Preparation method of NK cells and their application in cancer treatment

Abstract

The present invention relates to a method for preparing NK cells and their application in treating cancer. Specifically, a monoclonal antibody specific for PD-1 is provided, which can significantly improve the killing effect of cancer cells after being used in combination with NK cells, and has good application prospects.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a method for preparing NK cells and their application in treating cancer. Background Art

[0002] Well-known cancer risk factors include smoking, unhealthy diet, overweight and lack of exercise, which may further increase the burden of breast cancer, lung cancer, colorectal cancer and other cancers. Although there are many cancer prevention measures, cancer prevention is uncertain, so treatment is the key.

[0003] Tumor immunotherapy achieves long-term anti-tumor effects by improving the patient's own immune system, providing new treatment strategies and powerful assistance for patients who have not responded well to traditional anti-tumor therapies. Currently, the cell immunotherapy that has been carried out is mostly focused on genetically modified immune cells for targeted treatment of malignant tumors. A variety of CAR-Ts have been approved by the FDA for clinical trials, but the therapy is expensive, prone to cytokine release syndrome and graft-versus-host disease, and has strong immune rejection and limited cell sources. As solid immune cells, NK cells can directly kill tumor cells and virus-infected cells through receptors for immune regulation; the mechanism of NK cells killing tumors requires little specific antibodies and will not cause immune rejection in patients; the safety of NK cells for allogeneic transfusion has been confirmed, and it has a variety of sources, and the cost of forming cell products will also be low. Based on this, researchers use cytokine induction, combined monoclonal antibodies, and engineering transformation methods to activate the anti-tumor activity of NK cells from different sources, and use them in preclinical and tumor effect studies and clinical trial studies.

[0004] NK cells participate in anti-tumor immune responses through a variety of mechanisms. The NK cell surface receptor CD16 kills tumor cells through the ADCC effect; NK cells can release perforin and granzyme to activate the apoptosis system to attack tumor cells; NK cells can express death ligand (FASL) to bind to target cell death receptor (FAS) to cause target cell apoptosis. Unlike T cells, which require antigen processing and presentation during the activation process, the process of NK cell immune response does not require prior sensitization of tumor cells or specific antibodies, but is directly controlled by the interaction between NK cell activation inhibitory receptors and target cells. When the NK surface KIR subpopulation binds to specific HLA molecules, it can block the role of NK cells in recognizing and attacking target cells; and for target cells that do not express KIR and can bind to HLA molecules, NK cells will be activated, thereby exerting cytotoxic effects. NK cells will also secrete a variety of cytotoxins and cytokines under the regulation of receptors to directly kill tumor cells and induce immune responses. In order to escape the recognition and attack of NK cells, tumor cells will downregulate the activation receptors of NK cells. Therefore, when NK cells are exposed to the tumor microenvironment (TME) in the patient's body, NK cell function is easily dysregulated, resulting in a reduction in cytotoxicity. Tumor cells can also bind to inhibitory receptors and produce immunosuppressive factors such as TGF-β, IL-10, PGE2, etc., to directly immunosuppress NK cells. Studies have shown that tumor patients whose NK cells are positive for the inhibitory receptor NKG2A have a poor prognosis. Not only that, there are a large number of immunosuppressive cells in the TME, such as regulatory T cells (Tregs), tumor-associated macrophages (TAMs), etc., which hinder NK cells from entering the core of the tumor. The accumulation of tumor metabolites such as adenosine lactate will inhibit the killing ability of NK cells.

[0005] At present, researchers are using NK cells to treat a variety of solid tumors, such as gastric cancer, colorectal cancer, pancreatic cancer, etc., and clinical trials are underway. The research results are not yet complete, but it has been preliminarily shown that NK cell infusion therapy can induce tumor suppression or shrinkage in some patients. NK cells are the "vanguard" fighting on the front line of anti-cancer. As the first line of defense for immune surveillance, they have rapid response and strong anti-tumor capabilities. They can be activated without the need for antigen presentation, spontaneously identify abnormal cells in the body (such as cancer cells, infected cells, etc.), and release killing mediators - "granzymes" and "perforins", causing malignant cells to apoptosis, leaving hidden cancer cells nowhere to hide.

[0006] Clinical trials for the treatment of tumors after engineering NK cells are gradually being established, and have shown significant efficacy in the treatment of hematological malignancies. The efficacy of NK cells combined with immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) is particularly evident in drug-resistant tumors. However, there are not enough studies on the combination of NK cells and PD-1 monoclonal antibodies for the treatment of lung cancer. Summary of the invention

[0007] The present invention provides a monoclonal antibody specifically targeting PD-1.

[0008] Specifically, the monoclonal antibody against PD-1 of the present invention is named 5F2, the sequence of its light chain variable region is shown in SEQ ID NO: 1, and the sequence of its heavy chain variable region is shown in SEQ ID NO: 2.

[0009] In some embodiments, an isolated antibody (a) has a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1; or a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2; or (b) has a VL sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:1; or a VH sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:2; or (c) has a VL sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:1; or a VH sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2; or (d) has a VL sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:1; or a VH sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:2; or (e) has a VL sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1; or a VH sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0010] The present invention provides a pharmaceutical composition for treating lung cancer, which contains the monoclonal antibody of the present invention and NK cells.

[0011] Furthermore, the monoclonal antibodies of the present invention can also be prepared into corresponding immunoconjugates.

[0012] The present invention also provides immunoconjugates comprising an anti-PD-1 antibody herein conjugated with one or more cytotoxic agents, such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes (i.e., radioconjugates). Immunoconjugates allow targeted delivery of the drug moiety to tumors, and in some embodiments, intracellular accumulation therein, where systemic administration of unconjugated drugs may cause unacceptable levels of toxicity to normal cells. Antibody-drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to tumor cells expressing antigens, thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity. ADC compounds of the present invention include those having anticancer activity. In some embodiments, the ADC compound includes an antibody conjugated (i.e., covalently attached) to a drug moiety. In some embodiments, the antibody is covalently attached to the drug moiety via a linker. The antibody-drug conjugate (ADC) of the present invention selectively delivers an effective dose of the drug to tumor tissue, thereby achieving greater selectivity (i.e., a lower effective dose) while improving the therapeutic index ("therapeutic window"). The drug moiety (D) of the antibody-drug conjugate (ADC) may include any compound, moiety, or group having a cytotoxic or cytostatic effect. The drug moiety may exert its cytotoxic and cytostatic effects through mechanisms including, but not limited to, tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drug moieties include, but are not limited to, maytansinoids, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracyclines, duocarmycin, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, elinafide, and stereoisomers, isosteres, analogs, and derivatives thereof having cytotoxic activity. Non-limiting examples of such immunoconjugates are discussed in more detail below.

[0013] In the aforementioned use, the pharmaceutical composition can also be prepared for treating other cell proliferative disorders, specifically cancer. In some embodiments, the cancer is selected from the group consisting of esophageal cancer, gastric cancer, small intestine cancer, large intestine cancer, colorectal cancer, breast cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), B cell lymphoma, B cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia, AML, chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic leukemia (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, unclassifiable splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variants, Waldenstrom's macroglobulinemia, heavy chain disease , alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphatic tissue (MALT lymphoma), nodal marginal zone lymphoma, childhood nodal marginal zone lymphoma, childhood follicular lymphoma, primary cutaneous follicle center lymphoma, T cell / histiocyte-rich large B cell lymphoma, primary DLBCL of the CNS, primary leg-type cutaneous DLBCL, senile EBV-positive DLBCL, chronic inflammation-associated DLBCL, lymphomatoid granuloma tumor, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma: B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt's lymphoma and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma.

[0014] Specifically, the pharmaceutical composition of the present invention may also include a chemotherapeutic agent. A chemotherapeutic agent is a chemical compound used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, metodepa and urethane imine; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trihydroxymethylmelamine; polyacetic acid (especially bratacin and bratacinone); -9-tetrahydrocannabinol (dronabinol, -lapachone; lapachol; colchicine; betulinic acid; camptothecin; bryostatin; carlistatin; podophyllotoxin; podophyllic acid; tenipoxib; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; multicarmine (including synthetic analogs); argan; water chlorambucil; stoloniferol; sponge inhibitor; nitrogen mustards such as chlorambucil, naphthyl nitrogen mustard, clofosamide, estramustine, ifosfamide, nitrogen mustard, oxychloramine hydrochloride, melphalan, new nitrogen mustard, phenyl mustard, phenyl mustard, three mustard cyclophosphamide, uracil nitrogen mustard; nitrosoureas such as carmustine, chlorozotocin, fortaine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin 1 and calicheamicin; danemycin, including danemycin; esperamicin; and new carcinogens and related pigment protein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin, Carrubicin, carminomycin, carmoamicin, chromomycin, dactinomycin, daunorubicin, platinum analogs such as cisplatin, oxaliplatin (e.g., and carboplatin; vinca alkaloids, which prevent tubulin polymerization to form microtubules, including vinblastine, vincristine, vindesine, and vinorelbine, etoposide; ifosfamide; mitoxantrone; leucovorin; noantrol; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitors; difluoromethylornithine; retinoids such as retinoic acid, including bexarotene, bisphosphonates (e.g., or etidronate-58095, zoledronic acid / zoledronate, alendronate, pamidronate, tiludronate, or risedronate, and troxacitabine (1,3-dioxolane nucleoside cytosine) analogs); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation; topoisomerase 1 inhibitors 439006 (sorafenib; sunitinib; perifosine, celecoxib or etoricoxib), proteosome inhibitors; bortezomib-779; tipifarnib; sorafenib; tyrosine kinase inhibitors; farnesyl transferase inhibitors, such as lonafarnib and any pharmaceutically acceptable, acid or derivative thereof; and combinations of two or more of the above, such as (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone) and (abbreviation for treatment regimen of oxaliplatin combined with 5-FU and folinic acid); and combinations of two or more of the above.

[0015] As a general proposition, whether or not it is carried out by one or more administrations, the therapeutically effective amount of the anti-PD-1 antibody administered to a human will be in the range of about 0.01 mg / kg to about 100 mg / kg of the patient's body weight. In some embodiments, the antibody used is, for example, about 0.01 mg / kg to about 45 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 35 mg / kg, about 0.01 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 25 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, or about 0.01 mg / kg to about 1 mg / kg, for example, daily administration. In one embodiment, on the first day of a 21-day cycle, the anti-PD-1 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as an infusion. For repeated administration over several days or longer, depending on the condition, treatment should generally continue until the symptoms of the disease are suppressed as desired. An exemplary dose of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., so that the patient receives about two to about twenty doses, or, e.g., about six doses of the anti-PD-1 antibody). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and assays.

[0016] Specifically, the present invention also provides the use of the monoclonal antibody 5F2 of PD-1 and NK cells in the preparation of a drug kit for treating cancer, wherein the sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 1, and the sequence of the heavy chain variable region thereof is shown in SEQ ID NO: 2.

[0017] Specifically, the NK cells are isolated directly from blood, or expanded from commercial cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the specific identification result of 5F2 monoclonal antibody

[0019] Figure 2 This is the growth inhibition effect of 5F2 monoclonal antibody on cancer cells

[0020] Beneficial Effects

[0021] The present invention relates to a method for preparing NK cells and their application in treating cancer. Specifically, a monoclonal antibody specific to PD-1 is provided, which can significantly improve the killing effect of cancer cells after being used in combination with NK cells, and has good application prospects. DETAILED DESCRIPTION

[0022] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0023] Example 1 Preparation of PD-1 monoclonal antibody

[0024] After two 5-week-old female BALB / c mice were temporarily kept for one week, the prepared PD-1 recombinant protein was taken every 7 days, purchased from Biolab, item number JN0871 (60 μg), mixed with an equal volume of Freund's complete adjuvant and emulsified, and injected intraperitoneally into BALB / c mice (0.2 mL / mouse). After an interval of 14 days, the same amount of protein was emulsified with Freund's incomplete adjuvant and continued to be injected, for a total of 4 times. Blood was collected 3 days after immunization, and the serum titer was determined by indirect ELISA. Mouse No. 1 with a high titer was selected, and 100 μg of recombinant protein was boosted intravenously in the tail 3 days before fusion. After boosting immunization, the mice were killed and the mouse spleen cells were taken; then the cells were fused with mouse myeloma cells (SP2 / 0) using sterile PEG1500, and the hybridoma cells formed after fusion were placed in HAT medium and cultured in a 5% CO2 incubator at 37°C. When the number of cells reached two-thirds of the bottom area of ​​the well, the cell culture supernatant was taken for indirect ELISA detection. 75 positive hybridoma cell lines were screened by indirect ELISA. The positive wells were transferred to 24-well plates for expansion and then cloned. When the positive wells in the 24-well plates grew to 3 / 4 of the bottom of the wells, the positive hybridoma cells were diluted by limiting dilution and placed in HT culture medium and cultured in a 37°C, 5% CO2 incubator. When the cells grew to 2 / 3 of the bottom of the wells, indirect ELISA was performed. The wells with positive test results were expanded and then cloned for the second time, and finally the hybridoma cell line 5F2 with the best positive reaction and the most stable passage was obtained.

[0025] Female 6-week-old BALB / c mice were selected and fed for 8 days before intraperitoneal injection of 500 μL incomplete Freund's adjuvant. 6 The hybridoma cell line 5F2 was injected into the abdominal cavity of mice. When the abdomen of the mice was obviously swollen, the ascites was collected. The ascites was centrifuged at 4°C and 3000r / min for 15min to obtain the supernatant. After desalting using a PD-10 desalting column, the monoclonal antibody was purified by a Protein-G affinity chromatography column and stored at -80°C. The concentration of the monoclonal antibody was 3.07mg / mL using the BCA method, and the antibody was relatively pure using SDS-PAGE. The titer of the purified antibody was determined by indirect ELISA. The optimal antigen concentration of 1.2mg / mL was used as the coating concentration, the monoclonal antibody after multiple dilution was used as the primary antibody, HRP-labeled goat anti-mouse IgG was used as the secondary antibody, and the wells with PBS as the primary antibody were recorded as negative controls. Each experimental well was recorded as ODt, and the corresponding dilution ratio of ODt / OD0≥2.1 was used as the titer detection result. The results showed that the titer of the 5F2 monoclonal antibody was 2.56×10 5 .

[0026] Example 25 Identification of the specificity of the F2 monoclonal antibody

[0027] The PD-1 recombinant protein, human NCI-H446 small cell lung cancer cells, and mouse SP2 / 0 cells were boiled and denatured and then subjected to SDS-PAGE, with a separation gel concentration of 12% and a concentration gel concentration of 5%. The electrophoresis gel was stained with Coomassie Brilliant Blue staining solution and then decolorized until the protein bands were clear. Western blot analysis was performed by transferring the electrophoresis gel to a membrane at 200 mA for 20 minutes, and then blocking it in 5% skimmed milk powder for 1 hour. After washing three times with PBST, 5F2 monoclonal antibody (50 ng / mL) was used as the primary antibody, and goat anti-mouse HRP-IgG antibody (1:2500) was used as the secondary antibody, and ECL was used for color development after washing. The results are as follows Figure 1 shown.

[0028] from Figure 1 The results show that the 5F2 monoclonal antibody can specifically bind to the PD-1 recombinant protein and the PD-1 protein in human NCI-H446 small cell lung cancer cells, but not to any protein in mouse SP2 / 0 cells, showing good specificity.

[0029] Example 35 Affinity and sequence identification of F2 monoclonal antibody

[0030] The coating solution was diluted with recombinant PD-1 recombinant protein to 2μg / ml, 100μl / well, and coated on a 96-well ELISA plate at 4°C overnight. Prepare a mixture of monoclonal antibodies for antigen competition, dilute the antigen to 2μg / ml, 1μg / ml, 0.5μg / ml, 0.25μg / ml, 0.125μg / ml, 0.0625μg / ml, 0.03125μg / ml, 0μg / ml, take 50μl of each and incubate with 50μl 5F2 monoclonal antibody at 4°C overnight (the control antibody is pembrolizumab). Wash 3 times with PBST, block with 1% BSA, and incubate at 37°C for 2h. Wash 3 times with PBST, add 100μl of the incubation solution of each concentration of antigen and antibody to each well, set PBS as the zero well, and incubate at 37°C for 1h. Wash 3 times with PBST, add 100 μl of goat anti-mouse antibody labeled with horseradish peroxidase (HRP) to each well (1:10000 dilution), and incubate at 37°C for 1 hour. Wash 3 times with PBST, add TMB substrate solution, 100 μl / well, color development for 15 minutes in the dark, add stop solution (1M HCl) 100 μl / well, and immediately measure with a microplate reader, read the absorbance at 450 nm (OD450nm) and 630 nm (OD630nm), and calculate ΔOD450nm=

[0031] OD450nm-OD630nm. The formula for calculating the antibody affinity constant is A0 / (A0-A)=1+Kd / a0, where A0 is the ΔOD450nm when the competing antigen is 0, A is the ΔOD450nm corresponding to each competing antigen concentration, a0 is the total amount of antigen, and Kd is the dissociation constant. The results are shown in Table 1.

[0032] Table 1 Antibody affinity properties results

[0033] Antibody Name Dissociation constant 5F2 monoclonal antibody <![CDATA[(2.28±0.03)×10 -9 (M)]]> Pembrolizumab <![CDATA[(4.19±0.04)×10 -9 (M)]]>

[0034] The results in Table 1 show that the 5F2 monoclonal antibody of the present invention has good affinity with PD-1.

[0035] The 5F2 hybridoma cells were entrusted to Dion Biotechnology for antibody variable region gene sequencing services. After comparison and identification, the sequence of the light chain variable region was shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region was shown as SEQ ID NO: 2.

[0036] Example 45 Activity Analysis of F2 Monoclonal Antibody

[0037] Detection of the growth inhibitory activity of 5F2 monoclonal antibody on human NCI-H446 small cell lung cancer cells: Human NCI-H446 small cell lung cancer cells in the logarithmic growth phase were inoculated into 96-well plates, adhered to the plate for 24 hours, and the cell culture medium containing different concentrations of 5F2 monoclonal antibody was replaced. A total of 5 monoclonal antibody concentration gradients of 0, 10, 50, 100, and 200 mg / L were set, and 4 parallel samples in each group were cultured for 48 hours. The absorbance (A) value was measured by the methylthiazolium tetrazodone (MTT) colorimetric method, and the growth inhibition rate of cells after treatment with different concentrations of monoclonal antibodies was calculated. The treatment condition of 100 mg / L of pembrolizumab was used as a positive control. Growth inhibition rate = 1-(A value of the drug-added group / A value of the control group). The results are as follows Figure 2 shown.

[0038] from Figure 2 It can be seen that the 5F2 monoclonal antibody of the present invention can effectively inhibit the growth of human NCI-H446 small cell lung cancer cells, and has a certain dose dependence. Compared with the negative control group, the difference is extremely significant (P<0.01). Under the same concentration conditions, the inhibitory effect is also better than that of the positive control.

[0039] At the same time, Western blot was used to detect the expression of PD-1 protein in each group of cells, and the gray value of β-acitn was used as the basis for relative expression to calculate the gray value of PD-1. The results are shown in Table 2.

[0040] Table 2 Relative expression of PD-1 protein in each group

[0041] Group PD-1 relative expression value No mAb treatment group 0.81±0.10 10mg / L monoclonal antibody treatment group 0.24±0.06# 50mg / L monoclonal antibody treatment group 0.18±0.04# 100mg / L monoclonal antibody treatment group 0.12±0.05# 200mg / L monoclonal antibody treatment group 0.05±0.01# Positive control group (100 mg / L) 0.15±0.02#

[0042] # indicates significant difference compared with the group without mAb treatment (P<0.05).

[0043] It can also be seen from the results in Table 2 that the monoclonal antibody of the present invention has good activity in inhibiting the expression of PD-1 protein.

[0044] Example 5 Preparation of NK cells

[0045] The spleen of C57BL / 6 nude mice was extracted and ground into a single cell suspension. The suspension was centrifuged at 1200 rpm for 5 min to dissolve the red blood cells and then the spleen cell suspension was counted and counted as 1×10 7 10 cells / ml were added to the culture medium and cultured in IL-26000U / ml. After 2 days, NK cells were purified by anti-CD3 antibody magnetic bead method, and then IL-26000IU / mL was added and cultured for 24h. IL-1210ng / ml and IL-18100ng / mL were added and cultured for another 48h before harvesting the cells. The cells were centrifuged to obtain activated NK cells. Identification: Take a small amount of precipitate and add antibody NK1.1 + ,NKp46 and CD3, CD4 and CD8, flow cytometry (FACS) was used to detect the phenotype of NK cells, and the results showed that NKp46>96%, NK1.1 + >92%,CD8 + <1.5%, CD4 + <2%, indicating that activated NK cells were successfully prepared.

[0046] Example 6 NK cell and antibody mouse experiment

[0047] Human NCI-H446 small cell lung cancer cells in the exponential growth phase were prepared into a single cell suspension and subcutaneously inoculated into the flank of NCG mice at a number of 1*10 7 When the tumor grows to about 80 mm 3 At 4 pm, the mice were randomly divided into groups according to the tumor volume, with 10 tumor-bearing mice in each group. Drug administration began on the day of grouping. The grouping and drug administration schedule are detailed in Table 3.

[0048] Table 3 Grouping

[0049]

[0050] The tumor volume V0 was measured after the start of drug administration, and the tumor volume Vt was measured again on the 35th day after drug administration. The tumor growth inhibition rate was calculated = (tumor volume V0 before treatment - tumor volume Vt after treatment) / tumor volume V0 before treatment × 100%. The results are shown in Table 4.

[0051] Table 4: Tumor growth inhibition rate of each group

[0052]

[0053] As can be seen from Table 4, the combined use of NK cells and the 5F2 monoclonal antibody of the present invention can significantly improve the killing activity of NK cells, which shows that the PD-1 monoclonal antibody can block the immune checkpoint inhibition of tumor cells on NK cells and ensure the killing effect of NK cells.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it; although the present invention is described in detail with reference to the above embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above embodiments, or replace part or all of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against PD-1, characterized in that The sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

2. Use of the monoclonal antibody against PD-1 according to claim 1 in the preparation of a drug for inhibiting small cell lung cancer.

3. Use of the monoclonal antibody against PD-1 and NK cells according to claim 1 in the preparation of a drug kit for inhibiting small cell lung cancer.

4. The use according to claim 3, wherein the small cell lung cancer cells are NCI-H446 small cell lung cancer cells.

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