CN111777677A

CN111777677A - EGFR T790M new antigen epitope peptide and application thereof in treating tumors

Info

Publication number: CN111777677A
Application number: CN201910437051.3A
Authority: CN
Inventors: 杜学明; 李凤娥; 霍冲; 邓丽刚; 邹庆薇; 王亚玲
Original assignee: Tianjin Hengjia Biotechnology Development Co ltd
Current assignee: Tianjin Hengjia Biotechnology Development Co ltd
Priority date: 2019-04-04
Filing date: 2019-05-24
Publication date: 2020-10-16

Abstract

The invention provides an EGFR T790M new antigen epitope peptide and application thereof in treating tumor, wherein the EGFR T790M new antigen epitope peptide sequence contains one or two different amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, or contains an amino acid sequence formed by adding, deleting or replacing one or more amino acids of the amino acid sequence shown in one or two different amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2. The new epitope peptide can activate T lymphocytes, specifically kill HLA-C1502 tumor cells with EGFR T790M mutation, and can be used for preparing cancer drugs for treating non-small cell lung cancer.

Description

EGFR T790M new antigen epitope peptide and application thereof in treating tumors

Technical Field

The invention belongs to the field of molecular immunology, relates to an epitope peptide, and particularly relates to an EGFR T790M new epitope peptide and application thereof in tumor treatment.

Background

Lung cancer is a common high-grade malignant tumor in clinic, the prognosis is very poor, and the five-year survival rate is less than 10%. According to histological classification, lung cancer can be classified into small cell lung cancer and non-small cell lung cancer, wherein the incidence rate of non-small cell lung cancer accounts for about 80%. Surgery is the first and most important treatment for lung cancer. However, more than half of patients with non-small cell lung cancer have long-range metastasis during the first diagnosis, and the chance of surgical treatment is lost. The appearance of tumor-targeted drugs changes the traditional treatment mode of tumors to a great extent, greatly improves the prognosis of patients, and remarkably prolongs the progression-free survival period and the overall survival period, particularly the appearance of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). Epidermal Growth Factor Receptor (EGFR) mutations can occur in about half of non-small cell lung cancer patients, and the activation of signal pathways mediated thereby plays an indispensable role in the development and progression of non-small cell lung cancer. The non-small cell NCCN guidelines recommend targeted drugs as a first-line treatment regimen for patients with advanced lung cancer with mutations in the EGFR gene. The EGFR gene is composed of 28 exons, the tyrosine kinase functional region of the EGFR gene is coded by the exons 18-24, wherein the exons 18-21 are the most common gene mutation points, and for patients with advanced non-small cell lung cancer with sensitive mutation of the EGFR gene, the EGFR gene has obviously prolonged survival rate and effective rate in the aspects of no progress, compared with the traditional chemotherapy, and meanwhile, the EGFR gene has better survival quality and tolerance. However, often after targeted therapy, resistance inevitably occurs. Among them, the T790M mutation in exon 20 is the most common, and about 60% of patients clinically counted at present have EGFR-TKI acquired drug resistance. The T790M mutation theory suggests that the 20 th exon of the EGFR gene undergoes secondary mutation during the treatment process of applying EGFR-TKI, and as a result, the formation of the steric hindrance affects the formation of hydrogen bonds between tyrosine kinase and EGFR-TKI, and finally the EGFR-TKI cannot be combined with the tyrosine kinase, namely, first-line targeted drugs such as Iressa cannot be continuously effective, and the mutation of T790M is the most important factor of EGFR-TKI secondary drug resistance.

With the development of immunology and biotechnology, tumor immunotherapy has become another innovation of tumor treatment mode after surgery, chemotherapy, radiotherapy, targeted therapy. The immunotherapy utilizes the immune system to recognize tumor-associated antigens, regulate the ability of an organism to attack tumor cells, improves the immunogenicity and the sensitivity of the tumor cells to killing of effector cells by intervention means, stimulates or enhances the anti-tumor immune response of the organism, and plays roles of killing tumors and inhibiting the growth of the tumors by cooperating with the immune system of the organism. The protein with specific amino acid sequence variation generated by tumor cells based on gene variation is called neoantigen (neoantigen), and if the abnormal protein is degraded into short peptide fragments (antigen epitope) in cells (tumor cells or APCs), the short peptide fragments are combined with MHC-I or MHC-II molecules with high affinity and presented on the cell surface in a complex form, and the abnormal protein is recognized by T lymphocyte receptor (TCR) expressed on the surface of T cells to cause the activation of the T cells, thereby attacking and eliminating the tumor cells. The new antigen epitope peptide caused by gene mutation is artificially synthesized to construct personalized tumor vaccine, and the personalized tumor vaccine is returned to the body to activate immune cells, so that the tumor cells with the antigen can be killed. The novel antigens are ideal targets for tumor immunotherapy. Because the neoantigen is specifically expressed only in tumors, it is less likely to induce tolerance, damaging normal tissue cells.

Disclosure of Invention

The invention aims to provide an EGFR T790M new antigen epitope peptide and application thereof in treating tumors, wherein the new antigen epitope peptide has the function of activating T cells to kill tumor cells in a targeted manner.

The amino acid sequence of the new antigen epitope peptide is selected from any one of the following items:

1) peptide comprising one or two different amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2;

2) a peptide consisting of an amino acid sequence formed by adding, deleting or replacing one or more amino acids to, or from two different amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2,

the new antigen epitope peptide can be used alone, or can be used in combination with other epitope peptides.

The new antigen epitope peptide can form a new antigen epitope peptide-HLA-C1502 compound with an HLA-C1502 molecule. HLA-C1502 molecules may be derived from antigen presenting cells or target cells (tumor cells).

The complex of the neo-epitope peptide-HLA-C1502 can be recognized by HLA-C1502 restricted T lymphocytes or induce HLA-C1502 restricted cytotoxic T lymphocytes.

The new epitope peptide can be incubated with antigen presenting cells to induce the antigen presenting cells to mature and form a new epitope peptide-HLA-C1502 complex on the cell surface.

The new antigen epitope peptide, the new antigen epitope peptide-HLA-C1502 compound and the antigen presenting cell co-incubated with the new antigen epitope peptide can induce the activation of T lymphocyte to form cytotoxic T lymphocyte and kill the target cell presenting the new antigen epitope peptide.

The new antigen epitope peptide, the new antigen epitope peptide-HLA-C1502 compound and the antigen presenting cell of the co-incubation epitope peptide can be applied to the field of antitumor drugs, such as active ingredients of therapeutic tumor vaccines.

The therapeutic tumor vaccine or immunogenic composition can induce specific cytotoxic T cell response, and the vaccine or immunogenic composition comprises one or more of a new antigen epitope peptide, an adjuvant and a carrier, and further, the new antigen epitope peptide can be combined with the carrier or antigen presenting cells (such as dendritic cells).

The anti-tumor drug can treat HLA-C1502 patients containing EGFR T790M. Preferably, the patient is a non-small cell lung cancer patient.

The novel epitope peptides can be used for preparing immunotherapy drugs or methods for treating tumors, including adoptive cell therapy, such as chimeric antigen receptor T cell technology (CAR-T) and T cell receptor chimeric T cell technology (TCR-T).

The new antigen epitope peptide and the new antigen epitope peptide-HLA-C1502 compound can be used for screening a specific T lymphocyte receptor (TCR), and the specific TCR recognizes the epitope peptide-HLA-C1502 compound and mediates T lymphocytes to play an anti-tumor cytotoxic effect.

The antitumor drug can be used alone or in combination with an immunologic adjuvant or in combination with other antitumor drugs or treatment methods, and the combined drugs can comprise a chemical drug, a targeting drug and an immune checkpoint inhibitor; the treatment method comprises chemotherapy. The methods of use may be administered simultaneously or sequentially in any order.

The anti-tumor drug can be a pharmaceutical composition, comprising one or more of the new antigen epitope peptide, optionally pharmaceutically acceptable carriers, excipients and additive combinations, and further, the new antigen epitope peptide comprises pharmaceutically acceptable salts thereof.

The invention has the beneficial effects that:

the EGFR T790M mutation is a common drug resistance mutation after a first-line EGFR targeted drug is used for treating lung cancer, and the novel epitope peptide vaccine can provide a promising immunotherapy method which can further aim at EGFR targeted drug resistance for patients with HLA-C1502 type EGFR T790M mutation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1A flow cytometry analysis of specific CTL ratios induced by neoantigenic peptide LTSTVQLIM.

FIG. 1B flow cytometry analysis of specific CTL ratios induced by neoantigenic peptide STVQLIMQL.

Figure 2A flow cytometry analyses HLA expression rates of T2 cells not transfected with HLA-C1502 (control).

Figure 2B flow cytometry analysis of HLA expression rate of T2 target cells transfected with HLA-C1502.

FIG. 3A ELISA was performed to examine the IFN-. gamma.release amount of the neoantigenic peptide LTSTVQLIM-specific CTL incubated with T2 target cells.

FIG. 3B ELISA was performed to examine the amount of IFN-. gamma.released from the co-incubation of T2 target cells with neoantigenic peptide STVQLIMQL-specific CTLs.

FIG. 4 ELISA assays IFN-. gamma.release from PBMCs of patients treated with neoantigenic peptide LTSTVQLIM.

FIG. 5 ELISA assays IFN-. gamma.release from PBMC of patients treated with neoantigenic peptide STVQLIMQL.

The specific implementation mode is as follows:

example 1 isolation of peripheral blood mononuclear cells

20ml of venous blood from HLA-C1502 healthy donors were removed, blood samples were diluted with equal volumes of sterile PBS and lymphocyte isolates (Stemcell, 07861) were transferred to a new 50ml centrifuge tube ensuring a volume ratio to blood of 3: 4, carefully adding the diluted blood to the surface of the separating medium, operating as gently as possible, avoiding mixing, and clearly seeing the boundary between the two liquids. Centrifuge at 400g for 30min at room temperature. After centrifugation, the mononuclear cell layer was aspirated, transferred to a new sterile centrifuge tube, and washed twice with PBS. The cells were resuspended in RPMI-1640 medium and counted.

Example 2 neoepitope peptide activates specific T lymphocytes

2.5 × 10⁶One cell is suspended in 90% RPMI-1640 +10% FBS (containing IL-2, 20 ng/ml), 2.5 × 10 was added⁵Dynabeads^®Human T-Activator CD3/CD28 magnetic beads (Gibco, 11131D) were seeded into 6-well plates at 1.25 × 10 per well⁶The neoantigen peptide LTSTVQLIM (2.5 μ g/ml) and the neoantigen peptide STVQLIMQL (2.5 μ g/ml) were added to each cell and each well, and the mixture was incubated at 37 ℃ and 5% CO₂And (4) incubating. After 24 hours, the cells and the magnetic beads were resuspended, placed on a magnetic frame, the magnetic beads were discarded, the supernatant cells were collected and cultured for one week, and the activated specific Cytotoxic T Lymphocytes (CTLs) were detected using the neoantigen-specific tetramer, as shown in fig. 1, where fig. 1A shows that the expression rate of CTLs caused by the specificity of neoantigen peptide LTSTVQLIM was 31.4%, and fig. 1B shows that specific CTLs caused by the expression rate of neoantigen peptide STVQLIMQL was 24.1%.

EXAMPLE 3 killing of target cells by specific CTLs

1. Construction of T2 target cells for HLA-C1502

The full-length HLA-C1502 gene was cloned into a lentiviral expression vector pCDH (purchased from SBI) using restriction endonucleases EcoRI and BamHI, resulting in recombinant plasmid construction of the lentiviral expression vector pCDH-C1502 overexpressing HLA-C1502 molecules.

293T cells (purchased from basic medical research institute of Chinese academy of medical sciences) in a logarithmic growth phase are taken and inoculated into a T25 cell culture flask containing 10% FBS DMEM medium, and transfection is carried out when the cell confluence reaches 80-90%. The recombinant plasmid and the mixed packaging vector plasmid pPACKH1 (from SBI) were mixed well, and after mixing well, 500. mu.l of 1. mu.g/. mu.l Lipofectin Liposome transfection reagent Lip2000 (Invitrogen, 11668-. The DNA/liposome complex is added into a culture dish drop by drop and mixed evenly. Placing the culture dish at 37 ℃ and 5% CO₂The incubator, after 6-8 hours of incubation, removes the medium containing the transfection reagent and replaces it with fresh complete medium. After 48 hours, the culture medium was collected into a sterile centrifuge tube, centrifuged at 4 ℃ and 2000g for 10 minutes, the supernatant was filtered through a 0.45 μm PES membrane, a new sterile ultracentrifuge tube was added to the virus-containing culture medium supernatant (i.e., filtrate), and centrifuged at 4 ℃ and 20,000g for 3 hours. Carefully centrifuge the tubeAnd (4) sucking the liquid in the step (a), and obtaining the precipitate as the target lentivirus.

The T2 cell line was virus transduced, polybrene (Santa Cruz, sc-134220) was added to the cultured T2 cells to a final concentration of 6. mu. to/ml, the above lentivirus was added, blown gently and mixed well, and centrifuged at 800g at room temperature for 1 hour. Then placing at 37 ℃ and 5% CO₂The culture chamber of (1) was maintained for 24 hours, the virus-containing medium supernatant was removed, the cell pellet was resuspended in fresh medium, the cells were transferred to a new culture vessel and maintained for further culture, and on day 4 the cells were labeled with a labeled antibody and tested for expression of HLA-C1502 by flow assay, see figure 2. Fig. 2A shows T2 cells that were not transfected with HLA-C1502, fig. 3B shows T2 target cells that were transfected with HLA-C1502, and the transfection efficiency of HLA-C1502 was 98%.

2. Co-incubation experiment of specific CTL and target cell

Target cells T2-C1502 adjusted cell density to 2 × 10⁵Per mL, according to 2 × 10⁴The target cells were inoculated in a 96-well plate per well, and 100. mu.l of 1640 medium (containing 10% FBS) was added, and 10uM neoantigen peptide LTSTVQLIM and 10uM neoantigen peptide STVQLIMQL were added, respectively. Place 96-well plates at 37 ℃ in 5% CO₂Incubate in incubator for 4 h.

The CTL cells obtained in example 2 were collected by centrifugation, resuspended in 1640 medium (containing 10% FBS), and then added to T2 target cells in which neoantigen peptide LTSTVQLIM was incubated and T2 target cells in which neoantigen peptide STVQLIMQL was incubated in a 96-well plate at an effective target ratio of 1:1, 2.5:1, 5:1, 10:1, respectively, and the 96-well plate was placed at 37 ℃ with 5% CO₂And (5) culturing for 24 hours in an incubator. After the culture was completed, the 96-well plate was removed, centrifuged at 800g for 10min at room temperature, and the expression of the cytokine IFN-. gamma.in the co-culture supernatant was examined using a Human IFN-. gamma.ELISA Kit (purchased from Davidae). The results are shown in fig. 3, and fig. 3A shows the killing effect of the neoantigenic peptide LTSTVQLIM-specific CTL on T2 target cells, at an effective-to-target ratio of 10:1, the IFN-gamma secretion amount of CTL co-incubated with target cells can reach 1805pg/ml, and FIG. 3B shows the killing effect of the neoantigen peptide STVQLIMQL specific CTL on T2 target cells, wherein the effective-to-target ratio is 10:1, the CTL co-incubated with the target cells can secrete IFN-gamma in an amount of 1440 pg/ml.

Example 4 treatment of HLA-C1502 patients with EGFR T790M with the neoantigenic peptide LTSTVQLIM

Patient a, female, 48 years old, non-small cell lung cancer stage IV, adenocarcinoma, right lung single-haired, pleural metastases, bone metastases, liver metastases. Vinorelbine and cisplatin progress after chemotherapy, erlotinib and ocitinib resist drug after targeted therapy, and diseases progress. Lung lesion tissue biopsies were taken and subjected to the detection of genes related to tumorigenesis development and the detection of HLA typing, which showed that patients carried EGFR T790M mutation, HLA typing was HLA-a 1102A 2402, HLA-B1501B 4501, HLA-C1502C 0103, HLA-DRB1 0701DRB1 1454, and HLA-DQB1 0202 DQB1 0502. The neoantigenic peptide LTSTVQLIM of the present invention was used for therapy, and 200. mu.g of the neoantigenic peptide was dissolved in 1ml of PBS and injected subcutaneously into the upper arm 1 time per week for 12 consecutive weeks.

Collecting blood before treatment and 4 weeks, 8 weeks and 12 weeks after treatment, separating mononuclear cells (PBMC) according to cell size of 2-5 × 10 per well⁵Individual cells, 250 μ l total volume, were added to 96-well plates. Adding LTSTVQLIM (12.5 μ g/ml) and IL-2 (500 u/ml) to corresponding wells, using wells without antigen peptide as controls, at 37 deg.C and 5% CO₂Culturing for 3 days, taking cell culture supernatant to perform IFN-gamma level detection, taking a human interferon-gamma ELISA detection kit (Beijing Dake is biotechnology limited) to balance for 20min at room temperature, taking 100 mul of cell culture solution supernatant, adding 100 mul of standard products with different concentrations after dilution to corresponding holes, adding 100 mul of dilution buffer to a blank control, adding 50 mul/hole of an antibody diluted by 1:50 of the dilution buffer, covering a sealing membrane to react for 2h at room temperature, and washing for three times by 300 mul/hole of washing liquor, wherein the used standard products, the dilution buffer, the antibody and the 1 × washing liquor are all reagents in the human interferon-gamma detection kit, the result is shown in a figure 4, the IFN-gamma secretion capacity of peripheral blood specific T cells of a patient is increased after 4 weeks, 8 weeks and 12 weeks of treatment, and the specific immunoreaction of the patient is activated by peptide immunotherapy.

The patient has no serious adverse reaction during the treatment period, stable diseases before and after treatment, no obvious progress, better life quality of the patient and clinical benefit.

Example 5 treatment of HLA-C1502 patients with EGFR T790M with the neoantigenic peptide STVQLIMQL

Patient B, female, age 61, non-small cell lung cancer stage IIIB, adenocarcinoma, right lung single-shot, pleural metastases. Pemetrexed + carboplatin 5 cycle, docetaxel + carboplatin 1 cycle, drug resistance after gefitinib and ocitinib targeted therapy, and disease progression. Lung lesion tissue biopsy is taken, and tumor development related gene detection and HLA typing detection are carried out, and the results show that the patient carries the EGFRT790M mutation, and the HLA typing is HLA-A1102A 1101, HLA-B3501B 4001, HLA-C1502C 0401, HLA-DRB1 0301 DRB1 0406 and HLA-DQB1 0201 DQB1 0302. The neoantigenic peptide STVQLIMQL of the present invention was used for therapy, and 200. mu.g of the neoantigenic peptide was dissolved in 1ml of PBS and injected subcutaneously into the upper arm 1 time per week for 12 consecutive weeks.

Collecting blood before treatment and 4 weeks, 8 weeks and 12 weeks after treatment, separating mononuclear cells (PBMC) according to cell size of 2-5 × 10 per well⁵Individual cells, 250 μ l total volume, were added to 96-well plates. Adding STVQLIMQL (12.5 μ g/ml) and IL-2 (500 u/ml) to corresponding wells, using wells without antigen peptide as controls, at 37 deg.C and 5% CO₂Culturing for 3 days, taking cell culture supernatant to perform IFN-gamma level detection, taking a human interferon-gamma ELISA detection kit (Beijing Dake is biotechnology limited) to balance for 20min at room temperature, taking 100 mul of cell culture solution supernatant, adding 100 mul of standard products with different concentrations after dilution to corresponding holes, adding 100 mul of dilution buffer to a blank control, adding 50 mul/hole of an antibody diluted by 1:50 of the dilution buffer, covering a sealing membrane to react for 2h at room temperature, and washing for three times by 300 mul/hole of washing liquor, wherein the used standard products, the dilution buffer, the antibody and the 1 × washing liquor are all reagents in the human interferon-gamma detection kit, the result is shown in a figure 5, the IFN-gamma secretion capacity of peripheral blood specific T cells of a patient is increased after 4 weeks, 8 weeks and 12 weeks of treatment, and the specific immunoreaction of the patient is activated by peptide immunotherapy.

The EGFR T790M new antigen epitope peptide provided by the invention and the application thereof in treating tumors are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these changes and modifications also fall into the protection scope of the appended claims.

Sequence listing

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Claims

1. A neo-epitope peptide, or a combination thereof, selected from any one of:

2) and (b) a peptide consisting of an amino acid sequence formed by adding, deleting or replacing one or more amino acids to, from or to one or two different amino acid sequences represented by SEQ ID No. 1 and SEQ ID No. 2, wherein the epitope peptide is capable of forming a complex with HLA-C1502 molecules and recognizing or inducing HLA-C1502-restricted T lymphocytes.

2. A nucleotide sequence encoding the amino acid sequence of the neo-epitope peptide according to claim 1.

3. A major histocompatibility antigen complex comprising an HLA-C1502 molecule and the neoepitope peptide of claim 1.

4. An antigen presenting cell capable of recognizing and presenting the neo-epitope peptide according to claim 1.

5. A T lymphocyte inducer, characterized in that the active ingredients of the T lymphocyte inducer comprise:

1) the neo-epitope peptide according to claim 1;

2) a major histocompatibility antigen complex according to claim 3;

3) the antigen presenting cell of claim 4;

one or more of them.

6. The tumor vaccine is characterized in that the tumor vaccine is a therapeutic vaccine, and the active ingredients of the tumor vaccine comprise:

1) the neo-epitope peptide according to claim 1;

2) a major histocompatibility antigen complex according to claim 3;

3) the antigen presenting cell of claim 4;

against tumors carrying the EGFR T790M gene mutation and HLA-typed HLA-C1502.

7. Use of the neo-epitope peptide according to claim 1 for the preparation of a medicament for the treatment of cancer, wherein the cancer is a tumor having the EGFR T790M gene mutation and HLA-typed with HLA-C1502.

8. Use of the neoepitope peptide of claim 1 for the preparation of an immune drug or method for the treatment of cancer, comprising adoptive cell therapy such as chimeric antigen receptor T cell technology (CAR-T) and T cell receptor chimeric T cell technology (TCR-T).

9. Use of the MHC antigen complex of claim 3 for screening and preparing a T lymphocyte receptor that specifically recognizes the MHC antigen complex of claim 3 and mediates the T lymphocytes to exert anti-tumor cytotoxic effects.