A diagnostic reagent and a therapeutic reagent for human head and neck squamous cell carcinoma

Abstract

The application discloses a human head and neck squamous cell carcinoma clinical diagnosis reagent, which is a reagent for detecting the expression amount of a human NCAPH gene. The expression level of the human NCAPH gene is negatively correlated with the clinical prognosis of human head and neck squamous cell carcinoma. Experimental results show that the expression of the human NCAPH gene in a human head and neck squamous cell carcinoma cell line is higher than that in normal nasopharyngeal epithelial cells. Knocking down the expression of the human NCAPH gene can significantly inhibit the proliferation and colony sphere formation ability of the head and neck squamous cell carcinoma cells. The application also provides a drug for inhibiting the expression of the human NCAPH gene or a NPIDP small peptide drug for inhibiting the expression of the human PD-L1 gene. The NPIDP small peptide drug is used on human head and neck squamous cell carcinoma cells, can significantly compete to block the combination of NCAPH and HIP1R, promote the degradation of the PD-L1 protein, and thus enhance the T cell killing effect. The application provides a new diagnosis and treatment drug for the treatment of human head and neck squamous cell carcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor treatment, in particular to a diagnostic reagent and a therapeutic reagent for human head and neck squamous cell carcinoma, and belongs to the technical field of biological medicine. BACKGROUND

[0002] Head and neck squamous cell carcinoma (HNSCC) originates from the mucosal epithelial cells of the oral cavity, pharynx, larynx and nasal sinus, and is the most common malignant tumor in the head and neck. Epidemiological studies have shown that the main risk factors for HNSCC include smoking, drinking, exposure to environmental pollutants and infection with viral factors (i.e. HPV and EBV), among which the increase in HPV infection rate is one of the important reasons for the high incidence of HNSCC in recent years. In the past nearly two decades, surgical resection combined with radiotherapy and chemotherapy has improved the survival rate of HNSCC patients to a certain extent, and platinum, 5-fluorouracil, docetaxel and cetuximab have also become first-line drugs for systemic treatment of HNSCC. The most common factors affecting the occurrence and development of HNSCC are high-frequency mutations of some tumor suppressor genes, including TP53, CDKN2A and FAT1, but it is still very difficult to develop treatment targeting these inactivated tumor suppressor genes. Recent studies suggest that the PI3K / AKT / mTOR signaling pathway and the cell cycle signaling pathway can be used as new therapeutic targets for HNSCC, but clinical studies of drugs targeting these signaling pathways have been proven to be less effective.

[0003] Tumor immunotherapy has become a new and popular treatment for cancer because of its specificity and small side effects. The tumor immune microenvironment is mainly composed of tumor infiltrating lymphocytes (T cells, B cells and NK cells) and myeloid cells (macrophages, neutrophils, dendritic cells and myeloid-derived suppressor cells). Under physiological conditions, the activity of T cells is regulated by a complex system that selectively eliminates pathogens and abnormal cells, but avoids attacking normal cells to maintain immune homeostasis. Although HNSCC tumors are highly infiltrated by immune cells compared to other solid tumors, a number of studies have confirmed that the tumor microenvironment (TME) in most HNSCCs plays a highly immunosuppressive role. PD-1 (encoded by PDCD1) / PD-L1 (encoded by CD274) is an important protein for maintaining immune homeostasis. In the TME, the PD-1 / PD-L1 axis is hijacked by tumor cells. Upregulation of PD-L1 expression by tumor cells in advanced HNSCC and upregulation of PD-L1 and CTLA4 by MDSC and Treg cells recruited to the TME counteract the TCR signaling cascade through phosphorylated SHP-2 to evade immune detection, greatly weakening the lytic activity of T cells against tumor cells. Therefore, targeting and blocking the PD-1 / PD-L1 axis is one of the main solutions to avoid tumor cell immune escape and improve the effectiveness of immunotherapy. Although some immunotherapy drugs targeting the PD-1 / PD-L1 signaling axis, such as Keytruda (pembrolizumab), have entered clinical trials for HNSCC, the response rate is about 15%. Therefore, it is urgent to further analyze the expression regulation mechanism of PD-L1 in HNSCC and find new targeted drugs.

[0004] Currently, there is no related report on the correlation between the expression level of NCAPH gene and human head and neck squamous cell carcinoma, and the function of NCAPH in regulating PD-L1 of tumor cells. SUMMARY

[0005] The present application provides a human head and neck squamous cell carcinoma clinical diagnostic reagent, which is prepared from a reagent for detecting the expression level of human NCAPH gene, and the reagent for detecting the expression level of human NCAPH gene is a reagent for detecting high expression level of human NCAPH gene.

[0006] The present application uses human NCAPH gene sequence to design RNA primer sequence of human NCAPH, and detects the level of RNA of human NCAPH by real-time quantitative PCR method; the primer sequence for detecting the level of RNA of human NCAPH is AAACACGCAGATTACGGAACA, GTTGGTTGGTTCGGTGTCTTT.

[0007] The diagnostic reagent of the present application also includes conventional commercially available reagents used in real-time quantitative PCR.

[0008] Another object of the present application provides a drug for treating human head and neck squamous cell carcinoma, which is a drug screened for the purpose of inhibiting the expression of human NCAPH gene.

[0009] The present application found that the NPIDP small peptide drug binds to PD-L1 on human head and neck squamous cell carcinoma cells, simulates the binding of NCAPH and PD-L1, effectively blocks the binding of NCAPH and PD-L1, promotes PD-L1 to enter the lysosomal degradation pathway, thereby promoting immune activation and inhibiting the development of human head and neck squamous cell carcinoma, so as to achieve the purpose of treating human head and neck squamous cell carcinoma.

[0010] Another object of the present application provides a drug for treating human head and neck squamous cell carcinoma, which is a drug screened for the purpose of inhibiting the expression of human PD-L1 gene.

[0011] The NPIDP small peptide drug is a molecular small peptide composed of cell-penetrating peptide iRGD (CRGDKGPDC) and amino acid sequence 271-360 of NCAPH molecule, and the amino acid sequence is shown as SEQ ID NO: 1.

[0012] The present application found that the NPIDP small peptide drug binds to PD-L1 on human head and neck squamous cell carcinoma cells, simulates the binding of NCAPH and PD-L1, effectively blocks the binding of NCAPH and PD-L1, promotes PD-L1 to enter the lysosomal degradation pathway, thereby promoting immune activation and inhibiting the development of human head and neck squamous cell carcinoma, so as to achieve the purpose of treating human head and neck squamous cell carcinoma.

[0013] The drug for treating human head and neck squamous cell carcinoma of the present application takes the drug for inhibiting the expression of human NCAPH gene or NPIDP small peptide as active ingredient, which can also add one or more pharmaceutically acceptable adjuvants, or be compounded with other active ingredients to play a synergistic role in inhibiting human head and neck squamous cell carcinoma; and can be prepared into a pharmaceutically suitable dosage form.

[0014] Advantages and technical effects of the present application:

[0015] This invention provides a new approach for the clinical diagnosis and treatment of human head and neck squamous cell carcinoma. The invention uses the detection of high expression of the human NCAPH gene for clinical diagnosis; it discovers that the NCAPH gene is highly expressed in human head and neck squamous cell carcinoma, and aims to treat this carcinoma by targeting and inhibiting its expression. Furthermore, the invention experimentally demonstrates that NPIDP small peptide drugs can significantly and specifically degrade PD-L1, thereby activating the immune system and inhibiting the progression of human head and neck squamous cell carcinoma. Moreover, treatment with NPIDP small peptide drugs significantly reduces the protein level of PD-L1 in human head and neck squamous cell carcinoma cell lines, and the protein level of PD-L1 on the cell membrane also decreases accordingly, resulting in a significant reduction in the number of PD-L1-positive cells. When NPIDP small peptide drugs are co-cultured with T cells, the T cell killing effect is significantly higher than that of T cells not treated with NPIDP small peptide drugs. In vivo animal experiments show that treatment with PD-1 monoclonal antibody and NPIDP small peptide drugs alone can effectively inhibit the growth of xenografts in nude mice, and the combination of the two can more effectively inhibit tumor growth. Furthermore, NPIDP small peptide treatment showed no significant toxic side effects in mice. This invention provides a novel approach for the diagnosis and treatment of human head and neck squamous cell carcinoma, possessing clinical application value and promising market prospects. Attached Figure Description

[0016] Figure 1 The expression of NCAPH in head and neck squamous cell carcinoma and its relationship with prognosis are shown in Figure A, which shows the expression of NCAPH in normal and tumor tissues; and Figure B shows the relationship between NCAPH expression level and prognosis.

[0017] Figure 2 The expression levels of the human NCAPH gene in normal human nasopharyngeal epithelial cells (NP69) and head and neck squamous cell carcinoma cell lines (CNE1, CNE2, SUNE1, 5-8F, SCC25, CAL27, and FADU) are shown in the figure above, which is the result of qPCR detection, and the figure below is the result of immunoblotting detection.

[0018] Figure 3 To detect the efficiency of NCAPH gene knockdown in human head and neck squamous cell carcinoma lines CAL27 (left) and CNE2 (right); the top figure shows the RNA expression level detection results, and the bottom figure shows the protein detection results.

[0019] Figure 4 Immunoblotting experiments of EMT-related N-cadherin (N-cad) and Vimentin proteins after stable knockdown of NCAPH in head and neck squamous cell carcinoma cell lines CAL27 and CNE2; the top figure shows the immunoblotting results of N-cadherin and Vimentin proteins in each stable CAL27 cell line, and the bottom figure shows the immunoblotting results of N-cadherin and Vimentin proteins in each stable CNE2 cell line.

[0020] Figure 5 The growth curve results of knockdown stable cell lines of head and neck squamous carcinoma cells CAL27 and CNE2, the left graph is the growth curve of each stable cell line of CAL27, and the right graph is the growth curve of each stable cell line of CNE2;

[0021] Figure 6 The results of clone sphere formation experiment after stably knocking down NCAPH gene in head and neck squamous carcinoma cell lines CAL27 and CNE2, the left graph is the clone sphere growth of each stable cell line of CAL27 and CNE2, and the right graph is the clone sphere growth statistics of each stable cell line of CAL27 and CNE2;

[0022] Figure 7 The results of transwell chamber migration experiment after stably knocking down NCAPH gene in head and neck squamous carcinoma cell lines CAL27 and CNE2, A is the chamber migration of each stable cell line of CAL27 and CNE2, and B is the chamber migration statistics of each stable cell line of CAL27 and CNE2;

[0023] Figure 8 For simultaneous overexpression of NCAPH-Myc and PD-L1-Flag or N-terminal (PD-L1-N-Flag) or C-terminal (PD-L1-C-Flag) truncated body protein of PD-L1-Flag in 293T cells, the interaction region of NCAPH protein and PD-L1 was detected by immunoprecipitation method, and the results are shown in the figure;

[0024] Figure 9 For overexpression of PD-L1-Flag and different truncated body proteins of NCAPH-Myc in 293T cells, the interaction region of PD-L1 protein and NCAPH was detected by immunoprecipitation method, and the results are shown in the figure;

[0025] Figure 10 For the design of molecular small peptides for the screened NCAPH minimal segment N3 interacting with PD-L1, the purification and detection of molecular small peptides were carried out, wherein A is the design schematic diagram of molecular small peptides and its control, B is the Coomassie brilliant blue staining result diagram of purified molecular small peptides, and C is the Ni column protein pull down experiment result diagram;

[0026] Figure 11 The results of the influence of PBS, Sumo, and NPIDP small peptides on the growth of head and neck squamous carcinoma cell CAL27 cell line:

[0027] Figure 12 The results of the influence of PBS, Sumo, and NPIDP small peptides on the clone sphere formation of head and neck squamous carcinoma cell CAL27 cell line;

[0028] Figure 13The expression levels of NCAPH, HIP1R and PD-L1 in CAL27 and CNE2 cells of head and neck squamous cell carcinoma treated by NPIDP were detected. The left graph shows the expression levels of NCAPH, HIP1R and PD-L1 in CAL27 cells treated by PBS, Sumo and NPIDP under the stimulation of IFNγ. The right graph shows the expression levels of NCAPH, HIP1R and PD-L1 in CNE2 cells treated by PBS, Sumo and NPIDP under the stimulation of IFNγ.

[0029] Figure 14 The effect of PBS, Sumo and NPIDP on the competitive binding of NCAPH and HIP1R to PD-L1 was detected by IP experiment using PD-L1 antibody.

[0030] Figure 15 The cell killing experiment of CAL27 or CNE2 cells co-cultured with or without PBMC after treatment with PBS, Sumo and NPIDP was performed. The upper graph shows the cell killing experiment results of CAL27 stable cell strains. The lower graph shows the cell killing experiment results of CNE2 stable cell strains.

[0031] Figure 16 The inhibitory effect of different drugs (negative control vehicle, PD-1 antibody, NPIDP, PD-1 antibody and NPIDP) on tumor in vivo was detected. The left graph shows the drug administration scheme of mice. The middle graph shows the growth of tumor after administration. The right graph shows the statistical results of tumor growth curve. The lower left graph shows the statistical results of tumor weight.

[0032] Figure 17 The expression of immune-related molecules PD-L1, CD8 and GZMB in tumor treated by different drugs (negative control vehicle, PD-1 antibody, NPIDP, PD-1 antibody and NPIDP) in vivo was detected by immunohistochemical staining. The left graph shows the immunohistochemical staining results of different drug treatments. The right graph shows the statistical results of PD-L1+, CD8+ and GZMB+ cells.

[0033] Figure 18 The toxicity of small peptide NPIDP to mice was detected. The left graph shows the body weight of mice. The right graph shows the immunohistochemical staining of different internal organs after administration. DETAILED DESCRIPTION

[0034] The application will be further described in detail by examples, but the protection scope of the application is not limited to the content described. The methods in the examples are all conventional methods unless otherwise specified. The reagents used are all conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified.

[0035] Example 1: Bioinformatics analysis

[0036] RNAseq data and clinical data of TCGA-HNSCC (head and neck squamous cell carcinoma) project were downloaded and sorted from TCGA database (https: / / portal.gdc.cancer.gov) as analysis materials; the software was R (4.2.1) version, the expression difference analysis was visualized by using ggplot2 package, Wilcoxon rank sum test statistical analysis was performed by using stats package and car package; survival analysis was performed by using survival package to test the proportional risk hypothesis and fit survival regression, the analysis results were visualized by using survminer package and ggplot2 package, and Logrank test statistical analysis was performed.

[0037] The results are shown in Figure 1 From the results in the figure, it can be seen that the expression of human NCAPH gene in head and neck squamous cell carcinoma tissue is higher than that in normal tissue, the expression of NCAPH is negatively correlated with the survival of patients, the survival time of patients with high expression is short, and the survival time of patients with low expression is longer.

[0038] Example 2: Fluorescent quantitative PCR experiment

[0039] When the target cells are cultured to the logarithmic growth phase, the culture medium is discarded, the cells are rinsed with PBS and then discarded, 1 mL of Trizol lysis solution is added, the cells in the dish are gently blown with a pipette, and after complete lysis, the Trizol lysis solution sample is transferred to a 1.5 mL collection tube; the Trizol lysis solution sample is centrifuged at 12000 g and 4°C for 5 minutes, and then the supernatant is transferred to a new 1.5 mL collection tube, and the lower precipitate is the incompletely lysed cells which can be discarded. 200 μL of chloroform is added to the Trizol lysis solution, vortexed and mixed, then incubated at room temperature for 5 minutes, and then centrifuged at 12000 g and 4°C for 15 minutes. Carefully transfer the uppermost liquid after centrifugation to a new RNase-free 1.5 mL collection tube, add 750 μL of isopropanol, mix gently, incubate at room temperature for 10 minutes, and then centrifuge at 7500 g and 4°C for 5 minutes. Discard the isopropanol supernatant, add 1 mL of 75% ethanol prepared with DEPC water to wash the precipitate, then centrifuge at 7500 g and 4°C for 5 minutes, discard the 75% ethanol supernatant, invert the collection tube, and when the RNA precipitate is dry and translucent, add an appropriate amount of DEPC water to dissolve the RNA, and use Nano-Drop to determine the RNA concentration. 1 μg of RNA is reversely transcribed into cDNA using a commercial reverse transcription kit, and the commercial SYBR, NCAPH primers (Human NCAPH_F: AAACACGCAGATTACGGAACA; Human NCAPH_R: GTTGGTTGGTTCGGTGTCTTT) are analyzed according to the corresponding reaction system (SYBR 20 μL, 2.5 μM Primer 2.4 μL, cDNA 1 μL, ddH2O 6.6 μL).

[0040] The results are shown in Figure 2 The expression of human NCAPH gene in head and neck squamous cell carcinoma (CNE1, CNE2, SUNE1, 5-8F, SCC25, CAL27, FADU) cell lines was significantly higher than that in normal immortalized nasopharyngeal epithelial cells NP69.

[0041] Example 3: Western blotting experiment

[0042] 1. Once the target cells have grown to the logarithmic growth phase, place them on ice, discard the culture medium, rinse the cells with PBS and discard the PBS, add an appropriate amount of RIPA lysis buffer, and incubate on ice for 5 minutes. Use a cell scraper to transfer the lysis buffer to a 1.5 mL collection tube. After three freeze-thaw cycles in liquid nitrogen, centrifuge at 15000g, 4℃ for 25 minutes (generally the highest speed of a refrigerated centrifuge). Transfer the supernatant to a clean 1.5 mL collection tube for subsequent concentration determination. Add ddH2O and 5×SDS protein buffer to the protein supernatant to prepare a protein sample of a specific concentration. Gently tap the bottom of the tube to mix, heat in a 95℃ metal bath for 2-5 minutes, and then briefly centrifuge before proceeding with electrophoresis. Assemble the protein SDS-PAGE gel in the electrophoresis tank and add electrophoresis buffer. Electrophoresis conditions are constant voltage 150 V for 1 hour. For the transfer, first soak the cut PVDF membrane in methanol for 5 minutes, then assemble the gel in a "sandwich" configuration (sponge mesh-filter paper-gel-PVDF membrane-filter paper-sponge mesh) in transfer buffer. Perform the transfer at a constant current of 200-500 mA for 1-2 hours. After the transfer, cut the transferred PVDF membrane according to the desired experimental purpose. Immerse the cut PVDF membrane in 5% skim milk (prepared with 1×TBST wash buffer) and incubate at room temperature on a shaker for 1 hour. Wash away excess milk using TBST wash buffer. Add the corresponding primary antibody (prepared with new cymet antibody dilution buffer), incubate overnight on a shaker at 4°C; wash three times with TBST washing buffer, 10 minutes each time; add the secondary antibody corresponding to the primary antibody species (prepared with 5% skim milk), incubate on a shaker at room temperature for 2 hours; wash three times with TBST washing buffer, 10 minutes each time; mix HRP developing solution according to the ratio (solution A:solution B = 1:1), immerse the PVDF membrane (which has been blotted dry with special paper towels) in the solution, react for about 1 minute, and then place the membrane in a developing apparatus for development.

[0043] The results are as follows Figure 2 As shown, the expression level of the NCAPH gene in head and neck squamous cell carcinoma (CNE1, CNE2, SUNE1, 5-8F, SCC25, CAL27, FADU) cell lines was significantly higher than that in normal immortalized nasopharyngeal epithelial cells NP69.

[0044] 2. Head and neck squamous cell carcinoma cells CAL27 and CNE2 were first treated with 10 ng / mL IFNγ for 12 hours, then treated with PBS, Sumo (10 μg / mL, for solubilization), and NPIDP peptide (10 μg / mL, NPIDP peptide sequence is as follows: CRGDKGPDCGGGGSGGGGSGGGGSHHHHHHRSELLFPSDVQTLSTGEPLELPELGCVEMTDLKAPLQQCAEDRQICPSLAGFQFTQWDSETHNESVSALVDKFKKNDQVFDINAEVDESD) for another 12 hours. After lysis with IP buffer containing 0.5% NP-40, the protein samples were subjected to three freeze-thaw cycles in liquid nitrogen. After BCA protein concentration quantification, an appropriate amount of 5×SDS protein buffer was added and mixed with magnetic beads. The mixture was heated in a 95°C metal bath for 10 minutes and then briefly centrifuged before proceeding with electrophoresis experiments.

[0045] The results are as follows Figure 13 As shown, the protein levels of NCAPH and HIP1R did not change significantly after drug treatment, and the protein level of PD-L1 did not change significantly under PBS and 10 μg / mL Sumo treatment, but decreased significantly after NPIDP peptide treatment.

[0046] Example 4: Construction of NCAPH knockdown cell lines

[0047] 1. The target gene NCAPH was knocked down using the pLKO.1-shRNA lentiviral expression vector. The mRNA sequence of the NCAPH gene was obtained from the NCBI database, and shRNA target sequences were designed and synthesized (Human NCAPH sh#1: TCAGAGATTCTTAAACAGAAA; Human NCAPH sh#2: TCTCCTAAATTGATCTGTTAT). Nhe I and EcoRI restriction enzyme to pLKO.1-puro vector, and identified by enzyme digestion and sequencing, the identified correct NCAPH knockdown plasmid was prepared by lentivirus packaging system: first, the plasmid with shRNA target sequence and packaging plasmid (PMD2.G, PSPAX2) were co-transfected into HEK-293T cells by calcium phosphate transfection method, 8 hours later, the normal culture medium was replaced, then the supernatant culture medium containing lentivirus was collected at 48 hours and 72 hours, Polybrene (10 μg / mL) was added and different HNSCC tumor cell lines were infected for 12 hours and the normal culture medium was replaced, 48 hours after infection, the normal culture medium containing Puromycin (2 μg / mL) was replaced to screen the stable cell lines after lentivirus infection, after 3 passages, the expression of RNA and protein in the cells after NCAPH knockdown was detected by qRT-PCR and immunoblotting technology (same as examples 2, 3);

[0048] Results are shown in Figure 3 The RNA and protein expression levels of NCAPH gene in CAL27 and CNE2 knockdown stable cell lines of head and neck squamous cell carcinoma were significantly reduced;

[0049] And the expression levels of EMT-related N-cad and Vimentin proteins in CAL27 and CNE2 knockdown stable cell lines of head and neck squamous cell carcinoma were significantly reduced compared with control cells Figure 4 ).

[0050] Example 5: Growth curve experiment

[0051] 1. When the head and neck squamous cell carcinoma CAL27 and CNE2 cell lines were grown to the logarithmic growth phase, they were digested into single cells with 0.25% trypsin, and then 2 repeated holes were inoculated into 12-hole cell culture plates at a density of 1×10 4 per hole, then the cells were digested with 0.25% trypsin and counted every day, and each hole was counted 3 times, and the cell growth curve was drawn after 6 days of statistics.

[0052] Results are shown in Figure 5 The growth rate of CAL27 (left) and CNE2 (right) knockdown NCAPH stable cell lines was significantly lower than that of the control cell lines.

[0053] 2. Effect of NPIDP small peptide on growth of head and neck squamous cell carcinoma CAL27 cell line

[0054] The experimental setup negative control (PBS buffer), Sumo treatment group (add 10 μg / mL of Sumo, solubilizing tag), NPIDP small peptide treatment group (add 10 μg / mL of NPIDP small peptide, NPIDP small peptide sequence as follows: CRGDKGPDCGGGGSGGGGSGGGGSHHHHHHRSELLFPSDVQTLSTGEPLELPELGCVEMTDLKAPLQQCAEDRQICPSLAGFQFTQWDSETHNESVSALVDKFKKNDQVFDINAEVDESD); the operation process is the same as above.

[0055] The results are shown in Figure 11 As can be seen from the figure, CAL27 cells under NPIDP small peptide treatment have no significant difference in growth with negative control group PBS group and Sumo group.

[0056] Example 6: Clonal formation experiment

[0057] NCAPH knockdown stable cell line clonal sphere formation experiment

[0058] After the target cells grow to the logarithmic growth phase, 600 target cells are inoculated into a 6-well plate containing 2 mL of normal culture medium after being digested into single cells with 0.25% trypsin, and fresh culture medium is replaced every other day. After about 2 weeks of culture, wash twice with PBS, fix the cells with 4% paraformaldehyde for 20 minutes, wash twice with PBS, and stain with 0.5% crystal violet staining solution. Distilled water is used to wash off excess staining solution and air dry. The number of colonies formed in each well is counted and analyzed.

[0059] The results are shown in Figure 6 As shown, the number of clonal sphere formation of the knockdown stable cell line of head and neck squamous cell carcinoma cells CAL27 and CNE2 is significantly lower than that of the control group (head and neck squamous cell carcinoma cell CAL27 and CNE2 SC group).

[0060] 2, NPIDP small peptide on head and neck squamous cell carcinoma cell CAL27 cell clonal sphere formation experiment

[0061] The experimental setup negative control (PBS buffer), Sumo treatment group (add 10 μg / mL of Sumo, solubilizing tag), NPIDP small peptide treatment group (add 10 μg / mL of NPIDP small peptide, NPIDP small peptide sequence as follows: CRGDKGPDCGGGGSGGGGSGGGGSHHHHHHRSELLFPSDVQTLSTGEPLELPELGCVEMTDLKAPLQQCAEDRQICPSLAGFQFTQWDSETHNESVSALVDKFKKNDQVFDINAEVDESD); the operation process is the same as above.

[0062] Results are shown in Figure Figure 12 As shown in Figure, there was no significant difference in the ability of CAL27 cell clone to form spheroids between the group with added NPIDP small peptide and the Sumo treatment group and the control (PBS buffer) group.

[0063] Example 7: Transwell chamber migration experiment

[0064] When the target cells grow to the logarithmic growth phase, 3x10 5 cells are taken out, centrifuged to remove the supernatant, and resuspended in 1 mL of serum-free medium. 100 μL of cell suspension is inoculated into the upper chamber of the Transwell chamber, 600 μL of complete medium is added to the lower chamber, and cultured for 24 hours. The culture medium above the chamber is discarded, and the cells that have not passed through the chamber are gently wiped off with a cotton swab soaked in PBS. 600 μL of 4% paraformaldehyde is added to the lower chamber to fix the cells for 20 minutes. 600 μL of 0.5% crystal violet staining solution is added to the lower chamber for 1 hour. Excess staining solution is washed with distilled water and air-dried. The number of migrated cells is recorded by taking pictures under a microscope.

[0065] Results are shown in Figure Figure 7 As shown in Figure, the number of migrated cells in the stable cell lines with knockdown of NCAPH in CAL27 and CNE2 was significantly lower than that in the control cell lines.

[0066] Example 8: Co-immunoprecipitation experiment

[0067] 1. In this study, the full-length (NFL and PD-L1-Flag) and truncated (N1, N2, N3, N4, N5, N6, PD-L1-N-Flag, PD-L1-C-Flag) cDNAs encoding human NCAPH, HIP1R, and PD-L1 were synthesized at Shanghai Jereh Biotechnology Co., Ltd., and cloned into the pCDNA3.1 vector using NheI and KpnI (NCAPH), NheI and EcoRI (PD-L1), and NheI and NotI (HIP1R). The constructed target plasmids were transfected into HEK-293T cells, and target cells were collected at 48 h and 72 h post-transfection. Lysis was performed using IP buffer containing 0.5% NP-40. Protein samples were subjected to three freeze-thaw cycles in liquid nitrogen before BCA protein concentration quantification. After concentration determination, a protein-antibody mixture was prepared at a ratio of 1 mg protein to 1 μg antibody. This ratio is a standard practice and can be adjusted appropriately based on experimental results. Incubate the protein-antibody mixture overnight at 4°C on a vertically rotating shaker. Place the Protein A / G magnetic beads on a magnetic rack and wash three times with IP buffer containing 0.2% NP-40 to remove residual magnetic bead storage solution components. Add 50 μL of magnetic beads to each protein-antibody mixture and incubate at 4°C for 2 hours on a vertically rotating shaker. After the reaction, place the magnetic bead-protein-antibody mixture on a magnetic rack, discard the supernatant, and wash three times with IP buffer containing 0.2% NP-40, removing as much supernatant as possible on the last wash. Then add 50 μL of 1.5×SDS protein buffer and mix well with the magnetic beads. Heat in a 95°C metal bath for 10 minutes, then briefly centrifuge before proceeding with electrophoresis.

[0068] The amino acid sequences of the full-length NCAPH-Myc protein, N1, N2, N3, N4, N5, and N6 are shown in SEQ ID NO:2-8;

[0069] The results are as follows Figure 8 As shown, immunoprecipitation experiments using Myc antibodies revealed that both the full-length PD-L1-Flag protein and the C-terminal truncated protein of PD-L1-C-Flag could interact with the full-length NCAPH-Myc protein, while the N-terminal truncated protein of PD-L1-N-Flag could not interact with the full-length NCAPH-Myc protein.

[0070] The results are as follows Figure 9 As shown, immunoprecipitation experiments using Flag antibodies revealed that the full-length PD-L1-Flag protein can bind to the full-length NCAPH-Myc protein, N1, N3, N4, and N5, but cannot bind to N2 and N6, thus narrowing the interventional functional peptide to the N3 peptide region.

[0071] 2. After treating CAL27 cells with 10 ng / mL IFNγ for 12 hours, supplement with 100 μM chloroquine and 10 μg / mL Sumo or NPIDP peptide for another 12 hours. Lyse the cells with IP buffer containing 0.5% NP-40. Perform BCA protein concentration quantification after three freeze-thaw cycles in liquid nitrogen. After concentration determination, prepare a protein-antibody mixture at a ratio of 1 mg protein to 1 μg anti-PD-L1 antibody. This ratio is a standard practice and can be adjusted based on experimental results. Incubate the protein-antibody mixture overnight at 4°C on a vertical shaking incubator. Place Protein A / G magnetic beads on a magnetic rack and wash three times with IP buffer containing 0.2% NP-40 to remove residual magnetic bead storage solution components. Add 50 μL of magnetic beads to each protein-antibody mixture and incubate on a vertically rotating shaker at 4°C for 2 hours. After the reaction, place the magnetic bead-protein-antibody mixture on a magnetic rack, discard the supernatant, and wash three times with IP buffer containing 0.2% NP-40, removing as much supernatant as possible during the last wash. Then add 50 μL of 1.5×SDS protein buffer and mix well with the magnetic beads. Heat in a 95°C metal bath for 10 minutes, then briefly centrifuge before proceeding with electrophoresis.

[0072] The results are as follows Figure 14 As shown, under the conditions of chloroquine inhibition of autophagy and inflammatory stimulation, PD-L1 interacts with both NCAPH and HIP1R under PBS and Sumo treatment. However, after the addition of NPIDP peptide, the total expression level of the protein did not change, but the binding of PD-L1 to HIP1R was enhanced, while the binding to NCAPH was significantly inhibited.

[0073] Example 9: In vitro purification experiment of small peptides

[0074] The cell penetrating peptide iRGD (CRGDKGPDC) and the DNA sequence of the 271-360 aa region of NCAPH were synthesized commercially and cloned into the prokaryotic expression plasmid pET-30a, with the help of the fusion tag Sumo; 2 μL of the plasmid was used to transform BL21 (DE3) competent cells, which were then plated on Kana-resistant LB plates and incubated at 37°C for 16-18 h. A single colony was picked and inoculated into a test tube containing 4 mL of LB medium with the corresponding resistance, and incubated at 37°C and 220 rpm until the OD600 of the bacterial cells was 0.6-0.8; 0.3 mM IPTG was added to the test tube, and the expression was induced at 16°C and 220 rpm; the bacterial slurry was collected by centrifugation at 8000 rpm for 5 min; the bacterial slurry was resuspended in 25 mL of PBS buffer per 500 mL of bacterial solution, and then broken in an ice bath for 30 min using an ultrasonic disrupter; the supernatant was obtained by centrifugation at 10000 rpm for 35 min, and then filtered using a 0.45 μm filter; 4 mL of Ni-Beads was used to pack the column, and the packing material was washed with ultrapure water for 5-10 column volumes; then the packing material was equilibrated with resuspension buffer (containing 20 mM PB, 300 mM NaCl, 5% glycerol, 20 mM imidazole, pH 8.0) for 5-10 column volumes; the filtered sample was purified by chromatography, and the loading flow rate was 2 mL / min; after loading, the column was washed with resuspension / equilibration buffer for 5-10 column volumes; then the protein was eluted using resuspension buffer containing 50 mM imidazole; the Ni column-purified sample was dialyzed into storage buffer (containing 20 mM PB, 300 mM NaCl, 5% glycerol, pH 8.0), and the protein concentration was determined; 10 μL of SUMO enzyme was added to 1 mg of protein, and the sample was incubated at 4°C for 16 h; the cleaved sample was purified using Ni-Beads; the loading flow rate was 2 mL / min; after loading, the column was washed with equilibration buffer for 5-10 column volumes; then the protein was eluted using resuspension buffer containing 250 mM imidazole; the eluted protein sample was dialyzed into storage buffer, with a dialysis ratio of >1000 times; and the sample was filtered and stored using a 0.22 μm filter.

[0075] The results are shown in Figure 10 Figure 10 A shows the structure of the small peptide purified in vitro, Figure 10 B shows the in vitro purification result of the small peptide.

[0076] Example 10: Ni pull down experiment

[0077] ​The CAL27 cells treated with 10 ng / mL of IFNγ for 24 h were lysed on ice for 10 min using RIPA lysis buffer, the lysis mixture was collected into a 1.5 mL centrifuge tube, centrifuged at 4 ℃, 15000 rpm for 25 min, and the supernatant was taken as the total cell protein; 50 μL of Ni-Beads was washed with PBS for 3 times, resuspended in 500 μL of PBS, and 100 μg of the target protein was added, which was incubated for 30 min by upside-down mixing at 4 ℃, centrifuged at 4 ℃, 3000 rpm for 1 min, and the supernatant was removed, 1 mg of total cell protein (lysis buffer was diluted to 500 μL) was added, which was incubated for 1 h by upside-down mixing at 4 ℃, washed with 500 μL of PBS for 3 times, resuspended the beads with 50 μL of Loading Buffer, and heated at 100 ℃ for 10 min in a metal bath for subsequent immunoblot experiment.

[0078] The results are shown in FIG. 11C, and the results of the nickel column pull-down experiment showed that only PD-L1 protein could be pulled down on the nickel column combined with NPIDP, and Sumo could not pull down PD-L1 protein. Figure 10

[0079] Example 11: T cell killing experiment

[0080] Extraction of human PBMC cells (peripheral blood mononuclear cells): about 10 mL of blood was taken from a healthy person, Fico separating solution was added to a centrifuge tube and restored to room temperature, the blood was slowly added to the centrifuge tube (without breaking the boundary between the separating solution and the blood), and centrifuged at 1500 rpm for 30 minutes at room temperature. The cells in the white layer in the middle after centrifugation were PBMC cells. The PBMC cells were slowly sucked out and added to PBS, centrifuged at 1500 rpm for 10 minutes at room temperature, repeated twice, and then the cell precipitate was resuspended with RPMI-1640 complete culture medium, added to a cell culture dish, and placed in a cell incubator for 2 hours of precipitation. The non-adherent cells in the culture medium were transferred to a new culture dish, and then CD3 (100 ng / mL) and IL-2 (10 ng / mL) were added for activation for 48 hours. After counting the target cells, 1×10 5 Each well was inoculated in a 24-well plate, and after the cells adhered, PBS control group, Sumo group and NPIDP small peptide group (10 μg / mL each) were added for pretreatment for 12 hours. Then 5×10 5 activated PBMC cells and target tumor cells (CAL27 and CNE2) were co-cultured in each well, and after 48 hours of culture, the supernatant was discarded, washed once with PBS, fixed with 4% paraformaldehyde for 20 minutes, washed twice with PBS, and 300 μL of 0.5% crystal violet was added to each well for staining for 5 minutes. After washing the excess crystal violet with distilled water and drying, the photograph was taken. ​

[0081] Results are shown in Figure 15 Figure 2. Killing effect of PBMC on tumor cells after co-culturing with CAL27 and CNE-2 cells. The killing effect of PBMC on tumor cells after co-culturing with CAL27 and CNE-2 cells was significantly higher when the PBMC was treated with NPIDP small peptide than when the PBMC was treated with PBS or Sumo.

[0082] Example 12: Animal model experiment

[0083] Head and neck squamous cell carcinoma cells SCCVII were transplanted subcutaneously into the inguinal region of 5-6 week old C57BL / 6 mice. The tumor volume and body weight of the mice were monitored every other day. When the tumor volume reached 100 mm 3 The mice were given intraperitoneal injection of drug treatment (PD-1 monoclonal antibody: 100 μg; NPIDP small peptide: 200 μg; a total of 5 times) when the tumor volume reached 100 mm

[0084] Results are shown in Figure 16 Figure 3. A, schematic diagram of drug administration to mice; B, schematic diagram of tumor in mice under different drug administration conditions. It can be seen that administration of PD-1 monoclonal antibody or NPIDP small peptide alone can inhibit tumor growth to a certain extent, and PD-1 monoclonal antibody combined with NPIDP can more effectively inhibit tumor growth; C, statistical results of tumor volume under different drug treatment conditions; D, statistical results of tumor weight under different drug treatment conditions.

[0085] Example 13: IHC experiment

[0086] Formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were dewaxed in a 65°C oven for 1 hour, followed by dewaxing with xylene I for 10 minutes, xylene II for 10 minutes, xylene III for 10 minutes, and then rehydrated with a gradient of ethanol: 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 100% ethanol III for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes. The slides were then immersed in pH 9.0 EDTA antigen retrieval buffer or pH 6.0 citrate tissue antigen buffer and autoclaved for 8 minutes for antigen retrieval. After rinsing in PBS, the slides were permeabilized with PBST (0.3% Triton X-100) for 15 minutes, followed by hydrogen peroxide blocking for 10 minutes. The tissue slides were then incubated with the primary antibody overnight at 4°C. Finally, biotinylated universal secondary antibody was added and incubated at room temperature for 30 minutes. After rinsing in PBS for 5 minutes, the slides were developed using a 3,5-diaminobenzidine (DAB) substrate kit (DAB staining solution), followed by hematoxylin counterstaining, clearing with graded ethanol and xylene, and mounting with neutral resin.

[0087] The results are as follows Figure 17 As shown in the figure, compared with the Vehicle (PBS) group, administration of PD-1 monoclonal antibody or NPIDP peptide alone can inhibit PD-L1 expression to a certain extent and promote the infiltration of CD8+ T cells and GZMB positive cells. PD-1 monoclonal antibody combined with NPIDP peptide can further inhibit PD-L1 expression and promote the infiltration of CD8+ T cells and GZMB positive cells.

[0088] Example 14: Drug toxicity experiment in animal models

[0089] Mice treated with different drugs (vehicle, PD-1 antibody, NPIDP peptide, and co-treatment with PD-1 antibody and NPIDP peptide) were weighed to detect changes in body weight. Different tissues from the mice were fixed in formalin, and the paraffin-embedded (FFPE) tissue sections were dewaxed in a 65°C oven for 1 hour, followed by dewaxing with xylene I for 10 minutes, xylene II for 10 minutes, xylene III for 10 minutes, and then rehydrated with a series of ethanol solutions: 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 100% ethanol III for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes. After rehydration, hematoxylin was stained for 5 minutes, differentiation was performed using differentiation solution, rinsed with water to regain blue color, eosin stained for 20 seconds, dehydrated with a series of ethanol solutions, cleared with xylene, and mounted with neutral resin.

[0090] Figure 18 Results A showed that different drug treatments (vehicle, PD-1 antibody, NPIDP peptide, and co-treatment with PD-1 antibody and NPIDP peptide) had no effect on mouse body weight.Figure 18 BResults show that the small peptide NPIDP does not cause significant abnormalities in the structure of the visceral tissues (heart, liver, spleen, lung, kidney) in mice.

Claims

1. A human head and neck squamous cell carcinoma clinical treatment drug, which is a NPIDP small peptide drug for inhibiting expression of a human PD-L1 gene. The amino acid sequence of the NPIDP small peptide drug is shown as SEQ ID NO:

1.

2. A human head and neck squamous carcinoma clinical treatment drug, characterized in that: The NPIDP small peptide drug is used in combination with an immunotherapy drug. The amino acid sequence of the NPIDP small peptide drug is shown as SEQ ID NO:

1. The NPIDP small peptide drug is used in combination with an immunotherapy drug. The amino acid sequence of the NPIDP small peptide drug is shown as SEQ ID NO: 1.

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