] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

[0021] Furthermore, the preparation may be one or more of a conventional preparation, a sustained-release preparation and / or a controlled-release preparation.

[0022] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0023] Furthermore, the pharmaceutical composition can be administered via injection, cavity administration or respiratory tract administration.

[0024] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0025] In a sixth aspect, the present invention provides the use of the tumor neoantigen polypeptide described in the first aspect in the preparation of an immune activity regulator, wherein the immune activity regulator can activate and / or activate T cells.

[0026] Furthermore, the T cells include, but are not limited to, one or more of naive T cells, specific cytotoxic T cells, killer T cells and / or memory T cells.

[0027] Furthermore, the activation of the immune activity regulator requires the participation of antigen presenting cells (APC).

[0028] Furthermore, the APCs include but are not limited to dendritic cells (DCs), monocytes and / or B cells.

[0029] Preferably, the APC is a dendritic cell (DC), which is derived from a patient with L858R mutation in EGFR.

[0030] Furthermore, the DC cells contain human leukocytes whose HLA typing is HLA-A*02:06.

[0031] In a seventh aspect, the present invention also provides the use of the tumor neoantigen polypeptide described in the first aspect in the preparation of a drug for preventing and / or treating tumors.

[0032] Furthermore, the tumor is lung cancer.

[0033] Furthermore, the lung cancer is lung cancer accompanied by a high-frequency mutation of EGFR.

[0034] Furthermore, the high-frequency EGFR mutation is L858R mutation.

[0035] Furthermore, the lung cancer is lung cancer suffered by patients whose HLA typing of human leukocytes is HLA-A*02:06.

[0036] Beneficial Effects

[0037] 1. The present invention provides an effective method for predicting and screening tumor neoantigens using a tumor neoantigen prediction platform in a real tissue specimen transcriptome sequencing database, obtains unique mutations in tumor tissues, and more accurately predicts neoantigen polypeptides with high immunogenicity, high affinity, and high activation effect; the neoantigen polypeptides predicted and screened by this method have high affinity and can directly activate effector T cells without presentation through HLA molecules.

[0038] 2. The new antigen polypeptide provided by the present invention can stimulate and activate human T cells specific to the EGFR gene L858R mutation in vitro and amplify them in large quantities, and can be used as a tumor vaccine for the treatment of lung cancer patients.

[0039] 3. The new antigen polypeptide synthesized by the EGFR gene L858R mutation of the present invention can activate a large number of T lymphocytes specific to the EGFR gene L858R mutation peptide in vitro and release the cytokine IFN-γ, indicating that the polypeptide has obvious immunogenicity and can enhance the killing ability of T cells against cancer cells in lung cancer NSCLC patients with EGFR gene L858R mutation.

[0040] 4. The new antigen polypeptide of the present invention fills the gap of personalized antigen peptide in the treatment of NSCLC patients with EGFR-L858R somatic mutation. At the same time, the new antigen polypeptide of the present invention can be synthesized on a large scale and used in the subsequent standardized and personalized immunotherapy of patients with EGFR-L858R mutation tumors, and has a good clinical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Immunohistochemical staining of some lung cancer tissues. Note: Pt1-4 represent lung cancer tumor tissue samples from different patients.

[0042] Figure 2 This is to evaluate the effectiveness of the synthetic peptides No. 2 and No. 3. Note: MT.2 is the synthetic peptide No. 2, and MT.3 is the synthetic peptide No. 3; lectin (PHA, phytohemagglutinin) is the positive control, and the working concentration of PHA is 5μg / mL; Medium is an equal volume of sterile water, which is the negative control group.

[0043] Figure 3 This is the statistical chart of ELISPOT results of the synthetic peptide sequences No. 2 and No. 3. Note: SFU / 10 6 The cells are spot-forming units / million cells, based on the number of cells plated at 2×10 5 Multiply by 5 to get (i.e. average number of spots multiplied by 5).

[0044] Figure 4 The killing rate of CTLs specific to the synthetic polypeptide sequences No. 2 and No. 3 against lung cancer cells in vitro. DETAILED DESCRIPTION

[0045] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0047] Example 1 Prediction and screening of tumor neoantigens using the tumor neoantigen prediction platform

[0048] 1.1 Methods

[0049] 1. A total of 26 lung cancer tumor tissue samples and corresponding adjacent tissues were collected clinically, genomic DNA was extracted, and whole exome sequencing and transcriptome sequencing were performed. Through paired analysis of whole exome and adjacent tissues (normal tissues), unique mutations of tumor tissues can be obtained, and the peptides of these unique mutations constitute the first phase of the tumor neoantigen database.

[0050] 2. Calculate gene expression levels (FPKM) based on the transcriptome sequencing data of tumor samples. By comparing with the reference genome, filter out the new antigens with FPKM < 10 in the first-stage tumor new antigen database to form the second-stage tumor new antigen database.

[0051] 3. Perform HLA genotyping on each patient, predict antigen affinity based on the HLA typing of each patient and the second-stage tumor neoantigen database, and select tumor neoantigens with IC50 values ​​<500nM to form the final tumor neoantigen database.

[0052] 1.2 Results

[0053] A total of 5 candidate tumor neoantigen peptides were screened out (see Table 1).

[0054] Table 1 Information of candidate tumor neoantigen peptides

[0055]

[0056] Example 2 Tumor Immune Infiltration Assessment

[0057] The tumor microenvironment immune infiltration of lung cancer patients' tumor tissues was evaluated. Through tumor immune infiltration analysis, the malignancy and immune status of tumor tissues were judged, providing reference information for further screening of tumor neoantigens. The greater the degree of tumor immune infiltration, the stronger the immunogenicity of tumor neoantigens.

[0058] 2.1 Methods

[0059] 2.1.1 Immunohistochemical staining of four molecular markers CD3 / CD8 / CD45RO / FOXP3 in tumor tissue samples from 4 lung cancer patients.

[0060] 1. Sample Fixation and Sectioning

[0061] Tumor tissue samples were paraffin-embedded, sectioned, and mounted on glass slides.

[0062] 2. Dewaxing and hydration

[0063] The sections were first immersed in xylene to dissolve and remove the paraffin on the sections, and then washed in absolute ethanol (repeated twice), 95% ethanol, 75% ethanol and deionized water in sequence.

[0064] 3. Antigen Retrieval

[0065] High-pressure repair is used to open the cross-linking between tissue antigen proteins and formaldehyde, thereby improving the detection rate of tissue antigens.

[0066] 4. Removal of endogenous peroxidase

[0067] Endogenous peroxidase blocking agent was added to completely cover the tissue, incubated in the dark, and washed with PBST (0.1% Tween 20 dissolved in PBS buffer).

[0068] 5. Blocking and incubation of primary and secondary antibodies

[0069] Add an appropriate amount of sheep serum blocking solution, block at room temperature, add primary antibodies CD3 (1:500), CD8 (1:300), CD45RO (undiluted), FOXP3 (1:250) diluted with serum blocking solution, and incubate overnight. Wash repeatedly with PBST, add rabbit / mouse universal secondary antibody, incubate at room temperature, and wash with PBST. Add an appropriate amount of DAB reaction solution, and put it into deionized water to terminate the reaction after the reaction.

[0070] 6. Hematoxylin Counterstaining

[0071] Stain with hematoxylin, wash off excess dye, differentiate with hydrochloric acid and ethanol, wash with water, blue with ammonia water, and wash with water.

[0072] 7. Dehydration and sealing observation

[0073] The sections were soaked in 70%, 85%, 95% ethanol, anhydrous ethanol, and xylene, respectively. Neutral resin was added, the sections were sealed, and the sections were observed under a microscope after drying.

[0074] 2.1.2 Analysis of the infiltration levels of 10 types of cells in tumor tissue

[0075] The infiltration levels of 10 types of cells in tumor tissues were analyzed, including T cells, CD8+T cells, cytotoxic lymphocytes, natural killer cells (NK cells), B lymphocytes (B lineage), monocytes (Monocytic lineage), myeloid dendritic cells (Myeloid dendritic cells), neutrophils, endothelial cells (Endothelial cells) and fibroblasts.

[0076] Using the MCP-counter algorithm, based on a set of specific marker genes, the expression levels of immune-related genes are converted into the number of specific types of immune cells through a model, and an estimated value of the number of each immune cell type in a single sample is obtained, which is used to quantitatively evaluate the relative abundance of multiple cell types in the tumor microenvironment. The higher the value, the higher the corresponding immune cell content.

[0077] 2.2 Results

[0078] Depend on Figure 1 It can be seen that immune cells expressing CD3 / CD8 / CD45RO have a high degree of infiltration in tumor tissues, while immune cells expressing FOXP3 have almost no infiltration. As an immunomodulatory molecule, the high expression of FOXP3 indicates that the tumor environment is in a suppressive state. The high expression of CD3 / CD8 / CD45RO molecules indicates that there are more memory effector T cells in the tumor environment. Different NSCLC patients have a certain degree of immune infiltration, but the infiltration level varies.

[0079] The abundance of 10 types of immune cells (8 immune cells and 2 stromal cells) in the tumor tissues of 26 lung cancer patients was calculated by MCP-counter scores. As shown in Table 2, it can be seen that the content of fibroblasts in the cancer tissues of all 26 patients was generally increased, and the content of B lymphocytes in the patients was significantly increased, indicating that the infiltration degree of these immune cells in the tumor microenvironment was high.

[0080] Table 2 MCP-Counter Score results

[0081]

[0082] Example 3 In vitro cell experiment to evaluate the effectiveness of tumor neoantigen polypeptide sequences

[0083] 3.1 Methods

[0084] 1. Collection of peripheral blood mononuclear cells (PBMC)

[0085] Take the patient's peripheral blood and add an equal volume of PBS buffer. Take another centrifuge tube, add Ficoll (lymphocyte separation solution), add the diluted blood sample, centrifuge, remove the upper plasma layer, collect the middle buffy coat layer (PBMC), and freeze.

[0086] 2. T cell incubation

[0087] Take out the frozen PBMC cells and thaw the cells; centrifuge to remove the supernatant, resuspend the cells in a culture medium containing 5% fetal bovine serum, mix well, and count the number of live cells by trypan blue staining; after counting, adjust the cell concentration to 2×10 5 / mL and continue culturing; add the 5 synthetic peptides in Example 1 and cell growth factors IL-2 / IL-7 (working concentration 50 ng / mL) and culture at 37°C; collect the cells after ten days, wash the cells and set aside.

[0088] 3. ELISPOT detection and counting

[0089] First, add 100 μL of coated IFN-γ antibody to each well of the ELISPOT plate and incubate overnight; secondly, add 200 μL of 1640 culture medium and incubate at room temperature; respectively add the peptides in Example 1 (working concentration is 5 μg / mL), set up positive and negative control groups (water) for each, place in an incubator (5% CO2) and culture; finally, perform a color reaction, add detection antibody and streptavidin to the wells, wash away excess antibody, add color substrate, wash with sterile water, dry the plate, and detect and count the number of spots under a dissecting microscope.

[0090] 3.2 Results

[0091] The activation effects of the five selected tumor neoantigen peptides on the patient's T cells were systematically analyzed by ELISPOT plate spot counting. Figure 2 The ELISPOT results of antigen peptides No. 2 and 3 are shown. The spot counts are shown in Figure 3 and Table 3. After calculation, the positive spot forming unit (SFU) / million cells of the peptide No. 2 is 390, which is greater than the positive threshold of 50 (Porter M et al., J Invest Dermatol. 2022), indicating that the peptide can significantly activate T lymphocytes and release the cytokine IFN-γ, that is, the peptide produced by the EGFR gene carrying the L858R mutation can stimulate the immune response and increase the killing ability of T cells against cancer cells with the EGFR gene L858R mutation. In contrast, the positive spot forming unit (SFU) / million cells of the peptide No. 3 is 15, which is less than the positive threshold, indicating that it cannot stimulate T lymphocyte activation and release cytokine IFN-γ and is not immunogenic. Peptide No. 2 is a polypeptide derived from the L858R mutation of the EGFR gene.

[0092] The experimental results show that the peptide VSSELLCPV (No. 2) targeting the EGFR-L858R somatic mutation can significantly activate T lymphocytes in lung cancer patients and release cytokine IFN-γ, and the peptide has obvious immunogenicity. The new antigen peptide targeting the EGFR gene L858R mutation site can be used in standardized and personalized tumor immunotherapy in the future.

[0093] Table 3 ELISPOT plate spot counts

[0094] Average number of spots in negative control <![CDATA[Negative SFU / 10 6 cells]]> Average number of spots in positive control <![CDATA[Positive SFU / 10 6 cells]]> Peptide No. 1 0.6 3 3.7 18.5 Peptide No. 2 0 0 78 390 Peptide No. 3 3 15 3 15 Peptide No. 4 0 0 2.6 13 Peptide No. 5 1.5 7.5 3.7 18.5

[0095] Example 4 Neoantigen-specific CTL kills tumor cells in vitro

[0096] 4.1 Methods

[0097] 1. Isolation of PBMCs

[0098] PBMCs from positive lung cancer patients were collected according to the method in Example 3, and mononuclear cells were obtained by magnetic bead sorting.

[0099] 2. Isolation and culture of dendritic cells (DC) in vitro

[0100] The concentration of enriched monocytes was adjusted to 5×10 using RPMI-1640 culture medium (containing 10% FBS). 5 cells / mL, plated on a 24-well plate, added recombinant human IL-4 (20 ng / mL) and GM-CSF (20 ng / mL) at the same time, and incubated in 5% CO2 for 5 days to obtain monocyte-derived dendritic cells.

[0101] 3. Induction of CTL effector cells

[0102] The synthetic peptide 2 and peptide 3 in Example 1 of the present invention were added to stimulate DC. On the 6th day of differentiation, 10 ng / mL TNF-alpha and 10 ng / mL IL-6 were added to the DC with the added peptides to promote DC maturation. A polyclonal CTL killer cell population was obtained by amplification, and the antigen peptide 2 and antigen peptide 3 specific CTL cells were obtained.

[0103] 4. In vitro killing experiment

[0104] The new antigen peptide 2 / 3-DC-CTL in Example 1 was used as effector cells, and the human non-small cell lung cancer cell line A549 was used as target cells, and the effector-target ratios were set to 1:1 and 4:1, respectively. The LDH release experiment was used to detect the killing level of CTL on tumor cells, and the killing efficiency of CTL on target cells was calculated.

[0105] 4.2 Results

[0106] like Figure 4 As shown in Table 4, the results indicate that the new antigen peptide 2 (VSSELLCPV) can promote CTL to effectively kill tumor cells at different effector-target ratios, indicating that DC-CTL cell immunotherapy targeting VSSELLCPV is effective in the treatment of lung cancer.

[0107] Table 4 Statistics of killing rate of tumor cells by neoantigen-specific CTL cells (%)

[0108] Effect-target ratio 1:1 Effect-target ratio 4:1 Peptide No. 2 22.3 62.5 Peptide No. 3 8.9 20.1

Claims

1. A tumor neoantigen polypeptide, the amino acid sequence of which is SEQ ID No. 1, and the neoantigen polypeptide is a neoantigen polypeptide with an antigenic recognition region determined based on the EGFR gene L858R mutation.

2. A nucleic acid molecule encoding the tumor neoantigen polypeptide according to claim 1.

3. A gene expression vector comprising a nucleotide encoding the tumor neoantigen polypeptide as claimed in claim 1.

4. A host cell comprising the nucleic acid molecule according to claim 2 and / or the expression vector according to claim 3, wherein the host cell is transformed or transfected with the nucleic acid molecule according to claim 2 and / or the expression vector according to claim 3.

5. A pharmaceutical composition comprising the new antigen polypeptide according to claim 1 and optional pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, which can be prepared into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

7. Use of the new antigen polypeptide as claimed in claim 1 in the preparation of an immune activity regulator, wherein the immune activity regulator can activate and / or activate T cells.

8. The use according to claim 7, wherein the T cells include but are not limited to one or more of naive T cells, specific cytotoxic T cells, killer T cells and / or memory T cells.

9. Use of the new antigen polypeptide as claimed in claim 1 in the preparation of a drug for preventing and / or treating tumors, wherein the tumor is lung cancer accompanied by a high-frequency mutation of EGFR, and the high-frequency mutation of EGFR is L858R mutation.

10. The use according to claim 9, wherein the tumor is lung cancer suffered by a patient whose HLA typing of human leukocytes is HLA-A*02:06.

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