Application of LMK-235 in preparation of medicine for inducing generation of tumor neoantigen

Abstract

The invention discloses an application in preparation of a medicine for inducing generation of a tumor neoantigen. The LMK-235 is utilized to actively induce the generation of the tumor neoantigen, so that the generation probability of effective neoantigen epitopes is remarkably improved, and meanwhile, the risk of loss of the neoantigen epitopes is avoided. Compared with a traditional new antigen screening strategy based on somatic mutation, the method for inducing the new antigen by using the LMK-235 is more convenient and efficient, has very high feasibility and credibility, and is lower in cost and wide in applicability.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to the use of LMK-235 in the preparation of a drug for inducing the generation of new tumor antigens. Background Art

[0002] Bladder cancer is usually treated with a combination of surgery, radiotherapy, and chemotherapy. For non-muscle invasive bladder cancer, the main treatment is transurethral resection of bladder tumor (TURBT). Immediate intravesical instillation therapy is usually performed after surgery, using drugs such as Bacillus Calmette-Guérin (BCG), mitomycin C (MMC), or epirubicin to prevent recurrence. For resectable muscle-invasive bladder cancer, the standard treatment is radical cystectomy combined with bilateral pelvic lymph node resection. In addition, cisplatin-based neoadjuvant chemotherapy has been shown to significantly improve patient survival. For patients with unresectable or advanced metastatic bladder cancer, systemic chemotherapy is the main treatment, combined with palliative surgery or radiotherapy to relieve symptoms. Although surgery and chemoradiotherapy remain the main treatments for bladder cancer, surgical trauma, toxic side effects of chemoradiotherapy, and drug resistance remain difficult to overcome. In addition, the low response rate to treatment and the high recurrence rate after surgery make it urgent to explore new treatment methods to improve the efficacy.

[0003] As an emerging cancer treatment method, immunotherapy has the advantages of long-lasting efficacy, strong targeting and few side effects. At present, various immunotherapies such as monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, adoptive cell therapy and small molecule inhibitors have been widely used in the adjuvant treatment of various cancers. In the treatment of bladder cancer, a variety of immune checkpoint inhibitors have been approved as first-line drugs for patients with cisplatin intolerance and second-line drugs for patients with advanced and metastatic disease. In addition, immunotherapies such as interleukin-2 (IL-2) cytokine therapy, chimeric antigen receptor T-cell (CAR-T) therapy, oncolytic viruses and bispecific antibodies have also achieved good results in clinical trials of bladder cancer. However, although immunotherapy has good application prospects in bladder cancer, its population response rate is low, only 20% to 30%, and the clinical efficacy shows obvious individual differences. Therefore, improving the treatment efficiency of immunotherapy has become a difficult problem to be solved in the current field of bladder cancer immunotherapy.

[0004] With the development and widespread application of high-throughput omics technology, immunotherapy based on neoantigens has become a research hotspot. Neoantigens are antigenic epitope peptides unique to tumor cells. Their main sources include non-synonymous single nucleotide variations, DNA insertions or deletions, and gene fusion events at the gene level; RNA splicing changes and endogenous retrotransposition at the transcription level; and non-synonymous codon preference, translation start site changes, and peptide amidase activity changes at the translation level. These abnormal DNA, RNA, or protein products will be degraded by proteasomes to produce peptide epitopes that do not exist in the cell itself, and will be specifically presented to T lymphocytes by the major histocompatibility complex (MHC) on the surface of tumor cells, thereby activating anti-tumor immune responses. Neoantigens are only expressed in tumor cells and have extremely high targeting. Because they are non-self antigens, they are not easily affected by immune tolerance mechanisms, so they can become a very ideal immunotherapy target.

[0005] Tumor neoantigens are currently mainly used for cancer treatment through personalized vaccines and adoptive T cell therapy, and the screening of neoantigens is the first and key step in this process. The existing screening strategy usually involves first obtaining normal tissue and tumor tissue samples from patients, and identifying tumor mutation genes by comparing the results of whole exome sequencing (WES) of these two groups of samples. Subsequently, their expression levels are analyzed using cDNA chips or RNA sequencing (RNA-sequencing, RNA-seq), and their affinity to human leukocyte antigen (human leukocyte antigen, HLA) is predicted by computer analysis and mass spectrometry, and finally candidate neoantigens with moderate expression and strong binding to MHC are screened. However, the screened neoantigens must undergo a series of validations, such as enzyme-linked immunospot assay (ELISpot), T cell activation analysis, and pMHC tetramer analysis, to ensure that they can be recognized by T cell receptors (TCR) before they can be used in clinical treatment.

[0006] However, this traditional screening method has many limitations. First, the neoantigen spectrum between patients is highly heterogeneous, and shared neoantigens are extremely rare; the development of personalized vaccines based on tumor neoantigens not only requires high R&D costs, but also faces a long preparation cycle, which seriously hinders its clinical transformation process; the effective screening of neoantigens is heavily dependent on the tumor mutation load. Even in tumors with a high mutation load, the proportion of neoantigens that can be ultimately verified as having clinical application value is still extremely low; tumor development has significant spatiotemporal dynamic characteristics. As the tumor continues to evolve, the neoantigens predicted based on the primary tumor tissue may be lost or changed, making it difficult for the corresponding treatment strategy to achieve the expected effect.

[0007] Therefore, there is an urgent need to develop new strategies to break through the technical bottlenecks faced by traditional neoantigen therapies, including inherent problems such as long screening cycles, high R&D costs, and low screening efficiency. It is also necessary to address key challenges such as the fact that the screened neoantigens are only suitable for personalized treatment of patients and lack universality, so as to give full play to the therapeutic potential of tumor neoantigens and provide intervention strategies with more clinical translation prospects for the field of tumor immunotherapy. Summary of the invention

[0008] In view of the defects in the prior art, the present invention proposes the use of LMK-235 in the preparation of drugs that induce the generation of neo-tumor antigens.

[0009] The present invention provides the use of LMK-235 in preparing a drug for inducing the generation of new tumor antigens.

[0010] LMK 235 is a potent and selective HDAC4 / 5 inhibitor with the chemical formula C 15 H 22 N2O4, LMK-235 induce the generation of tumor neoantigens by regulating RNA splicing.

[0011] In some embodiments, the drug further comprises pharmaceutically acceptable excipients; the excipients include but are not limited to any one or more of diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, solubilizers, preservatives, pH regulators, osmotic pressure regulators, surfactants, coating materials, antioxidants or buffers.

[0012] In some embodiments, the drug further includes any one or more of epigenetic regulators, immune checkpoint inhibitors and tumor vaccines; the epigenetic regulators include but are not limited to histone deacetylase (HDAC) inhibitors, DNA methyltransferase (DNMT) inhibitors, histone methyltransferase inhibitors and histone acetyltransferase agonists; the immune checkpoint inhibitors include but are not limited to PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors; the tumor vaccines include tumor cell vaccines, DNA vaccines and viral vector vaccines, etc.

[0013] In some embodiments, LMK-235 is the only active ingredient in the medicament.

[0014] In some embodiments, the concentration of LMK-235 is greater than 1 μM, preferably 1 to 3 μM.

[0015] In some embodiments, the tumor is any one of bladder cancer, liver cancer, lung adenocarcinoma, prostate cancer, breast cancer, pancreatic cancer, colon cancer, oral squamous cell carcinoma, melanoma, kidney cancer, bile duct carcinoma and glioblastoma.

[0016] The present invention also provides a new tumor antigen, which is induced to be produced after tumor cells are treated with an effective amount of LMK-235.

[0017] In some embodiments, the effective amount of LMK-235 refers to a concentration of LMK-235 greater than 1 μM, preferably 1 to 3 μM.

[0018] In some embodiments, the amino acid sequence of the tumor neoantigen is shown in any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19.

[0019] The present invention also provides the use of the neotumor antigen in the preparation of anti-tumor drugs.

[0020] In some embodiments, the tumor is any one of bladder cancer, liver cancer, lung adenocarcinoma, prostate cancer, breast cancer, pancreatic cancer, colon cancer, oral squamous cell, carcinoma melanoma, kidney cancer, bile duct cancer and glioblastoma.

[0021] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0022] 1. The present invention provides a new use of the HDAC4 / 5 inhibitor LMK 235 in inducing the generation of tumor neoantigens. LMK-235 is used to actively induce the generation of tumor neoantigens, which significantly increases the probability of generating effective neoantigen epitopes and avoids the risk of neoantigen epitope loss. Compared with the traditional neoantigen screening strategy based on somatic mutations, the use of LMK-235 to induce neoantigens is more convenient and efficient, has high feasibility and credibility, is lower in cost, and has wide applicability.

[0023] 2. LMK 235 can stimulate tumor cells to express specific neoantigens, providing personalized immunotherapy plans that adapt to the tumor characteristics of different patients, thereby improving the accuracy and effectiveness of treatment.

[0024] 3. The present invention induces the generation of new antigens through LMK-235, which helps to reduce the risk of tumor recurrence and reduce patients' resistance to existing treatments.

[0025] 4. LMK-235 has broad application potential in a variety of solid tumors and can be applied to different types of cancer to improve the overall effect of cancer treatment.

[0026] 5. Compared with Class I HDAC inhibitors, the targeted inhibition of Class IIa HDAC activity by LMK-235 does not interfere with megakaryocyte differentiation and platelet production, avoids common side effects such as fatigue, nausea and blood toxicity, and is safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 This is a graph showing the IC50 results of LMK-235 in MB49 cells according to Example 1 of the present invention;

[0029] Figure 2The results of MB49 cell proliferation levels after treatment with gradient concentrations of LMK-235 in Example 1 of the present invention are shown in Figure A; Figure A is a proliferation curve chart detected by flow cytometry, and Figure B is a bar chart;

[0030] Figure 3 The results of the changes in the apoptosis level of MB49 cells after treatment with gradient concentrations of LMK-235 in Example 1 of the present invention are shown in Figure A; Figure A is a flow cytometry apoptosis detection result, and Figure B is a bar chart;

[0031] Figure 4 The results of subcutaneous tumor formation of MB49 cells treated with LMK-235 in Example 2 of the present invention in fully immunized mice and immunodeficient mice are shown in Figure A; Figure B is a tumor formation result in fully immunized mice, and Figure A is a tumor formation result in immunodeficient mice;

[0032] Figure 5 This is a result diagram of ELISpot validation of MB49 cells treated with LMK-235 to activate immune response in vitro in Example 3 of the present invention;

[0033] Figure 6 Figure 4 is a diagram showing the results of IHC staining of tumor tissues after the MB49 cells treated with LMK-235 in Example 4 of the present invention formed tumors in C57BL / 6 mice; Figure A is a tissue staining diagram, and Figure B is a statistical analysis diagram of CD8 and CD11c positive signals;

[0034] Figure 7 The results of the changes in the proportion of tumor infiltrating lymphocyte (TIL) subsets after LMK-235 treatment in mice according to Example 5 of the present invention are shown in Figure A; Figure B is a DC cell, Figure C is a M1 macrophage, and Figure D is a CD8 + T cells, Figure D shows effector T cells that are positive for interferon-γ (IFN-γ) and granzyme B (GrB);

[0035] Figure 8 This is a result diagram of the change levels of major histone acetylation sites in MB49 cells after LMK-235 treatment in Example 6 of the present invention;

[0036] Fig. 9This is an analysis summary diagram of the sequencing results of RNA-seq and transposase-accessible chromatin sequencing (ATAC-seq) of MB49 cells treated with LMK-235 in Example 7 of the present invention; Figure A is a flowchart of new antigen prediction based on RNA-seq and 6 abnormal splicing events obtained by the final screening, B is the type and gene of the abnormal splicing event, Figure C is a volcano map of the up-regulated genes belonging to the 6 abnormal splicing events, Figure D is an openness analysis diagram of the chromatin corresponding to the genes belonging to the abnormal splicing events, and Figure E is a new antigen and H2-K b or H2-D b Affinity statistics of two MHC subtypes;

[0037] Fig.10 The results of ELISpot detection of in vitro immunogenicity of new antigens in Example 8 of the present invention are shown in Figure A, and Figure B is a spot statistical graph;

[0038] Fig.11 This is a result diagram of the ELISpot detection of the in vivo immunogenicity of the new antigen in Example 9 of the present invention;

[0039] Fig.12 Figure 1 is a graph showing the tumor formation results of MB49 cells pretreated with LMK-235 in Example 10 of the present invention in C57BL / 6 mice immunized with the new antigen polypeptide; Figure A is a graph showing the subcutaneous tumor volume change trend at various time points in mice immunized with different polypeptides, Figure B is a graph showing the actual tumors at the detection endpoints of different treatment groups, and Figure C is a graph showing the statistical results of tumor weight;

[0040] Fig.13 MB49 cell lysate pretreated with LMK-235 in Example 11 of the present invention and mouse lymph node CD8 + ELISpot results of T cell co-incubation;

[0041] Fig.14 Figure 1 is a summary of the results of the combined treatment of LMK-235 and anti-PD-1 in Example 12 of the present invention; Figure A is a volume change trend diagram of the subcutaneous tumors of each group of mice at each time point, Figure B is a statistical result diagram of the weight of the subcutaneous tumors of each group of mice at the end of the experiment, and Figure C is a physical diagram of the tumors in different treatment groups; Figures D to H are statistical diagrams of the changes in the proportion of TIL subpopulations of subcutaneous tumors of each group of mice, wherein Figure D is DC cells, Figure E is M1 macrophages, and Figure F is CD8 + T cells, Figure G is an effector T cell that secretes IFN-γ, and Figure H is an effector T cell that secretes GrB;

[0042] Fig.15 The results of the combined treatment of LMK-235 and anti-PD-1 in Example 12 of the present invention are summarized in Figure A and Figure B are central memory T cells (Central Memory T cells, T cm ) and effector memory T cells (EffectorMemory T cells, T em ) ratio change statistical graph; Figure C is a weight change trend graph of mice in each group during the experiment, Figures D and E are statistical results of the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (U / L) indicating liver toxicity after treatment, and Figures F, G, H, and I are weight statistical graphs of the heart, liver, spleen, and kidney of each group, respectively;

[0043] Fig.16 This is a summary of the hematoxylin-eosin staining (H&E) of various organs of mice treated with the above combination therapy. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0045] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0046] The basic contents of the technical solution of the present invention are as follows:

[0047] The present invention firstly measures the IC50 value of LMK-235 in the mouse bladder cancer cell line MB49 cells by the CCK-8 method, then sets a concentration gradient of LMK-235 based on the IC50 value to treat the MB49 cells in vitro and detects the proliferation and apoptosis levels thereof, thereby determining 2 μM as the working concentration of LMK-235 which has little effect on the proliferation and apoptosis of MB49 cells. After determining the working concentration of LMK-235, MB49 cells treated with LMK-235 in vitro and untreated were inoculated subcutaneously in C57BL / 6 mice and NSG mice, respectively. By comparing the tumor volumes of fully immunized mice and immunodeficient mice, it was shown that LMK-235 could inhibit the tumor formation of MB49 cells in mice with the participation of the immune system. Then, the MB49 cell lysate treated with LMK-235 in vitro was co-cultured with mouse spleen cells, and the activation of immune cells in vitro was detected by ELISpot. At the same time, subcutaneous tumor tissues of C57BL / 6 mice in the control group and the treatment group were taken, and the changes in the proportions of each subpopulation in TIL were analyzed by IHC staining and flow cytometry. The results showed that LMK-235 has a certain ability to activate the immune system both in vivo and in vitro. After confirming that LMK-235 can activate the immune response, RNA-seq and ATAC-seq were performed on untreated and LMK-235-treated MB49 cells. Six abnormal splicing events were screened based on the RNA-seq results, and the possible new antigenic peptides were predicted by bioinformatics analysis. The artificially synthesized new antigen peptides were co-cultured with DCs, and their effects on control CD8 + T cells and CD8 T cells derived from lymph nodes of mice immunized with peptides in vivo + Then, MB49 cells treated with LMK-235 in vitro were inoculated subcutaneously into mice immunized with peptides. In addition, DC cells co-cultured with MB49 cell lysate treated with LMK-235 and CD8 T cells in lymph nodes of mice immunized with peptides were cultured. + T cell co-culture, by comparing the size of subcutaneous tumors in the untreated group and the peptide-treated group and the CD8 + The activation of T cells was used to screen the most likely neoantigen peptides to be presented. Then LMK-235 and the immune checkpoint inhibitor anti-PD-1 were used in combination, and the growth trend of MB49 cells under the skin of C57BL / 6 mice was measured to compare the therapeutic effect of LMK-235 and verify the synergistic effect of LMK-235 and anti-PD-1 combined therapy. At the same time, the preliminary safety evaluation of LMK-235 as a neoantigen induction and anti-tumor treatment drug was carried out by detecting serum toxicological indicators and observing the changes in the structure of major organs.

[0048] Example 1 Effect of LMK-235 on proliferation and apoptosis of MB94 cells

[0049] 1. IC50 determination:

[0050] MB94 cells, a mouse bladder cancer cell line, were cultured at 4 × 10 3 The number of cells was inoculated in a 96-well plate, and after the cells adhered, the MB49 cells were treated in vitro with gradient concentrations of LMK-235 (0, 10nM, 50nM, 100nM, 500nM, 1000nM, 5000nM, 10000nM), with four replicates for each concentration. Then, 10% CCK8 developer prepared in culture medium was added, and the plates were placed in an incubator at 37°C for 2 to 4 hours, and then read using an enzyme reader.

[0051] The results are as follows Figure 1 As shown, the IC50 value of LMK-235 in MB49 cells was 3148 nM as detected by CCK8 for cell viability.

[0052] MB49 cells were treated with gradient concentrations of LMK-235 (0, 0.1 μM, 1 μM, 10 μM, 50 μM) in vitro. After 96 h, the cells in each concentration treatment group were plated at 7.5 × 10 5 / well inoculated in a 24-well plate, three replicate wells of each concentration were cultured in a 37°C incubator overnight. After adherence, the culture medium was replaced with a culture medium containing EdU (1:2000), and then incubated in a 37°C incubator for 1 hour. Then the cells were washed once with PBS, digested with trypsin without EDTA, centrifuged at 1100rpm for 5min, and the supernatant was discarded. The cells were fixed with 4% paraformaldehyde at room temperature for 20min, and the speed was increased to 600g and centrifuged for 10min and the fixative was discarded. 2mg / mL glycine was added, incubated at room temperature for 5min to neutralize the residual aldehyde group, and the glycine solution was discarded; the permeabilization agent (PBS containing 0.5% TritonX-100) was added and incubated at room temperature for 10min to break the membrane, and the permeabilization agent was discarded. After washing once with PBS, 200μL 1×Apollo staining reaction solution was added to the cells, incubated at room temperature in the dark for 30min, and the staining reaction solution was discarded. After washing once with the permeabilization agent and once with PBS, the cells were resuspended with 500 μL PBS, and finally the proliferation levels of MB49 cells at each gradient concentration were detected by flow cytometry.

[0053] The results are as follows Figure 2 As shown in AB, with the increase of LMK-235 concentration in the culture medium, the proliferation ratio of MB49 cells gradually decreased, indicating that LMK-235 can inhibit the proliferation of MB49 cells.

[0054] 3. Cell apoptosis detection:

[0055] MB49 cells were treated in vitro with gradient concentrations of LMK-235 (0, 0.1μM, 1μM, 10μM, 50μM), with 3 replicates for each concentration. After 96 hours, the cells were digested with EDTA-free trypsin, centrifuged at 4℃ and 1100rpm for 5 minutes, and the supernatant was discarded. The cells were washed with 4℃ pre-cooled PBS and the PBS was discarded. The staining buffer was prepared by adding 3μL FITC-Annexin V and 5μL PI dye to 100μL 1×Annexin V Binding Buffer. 100μL pre-cooled staining buffer was added and incubated at room temperature in the dark for 10 minutes. Finally, 500μL pre-cooled 1×Annexin V Binding Buffer was added to terminate the staining, and the apoptosis level of MB49 cells at each gradient concentration was detected by flow cytometry within 1 hour.

[0056] The results are as follows Figure 3 As shown in A and B, with the increase of LMK-235 concentration in the culture medium, the apoptosis rate of MB49 cells gradually increased, indicating that LMK-235 can induce apoptosis of MB49 cells.

[0057] Example 2 Effect of LMK-235 on subcutaneous tumor formation in C57BL / 6 and NSG mice

[0058] 6-8 weeks old C57BL / 6 mice and NSG mice were randomly divided into two groups, drug control group and drug treatment group, with 10 mice in each group. MB49 cells treated with DMSO (drug control group) or 2 μM LMK-235 (drug treatment group) were subcutaneously inoculated on both sides of the abdomen of each mouse in supine position, with 5×10 5 cells; the length (L, mm) and width (W, mm) of the subcutaneous tumor were measured on days 7, 10, 13, 16, 19, and 22 after inoculation, and the length (L×W 2 ) to calculate tumor volume.

[0059] The results are as follows Figure 4 As shown, Figure 4 A is the result of MB49 cell tumor formation in fully immunized mice. As can be seen from the left figure, after LMK-235 treatment, the tumor volume of MB49 cells in C57BL / 6 mice at different time points was smaller than that of the control group. In the right figure, on the 23rd day, the tumor volume of the LMK-235 treatment group was significantly smaller than that of the DMSO control group. Figure 4Figure B shows the results of MB49 tumor formation under the skin of immunodeficient mice. As can be seen from the left figure, the tumor volume of the LMK-235-treated group was slightly larger than that of the DMSO group at the same time point. As can be seen from the right figure, on the 23rd day, the tumor volume of the LMK-235-treated group was slightly larger than that of the control group, but the difference was not as obvious as that of fully immunized mice. The above results show that LMK-235 has a certain inhibitory effect on the tumor formation of MB49 in vivo, and this inhibitory effect depends on the participation of the immune system.

[0060] Example 3 Effect of LMK-235 on Immune Cell Activation in Tumor Cells in Vitro

[0061] Femurs and tibias of C57BL / 6 mice were minced to isolate bone marrow cells. Red blood cells in the bone marrow cells were lysed with 1× red blood cell lysis buffer, and then cultured in a 37°C incubator for 5 consecutive days with DMEM containing 10 ng / mL mouse recombinant interleukin-4 (IL-4), 20 ng / mL mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10% FBS. The culture medium was changed once a day during this period to induce DC differentiation. 1×10 7 MB49 cells in the drug control group (DMSO), drug treatment group (LMK-235) and positive control group (overexpression of chicken ovalbumin OVA) were cultured in triplicate. Cell lysates were obtained after five freeze-thaw cycles. The supernatant of the lysate after centrifugation was taken and co-incubated with the differentiated DCs for 24 hours, and then washed once with PBS to remove the residual lysate; then DCs and mouse spleen cells were placed in an ELISpot 96-well plate for co-culture at a ratio of 1:2; after 48 hours, the cells in the 96-well plate were removed and washed 5 times with PBS (200 μL / well); 1 μg / mL R4-6A2-biotin was added and incubated at room temperature for 2 hours, and then washed 5 times with PBS (200 μL / well); then 100 μL / well 1×Streptavidin-ALP was added, incubated at room temperature for 1 hour, and then washed 5 times with PBS (200 μL / well); finally, 100 μL / well of BCIP / NBT plus substrate was added for color development, and the 96-well plate was washed with running water after 5 to 10 minutes to terminate the color development, and the plate was read and photographed after being placed in a dark place to dry.

[0062] The results are as follows Figure 5 As shown, the spots induced by the MB49 cell lysate in the LMK-235-treated group were significantly more than those in the control group (DMSO), demonstrating that the MB49 cell lysate treated with LMK-235 can significantly induce immune cell activation.

[0063] Example 4 IHC staining to identify the effect of LMK-235 on TIL recruitment in mouse tumors

[0064] Wild-type C57BL / 6 mice aged 6 to 8 weeks were randomly divided into a control group and a treatment group, with 5 mice in each group. The mice were subcutaneously inoculated with 5×10 MB49 cells of the control group (DMSO) or the treatment group (LMK-235) on both sides in the supine position. 5 When the tumor reached a certain volume, the mice were euthanized and the tumor was isolated, washed with PBS, fixed with 4% paraformaldehyde, and then embedded in tissue and sectioned in paraffin.

[0065] Place the slide in xylene for 10 min → xylene for 10 min → 100% ethanol for 3 min → 95% ethanol for 3 min → 85% ethanol for 3 min → 70% ethanol for 3 min. Place in PBS and wash on a shaker for 5 min. Prepare citric acid buffer (1×), place in a microwave oven for 3 min to boil, place the slices, adjust to low heat and cook for 25 min, cool naturally to about 50°C; place in PBS and shaker for 3 min, wash three times. Remove the slide, use lens paper to absorb the PBS around the tissue, use a tissue pen to draw a circle around the tissue, then add endogenous peroxide blocker, and let it stand for 10 min. Place in PBS and wash for 3 min, and repeat the wash three times. Add 5% BSA solution to block at room temperature for 15 min to block non-specific sites. Drain BSA, add primary antibody to completely cover the tissue, incubate in a 4°C refrigerator overnight, take it out from 4°C, warm it up to room temperature for 10 min, place it in PBS and shaker for 5 min, and wash four times. Remove the slide, wipe the PBS solution around the tissue, add the reaction enhancement solution, incubate for 30 minutes, wash three times, remove the slide, wipe the PBS solution around the tissue, add the secondary antibody, incubate at room temperature for 30 minutes, wash four times, dilute the DAB stock solution (20×) to 1×, and color under a microscope. Put the colored slide into PBS, shake it for 5 minutes, soak it in hematoxylin for 27 seconds, wash it with water for 2 minutes, and then put it in 1% hydrochloric acid alcohol for differentiation for 8 seconds, and wash it in tap water for 2 minutes. Put the slide into 70% ethanol for 3 minutes → 80% ethanol for 3 minutes → 90% ethanol for 3 minutes → 95% ethanol for 3 minutes → 100% ethanol for 3 minutes → 100% ethanol for 3 minutes → xylene for 2 minutes → xylene for 2 minutes. Drop neutral gum diluted with xylene next to the tissue, then cover it with a clean cover glass, and place the sealed slide in a fume hood to dry naturally. Scan the slide with a slide scanner and use Image J for statistical analysis of positive signals.

[0066] The results are as follows Figure 6 As shown in A and B, the tumor tissues in the LMK-235-treated group were infiltrated with more CD8 +T cells and DC cells, indicating that MB49 cells treated with LMK-235 can recruit more TILs in the tumor microenvironment.

[0067] Example 5 Flow cytometry analysis of the effect of LMK-235 on TIL recruitment and activation

[0068] Wild-type C57BL / 6 mice aged 6 to 8 weeks were randomly divided into a control group and a treatment group, with 5 mice in each group. The MB49 cells of the control group (DMSO) or the treatment group (LMK-235) were subcutaneously inoculated bilaterally in the abdomen in the supine position, with 5×10 5 After the tumor reached a certain volume, the mouse was euthanized and the tumor tissue was isolated; the tumor tissue was cut into pieces with scissors and placed in a Miltenyi C tube containing 5 mL of digestive enzymes, and the digestion program was run on the Miltenyi tissue dissociation instrument. After the digestion was completed, the digestion was terminated with a culture medium containing 10% FBS and 2% EDTA; the residual tumor tissue was ground and filtered on a 70 μm filter membrane with the tail of a 1 mL syringe, and rinsed with PBS while grinding, and the cell pellet was collected after centrifugation at 500 g for 5 minutes; 10 mL of red blood cell lysis buffer was used for lysis at room temperature for 15 minutes, and 5 times the volume of PBS was added to stop the lysis of red blood cells, and PBS was used for washing once; the cells were resuspended with 5 mL of 40% Percoll, and the cells were evenly and slowly spread on the wall of the 15 mL centrifuge tube on the 5 mL of 70% On the surface of Percoll, centrifuge at 350g for 25min, adjust the speed to 0 up and 0 down; after centrifugation, aspirate the cell layer at the interface into a new 15mL centrifuge tube and wash once with PBS; after counting the cells, prepare cell death and viability dye according to the number, incubate at room temperature in the dark for 15min, stop staining, and wash once with PBS; after blocking with CD16 / 32 antibody at room temperature for 15min, add no less than 2×10 7 The cells / well were evenly divided into three groups and placed in a U-shaped 96-well plate; flow cytometry staining procedures were performed according to the surface markers of the immune cell subsets detected: DC (CD45 + CD11c + CD86 + MHCII + ); M1(CD45 + CD11b + F480 + CD86 + CD206 + ); CD8 + T(CD45 + CD3 + CD8 + IFN-γ + / GrB + ) After staining, flow cytometry was used to detect the infiltration of immune cells.

[0069] The results are as follows Figure 7 As shown, compared with the DMSO control group, the DC cells, M1 macrophages, and CD8 + The proportion of T cells and effector T cells secreting IFN-γ and GrB increased significantly, indicating that LMK-235 treatment of MB49 can significantly increase the infiltration of pro-inflammatory immune cells in the tumor microenvironment and significantly induce CD8 + T cell activation.

[0070] Example 6 Effect of LMK-235 treatment on histone acetylation levels in MB49 cells

[0071] MB49 cells of the control group (DMSO) and the treatment group (0.5μM, 2μM LMK-235) treated for 96h were collected, and the appropriate amount of RIPA lysis buffer was added, and the cells were lysed on ice for 30min. The supernatant was taken after centrifugation at 4℃ and 12000rpm. The concentration was quantitatively adjusted by BCA, and 5× loading buffer was added according to the amount. The protein sample was obtained by metal bath at 100℃ for 10min. An appropriate amount of protein sample was loaded, electrophoresed at 120V for 60min, transferred to the membrane at 120V ice bath for 60min, blocked with 5% milk for 2h, incubated with primary antibody at 4℃ overnight, rinsed with TBST 4 times, 10min each time, incubated with secondary antibody for 50min, rinsed with TBST 4 times, 10min each time, prepared developer, evenly covered the membrane surface and exposed to image.

[0072] The results are as follows Figure 8 As shown in the figure, after LMK-235 treatment, the acetylation levels of the main sites of histone H3 and H4 in MB49 cells, H3K9ac, H3K18ac, H3K27ac; H4K5ac, H4K8ac, and H4K12ac, were significantly upregulated, and showed a clear dose-dependent trend. This indicates that LMK-235 can inhibit the activity of histone deacetylase (HDAC) in MB49 cells, thereby possibly participating in the regulation of RNA alternative splicing and the generation of new antigens.

[0073] Example 7 LMK-235 induces the generation of new antigen epitopes in bladder cancer cells by regulating RNA splicing

[0074] Collect appropriate amount of MB49 cells from control group (DMSO) and treatment group (LMK-235), add 500 μL Trizol reagent, mix by pipetting, and let stand for 10 min; add 100 μL chloroform, shake vigorously to mix, let stand for 3 min, centrifuge at 4°C, 12000g, 15 min; draw 200 μL supernatant into EP tube with 250 μL isopropanol, gently invert to mix, let stand for 10 min, centrifuge for 10 min; discard the supernatant, wash twice with 1mL 75% ethanol, centrifuge for 3 min; open the lid for 3 min to dry, add DEPC water preheated to 40-50°C to dissolve RNA, take appropriate amount of RNA for RNA-seq.

[0075] Appropriate amounts of MB49 cells from the control group (DMSO) and the treatment group (LMK-235) were taken, with three replicates in each group. Libraries were established using the TruePrep Index Kit V2 for Illumina and the Hyperactive ATAC-Seq Library Prep Kit for Illumina kits, and then ATAC-seq was performed.

[0076] Table 1 Neoantigen sequences predicted by 6 abnormal splicing events

[0077]

[0078]

[0079] The results are shown in Table 1 and Fig. 9 As shown in Table 1, the new antigen sequences predicted by 6 abnormal splicing events are shown. Fig. 9 A shows the results of new antigen screening based on RNA-seq and the six abnormal splicing events finally screened; Fig. 9 B shows the type and gene of the abnormal splicing event. Fig. 9 C is the annotation of the genes where these six abnormal splicing events are located in the volcano map; Fig. 9 D is the openness analysis diagram of the chromatin corresponding to the gene where the abnormal splicing event is located. Fig. 9 E is the new antigen in Table 1 and MHC H2-K b or H2-D b The above results show that LMK-235 treatment can inhibit HDAC activity, increase the level of histone acetylation in MB49 cells, and increase the openness of the chromatin region where the abnormally spliced ​​genes are located, thereby inducing the generation of new tumor antigen epitopes.

[0080] Example 8 In vitro immunogenicity testing of new antigens

[0081] Femurs and tibias of C57BL / 6 mice were cut and minced to separate bone marrow cells. After lysing the red blood cells with red blood cell lysis solution, the cells were cultured in a 37°C incubator with DMEM containing 10ng / mL mouse recombinant IL-4, 20ng / mL mouse recombinant GM-CSF and 10% FBS for 5 consecutive days. The culture medium was changed once a day during this period to induce DC maturation. The predicted new antigen peptides and positive control peptides (OVA) were co-incubated with differentiated DCs at a concentration of 10μg / mL for 48h and then washed with PBS to remove residual peptides; DCs were then incubated with CD8 + T cells were resuspended in 1640 medium without FBS at a ratio of 1:1 and co-cultured in an ELISpot 96-well plate for 48 hours, then washed 5 times with PBS (200 μL / well) to remove the cells in the 96-well plate; then 1 μg / mL R4-6A2-biotin was added and incubated at room temperature for 2 hours, and washed 5 times with PBS (200 μL / well); then 100 μL / well 1×Streptavidin-ALP was added, incubated at room temperature for 1 hour, and washed 5 times with PBS (200 μL / well); finally, 100 μL / well of BCIP / NBT plus substrate was added for color development, and after 5 to 10 minutes, the 96-well plate was washed with running water to terminate the color development, and the plate was read and photographed after being placed in a dark place to dry.

[0082] The results are as follows Fig.10 As shown in Figure 2, most of the new antigen peptides induced significantly more spots than the positive control group (OVA), proving that these peptides can effectively activate CD8 + T cells, which have certain immunogenicity; Fig.10 A is a spot diagram, Fig.10 B is a spot statistics chart, and the results show that among the 19 predicted neoantigen peptides, 14 peptides produced significantly more spots than the OVA group.

[0083] Example 9 In vivo immunogenicity testing of new antigens

[0084] Wild-type C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 11 groups, including control group (adjuvant), positive control group (OVA), and peptide treatment group (C, H, J, K, L, O, P, Q, R), with four mice in each group. The peptide was dissolved in adjuvant at a concentration of 1 μg / μL and injected intramuscularly into the bilateral hind limbs of C57BL / 6 mice, 10 μL per side, once every 2 days, for a total of 3 times. 72 hours after the injection, the bilateral inguinal lymph nodes of mice in each group were cut and digested for 15 minutes, ground after termination and filtered through a 40 μM cell strainer, washed twice with PBS and resuspended at 1×10 6 / mL, 200 μL was spread on an ELISpot 96-well plate; bone marrow-derived DC cells that had been incubated with DMSO, OVA, and candidate peptides (C, H, J, K, L, O, P, Q, and R) for 48 h were also taken, washed once with PBS, and then incubated with corresponding lymph node-derived CD8 + T cells were resuspended in the ELISpot 96 plate at a ratio of 1:1 and cultured for 48 h before color development.

[0085] The results are as follows Fig.11 As shown, compared with the control group, DCs pretreated with peptides and lymph node-derived CD8 + After co-incubation with T cells, both showed a certain ability to activate antigen-specific T cells. Compared with the positive control group, polypeptides L and P had stronger activation abilities and higher in vivo immunogenicity.

[0086] Example 10 Peptide immunization in mice can significantly inhibit the in vivo tumorigenesis of MB49 cells treated with LMK-235

[0087] Wild-type C57BL / 6 mice aged 6 to 8 weeks were randomly divided into an untreated group, a control group (adjuvant), a positive control group (OVA), and a treated group (neoantigen peptide), with three mice in each group. The peptide was dissolved in the adjuvant at 1 μg / μL and injected intramuscularly into the hind limbs of C57BL / 6 mice three times every two days, with 10 μL on each side. 72 hours after the inoculation, MB49 cells treated with LMK-235 in vitro for 96 hours were inoculated subcutaneously on both sides in the supine position, with 5×10 5 When the tumor reached a certain volume, the length (L, mm) and width (W, mm) of the subcutaneous tumor were measured with a vernier caliper. The length (L, mm) and width (W, mm) of the subcutaneous tumor were calculated according to the ratio of 1 / 2 (L×W 2 ) Calculate the tumor volume. After the measurement, weigh the tumor and take a photo.

[0088] The results are as follows Fig.12 As shown, Fig.12 A represents the volume change trend of subcutaneous tumors in mice immunized with different peptides at various time points; Fig.12 B shows the tumor images of the measured endpoints in different treatment groups; Fig.12 The C is right Fig.12 The statistical results of tumor weight are shown in B. The monitoring results of tumor volume and weight showed that compared with the adjuvant group, subcutaneous tumor formation in 9 groups of mice was significantly inhibited, including the C, H, J, K, L, O, P, Q, and R polypeptide immunization groups, which indicated that the above antigen polypeptides had significant immunogenicity in mice, could activate anti-tumor immune response, and had the ability to target and kill MB49 tumors. Moreover, this targeted killing effect may be related to the production of corresponding antigen epitopes in MB49 cells caused by LMK-235.

[0089] Example 11 Neoantigens induced by LMK-235 can activate the immune response of the corresponding immunized mice

[0090] Wild-type C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 11 groups, including a control group (adjuvant), a positive control group (OVA), and a peptide-treated group (C, H, J, K, L, O, P, Q, R), with four mice in each group. The peptide was dissolved in an adjuvant at 1 μg / μL and injected intramuscularly into the hind limbs of C57BL / 6 mice at 10 μL once every two days for a total of 3 times. 72 hours after the last injection, the bilateral inguinal lymph nodes and spleens of each group of mice were obtained. The spleen was ground and passed through a 70 μM cell strainer, lysed, and passed through a 40 μM cell strainer. After washing twice with PBS, it was resuspended in 1640 culture medium to 1×10 6 / mL, 200 μL was spread on an ELISpot 96-well plate coated with anti-mouse IFN-γ. The lymph nodes were minced and digested for 15 min, then ground through a 40 μM cell strainer, washed twice with PBS, and resuspended at 1×10 6 / mL, differentiated DC cells were incubated with DMSO, LMK-235-treated and OVA-overexpressing MB49 cell lysates for 48 h, washed with PBS, and DC and corresponding lymph node cells were resuspended in 1640 culture medium in a 1:1 ratio in ELISpot 96 plates, and color was developed after 48 h of culture.

[0091] result Fig.13 As shown, compared with the control group, DCs pre-incubated with MB49 cell lysates treated with LMK-235 and lymph node-derived CD8 + After co-incubation with T cells, the antigen-specific T cells of polypeptides H, J, and K were almost not activated; compared with the positive control group, polypeptides L, O, P, and Q could significantly activate antigen-specific T cells. This indicates that antigen epitopes such as C, L, O, P, Q, and R may be produced in MB49 cells treated with LMK-235, and DCs can present them to the corresponding antigen-specific T cells, thereby activating immune responses.

[0092] Example 12 Combination therapy of LMK-235 and anti-PD-1 can significantly inhibit the tumorigenesis of MB49 cells in vivo

[0093] Wild-type C57BL / 6 mice aged 6 to 8 weeks were randomly divided into four groups: control group (Vehicle), LMK-235 alone treatment group, anti-PD-1 alone treatment group, and combination treatment group (anti-PD-1 & LMK-235), with 5 mice in each group. Wild-type MB49 cells were inoculated subcutaneously on both sides of the abdomen of the mice in the supine position, with 5×10 5cells, and the weight of the mice was recorded before tumor formation, and then recorded every 4 days, for a total of 6 times. The drug was administered on the 5th day after inoculation. LMK-235 was dissolved in adjuvant (10% DMSO + 40% PEG300 + 5% Tween80 + PBS) at a concentration of 4 mg / mL, and anti-PD-1 or IgG was dissolved in PBS at a concentration of 2 mg / mL, and 100 μL / mouse was intraperitoneally administered. The Vehicle group was injected with adjuvant and isotype control IgG, respectively; the LMK-235 monotherapy group was injected with LMK-235 and IgG, respectively; the anti-PD-1 monotherapy group was injected with anti-PD-1 and adjuvant, respectively; and the combination treatment group was injected with anti-PD-1 and LMK-235, respectively. LMK-235 or adjuvant was administered daily for 10 consecutive days; anti-PD-1 or IgG was administered every 3 days, for a total of 3 times. The tumor volume was measured every 2 days during the administration period, for a total of 7 times. At the end of the experiment, mice were anesthetized by intraperitoneal injection of 500 μL of Avertin, and a 1 ml syringe was used to draw heart blood from the mouse's intercostal space. After blood collection, the mouse was euthanized and the tumor and organs such as the heart, liver, spleen, and kidney were separated. After the blood sample was allowed to stand at room temperature for 6 hours, it was centrifuged at 4°C, 3000 rpm for 10 minutes, and serum was collected and sent for testing of AST and ALT levels in serum to evaluate liver function; after tumor separation, it was weighed and photographed, and then flow cytometry was used to analyze the various subpopulations of TIL (DC, M1 Macrophage, CD8 + T, staining strategy as before); each organ was weighed separately, part of the spleen was ground through a 40μM filter, and then washed twice with PBS after red lysis, and an appropriate amount of cells was taken for flow cytometry staining to evaluate the changes in memory T cells (T cm :CD44 + CCR7 + CD62L + ; T em :CD44 + CCR7 - CD62L - ) Then; the remaining tissues were fixed with 4% paraformaldehyde, then embedded in paraffin and sliced, and the slices were stained with H&E to evaluate whether there would be obvious histological lesions in the main organs after administration;.

[0094] The results are as follows Figures 14 to 16 As shown, Fig.14 Figure A shows the changes in tumor volume of each group of mice during the experiment. The results show that LMK-235 treatment alone can inhibit the tumor formation of MB49 cells in mice, which is consistent with the previous functional verification experimental results; anti-PD-1 treatment alone also showed a similar effect; while the tumor volume of mice in the LMK-235 and anti-PD-1 combined treatment group barely increased. Fig.14B and C are the weights and images of the tumors of mice in each group at the end of the experiment. Fig.14 The results of A were consistent with those of the combination group, and the tumors of mice in the combination group were the smallest, which indicates that the combination of LMK-235 and anti-PD-1 can more effectively inhibit the growth of MB49 cells in mice. Fig.14 D to H show the DC, M1 macrophages, and CD8 + T cells, and effector T cells (IFN-γ + CD8 + T.GrB + CD8 + As shown in the figure, compared with the single treatment group, more pro-inflammatory immune cells were infiltrated in the tumor tissue of the combined treatment group, which indicates that the tumor suppression effect of the combined treatment group is closely related to immune activation. Fig.15 A and B show that LMK-235 combined with anti-PD-1 therapy can effectively induce memory CD8 + T production, which can establish long-term protection against recurrence. Figure C shows the changes in body weight of mice in each group during the experiment, Figures D and E show the statistical results of the content of AST and ALT in serum (U / L), and Figures F to I show the statistical graphs of the weight of four important organs: heart, liver, spleen, and kidney among the groups. Fig.16 The results of H&E staining of major organs. During the treatment period, there was no significant difference in the body weight and major organ weight of mice in each group. Both single treatment and combined treatment can reduce the amount of AST and ALT in serum, indicating that drug treatment can reduce liver damage in mice caused by tumor load. The results of histological staining showed that the control group did not have obvious histological lesions compared with the single treatment group and the combined treatment group. After combined treatment, the recruitment of pro-inflammatory immune cells in the tumor microenvironment can be significantly increased, and tumor growth can be inhibited. Whether it is used alone or in combination, LMK-235 did not show obvious toxicological phenomena.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of LMK-235 in the preparation of drugs that induce the generation of neo-tumor antigens.

2. The use according to claim 1, characterized in that: The drug further includes any one or more of epigenetic regulators, immune checkpoint inhibitors and tumor vaccines.

3. The use according to claim 1, characterized in that: In the drug, LMK-235 is the only active ingredient.

4. The use according to claim 3, characterized in that: The concentration of LMK-235 is greater than 1 μM.

5. The use according to any one of claims 1 to 4, characterized in that: The tumor is any one of bladder cancer, liver cancer, lung adenocarcinoma, prostate cancer, breast cancer, pancreatic cancer, colon cancer, oral squamous cell carcinoma, melanoma, kidney cancer, bile duct cancer and glioblastoma.

6. A tumor neoantigen, characterized in that: The tumor neoantigen is induced by treating tumor cells with an effective amount of LMK-235.

7. The tumor neoantigen according to claim 6, characterized in that The effective amount of LMK-235 refers to LMK-235 at a concentration greater than 1 μM.

8. The tumor neoantigen according to claim 6, characterized in that The amino acid sequence of the tumor neoantigen is shown in any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 and SEQID NO.

19.

9. Use of the tumor neoantigen according to any one of claims 6 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is any one of bladder cancer, liver cancer, lung adenocarcinoma, prostate cancer, breast cancer, pancreatic cancer, colon cancer, oral squamous cell carcinoma, melanoma, kidney cancer, bile duct cancer and glioblastoma.

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