EGFR L858R mutation specific long peptide vaccine and application thereof

Abstract

The invention relates to the technical field of biological medicines, and provides an EGFR L858R mutation specific long peptide vaccine and application thereof. The long peptide vaccine provided by the invention comprises dendritic cells which are loaded by a long peptide antigen modified by a cell penetrating peptide and are induced to be mature. According to the invention, a long peptide sequence with good immunogenicity and affinity is screened out, and specific T cell immune response can be effectively induced. And the long peptide antigen is modified by adopting cell penetrating peptide (CPPs), so that efficient loading and delivery of the long peptide antigen in dendritic cells (DC) are realized. According to the vaccine, the presentation capacity of antigens is remarkably enhanced, CD8 + T cells can be effectively activated, the expression level of IFN-gamma is remarkably improved, and strong specific immune response is induced. In addition, when the vaccine is used in combination with a PD-1 antibody, the tumor immune microenvironment is further improved, and the immune activation effect is enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an EGFR L858R mutation-specific long peptide vaccine and application thereof. BACKGROUND

[0002] Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors in the world, and the incidence of EGFR gene mutation, especially L858R mutation, is higher in East Asian population. Although EGFR tyrosine kinase inhibitors (TKI) can significantly prolong the survival of patients, the EGFR L858R mutation type usually has earlier drug resistance. In addition, such patients have poor response to PD-1 / PD-L1 immune checkpoint inhibitors, which limits the effect of current immunotherapy strategies.

[0003] At present, the poor immunotherapy effect of tumors is mainly due to the poor antigen immunogenicity, presentation dysfunction or insufficient T cell infiltration of tumors. NeoAg is a personalized active immunotherapy, which uses specific antigens in tumor mutations to stimulate the immune system to attack cancer cells.

[0004] However, the existing NeoAg mostly uses short peptides or nucleic acid methods, and the delivery efficiency is limited and the immunogenicity is weak. Although traditional DC vaccines have been reported as delivery platforms, there is no public literature on the delivery of EGFR L858R-specific antigens. The application of CPP delivery enhancement to long peptide tumor vaccine research is also relatively rare. Therefore, the existing technology still lacks an efficient, deliverable, immunogenic and personalized immunotherapy strategy for EGFR L858R mutant NSCLC. SUMMARY

[0005] The purpose of the present application is to provide an EGFR L858R mutation-specific long peptide vaccine and application thereof, which uses a long peptide antigen modified by a cell penetrating peptide (CPP) to induce the maturation of dendritic cells (DC) through co-incubation, thereby forming a DC vaccine with high efficient antigen presentation capacity.

[0006] In a first aspect, the present application provides an EGFR L858R mutation-specific long peptide vaccine, which comprises dendritic cells loaded with a long peptide antigen modified by a cell penetrating peptide and induced to mature, and the amino acid sequence of the long peptide antigen is shown as SEQ ID NO: 1.

[0007] As an optional solution, the cell penetrating peptide is a TAT peptide, and the amino acid sequence thereof is shown as SEQ ID NO: 2.

[0008] In a second aspect, the present application provides a composition comprising the long peptide vaccine and an immune checkpoint inhibitor PD-1 monoclonal antibody.

[0009] In a third aspect, the present application provides a CPP-long peptide fusion, which is obtained by covalently linking a long peptide antigen and a cell penetrating peptide, wherein the amino acid sequence of the long peptide antigen is shown as SEQ ID NO: 1, and the amino acid sequence of the cell penetrating peptide is shown as SEQ ID NO: 2.

[0010] In a fourth aspect, the present application provides a method for constructing a dendritic cell vaccine, comprising the following steps: a) synthesizing an EGFR L858R specific long peptide antigen, wherein the amino acid sequence of the long peptide antigen is shown as SEQ ID NO: 1; b) covalently linking the long peptide antigen and a cell penetrating peptide to obtain a CPP-long peptide fusion; c) co-incubating the CPP-long peptide fusion with immature dendritic cells; d) inducing dendritic cell maturation to obtain a dendritic cell vaccine.

[0011] As an alternative, in the above method, the long peptide antigen and the cell penetrating peptide are covalently linked by a Sulfo-SMCC crosslinking agent; and the cell penetrating peptide is a TAT peptide, wherein the amino acid sequence of the TAT peptide is shown as SEQ ID NO: 2.

[0012] As an alternative, in the above method, the CPP-long peptide fusion is co-incubated with the immature dendritic cells at a concentration of 20 µg / mL for 12-24 hours.

[0013] In a fifth aspect, the present application provides a use of a peptide segment having an amino acid sequence shown as SEQ ID NO: 1 in the preparation of an immunotherapy preparation for treating EGFR L858R mutant non-small cell lung cancer.

[0014] In a sixth aspect, the present application provides a use of the long peptide vaccine, the composition, the CPP-long peptide fusion, and the dendritic cell vaccine prepared by the method in the preparation of a lung cancer treatment drug.

[0015] As an alternative, in the above use, the lung cancer includes EGFR L858R mutant non-small cell lung cancer.

[0016] The application provides an EGFR L858R mutation-specific long peptide vaccine. The application screens a long peptide sequence with good immunogenicity and affinity, which can effectively induce specific T cell immune response. And the long peptide antigen is modified by using cell penetrating peptide (CPP), so that efficient loading and delivery of the long peptide antigen in dendritic cells (DCs) is realized. The vaccine significantly enhances the antigen presentation capacity, can effectively activate CD8 ＋ T cells, significantly improves the expression level of IFN-γ, and induces strong specific immune response. In addition, when combined with a PD-1 antibody, the vaccine further improves the tumor immune microenvironment and enhances the immune activation effect. At the same time, the animal experiment results show that the vaccine has good safety, and no obvious toxic side effects are observed. The scheme of the application combines the design of mutation-specific long peptide, the enhancement of delivery by CPP and the DC vaccine platform, and constructs a safe and efficient, individualized and precise lung cancer immunotherapy strategy, which has good conversion application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The design and characteristics of the EGFR L858R long peptide vaccine of the application are shown. Among them, A. The amino acid sequence around the EGFR L858R mutation site has strong HLA I affinity; B. The hydrophilicity and other characteristics of the EGFR L858R long peptide sequence.

[0018] Figure 2 The construction and characterization of the DCs-EGFR L858R vaccine of the application are shown. Among them, A. Schematic diagram of construction of the DCs-EGFR L858R vaccine; B. Flow cytometry determines the optimal ratio of peptide segment to DC cells; C. HPLC detects the stability of the DCs-EGFR L858R vaccine; D. Confocal microscope shows the combination of the EGFR L858R long peptide and the DCs cells under the optimal concentration ratio (blue: DC cell nucleus, red: DC cell membrane, green: FAM-EGFR L858R long peptide).

[0019] Figure 3 The anti-tumor effect and safety of the DCs-EGFR L858R vaccine of the application are shown. Among them, A. Schematic diagram of construction of the mouse model and inoculation of the DCs-EGFR L858R vaccine; B. Tumor and weight of mice at the end of the experiment in the NS group, the DCs group, the EGFR L858R long peptide group and the DCs-EGFR L858R vaccine group; C. H&E staining and Ki67 immunohistochemistry of mouse tumor sections; D. Main blood indicators of mice after administration in the four groups; E. HE staining of main organs (such as heart, liver, spleen, lung and kidney) sections of mice at the end of the experiment.

[0020] Figure 4The anti-tumor effect of the DCs-EGFR L858R vaccine of the present application alone or in combination with PD-1 antibody and the influence on the survival of mice are shown. Among them, A. the tumor volume change curve of mice in the NS group, the Anti-PD1 group, the DCs-EGFR L858R vaccine group and the combination treatment group (Combo); B. the survival curve of mice in each group. DETAILED DESCRIPTION

[0021] The present application provides a specific long peptide vaccine against EGFR L858R mutation, which is a personalized cancer vaccine based on dendritic cell (DCs) delivery system. Through the delivery mode of peptide segment modified by cell penetrating peptide (CPP), efficient antigen loading and function induction of dendritic cells (DCs) are realized.

[0022] The long peptide vaccine of the present application includes mature dendritic cells loaded with long peptide antigens modified by cell penetrating peptides. For the "loading", it means that by contacting dendritic cells with long peptide antigens modified by cell penetrating peptides, the antigens enter or bind to DCs and are processed and presented by them, thereby endowing DCs with specific antigen presentation ability. Specifically, "loading" is usually achieved by in vitro incubation, for example, co-incubation of immature DCs with long peptide antigens modified by CPP, in which process DCs actively uptake the fusion peptide and process the antigen and load MHC molecules intracellularly. The implementation of loading is not limited to the above operation, but also includes but is not limited to various known antigen delivery means such as electroporation, liposome delivery, nano-carrier assistance, as long as the effective uptake and presentation of the antigen by DCs can be achieved, which are all considered within the scope of "loading" of the present application.

[0023] For the long peptide antigen, it is a polypeptide with a length of 26 amino acids, and its amino acid sequence is shown as SEQ ID NO: 1: VIVKTPQHVKITDFGRAKLLGAEEKE. It contains the L858R mutation site (i.e. the 858th amino acid is mutated from leucine to arginine) in the EGFR protein and the natural amino acid residues upstream and downstream of it. The peptide segment is analyzed and screened by bioinformatics platform (including NetMHCpan, IEDB) for the region before and after the L858R mutation site in the EGFR protein, which has good immunogenicity and affinity, can bind to MHC-I and MHC-II molecules of various HLA subtypes, thereby effectively activating CD8 ＋ T cells and inducing strong anti-tumor response.

[0024] To improve the delivery efficiency of the long peptide antigen in DC cells, the present application uses a cell penetrating peptide (CPP) to modify the antigen peptide, preferably a TAT peptide, the amino acid sequence of which is shown in SEQ ID NO: 2 (GRKKRRQRRRPQ). The CPP is covalently linked to the long peptide antigen under mild conditions by a heterobifunctional crosslinking agent Sulfo-SMCC to obtain a stable CPP-long peptide fusion. The fusion can be actively taken up by DCs, helping the antigen to enter the cytoplasm and participate in cross-presentation, significantly improving the antigen presentation efficiency.

[0025] For the specific method of constructing a DC vaccine, a specific scheme includes the following steps: 1. Design and synthesis of EGFR L858R specific long peptide antigen The region before and after the L858R mutation site in the EGFR protein is analyzed by bioinformatics tools (such as NetMHCpan, IEDB) to predict its binding affinity to MHC-I and MHC-II molecules, and 26 amino acid length long peptide sequences with good immunogenicity and affinity are screened. The final selected sequence is: VIVKTPQHVKITDFGRAKLLGAEEKE. This sequence contains the core residues of the L858R mutation site, which can effectively induce specific T cell immune response.

[0026] 2. Use of cell penetrating peptide (CPP) to enhance long peptide delivery efficiency The classic and efficient CPP sequence GRKKRRQRRRPQ (TAT peptide) is selected, which is covalently linked to the EGFR L858R long peptide under mild conditions by Sulfo-SMCC crosslinking agent (reaction buffer is 0.1M PBS pH7.2, reaction time is 6 hours, room temperature). The product is dialyzed and purified by HPLC, and the molecular weight is confirmed by ESI-MS mass spectrometry. The CPP-modified peptide has the effect of enhancing DC uptake efficiency and promoting endocytosis escape.

[0027] 3. Construction of dendritic cell vaccine (DC vaccine) PBMCs are isolated from the peripheral blood of healthy donors, and monocytes are obtained by CD14+ magnetic bead sorting. GM-CSF and IL-4 are added to induce the differentiation of monocytes into immature DCs (iDCs), and the culture period is 5-7 days. Then, the CPP-long peptide fusion peptide (20 µg / mL) is incubated with iDCs for 12-24 hours, and TNF-α and LPS are added to promote the maturation of DCs, obtaining mature DC vaccine (DCs-EGFR L858R vaccine) with high antigen presentation capacity, which can be used for active immunotherapy. The phenotype maturation is confirmed by flow cytometry (CD11c, CD80, CD83, HLA-DR).

[0028] The present invention further proposes a therapeutic approach combining the DC vaccine with a PD-1 immune checkpoint inhibitor. The DC vaccine can induce specific T cells to recognize the L858R mutant epitope, while the PD-1 antibody can enhance T cell activity by blocking immunosuppressive pathways, thereby creating a synergistic effect within the tumor immune microenvironment. Animal experiments have demonstrated that this combined treatment significantly enhances anti-tumor efficacy and has promising prospects for clinical translation.

[0029] Accordingly, the present invention also provides the CPP-long peptide fusion itself, which can be further used as an independent antigen delivery unit on the basis of preparing DC vaccine, or expanded to other immunotherapy pathways such as mRNA vaccine coding templates and polypeptide nano-delivery systems.

[0030] In addition, the long peptide antigen shown in SEQ ID NO: 1 is suitable for immunotherapy of EGFR L858R mutant non-small cell lung cancer, and can also be widely used in other tumor indications containing this mutation.

[0031] Therefore, the long peptide vaccine, the combination of the long peptide vaccine and the PD-1 immune checkpoint inhibitor, the CPP-long peptide fusion, etc. of the present invention can be used to prepare therapeutic drugs for EGFR L858R mutant non-small cell lung cancer.

[0032] The present invention provides a complete and highly convertible precision immunotherapy solution ranging from antigen screening, delivery methods, immune activation to combination therapy.

[0033] The solution of the present invention is further described below with reference to specific embodiments.

[0034] Example 1: Chemical covalent coupling of CPP and long peptide 1.1 Design and synthesis of EGFR L858R-specific long peptide antigen The regions before and after the L858R mutation site in the EGFR protein were analyzed using bioinformatics tools (NetMHCpan, IEDB) to predict its binding affinity on MHC-I and MHC-II molecules ( Figure 1 A) screened out a 26-amino acid long peptide sequence with good immunogenicity and affinity, and analyzed the hydrophilicity and other characteristics of the sequence ( Figure 1 B). The final selected sequence was VIVKTPQHVKITDFGRAKLLGAEEKE. This sequence contains the core residues of the L858R mutation site and is effective in inducing specific T cell immune responses.

[0035] 1.2 CPP-EGFR L858R long peptide fusion The cell penetrating peptide (CPP) and EGFR L858R long peptide sequence used in the vaccine of the present application were each prepared and purified by solid phase peptide synthesis. Subsequently, the CPP and the long peptide were covalently linked into a fusion peptide by using a heterobifunctional crosslinker in a buffer to form a CPP-EGFR L858R long peptide fusion. Specifically, the CPP was coupled with the long peptide containing a cysteine residue by using a common Sulfo-SMCC crosslinker (N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate).

[0036] The more specific process is as follows: the CPP peptide is dissolved in 0.1 M phosphate buffer (PBS, pH 7.2) to make the terminal amino group reactive. Sulfo-SMCC crosslinker (crosslinker to CPP peptide molecular ratio of about 10:1) is added thereto, and the reaction is allowed to proceed at room temperature for 30 minutes to allow the amino group on the CPP peptide to undergo nucleophilic substitution with the NHS ester group of Sulfo-SMCC to form an activated maleimide CPP. After the reaction is completed, the unreacted crosslinker is removed by dialysis (dialysis membrane with a molecular weight cut-off of 1 kDa, 4°C, PBS, dialysis overnight). Then, the long peptide containing a thiol (-SH) group is dissolved in PBS and added to the above maleimide-activated CPP solution (molar ratio of long peptide to CPP in the feed is about 1.2:1), and the reaction is allowed to proceed at room temperature for 2 hours to allow the thiol group of the long peptide to undergo cis addition with the maleimide group on the CPP to form a stable thioether bond covalent linkage product. After the reaction is completed, an excess of cysteine is added to terminate the reaction, and the un-coupled polypeptide is preliminarily separated by ultrafiltration. Finally, the fusion peptide coupling product is purified by reverse phase high performance liquid chromatography (HPLC). The HPLC purification conditions are, for example, a C18 column (particle size 5 µm, 250 x 10 mm) is used, the mobile phase A is 0.1% trifluoroacetic acid (TFA) aqueous solution, the mobile phase B is 0.1% TFA in acetonitrile, the organic phase is linearly increased from 5% B to 60% B over 30 minutes, the flow rate is 1.0 mL / min, and the detection wavelength is 214 nm. The CPP-long peptide fusion obtained by purification is eluted at about 18 minutes. The corresponding chromatographic fractions are collected and lyophilized under reduced pressure to obtain the target fusion peptide with a purity of >90%. The product molecular weight and sequence are confirmed to be correct by mass spectrometry and amino acid analysis.

[0037] Example 2: Source, culture and peptide segment loading of DC cells As shown in FIG. A, the construction process of the DCs-EGFR L858R vaccine is as follows. Figure 2

[0038] ​DC cell preparation: Fresh anti-coagulated peripheral blood from healthy donors was taken and mononuclear cells (PBMC) were isolated by density gradient centrifugation. Monocytes were enriched from PBMC using magnetic cell sorting technique (MACS) and CD14 + Monocytes. The sorted monocytes were suspended in RPMI-1640 complete medium containing recombinant human GM-CSF (1000 U / mL) and IL-4 (500 U / mL) and seeded in culture flasks and incubated in a 37 °C, 5% CO2 incubator for 5-7 days to allow the monocytes to adhere and differentiate into immature dendritic cells (iDC). Half of the medium was changed every 2 days and cytokines were added to maintain the induction conditions. On about the 5th day, the cell morphology changed from adherent monolayer to floating or semi-floating dendritic cell colonies with typical dendritic protrusions.

[0039] Antigen peptide loading: The iDC cells obtained in the previous step were washed with serum-free medium and adjusted to a density of 1×10^6 cells / mL. The prepared CPP-EGFR L858R long peptide fusion (obtained in Example One) was added according to the optimized dose ratio (20 μg of long peptide fusion per 1×10^6 DC cells) and incubated together. Incubate at 37 °C, 5% CO2 for about 24 hours to allow the DC cells to fully uptake and load the long peptide antigen. Figure 2 A).

[0040] Subsequently, the antigen-containing iDCs were induced to mature: human recombinant TNF-α (50 ng / mL), LPS (100 ng / mL), and other maturation stimulators were added, and the culture was continued for 48 hours. After stimulation, the iDC phenotype was transformed into mature dendritic cells (mDC), and the cell surface protrusions increased in morphology. Phenotype identification: flow cytometry was used to detect the phenotype and antigen loading of the mature DC vaccine Figure 2 B).

[0041] First, the expression of co-stimulatory molecules and MHC molecules on the surface of DCs was detected by specific antibody staining. Anti-human CD11c-APC labeled the DC cell population, and anti-HLA-DR-FITC (MHC-II molecules), anti-CD80-PE, and anti-CD83-PE labeled the DC maturation-related molecules. The results showed that the expression levels of CD80, CD83, HLA-DR, and other markers on the surface of DCs loaded with long peptides and stimulated by maturation were significantly higher than those of immature DCs, indicating that the DCs had successfully matured and possessed antigen presentation function. In the implementation process, the purity and stability of the CPP-long peptide fusion were detected by high-performance liquid chromatography (HPLC) Figure 2 C).

[0042] In addition, to verify that the DCs successfully loaded the antigenic peptides, a fluorescently labeled peptide segment was used to observe the DCs. The long peptide antigen was labeled with FITC fluorescent dye in advance, and after co-incubation with the DCs for a certain period of time, the green fluorescent signal in the DCs was observed using a fluorescence microscope. The results showed that the CPP-modified peptide segments were distributed in the DCs, and the fluorescence signals of the DCs and the peptide segments were highly co-localized under a confocal microscope, confirming that the CPP mediated the efficient entry of the long peptide into the DCs. In another scheme, the DCs and the long peptide were labeled with different colors of fluorescence (the DC cell membrane was stained with fluorescent red, and the peptide segment was labeled with fluorescent green), and the overlap of the two types of fluorescence was observed under a microscope after co-incubation, thereby directly showing the peptide segment delivery efficiency. The results showed that under the optimal loading conditions, most of the DCs successfully took up the fusion long peptide vaccine Figure 2 D). The mature DCs loaded with the EGFR L858R long peptide obtained by the above process are the DC tumor vaccine of the present application, which can be used in subsequent animal experiments.

[0043] Example Three: Animal experiment evaluation of the anti-tumor effect of the DC-EGFR L858R vaccine Animal model preparation and grouping: female NOD / SCID immunodeficient mice (4-6 weeks old) were selected and randomly grouped for tumor transplantation and vaccine efficacy observation experiments.

[0044] First, the human non-small cell lung cancer cell line H1975 (carrying EGFR L858R and T790M mutations and stably expressing the luciferase gene Luciferase) was prepared into a single cell suspension, and each mouse was inoculated with 5×10^6 tumor cells in the right axillary subcutis. About 7-10 days after inoculation, the mice locally formed palpable subcutaneous transplanted tumors (tumor volume about 100 mm³). At this time, each mouse was injected with 2×10^7 human peripheral blood PBMCs via the tail vein to establish a humanized tumor immune model (to provide a source of effector T cells for the DC vaccine). Subsequently, the mice were grouped according to different treatment schemes, with 5 mice in each group: the control group was inoculated with the same volume of PBS buffer; the DC cell group was inoculated with blank DC cells without antigen loading; the long peptide group was inoculated with the EGFR L858R long peptide antigen (containing 50 µg of the peptide segment, which was mixed with Freund's incomplete adjuvant and then subcutaneously injected); and the DC-long peptide vaccine group was inoculated with the DC cell vaccine loaded with the EGFR L858R long peptide (i.e., the mature DCs obtained in Example Two, containing 1×10^6 DCs per mouse). Each group was given the first treatment and administration one week after tumor inoculation, and then repeated every 7 days for a total of 3 times (once a week for 3 weeks). The administration was performed by subcutaneous multi-point peritumoral inoculation (inoculated subcutaneously around the tumor) to improve the accumulation of antigens and DCs in the tumor Figure 3 A). During the entire treatment period, the mice were in good general condition and showed no obvious adverse reactions.

[0045] Therapeutic effect observation and results: The long diameter and short diameter of the tumor of the mice were measured every 3 days from the first inoculation of the treatment, and the tumor volume was calculated to evaluate the tumor growth. The tumor volume calculation formula is: V = 0.5 × long diameter × (short diameter) 2 During the observation period (about 5 weeks). The tumor growth of the DC-EGFR L858R vaccine group was significantly inhibited. From the 2nd week, the tumor volume of the DC vaccine group increased significantly lower than that of the other control groups. By the end of the 5th week, the average tumor volume of the DC vaccine group was about 0.5 cm³, which was only about one fourth of the tumor volume of the PBS control group (about 2.3 cm³); it was also significantly reduced compared with the group only given DC cells (1.5 cm³) or the group only given long peptides (2.1 cm³), showing the good tumor inhibition effect of the DC tumor vaccine of the application Figure 3 B). The H&E staining and Ki67 immunohistochemical staining results of the tumor tissues of the mice showed that compared with the NS group, the necrotic area of the tumor tissue of the mice treated with the DCs-EGFR L858R vaccine significantly increased, the cell arrangement was disordered, the cell density was reduced, and the number of Ki67 positive cells was significantly reduced, indicating that the vaccine can significantly inhibit the proliferation activity of tumor cells Figure 3 C).

[0046] In terms of safety, the analysis of the peripheral blood indicators of the mice showed that there was no significant difference in red blood cell count (RBC), platelet count (PLT), white blood cell count (WBC), and liver and kidney function indicators (ALT, AST, BUN, and Creatinine) between the treatment groups and the NS control group, indicating that the vaccine has good biological safety and does not cause obvious blood system or liver and kidney function toxic side effects to the mice Figure 3 D). The pathological H&E staining results of the main organs (heart, liver, spleen, lung, and kidney) of the mice showed that there were no obvious pathological abnormal changes between the groups, the organ structure was complete, and there was no inflammatory cell infiltration or tissue damage signs, which further confirmed that the vaccine of the application has good safety Figure 3 E).

[0047] Example Four: Evaluation of the synergistic anti-tumor effect of the DC vaccine combined with PD-1 antibody This example further evaluates the anti-tumor effect of the combination of the DC-EGFR L858R vaccine and the immune checkpoint inhibitor PD-1 monoclonal antibody on the basis of Example Three.

[0048] The animal model and tumor inoculation method used in the experiment are the same as in Example Three. The tumor-bearing and humanized mice were randomly divided into the following four groups, 6 mice in each group: control group (PBS), DC vaccine group (only inoculated with DC-EGFR L858R vaccine, 1 × 106 DC each time), PD-1 antibody group (only treated with anti-PD-1 monoclonal antibody), and combination therapy group (DC-EGFR L858R vaccine + PD-1 monoclonal antibody). The DC vaccine was inoculated subcutaneously once a week for a total of 3 times. The PD-1 antibody was mouse anti-human PD-1 neutralizing antibody (Nivolumab), which was administered by tail vein injection once a week for a total of 3 times, with a single dose of 200 μg per mouse. The four groups were treated at the same time to ensure that the combination group received the DC vaccine and the PD-1 antibody at the same time. During the observation period, the body weight and behavior of the mice were monitored regularly, and no obvious toxic reactions were observed.

[0049] The tumor growth inhibition effect is shown in Figure 4 As shown in A, the application of PD-1 antibody alone only produces a slight inhibitory effect on tumor growth, and the tumor volume growth curve is close to that of the control group. The DC vaccine alone group shows a moderate tumor inhibition effect, and the tumor growth rate is slowed down. Most significantly, the combination therapy group has a tumor growth curve much lower than the other groups. At week 4, the average tumor volume of the combination group was about 0.6 cm³, which was significantly smaller than that of the DC vaccine group (1.5 cm³) and the PD-1 antibody group (2.1 cm³) at the same time, and was only about 1 / 4 of the tumor volume of the control group (2.5 cm³) Figure 4 A). It can be seen that the combination of DC vaccine and PD-1 antibody has a synergistic tumor growth inhibition effect.

[0050] Further survival follow-up results also support the improved efficacy of the combination of the two. As shown in Figure 4 B, the survival rate of mice in the combination therapy group was significantly higher than that in the control group and in any single-drug group. Statistics show that the median survival time of mice in the combination group was prolonged by about 30% and 60% compared with the DC vaccine alone group and the PD-1 antibody alone group, respectively; at 90 days after the start of treatment, about 40% of mice in the combination group were still alive, while about 10% and 0% of mice in the DC vaccine group and the PD-1 antibody group were still alive, respectively, and all mice in the control group died at about 60 days. The survival curve of the combination therapy group had a statistically significant difference compared with the control group (P < 0.01). As can be seen from the above results, the combination of DC vaccine and PD-1 antibody can effectively delay tumor progression and improve survival rate Figure 4 B).

Claims

1. An EGFR L858R mutation-specific long peptide vaccine, characterized in that: The method comprises loading dendritic cells with a long peptide antigen modified with a cell-penetrating peptide and inducing maturation, wherein the amino acid sequence of the long peptide antigen is shown in SEQ ID NO:

1.

2. The long peptide vaccine according to claim 1, characterized in that The cell penetrating peptide is TAT ​​peptide, and its amino acid sequence is shown in SEQ ID NO:

2.

3. A composition, characterized in that It comprises the long peptide vaccine according to any one of claims 1 or 2 and the immune checkpoint inhibitor PD-1 monoclonal antibody.

4. A CPP-long peptide fusion, characterized in that It is obtained by covalently linking a long peptide antigen and a cell-penetrating peptide. The amino acid sequence of the long peptide antigen is shown in SEQ ID NO: 1, and the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO:

2.

5. A method for constructing a dendritic cell vaccine, characterized in that: include: a) synthesizing an EGFR L858R-specific long peptide antigen, the amino acid sequence of which is shown in SEQ ID NO: 1; b) modifying the long peptide antigen with a cell-penetrating peptide by covalently linking it to obtain a CPP-long peptide fusion; c) co-incubating the CPP-long peptide fusion with immature dendritic cells; d) Induce dendritic cell maturation to obtain dendritic cell vaccine.

6. The method according to claim 5, characterized in that The long peptide antigen and the cell-penetrating peptide are covalently linked via a Sulfo-SMCC cross-linker; the cell-penetrating peptide is a TAT peptide, and its amino acid sequence is shown in SEQ ID NO:

2.

7. The method according to claim 5, wherein the CPP-long peptide fusion is incubated with immature dendritic cells at a concentration of 20 μg / mL for 12-24 hours.

8. Use of a peptide segment having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of an immunotherapeutic preparation for treating EGFR L858R mutant non-small cell lung cancer.

9. Use of the long peptide vaccine according to claim 1 or 2, the composition according to claim 3, the CPP-long peptide fusion according to claim 4, or the dendritic cell vaccine prepared by the method according to any one of claims 5 to 7 in the preparation of a drug for treating lung cancer.

10. The use according to claim 9, characterized in that The lung cancer includes EGFR L858R mutant non-small cell lung cancer.

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