Use of hegl, hegl monoclonal antibodies and use in tumor therapy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to the application of HEG1, HEG1 monoclonal antibody and the application in tumor treatment. It is proved that HEG1 is an immune checkpoint molecule with NK cell as an action target on the surface of tumor cells. The application provides the sequence of the HEG1 monoclonal antibody. The HEG1 monoclonal antibody has high specificity and high affinity for the HEG1 protein on the surface of tumor cells, can compete with HEG1 to bind and mediate the recognition and killing of natural killer cells NK cells on tumor cells, and block the immune escape. The application provides a new gene and direction for the treatment of tumors, provides an innovative idea for the development of precise targeted drugs, and has important conversion value and clinical application prospect in the field of tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of HEG1, HEG1 monoclonal antibodies, and their application in tumor treatment. Background Technology

[0002] NK cells are a type of natural killer cell. Activated NK cells can not only directly kill target cells but also rapidly release cytokines, inflammatory factors, and chemokines, thereby recruiting other immune cells and reshaping the microenvironment. NK cells have been a focus of development for novel therapeutic strategies to treat various human diseases, such as cancer, infectious diseases, and neuroinflammatory diseases. Immune checkpoint inhibitor (ICI) therapies, such as anti-PD-1 / PD-L1 and CTLA-4 therapies, have provided revolutionary options for cancer treatment in clinical practice. However, currently approved clinical ICIs mainly target T cells, are often associated with adverse reactions, and benefit only a small percentage of patients. Therefore, developing new immune checkpoints, such as NK cells, that target major tumor-associated immune cells is of great significance for the development of clinical cancer drugs.

[0003] HEG1 (Heart development protein with EGF-like domains 1) is a membrane protein highly expressed on the surface of various malignant tumor cells, playing an important role in tumor occurrence, development, and metastasis. HEG1 has been reported to have potential clinical applications in tumor therapy. For example, it can serve as a highly sensitive biomarker and therapeutic target for the diagnosis of malignant mesothelioma (Tsuji et al., 2017), and it has clinical value in liver cancer in terms of poor prognosis and metastasis (Zhao et al., 2019). Existing studies have also reported the targeted therapeutic ability of a monoclonal antibody (SKM9-2) against sialylated HEG1 for the treatment of malignant mesothelioma (Matsuura et al., 2018).

[0004] However, research on HEG1 in immunotherapy is currently limited, with only a few studies indicating that HEG1 can induce an immune response (Wang Zheng, 2023), but the molecular mechanism has not been elucidated. Furthermore, reported HEG1 antibodies exhibit low affinity for tumor cells and low sensitivity in recognizing tumor cells, limiting the potential of HEG1 in cancer treatment.

[0005] Therefore, it is essential to clarify the role of HEG1 in immunotherapy and to provide novel strategies for treating tumors based on HEG1. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention first provides the application of HEG1 as an immune checkpoint molecule on the surface of tumor cells, wherein the immune checkpoint molecule targets NK cells to regulate tumor cell immune escape.

[0007] Preferably, the tumor cells are HEG1 positive solid tumors, including but not limited to liver cancer cells, lung cancer cells, gastric cancer cells, malignant mesothelioma cells, and esophageal cancer cells, and more preferably lung cancer cells.

[0008] The present invention further provides the use of HEG1 monoclonal antibody or its antigen-binding fragment in the preparation of a drug for inhibiting tumor cell immune escape.

[0009] Preferably, the immune escape inhibitor is an immune checkpoint inhibitor.

[0010] Preferably, the HEG1 monoclonal antibody or its antigen-binding fragment targets NK cells and exerts its effect by competitively binding to HEG1, thereby blocking the HEG1-mediated immune checkpoint pathway.

[0011] The present invention again provides a HEG1 monoclonal antibody or its antigen-binding fragment thereof, wherein the HEG1 monoclonal antibody or its antigen-binding fragment has a light chain variable region and a heavy chain variable region, the light chain variable region comprising three antigen complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; the heavy chain variable region comprising three antigen complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.

[0012] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8.

[0013] The present invention also provides a polynucleotide encoding a light chain variable region and / or a heavy chain variable region of the HEG1 monoclonal antibody or its antigen-binding fragment as described above.

[0014] The present invention also provides an expression vector comprising the polynucleotides described above.

[0015] The present invention also provides a host cell comprising the expression vector described above.

[0016] Finally, this invention provides a tumor cell immune escape inhibitor, which is an immune checkpoint inhibitor comprising the HEG1 monoclonal antibody or its antigen-binding fragment as described above.

[0017] Preferably, the tumor cells include, but are not limited to, liver cancer cells, lung cancer cells, gastric cancer cells, malignant mesothelioma cells, and esophageal cancer cells, and are more preferably lung cancer cells.

[0018] Preferably, pharmaceutically acceptable carriers are also included.

[0019] Preferably, the carrier includes, but is not limited to, excipients that function as excipients, stabilizers, antioxidants, diluents, and sustained-release agents, such as starch, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, and hydroxyethyl cellulose.

[0020] Preferably, the pharmaceutical composition is an injection, suspension, emulsion, or powder.

[0021] The beneficial effects of this application are as follows: This application experimentally demonstrates that HEG1 is an immune checkpoint molecule on the surface of tumor cells that targets NK cells, expanding the scope of HEG1 protein function and application. This discovery also breaks through the existing framework of immune escape inhibitors primarily targeting T cells, addressing the limitations of traditional T cell-targeted therapy in terms of applicable populations and adverse reactions, thus enriching the target system for tumor immunotherapy.

[0022] This application uses the conserved sequence of HEG1 protein shared by humans and mice as an antigen to prepare HEG1 monoclonal antibodies. The prepared antibodies have high specificity and high affinity for HEG1 protein on the surface of tumor cells. They can competitively bind to HEG1, thereby mediating the recognition and killing of tumor cells by natural killer (NK) cells and blocking their immune escape, with significant effects.

[0023] This application not only provides new genes and directions for cancer treatment, but also offers innovative ideas for the development of precision targeted drugs, and has important translational value and clinical application prospects in the field of cancer treatment. Attached Figure Description

[0024] Figure 1 To illustrate the experimental results of knocking down the effect of HEG1 on lung cancer metastasis and survival in nude mice, figure a shows representative images of lung tumor metastases in nude mice from the shNC and shHEG1 groups, b shows the curves of the number of lung metastases in the shNC and shHEG1 groups, and c shows the survival curves of nude mice from the shNC and shHEG1 groups.

[0025] Figure 2To investigate the effect of HEG1 knockdown on subcutaneous tumor growth in mice with different immune backgrounds, the figures show the experimental results of immune healthy mice C57BL / 6J, T cell-deficient mice BALB / c Nude, and mice with combined T / B / NK cell deficiency and M-NSG deficiency, respectively. The images on the left side of the figures are photographs of tumor size changes over time, and the images on the right side are curves of tumor volume changes over time.

[0026] Figure 3 This figure shows the validation results of HEG1-mediated immune escape in lung cancer via NK cells. Figure a is an analysis of HEG1 expression and immune cell infiltration, with the vertical axis representing different immune cell subsets and the horizontal axis representing the correlation between HEG1 gene expression and the infiltration levels of various immune cells; positive numbers indicate positive correlation, and negative numbers indicate negative correlation. Figure b shows representative images of tumors in each group after the removal of B cells, granulocytes, and NK cells, respectively. Figure c shows the changes in tumor growth volume in each group after the removal of B cells, granulocytes, and NK cells, respectively. Figure d shows the tumor weight in each control group after the removal of B cells, granulocytes, and NK cells, respectively. In the figure, sh-NT is the negative control group without HEG1 knockdown, sh-HEG1+PBS is the control group with HEG1 knockdown, sh-HEG1+anti-CD20 is the control group with HEG1 knockdown and B cell removal, sh-HEG1+anti-Ly6G is the control group with HEG1 knockdown and granulocyte removal, and sh-HEG1+anti-asialo-GM1 is the control group with HEG1 knockdown and NK cell removal.

[0027] Figure 4 This is the validation result of HEG1 mediated by NK cells to induce immune escape in lung cancer. In the figure, a represents IHC-stained CD4+ in C57 mouse tumors. + Images of T cells and CD8+T cells, b is a quantitative map of T cell infiltration, and c is a single-cell sequencing map of lung tumors.

[0028] Figure 5 Electrophoresis image of purified HEG1 antibody from immunized mouse serum.

[0029] Figure 6 These are representative IHC staining images of four types of LLC cells: LLC shNC (control group), LLC shHEG1 (HEG1 knockdown group), LLC shHEG1 Vector (HEG1 knockdown and HEG1 additionback group), and LLC shHEG1 addback HEG1 (HEG1 knockdown and HEG1 additionback group).

[0030] Figure 7The results of in vitro and in vivo antitumor therapy with HEG1 monoclonal antibody (αHEG1 C4) are shown in the figure. Figure a shows the changes in tumor size over time in the control group and the treatment group; b shows the changes in tumor volume over time in the control group and the treatment group; c shows representative images of LLC cell Transwell invasion experiment in the control group and the treatment group (left image) and the number of metastatic cells (right image); d shows representative images of lung metastases in mice in the control group and the treatment group, with the arrows indicating the metastatic lesions.

[0031] Figure 8 The above rows show the staining results of HEG1 in clinical lung cancer tissues. The top row shows the staining of the negative control, and the bottom row shows the immunohistochemical staining. Detailed Implementation

[0032] The technical solution of the present invention will be described in more detail below with reference to experiments.

[0033] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, all experimental materials and reagents used in the experiments are commercially available. For experiments where specific techniques or conditions are not specified, the procedures described in the literature in this field or the product instructions should be followed.

[0034] The animals, cells, and reagents used in this experiment were obtained from the following sources: Female C57BL / 6J and Balb / c mice, aged 6-8 weeks, were purchased from Henan Skobes Company, while Balb / c Nude mice and M-NSG mice were purchased from Jicui Pharmaceutical Company.

[0035] Mouse lung cancer LLC cells were purchased from the Cell Bank of the Chinese Academy of Sciences, and SP2 / 0 cells were purchased from Pronosei.

[0036] DMEM medium and serum were purchased from Vicell (C3103, C04001), penicillin-streptomycin from Beyotime (C0222), Freund's complete adjuvant and Freund's incomplete adjuvant from Sigma (F5881-10ML, F5506-10ML), HAT medium (BL1314A) and HT medium (BL317A) from Biosharp, ascites adjuvant (KX0210048), Hybridoma Feeder additive (CM-2001) from Bio-Long, PEG1500 from Roche (10783641001), and syringes from Sugon.

[0037] 1. Animal husbandry Female C57BL / 6J and Balb / c mice, aged 6-8 weeks, were housed in a clean-grade animal facility at the School of Basic Medical Sciences, Anhui Medical University. Balb / c Nude and M-NSG mice were housed in an SPF-grade animal facility at the School of Basic Medical Sciences. Environmental conditions were controlled as follows: temperature 22±2°C, humidity 50%-60%, and a 12-hour light / 12-hour dark cycle. Five mice were housed in each cage, and sterilized feed and water were provided. All animals underwent acclimatization for one week before the experiment.

[0038] 2. Cell culture: LLC and SP2 / 0 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and placed in a constant temperature incubator at 37°C and 5% CO2.

[0039] Experiment 1 Effects of HEG1 gene knockdown on lung cancer metastasis and survival in mice To clarify the effect of HEG1 on tumor cell growth in mice, HEG1-knockdown LLC cell lines were obtained via shRNA lentivirus infection. Six- to eight-week-old female mice were randomly divided into a negative control group (shNC) and a HEG1 knockdown group (shHEG1). Cells were resuspended in PBS to a concentration of 3 × 10⁶ cells / mL. 6 Each mouse was injected with 100 μL of LLC cells or HEG1 knockdown LLC cells via tail vein injection. The mice were closely monitored and their survival time was recorded. Since the mice in the HEG1 knockdown group were in good condition, all experimental groups were sacrificed after 60 days. The lungs of the sacrificed mice were dissected, preserved in paraformaldehyde, fixed, embedded, and sectioned. The metastasis of the mice was detected by HE method.

[0040] The results are as follows Figure 1 As shown, compared with the control group, mice injected via tail vein to knock down LLC cells showed a significant reduction in lung tumor metastasis. Figure 1 In the negative control group (a), all mice died on day 46 after inoculation, while the survival rate of the knockdown group was significantly higher than that of the control group (a). Figure 1 (b) HE staining of mouse lungs showed that in mice injected with HEG1-normally expressing LLC cells, lung tissue exhibited obvious metastatic nodules, while the number and size of metastatic nodules in the HEG1 knockdown group were significantly reduced. Figure 1 (c)

[0041] Experiment 2 The influence of HEG1 on tumor growth To further clarify the regulatory role of HEG1 in tumor growth in vivo and its immune-dependent mechanism, subcutaneous xenograft experiments were conducted at 6-8 weeks in immunocompetent mice (C57BL / 6J), T-cell deficient nude mice (BALB / c Nude), and severely immunodeficient mice with combined T / B / NK cell deficiency (M-NSG).

[0042] Mice were randomly divided into a control group and a HEG1 knockdown group. LLC cells were subcutaneously injected into the control group or HEG1 knockdown group, with each mouse receiving 3 × 10-1 cells. 6 Once the mouse tumor is palpable, periodically measure the major axis (a) and minor axis (b) of the tumor using calipers, and calculate V using the following formula: V = (a × b) 2 ) / 2 to calculate tumor volume (V), when the tumor volume exceeds 1500 mm 3 If obvious ulceration occurs, the animal is euthanized in accordance with animal ethics guidelines. The mouse is dissected, the tumor tissue is removed, photographed, and the tumor is weighed.

[0043] The results are as follows Figure 2 As shown. In immunocompetent C57BL / 6J mice, the tumor volume in the HEG1 knockdown group was significantly smaller than that in the control group, suggesting that HEG1 can promote the growth of lung cancer cells in vivo. Figure 2 (a) In T-cell-deficient BALB / c Nude mice, HEG1 knockdown still significantly inhibited tumor growth, suggesting that this effect is independent of T cells (a). Figure 2 (b) In severely immunodeficient mice (M-NSG), there was no statistically significant difference in tumor growth between the HEG1 knockdown group and the control group, and the inhibitory effect of HEG1 knockdown on tumors was essentially eliminated. Figure 2 (c) The above results indicate that HEG1 can promote the growth of lung cancer cells in vivo, and this is dependent on immune cells other than T cells.

[0044] Experiment 3 Knocking down HEG1 suppresses tumors through natural killer (NK) cells. The differences in subcutaneous tumor growth among mice with different immune functions suggest that the immune system plays a role. Comparative analysis revealed that subcutaneous tumor growth in T-cell deficient nude mice was consistent with that in immune-normal mice, but inconsistent with that in mice with combined T / B / NK cell deficiencies. Therefore, the influence of T cells was ruled out, and NK cell involvement was suspected.

[0045] Correlation analysis of HEG1 expression and immune cell infiltration showed that HEG1 was positively correlated with B memory cells and negatively correlated with NK activated cells. Figure 3(a). Subsequently, before tumor transplantation, B cells, granulocytes, and NK cells were eliminated using CD20, anti-Ly6G, and anti-Asialo-GM1 antibodies, respectively. The experiment was repeated, and the results showed that only after NK cells were eliminated did the growth of tumor cells in the knockdown group recover. Figure 3 (As shown in b and c).

[0046] Immunohistochemical staining and single-cell analysis were performed on tumor cells from HEG1-injected mice. Subcutaneous tumor results showed CD4+. + and CD8 + Higher expression in tumors with low HEG1 expression ( Figure 4 In Figures a and b), single-cell sequencing results of lung tumors showed that NK cells expressed less HEG1 in tumors with high HEG1 expression than in tumors with low HEG1 expression. Figure 4 (c)

[0047] Based on the above experimental results, HEG1's regulation of tumors depends on the immune microenvironment. HEG1 mainly promotes the growth and progression of lung cancer in vivo by inhibiting NK cell-mediated tumor immune escape.

[0048] Experiment 4 Preparation of HEG1 monoclonal antibody 1. Design and synthesis of immunogen sequences Based on sequence alignment of human HEG1 protein (UniProt ID: Q9MLI3) and mouse HEG1 protein (UniProt ID: E9Q7X6), the polypeptide fragment "PKNPRSQEWGREA" (corresponding to amino acids 1299-1312 in human HEG1 and 1255-1268 in mouse HEG1), which shares a conserved continuous sequence region with high B-cell epitope prediction scores, was selected as the antigenic peptide. A cysteine ​​residue (Cys) was added to the N-terminus of this sequence to enhance immunogenicity. The peptide was purified to >95% purity by HPLC. It was then conjugated to the carrier protein KLH, and the KLH-peptide complex was synthesized by Taopu Pharmaceutical Co., Ltd.

[0049] 2. Evaluation of animal immunization and polyclonal antibody titers

[0050] Animal immunization: Five 6-8 week old BALB / c mice were immunized once with a 1:1 mixture of the above KLH-peptide complex and Freund's complete adjuvant, emulsified for 30 minutes, and then injected subcutaneously at multiple sites. Two weeks later, two booster immunizations were performed using Freund's incomplete adjuvant and emulsified antigen. Three days before blood collection, 50 μg of the peptide complex without adjuvant was injected intraperitoneally for a pulse immunization. Tail blood collection after the final immunization: A 2 mm tail was removed with sterilized scissors, and 100 μl of tail blood was collected. The tail was stopped with a dry cotton ball. After standing at room temperature overnight, the blood was centrifuged at 4000 rpm for 20 minutes, and the serum was collected.

[0051] ELASA potency test Taopu Biotechnology resynthesized BSA-PKNPRSQEWGREA for detecting the titer of HEG1 in mouse tail blood. The method was as follows: 1) Antigen coating: Dilute the antigen to 2 μg / ml with 10 mM PBS, and spread 100 μl / well into a 96-well plate using a multipipe, incubating overnight at 4°C; 2) Washing: Discard the liquid in the coated plate, blot dry, and then wash six times with 200 μl / well of PBST for 3 minutes each time, blot dry after each wash; 3) Blocking: Prepare 5% skim milk with PBST, and block 200 μl / well at 37°C for 2 h; 4) Incubation with primary antibody: Add 1 μl of serum to 200 μl of PBS, serially diluted to 9 wells. Serum from unimmunized mice served as a negative control, and a PBS blank control was also provided. Incubate at 37°C for 1.5 h. After incubation, wash 5 times and blot dry; 5) Incubation with secondary antibody: Add 100 μl of HRP-labeled secondary antibody to each well. Add μl to the corresponding well, incubate at 37℃ for 1 h, wash the plate 5 times and pat dry; 6) Develop color with 100 μl of TMB per well, incubate at 37℃ for 15 min; 7) Terminate, read the value, add 50 μl of stop solution to the well, and detect the OD value at 450 nm.

[0052] ELASA results showed that the antibody titer was 1:125600.

[0053] 3. Hybridoma cell construction and monoclonal line screening Spleens from mice with the highest titers of positive myeloma cells were selected. Splenic cells were isolated and mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1. The mixture was centrifuged at 1000 rpm for 10 min at 4°C. The supernatant was gently discarded, and the cells were placed in a 37°C water bath. 50% polyethylene glycol (PEG8000) was added dropwise over 1 min to induce cell fusion. 2 ml of pre-warmed DMEM was added over 2 min, and 8 ml of complete culture medium was added while mixing over 4 min to terminate fusion. The cells were centrifuged at 1000 rpm for 10 min at 4°C, resuspended in complete culture medium (20% FBS and 1% cell growth factor), and seeded in 1×10⁶ wells of 96-well plates. 5 / well, incubate at 37°C with 5% carbon dioxide.

[0054] On the second day after fusion, HAT medium was added. After 5 days, the medium was changed every 3 days using a half-replacement method. After two weeks, HAT was replaced with HT medium. When the hybridoma cell density reached 50%, the cell supernatant was collected for ELISA detection to screen for positive clones. The positive clones were further diluted to obtain single clones. The highest titer was found at position C4 in a 96-well plate and named αHEG1 C4.

[0055] 4. Ascites production and antibody purification Eight-week-old female Balb / c mice were selected. The abdomens of the mice were disinfected, and each mouse was injected with 0.5 ml of a specific ascites adjuvant to increase ascites production. 12-14 days later, C4 hybridoma cells in the logarithmic growth phase were injected into the mice at a dose of 5 × 10⁻⁶. 5 Ascites fluid was collected from each individual after 10 days. The abdomen was disinfected, and the ascites fluid was collected by puncture into a 50 ml centrifuge tube. The tube was centrifuged at 10000 g and 4℃ for 20 min to remove cell debris. After filtration through filter paper, the fluid was passed through a 0.45 μm filter and purified using a Protein A / G affinity chromatography column.

[0056] The specific purification steps are as follows: 1) Remove the top and bottom caps of the PAG-agarose gel column, allowing the liquid inside the column to flow down naturally due to gravity; 2) Wash the column with 10 ml of equilibration buffer; 3) Load the pre-treated ascites fluid onto the column, collect the ascites fluid flow-through in a 50 ml centrifuge tube, repeat the loading process three times, and collect the flow-through; 4) Wash the column with 10 ml of washing buffer; 5) Take 10 1.5 ml centrifuge tubes and add 100 μl of neutralization buffer to each tube; 6) Add 10 ml of elution buffer to elute the antibody, collect the sample into the above EP tubes, collecting 900 μl per tube, for a total volume of 1 ml; 7) After the elution buffer flows out naturally, wash the column with 10 ml of equilibration buffer; 8) After use, add 20% ethanol / PBS to the column, cap it, and store it upright at 4℃; 9) Store the eluted antibody at -20℃.

[0057] To verify antibody purity: Take 60 μl of the flow buffer and 1-10 of the elution buffer and place them in a 1.5 ml centrifuge tube. Add 15 μl of 5× loading buffer and denature at 100℃ for 10 min. Then, load 10 μl of the sample into each well and run a gel to verify the antibody purification effect.

[0058] Taking the preparation of two 10% SDS-PAGE separating gels as an example, the formulation is shown in Table 1: Table 1 Formulation of 10% SDS-PAGE Separating Gel

[0059] The flow buffer and elution buffer were loaded onto a 10% SDS-PAGE gel, respectively, and incubated at 80V for 30 min and 120V for 55 min. After incubation, the SDS-PAGE gel was stained with Coomassie Brilliant Blue. After staining, the gel was repeatedly destained with destaining solution until the background was clear and the bands were distinct.

[0060] The results are as follows Figure 5 As shown, the light and heavy chains of the monoclonal antibody can be observed at 25kD and 55kD. The antibody was dialyzed for subsequent in vitro and in vivo experiments.

[0061] 5. Immunohistochemistry (IHC) detection of antibody specificity and protein localization LLCshNC, LLC shHEG1, LLC shHEG1 Vetor (HEG1 knockdown LLC cells with added empty vector), and LLC shHEG1 addback HEG1 (HEG1 knockdown LLC cells with added HEG1) cells in logarithmic growth phase were seeded into 10 cm dishes. When the density reached 90%, the cells were scraped off with a cell scraper, fixed with paraformaldehyde, embedded in paraffin, and the tissue sections were sectioned to a sample thickness of 4 μm. Immunohistochemistry (IHC) staining was used to detect HEG1 protein expression, localization, and antibody specificity.

[0062] Immunohistochemistry: 1) Baking: Before dewaxing, place the slides in a 60℃ incubator for 1 hour to allow the tissue to adhere better to the slide; 2) Dewaxing paraffin sections to water: sequentially immerse the sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then wash with distilled water; 3) Antigen retrieval: Place the tissue sections in a retrieval box filled with citrate antigen retrieval buffer (pH 6.0), cover the box with plastic wrap to prevent evaporation, boil in boiling water for 1 hour, and after natural cooling, place the slides in PBS (pH 7.4) and wash three times on a shaker for 5 min each time; 4) Blocking endogenous peroxidase: first, use a histochemical pen to draw circles on the tissue, the movement should be quick to prevent the slide from drying out, add an appropriate amount of 3% hydrogen peroxide solution to the tissue, and incubate at room temperature in the dark for 25 minutes. 5) Serum blocking: Gently shake off excess PBS, add DMEM (containing 5% FBS) to the histochemistry zone to cover the tissue, and block at room temperature for 30 min; 6) Primary antibody addition: Slowly shake off the blocking solution, add the primary antibody covering the tissue to the slide at a dilution ratio of 1:50, and place in a humidified chamber with an appropriate amount of water to prevent antibody evaporation. Incubate overnight at 4°C; 7) Secondary antibody addition: Place the slide in PBS (pH 7.4), shake gently on a shaker, and wash three times for 5 min each time. Add the secondary antibody reagent covering the tissue. Incubate at room temperature for 1 h; 8) DAB color development: Place the slide in a box filled with PBS and wash three times for 5 min each time. After gently shaking the section dry, add DAB staining solution to cover the tissue. Observe under a microscope. The appearance of brown indicates a positive result. Rinse with tap water to stop the staining process. 9) Counterstain cell nuclei: Stain cell nuclei with hematoxylin for about 1 minute, then wash with tap water. 10) Dehydration and mounting: Place the slide in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and xylene I for 5 minutes to dehydrate and clear the slide. Remove the slide from the xylene and add a small amount of neutral resin to the slide for mounting. 11) After the neutral resin solidifies, observe under a microscope, scan with a slide scanner, and analyze the results.

[0063] The results are as follows Figure 6 As shown, the expression level of HEG1 in LLC shNC and LLC shHEG1 addback HEG1 is higher than that in LLC shHEG1 and LLC shHEG1 Vetor, and HEG1 is located on the cell membrane.

[0064] 6. Sequencing C4 hybridoma cells were sent to the company where cDNA sequences were obtained using RT-PCR. The light and heavy chain variable region base sequences were amplified by 5' RAGE and ligated into a T vector to form a recombinant plasmid. Sequencing and analysis were performed using M13-F. The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.8.

[0065] Specifically, the CDR amino acid sequences of the light chain variable region and the heavy chain variable region are shown in Table 2 below: Table 2. CDR amino acid sequences of the light chain variable region and the heavy chain variable region

[0066] Experiment 5 Therapeutic effects of HEG1 monoclonal antibody (αHEG1 C4) in mice with tumors To investigate the effect of the monoclonal antibody αHEG1 C4 on tumor growth in mice, C57BL / 6J mice were subcutaneously injected with 2 × 10⁶ wild-type LLC cells. 6 In each mouse, once the tumor volume reached 100 mm... 2 Mice were randomly divided into two groups and given either IgG (control group) or αHEG1 C4 (treatment group) at a concentration of 5 mg / kg, once every three days. During this period, the growth of the mice's tumors was measured regularly using a vernier caliper.

[0067] The results are as follows Figure 7 As shown in a and b, compared with the control group, αHEG1 C4 treatment significantly inhibited the growth of subcutaneous tumors in mice and significantly reduced the tumor volume.

[0068] Furthermore, to clarify the effect of αHEG1 C4 antibody on tumor cell invasion, LLC cells were treated with αHEG1 C4 and invasion experiments were conducted.

[0069] The results are as follows Figure 7 As shown in Figure c, compared with the control group, αHEG1 C4 treatment significantly inhibited the in vitro invasive ability of LLC cells.

[0070] Furthermore, to clarify the effect of αHEG1 C4 on tumor metastasis in mice, a mouse lung cancer metastasis model was established by tail vein injection of LLC cells. After modeling, the mice were treated with either IgG or αHEG1 C4 at a concentration of 25 mg / kg.

[0071] The results are as follows Figure 7 As shown in Figure d, compared with the control group, the number of lung metastases was significantly reduced after monoclonal antibody treatment.

[0072] The above experimental results demonstrate that HEG1 monoclonal antibodies have the effect of inhibiting tumor growth and metastasis in vivo, and have the potential to be developed into cancer treatment drugs.

[0073] Experiment 6 HEG1 expression is correlated in tissue sections of clinical oncology patients. To clarify the correlation between HEG1 and lung cancer patients, surgically resected lung cancer tissue specimens were collected from the Department of Pathology, First Affiliated Hospital of Anhui Medical University. All patients signed informed consent forms before surgery, and the samples were anonymized.

[0074] After ex vivo, the tissue was fixed in 10% neutral buffered formalin for 24 hours, followed by graded ethanol dehydration, xylene clearing, and paraffin embedding to prepare paraffin-embedded blocks, which were then dried at room temperature and protected from light. A 4 μm thick section was cut from the paraffin-embedded block and stained with hematoxylin and eosin (HE). Two independent pathologists confirmed the diagnosis of non-small cell lung cancer, indicating the reliability of the experimental sample.

[0075] Immunohistochemistry (IHC) staining was used to stain tumor patient tissues with IgG or αHEG1 C4, respectively, to detect the expression level and cellular localization of HEG1 protein.

[0076] See results Figure 8 The results showed that HEG1 expression was high in clinical lung cancer tumor samples, and HEG1 was located on the cell membrane. HEG1 expression was significantly correlated with lung cancer patients.

[0077] Although previous articles (Wang Zheng, 2023) have indicated that HEG1 can trigger an immune response, this study suggests that high HEG1 expression can promote increased infiltration of anti-tumor immune cells and reduce infiltration of immunosuppressive cells (MDSCs), concluding that HEG1 may promote immune surveillance rather than immune escape. The results shown in this application's experimental data contradict this article. The main reason is that the conclusions of this prior art are based on bioinformatics analysis rather than experimental verification. The article only revealed the correlation between HEG1 expression levels and the immune microenvironment from a bioinformatics perspective. However, correlation does not imply causation, and increased immune cell infiltration is not equivalent to enhanced anti-tumor immune function. Therefore, its research has certain limitations, ultimately leading to low reliability of its conclusions.

[0078] This application experimentally demonstrates that HEG1 is an immune checkpoint molecule on the surface of tumor cells that targets NK cells. The prepared HEG1 monoclonal antibody exhibits high specificity and affinity for the HEG1 protein, competitively binding to HEG1 and mediating the recognition and killing of tumor cells by natural killer (NK) cells, thus blocking their immune escape with significant efficacy. This application not only provides new genes and directions for tumor treatment but also offers innovative ideas for the development of precision targeted drugs, possessing significant translational value and clinical application prospects in the field of tumor therapy.

[0079] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of an HEG1 inhibitor in the preparation of a drug for inhibiting immune escape of tumor cells, wherein the HEG1 inhibitor inhibits immune escape of tumor cells by binding to HEG1 molecules on the surface of tumor cells, blocking the immunosuppressive effect of HEG1 molecules on NK cells; the tumor cells are lung cancer cells; the HEG1 inhibitor is a HEG1 monoclonal antibody or its antigen-binding fragment, the HEG1 monoclonal antibody or its antigen-binding fragment having a light chain variable region and a heavy chain variable region, the light chain variable region containing three antigen complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; the heavy chain variable region containing three antigen complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.

2. The application as described in claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

8.

3. A HEG1 monoclonal antibody or its antigen-binding fragment, characterized in that, It has a light chain variable region and a heavy chain variable region as described in claim 1 or 2.

4. A polynucleotide, characterized in that, Encoding the light chain variable region and heavy chain variable region of the HEG1 monoclonal antibody or its antigen-binding fragment as described in claim 3.

5. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, It includes the expression vector as described in claim 5.

7. A tumor cell immune escape inhibitor, characterized in that, The immune escape inhibitor is an immune checkpoint inhibitor, comprising the HEG1 monoclonal antibody or its antigen-binding fragment as described in claim 3; the tumor cells are lung cancer cells.

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