Lung cancer neoantigen polypeptide and its applications

Abstract

The present invention relates to the technical field of molecular immunology, and specifically discloses novel antigen polypeptides for lung cancer and their applications. The polypeptide comprises one or more amino acid sequences selected from those shown in, for example, PDGPPEKP (SEQ ID NO.1), MNKALLPAK (SEQ ID NO.2), and MYCELPDSF (SEQ ID NO.3); or is a polypeptide with the same or similar function obtained by substituting and / or deleting and / or adding at least one amino acid in the amino acid sequences of the above polypeptides. The novel antigen polypeptide for lung cancer provided by the present invention can bind to MHC class I molecules on human cells, can significantly stimulate the activation and expansion of T lymphocytes with tumor cell killing effects, and increase the killing ability of T cells against lung cancer cells. Therefore, the polypeptide can be used to prepare drugs for treating lung cancer, especially for preparing lung cancer cell vaccines. The antigen polypeptide can be used to treat lung cancer and prevent recurrence and metastasis in patients after surgery.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular immunology, and specifically to novel antigen polypeptides for lung cancer and their applications. Background Art

[0002] Lung cancer is one of the most malignant tumors globally and is the most common cause of tumor-related deaths. Most patients are in the middle and late stages when diagnosed with lung cancer, and the tumor often metastasizes. Advanced lung cancer patients experience metastasis of the tumor to various different organs, leading to poor prognosis. Currently, the efficacy of conventional treatment regimens (including surgery, radiotherapy, chemotherapy, targeted therapy, interventional therapy, traditional Chinese medicine, etc.) for advanced lung cancer has reached a bottleneck. Therefore, finding new treatment methods is crucial for improving the survival rate of advanced lung cancer patients.

[0003] Tumor immunotherapy is a new type of tumor treatment method and is considered the next-generation treatment after surgery, radiotherapy, chemotherapy, and small molecule targeted therapy. Compared with traditional treatment methods, immunotherapy focuses on treating tumors by enhancing the patient's own immune cells rather than directly killing tumor cells with drugs. This treatment method has many advantages, such as precise treatment, few side effects, and long-lasting effects. In addition, the body's immune system also has the characteristic of immune memory. Therefore, immunotherapy can help tumor patients form memory-type immunity and avoid the recurrence and metastasis of tumors.

[0004] With the in-depth study of the anti-tumor immune mechanism, tumor neoantigens have become a hot topic in immunotherapy research. Neoantigens are new protein molecules generated due to gene mutations during the process of cell malignant transformation and are generated by mutations (i.e., non-synonymous somatic mutations) that cause changes in the amino acid coding sequence. The peptide segments degraded from these abnormal proteins can bind to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum and are expressed on the cell surface. After being presented by DCs, they are recognized by CTLs, inducing specific killing of tumors. Neoantigens are only specifically expressed in tumor tissues, and normal tissues do not have these somatic mutations. Therefore, they are considered an ideal target for tumor immunotherapy.

[0005] Individualized neoantigen vaccines are based on gene sequencing and are personalized "advanced customized" vaccines targeting different mutation sites of each patient, including multiple sites. In recent years, with the rapid development of sequencing technology, the identification and application of neoantigens encoded by mutated genes in tumor cells has become a research hotspot in tumor immunology. Whole exome sequencing and transcriptome sequencing are performed using tumor patient tissues, and bioinformatics analysis is performed to obtain tumor cell-specific mutation sites and HLA typing and predict antigenic epitopes. Immunogenic tumor neoantigens are determined by in vitro stimulation of T cell activation experiments, which can be used for personalized treatment of tumor patients. Tumor neoantigens can be injected into patients as vaccines or combined with other biological agents to improve efficacy. Neoantigen vaccines have also been shown to be able to be used in combination with other treatments such as chemotherapy / radiotherapy, targeted drugs, immune checkpoint inhibitors, and cell stimulators, with more significant therapeutic effects. In 2018, an international research team led by researchers from the University of Pennsylvania published a study in Science Translational Medicine. The team used neoantigen vaccines to treat patients with advanced ovarian cancer. When 25 ovarian cancer patients were treated with tumor vaccines alone or in combination with other marketed drugs, they achieved better results than conventional treatments. In the same year, another article about neoantigen vaccines was published in Nature medicine. By performing whole exome sequencing and RNA sequencing on the subcutaneous lesions of the patient's right breast, 62 non-synonymous somatic mutations were found, of which only 4 mutations were potential attack lines, namely SLC3A2, KIAA0368, CADPS2 and CTSB mutant proteins. For these four mutations, the researchers designed vaccines to screen out various T cell populations with specific reactivity in patients, and then cultured and proliferated these T cell populations under appropriate conditions, and adoptively infused them. 22 months after the adoptive transfer of T cells, all target and non-target lesions in the patient had disappeared. A patient with advanced ovarian cancer was successfully cured by using neoantigen vaccines combined with adoptive T cell therapy. The above clinical research results successfully confirmed the feasibility of tumor treatment with tumor vaccines targeting neoantigens and their good clinical application prospects, and provided lung cancer neoantigen peptides and their applications. Summary of the invention

[0006] The purpose of the present invention is to provide a new lung cancer antigen polypeptide and its application in view of the defects of the prior art, so as to solve the problems raised by the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A novel lung cancer antigen polypeptide, which polypeptide comprises one or more amino acid sequences selected from those shown in PDGPPEKP (SEQ ID NO.1), MNKALLPAK (SEQ ID NO.2) and MYCELPDSF (SEQ ID NO.3);

[0009] Alternatively, it is a polypeptide with the same or similar function obtained by substituting and / or deleting and / or adding at least one amino acid in the amino acid sequences of the above polypeptides.

[0010] 2. The novel lung cancer antigen polypeptide according to item 1, wherein the polypeptide comprises modifications that increase in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, antigen presentation, or a combination thereof, and the modifications are conjugation with a carrier protein, conjugation with a ligand, conjugation with an antibody, PEGylation, polysialylation, HESylation, Fc fusion, albumin fusion, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, or addition of non-natural amino acids.

[0011] 3. A fusion protein or immunoconjugate, comprising the novel lung cancer antigen polypeptide according to any one of items 1-2, wherein the tumor antigen polypeptide is fused with a carrier protein, a ligand protein, an antibody, an Fc fragment, albumin, cholesterol, iron, or a protein targeting dendritic cells.

[0012] 4. An in vivo delivery system, comprising the tumor antigen polypeptide according to any one of items 1-2, and the delivery system further comprises a cell-penetrating peptide, nanoparticle encapsulation, virus-like particles, liposomes, or any combination thereof, and the cell-penetrating peptide is a TAT peptide, herpes simplex virus VP22, a transporter, Antp, or any combination thereof.

[0013] 5. A nucleic acid molecule encoding the tumor antigen polypeptide according to any one of items 1-2 or the fusion protein or immunoconjugate according to item 3.

[0014] 6. An in vivo delivery system comprising the nucleic acid molecule according to item 5, and the delivery system further comprises a plasmid, a virus, virus-like particles, or nanoparticles.

[0015] 7. A cell comprising or expressing the tumor antigen polypeptide according to any one of items 1-2 or the fusion protein or immunoconjugate according to item 3, or comprising a vector having the nucleic acid molecule according to item 5, or comprising the delivery system according to any one of items 4 or 6.

[0016] 8. An antibody against the tumor neoantigen polypeptide according to item 1, and the antibody is a polyclonal antibody or a monoclonal antibody.

[0017] 9. A pharmaceutical composition comprising the tumor antigen polypeptide described in any one of items 1-2, or the fusion protein or immunoconjugate described in item 3, or a vector comprising the nucleic acid molecule described in item 5, or the delivery system described in any one of items 4 or 6, or the cell described in item 7, or the antibody described in item 8, and optionally a pharmaceutically acceptable carrier or adjuvant.

[0018] 10. Use of the tumor antigen polypeptide described in any one of items 1-2, or the fusion protein or immunoconjugate described in item 3, or a vector comprising the nucleic acid molecule described in item 5, or the delivery system described in any one of items 4 or 6, or the antibody described in item 8, or the cell described in item 7 in the preparation of a peptide vaccine, gene vaccine or DC vaccine drug for preventing or treating tumors.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The neoantigen polypeptide of lung cancer provided by the present invention can bind to MHC class I molecules on human cells, can significantly stimulate the activation and expansion of T lymphocytes with tumor cell killing effects, and increase the killing ability of T cells against lung cancer cells. Therefore, this polypeptide can be used to prepare drugs for treating lung cancer, especially for preparing lung cancer cell vaccines. This antigen polypeptide can be used to treat lung cancer and prevent recurrence and metastasis in patients after surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the liquid chromatography detection map of polypeptides 1-3 of the present invention;

[0021] Figure 2 It is the result of the enzyme-linked immunospot assay of polypeptides 1-3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The following elaborates on the preferred embodiments of the present invention in conjunction with the drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making the protection scope of the present invention more clearly defined.

[0023] Example 1: Prediction and screening of neoantigens of lung cancer patients;

[0024] 1. Material preparation;

[0025] Peripheral blood and lung cancer tumor tissues of a lung cancer patient were collected through clinical sampling, DNA was extracted, and the protein-coding regions of 19,433 genes in the human genome were deeply sequenced using the Illumina high-throughput sequencing platform. According to the sequencing results and the reference genome (GRCh37 / hg19), tumor gene variant analysis and annotation were performed to analyze gene variant situations. Somatic mutation detection included: single nucleotide variants (SNVs), insertions and deletions (InDELs), copy number variations (CNVs), and gene fusions (Fusions). Germline mutation detection included: single nucleotide variants (SNVs), insertions and deletions (InDELs), and HLA typing was determined.

[0026] 2. Neoantigen screening;

[0027] The original fastq data of DNA sequencing was quality-controlled by the FastQC software to obtain the quality-controlled and filtered data, and the following procedures were carried out to identify candidate neoantigens:

[0028] (1) The original fastq data of DNA sequencing was quality-controlled by the FastQC software to obtain the quality-controlled and filtered data. The GATK software was used to quality-control the data, remove low-quality short sequences (reads), align the filtered reads to the reference genome, and generate a BAM-format alignment file;

[0029] (2) Variant screening was performed through the Mutect2 algorithm of GATK, the somatic mutations obtained were detected by VarScan, and based on the WES sequencing data, the variant allele frequency (VAF) was detected. Mutations with VAF > 10% and sequencing depth > 20x were retained;

[0030] (3) The ANNOVAR software was used to annotate the mutations, and mutations that changed the amino acid sequence were screened out.

[0031] (4) The IEDB website was used to predict the neoantigens generated by the mutations, and mutant polypeptides with an IC50 affinity for HLA molecules < 250 nM and a length of 9 - 11 amino acids were selected as candidate neoantigens. 50 < 250 nM and a length of 9 - 11 amino acids were selected as candidate neoantigens.

[0032] (5) Three candidate tumor neoantigens as shown in Table 1 were screened and prepared by chemical synthesis. The synthesized peptides of the prepared polypeptides were sterile and endotoxin-free. HPLC (C18 column, 220 nm) detection and analysis of the obtained polypeptides showed that the purity was > 98% ( Figure 1 ).

[0033] Table 1: Information of tumor neoantigen peptides

[0034]

[0035] Example 2: Detection of specific cytotoxic T cells in the patient's peripheral blood by Elispot;

[0036] (1) Isolation and culture of PBMC cells from the patient's peripheral blood: Transfer 10 ml of whole blood into a 50-ml centrifuge tube, add an equal volume of physiological saline for dilution, and mix well; Take a centrifuge tube and add 10 ml of Ficoll solution. Gently add the diluted blood to the upper layer of Ficoll in the centrifuge tube and centrifuge at 1500 rpm for 30 min. Use a pipette to aspirate the cells in the cell layer where PBMC is located into another clean centrifuge tube, add physiological saline to 30 ml, and centrifuge at 1500 rpm for 10 min. After centrifugation, discard the supernatant, and then add physiological saline for washing with the same operation; Resuspend the cells with the medium, plate them, and place them in the incubator for 2 h. The adherent cells are used for the induction culture of DC cells, and the non-adherent cells, T cells, and the remaining cells are frozen for subsequent T cell induction.

[0037] (2) Induction culture of DC cells: The adherent cells obtained in step (1) are cultured in human DC cell medium (RPMI1640 complete medium, 10 ng / ml rhIL-4, 500 U / ml rhGM-CSF) to induce the differentiation of monocytes into DC cells. Collect the DC cells derived from peripheral blood monocytes cultured for 6 days above, adjust the cell concentration to 2×10 5 cells / ml with human DC cell medium, divide them into 24-well plates, add 20 μM tumor polypeptide, and collect the cells 4 - 6 hours later. Discard the supernatant of the medium, centrifuge and wash the cells with RPMI1640 medium to remove the stimulants present in the original medium. Finally, suspend 2×10 5 cells in 0.5 ml of medium for stimulating allogeneic T lymphocytes.

[0038] (3) Induction culture of T cells: Resuscitate the cryopreserved T cells and add them to the peptide-sensitized autologous DC cells at a concentration of 4×10 6 cells / ml for co-culture. Add 20 U / ml rhIL-2 on the fifth day of co-culture, collect the lymphocytes 7 days after culture, and co-culture them with the peptide-sensitized allogeneic DC cells at a ratio of 10:1 for the second round of stimulation. The same stimulation is performed once a week for a total of three times. During this period, add 20 U / ml rhIL-2 every 3 days, change half of the medium every 3.5 days, and perform sub-well amplification of the cells as needed. Collect the cells 7 days after the last stimulation, and perform ELISPOT detection of IFN-γ on the collected T cells.

[0039] (4) Enzyme Linked Immunospot Assay (ELISPOT) is used to detect the secreted IFN-γ to evaluate the T cell immune response activated by the antigen peptide: Adjust the T cell concentration to 5×106 cells / ml. The cell suspension was directly transferred into an ELISPOT assay plate coated with anti-IFN-γ antibody, 100 μl per well. Two replicates were set up for each group, and irrelevant peptide control and PHA control were established, followed by overnight culture for 20 h. The T cell colonies secreting IFN-γ were detected according to the method described in the IFN-γ ELISPOT assay kit instruction manual, and the spots were analyzed using an instrument. The results are as Figure 2 shown.

[0040] The key to determining the success of neoantigen prediction is whether neoantigen-specific T cells can be effectively activated. Substances secreted by T cells activated by neoantigens, such as IFN-γ, TNF-α, granzyme B, and perforin, can be used to measure the level of antigen-specific T cells. Among them, the cytokine IFN-γ is often used to track specific CD8 + responding T cells and has become the most widespread form for evaluating the induction of antigen-specific T cell responses by candidate antigen peptides. Multiple immunological methods can be used to evaluate the T cell response to neoantigens in vitro, such as ELISPOT, mutant peptide-HLA molecule tetramer, enzyme-linked immunosorbent assay (ELISA), etc. ELISPOT has obvious advantages compared with other immunological assays, such as being HLA-independent, requiring relatively fewer cells, and having high sensitivity, and is currently the most commonly used method. When T cells are stimulated by antigen peptides to produce IFN-γ, the coated antibody on the ELISPOT plate in situ captures this factor produced by T cells, and through a color reaction, dark spots appear at the positions of IFN-γ secreted by the cells. Each spot can represent the "footprint" of specific T cells activated by antigen peptide stimulation. As Figure 2 can be seen, all three polypeptides have good immunogenicity and can significantly activate specific T cells against the mutant antigen peptides of ARID1A (p.R1906P), MAP3K6 (p.V527A), and NBPF12 (p.F1406C). Therefore, they can effectively increase the killing ability of T cells against lung cancer cells carrying this type of mutation.

[0041] The above embodiments only represent the implementation modes of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A novel antigen polypeptide for lung cancer, characterized in that: The amino acid sequence of the polypeptide is shown as MNKALLPAK (SEQ ID NO.2).

2. A nucleic acid molecule, characterized in that: It encodes the tumor antigen polypeptide described in claim 1.

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