Preparation method of immune cells and application thereof in treatment of tumors

Abstract

The application provides a preparation method of immune cells and application thereof in treating tumors. Specifically, a monoclonal antibody 3F-15 specifically targeting EGFR is provided, the antibody has good specificity and good inhibitory activity on colorectal cancer cells. After the monoclonal antibody is combined with NK cells, the combination has good synergistic effect on treating colorectal cancer. After the combination is prepared into a medicine box, the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular to a preparation method of immune cells and application thereof in treating tumors. BACKGROUND

[0002] Natural killer (NK) cells are a kind of natural immune cells with the ability to kill tumor cells, which can resist the invasion of virus or tumor cells through cytotoxicity and release of cytokines. As one of the important immune cells in the human body, NK cells play a crucial role in monitoring tumors and exhibit strong killing ability to abnormal cells in an antigen-independent manner.

[0003] Natural killer (NK) cells are also an important part of ILCs. NK cells mediate cytotoxicity by secreting interferon gamma (IFN-γ), expressing T-bet and EOMES, etc. Many studies have shown that NK cells can exert anti-tumor cytotoxicity, which depends on certain functional receptors on the surface of NK cells, such as NKG2 and killer cell inhibitory receptor (KIR) family, etc. In addition, NK cells also produce a series of cytokines and chemokines to regulate immune responses. By releasing CCL5, XCL1 and XCL2, NK cells can promote the aggregation of dendritic cells (DCs) to solid tumors, promote the anti-tumor immune effect of CD8+ T cells, and prolong the survival of patients. In addition, in the inflammatory response, NK cells enter the tumor-draining lymph nodes, affect T cell responses or regulate DCs to indirectly regulate T cell immunity. In the process of "DC editing", activated NK cells can kill immature DCs (iDCs) and retain mature DCs to form a quality control mechanism to ensure the smooth start of the T cell immune link. Therefore, in the tumor microenvironment, NK cells can recognize and kill target cells without specific antigen sensitization stimulation.

[0004] The recognition of tumor by NK cells mainly depends on the balance between the activation receptors and inhibitory receptors expressed by NK cells, which determines the killing effect of NK cells. Chemokines and adhesion factors in the tumor microenvironment recruit NK cells, increase the degree of NK infiltration in tumor tissue, and then make the activation receptors on the surface of NK cells recognize the corresponding ligands on the surface of tumor cells, release perforin and other killing mediators, and exert cytotoxicity against tumor cells. Studies have shown that the cytotoxicity of NK cells is a key factor in inhibiting tumor growth and improving patient prognosis. The infiltration of NK cells into tumor tissue is an important link regulated by chemokines and their related receptors and adhesion molecules. The degree of NK cell infiltration in the tumor site is related to the effect of immunotherapy, and targeting the recruitment of NK cells to the interior of the tumor can effectively improve the anti-tumor immune response.

[0005] Based on the good efficacy of NK cells, it is an important direction of research to use them for cancer treatment. Given the advantage that NK cells can kill without tumor-specific receptors, although NK cells can be isolated from the peripheral blood of patients and expanded in vitro, studies have shown that the expansion of NK cells is more complex than that of T cells. Although autologous NK cells were used to treat patients with digestive cancer, the NK cells had high lytic activity in vitro and strongly expressed functional markers (such as NKG2D and CD16) after in vitro expansion to 4720 times, no clinical response was observed. At the same time, the functional status and proliferation of autologous NK cells are often poor, which may be related to the treatment the patient received before the NK cells were isolated, making it one of the reasons for poor clinical efficacy. In response to this problem, various methods are currently being studied, including different combinations of activating cytokines (such as IL-2, JL-15, IL-18), the use of feeder cells to provide important cytokines during in vitro expansion, etc. The function of NK cells in cancer patients is highly disordered and the number is reduced, so in cancer immunotherapy, the adoptive transfer of allogeneic NK cells is widely explored to induce anti-tumor effects. In a clinical experiment of allogeneic NK cell transfusion, it was found that NK cells were transfused to immunosuppressed AML patients and treated with IL-2, and IL-15 increased and the number of NK cells increased, and 5 of 19 patients had remission of the disease: the human cell line NK-92 has been widely used in clinical research of allogeneic NK cell therapy. Clinical studies have shown that 15 patients with drug-resistant malignant tumors (lung cancer, leukemia, lymphoma) were transfused with NK-92 cells twice, 48 hours apart, and 75% of the cancer patients had some anti-tumor response, indicating that NK-92 cells have good efficacy. However, if NK-92 cells are to be used as a new method for cancer treatment, a large number of studies are still needed to verify their safety. Induced pluripotent stem cells (iPSCs) can also differentiate into NK cells, and NK cells derived from this source have been shown to have very high cytotoxicity against tumors of various origins, both in vitro and in vivo. Studies have also found that peripheral blood iPSCs-derived NK cells have low expression of human killer cell immunoglobulin-like receptors (KIRs) in vitro, but also have cytotoxicity against cancer cells and can produce ADCC, indicating that peripheral blood iPSCs can be used as an alternative source of NK cells. Numerous clinical studies have shown that allogeneic NK cells do not cause graft-versus-host disease (GVHD), indicating that allogeneic NK cell adoptive cell therapy is safe. Similar to autologous NK cell adoptive cell therapy, allogeneic NK cell adoptive cell therapy expands and activates NK cells in vitro, and they persist after being transfused into the body.

[0006] Although there are many studies on the use of existing NK cells for the treatment of cancer, the therapeutic effect of using NK cells alone to treat cancer is still weak, and there is a large space for improvement in treatment, so the use of NK cells in combination with other drugs to improve the therapeutic effect is an important research direction in the future. SUMMARY

[0007] The present application overcomes the defects of the prior art and provides an improved method for treating cancer.

[0008] Specifically, the present application provides a monoclonal antibody 3F-15 specifically targeting EGFR, the light chain variable region sequence of which is shown in SEQ ID NO: 1, and the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

[0009] More specifically, the present application also provides a pharmaceutical composition for treating colorectal cancer, characterized in that it contains a monoclonal antibody 3F-15 specifically targeting EGFR and a pharmaceutically acceptable carrier, the light chain variable region sequence of the monoclonal antibody being shown in SEQ ID NO: 1, and the heavy chain variable region sequence being shown in SEQ ID NO: 2.

[0010] More specifically, the present application also provides a kit for treating colorectal cancer, characterized in that it contains a monoclonal antibody 3F-15 specifically targeting EGFR and NK cells.

[0011] As discussed herein, it is contemplated that variations in the amino acid sequence of the antibody are encompassed by the present application, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. Certain percentages in between are included, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. In particular, conservative amino acid substitutions are included. Conservative substitutions occur when a member of one family of amino acids is substituted for another. Genetically encoded amino acids are generally divided into the following families: (1) acidic - aspartic acid, glutamic acid; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include: arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Other amino acid families include (i) serine and threonine, which are aliphatic hydroxyl family; (ii) asparagine and glutamine, which contain amide; (iii) alanine, valine, leucine and isoleucine, which are aliphatic; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartic acid with a glutamic acid, a threonine with a serine, a

[0012] Preferred amino acid substitutions are: (1) substitutions that decrease proteolytic susceptibility, (2) substitutions that decrease oxidative susceptibility, (3) substitutions that alter binding affinity of a protein complex, (4) substitutions that alter binding affinity, and (4) substitutions that impart or modify other physicochemical or functional properties of the analog. The analogs can include various muteins of sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in naturally occurring sequences (preferably in the portion of the polypeptide outside the domain that forms intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence.

[0013] In particular, the present application provides Cetuximab as a positive control in the experiment. Cetuximab is a murine / human chimeric monoclonal anti-EGFR antibody developed by Imclone Corporation. It has two N-linked glycosylation sites on both heavy chains and a molecular weight of approximately 152 kD (including the sugar moiety). Cetuximab can bind to the extracellular domain of EGFR with high affinity, thereby competitively antagonizing the binding of ligands to EGFR, blocking the binding of the receptor to ligands and then inhibiting the activation of EGFR tyrosine kinase mediated by ligands. The antibody is used as a positive control of the present application.

[0014] In some embodiments, the NK cells are used once a week and the antibody targeting human EGFR is once a week.

[0015] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytomas, classic Hodgkin's tumor / cross striated muscle tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid cancer, cardiac tumors, medulloblastoma, germ cell tumor, primary central nervous system lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), chronic myeloid neoplasms, colorectal cancer, craniopharyngioma, T-cell lymphocytic cancer, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (such as intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (such as non-small cell lung cancer, small cell lung cancer, chest lung blastoma, and bronchial tumors), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., childhood rhabdomyosarcoma, childhood vascular tumor, Ewing's sarcoma, Kaposi's sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer,hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tumours of the trachea and bronchus, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumours, vulval cancer and Wilms' tumour.

[0016] The antibodies of the present application (also referred to herein as "active compounds") and derivatives, fragments, analogs and homologs thereof can be incorporated into pharmaceutical compositions which can comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0017] Pharmaceutical compositions of the present application are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, buccal (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0018] Pharmaceutical compositions adapted for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, TMCremophor EL (BASF, Parsippany, N J.) or phosphate buffered saline (PBS). In all cases, the composition will be sterile and should be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like), and mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as a nopol, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, monostearate salts and gelatin.

[0019] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with the other ingredients, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution.

[0020] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier, for example, a liquid carrier which can be applied orally and which can be swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as a part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0021] In addition, as a method for delivering the NK cells manufactured by the method of the present application to a lesion, local transplantation using surgical means, intravenous administration, local injection administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intramuscular administration, intracerebral administration, intracerebroventricular administration, intraarterial administration, and the like can be considered.

[0022] The dose of the cell preparation of the present application is, for example, 10 5 -0 8 cells, preferably 5 x 10 8 cells, and the specific dose can be obtained by optimizing conventional experiments.

[0023] Advantages

[0024] The present application provides a preparation method of immune cells and its application in treating tumors. Specifically, a monoclonal antibody 3F-15 specifically targeting EGFR is provided, which has good specificity and good inhibitory activity on colorectal cancer cells. The monoclonal antibody has good synergistic effect on treating colorectal cancer when used in combination with NK cells, and the preparation of the two into a kit has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure of Western blot identification result of 3F-15 monoclonal antibody

[0026] Figure 2 Figure of growth inhibition effect of 3F-15 monoclonal antibody on SW620 colorectal cancer cell line DETAILED DESCRIPTION

[0027] Those skilled in the art can modify the process parameters according to the content herein to achieve. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology. The methods, devices and materials in the following examples, if not specifically stated, are conventional methods, devices and materials in the art, which can be purchased from the market.

[0028] Example 1 Preparation and identification of monoclonal antibody targeting EGFR

[0029] The purchased EGFR fragment (BES20668RP, Shanghai Bolisen Biotech Co., Ltd.) was used as an immunogen to immunize 4 BALB / c mice, intraperitoneally injected (90 mg per mouse), and immunized once every 3 weeks for a total of 3 times. Four to five days before fusion, the mice were intraperitoneally injected with 60 mg of the immunogen again to enhance the immune response. The mouse with the strongest immune response (No. 2) was used for subsequent experiments. The spleen cells of the No. 2 mouse were fused with mouse myeloma cells SP2 / 0 according to the conventional method. The hybridoma cells were cultured in DMEM complete medium containing 1% HAT and 10% calf serum. Half of the medium was replaced after 4 days. After 10 days, the DMEM complete medium containing 1% HT and 10% calf serum was used. After 2 weeks, the DMEM complete medium containing 10% calf serum was used. The hybridoma culture supernatant was obtained by indirect immunofluorescence labeling and flow cytometry screening to obtain positive clones. The medium was replaced in time and the above method was used for re-screening in time. The hybridoma cells secreting antibodies with high titer, single clone growth, good morphology, and an antibody secretion positive rate of more than 99.3% were selected for subcloning, culture, and preservation for future use. The light chain variable region sequence is shown in SEQ ID NO: 1 and the heavy chain variable region is shown in SEQ ID NO: 2 after sequence identification by Suzhou Jinweizhi Biotech Co., Ltd.

[0030] A 12-week-old BALB / c female mouse was intraperitoneally injected with 0.5 mL Freund's incomplete adjuvant. One week later, about 5x106 hybridoma cells 3F-15 in the logarithmic growth phase were injected into the abdominal cavity of the mouse. When the mouse's abdomen was significantly swollen, the ascites was extracted and centrifuged at 10,000 r / min for 10 min to collect the supernatant. The monoclonal antibody was purified by De Tai Biotech, and the purity of the antibody reached 99.5%. The antibody concentration was adjusted to 2 mg / mL for future use.

[0031] EGFR protein and control BSA protein samples were subjected to SDS-PAGE electrophoresis, wet transferred to a PVDF membrane, and blocked with 5% skim milk at room temperature for 1 h. The 3F-15 monoclonal antibody (1:1000 dilution) was used as the primary antibody, and incubated at 4°C for 12 h. The HRP-labeled goat anti-mouse IgG (1:5000 dilution) was used as the secondary antibody, and incubated at room temperature for 1 h. The color development and analysis of the test results are shown in Figure 1

[0032] As can be seen from Figure 1 , the 3F-15 monoclonal antibody of the present application can specifically bind to EGFR protein, has good specificity, and can be used for subsequent experiments.

[0033] Example 3 Characteristic detection of 3F-15 monoclonal antibody

[0034] ​Take 100 μL of EGFR protein with concentrations of 1, 0.5, 0.25, 0.125 mg / mL as coating antigens, respectively, and take 3F-15 monoclonal antibody and positive control Cetuximab as primary antibodies, which are diluted by 2 times from 5 μg / mL, to perform indirect ELISA. Take the logarithm value of the concentration (mol / L) of the monoclonal antibody as the abscissa, and take the corresponding D490nm value as the ordinate, to draw a sigmoid curve. The top of the sigmoid curve is D490nm, max, and the antibody concentration corresponding to 50% D490nm, max of each curve is found, respectively. The affinity constant of the monoclonal antibody is calculated according to the formula, and the result is shown in Table 1.

[0035] Table 1: Affinity detection result of the monoclonal antibody

[0036] Antibody name Affinity constant 3F-15 5.42 x 10 9 / M]] Cetuximab 2.17 x 10 9 / M]]

[0037] As shown in Table 1, the 3F-15 monoclonal antibody of the present application has good affinity.

[0038] Example 3: Inhibition activity of 3F-15 monoclonal antibody on colon cancer cells

[0039] The logarithmic growth period of human colon cancer cell line SW620 (HZC-50225, Shanghai Huazhen Industry Co., Ltd.) adherent cells is selected, and after trypsin digestion, 2×10 5 The cells are inoculated in a 96-well culture plate at 200 μl per well, and cultured at 37°C and 5% CO2.

[0040] The monoclonal antibody stock solution is prepared by incubating and shaking the sample 3F-15 monoclonal antibody in serum-free DMEM medium, and the diluted working solution is prepared with a final concentration of 500 μg / ml, 200 μg / ml, 100 μg / ml and 50 μg / ml. The positive control drug Cetuximab stock solution is prepared, and the diluted working solution is prepared with a final concentration of 500 μg / ml, 200 μg / ml, 100 μg / ml and 50 μg / ml.

[0041] After 24 h, the culture medium containing different concentrations of the sample to be tested is replaced, 3 parallel holes are set for each group, and each drug is repeated three times. The cells are cultured at 37°C and 5% CO2 for 48 h, and observed under an inverted microscope. After 48 h, the supernatant is discarded, and the cells are washed once with normal complete culture medium. 200 μl of complete culture medium is added to each well, and 20 μl of CCK-8 reagent is added to each well. The cells are cultured at 37°C for another 4 h. After mixing with a micro-ultrasonic oscillator, the optical density value is measured on an enzyme label instrument at a test wavelength of 450 nm. Taking the cell culture hole without adding the drug as a control, the monoclonal antibody drug cell death rate is calculated as (1-drug OD450 / control OD450) * 100%, and the result is shown in Table 2.Figure 2 .

[0042] from Figure 2 The results show that the monoclonal antibody of this invention has a stronger inhibitory effect on the growth of the SW620 colorectal cancer cell line than Cetuximab, and exhibits better biological activity. At a monoclonal antibody concentration of 200 μg / ml, the cell death rate of colorectal cancer cells reached (87.52 ± 1.03)%.

[0043] Example 3: Preparation of Highly Active NK Cells

[0044] Under aseptic conditions, spleens were harvested from 8-week-old C57BL6 mice. Spleen cells were washed with RPMI 1640 medium using a 23G needle. Red blood cells were lysed with ACK buffer and washed once with sorting buffer. Before sorting, cells were blocked with 2,4G2 antibody (eBioscience). T cells were removed using anti-Thy1.2 magnetic beads, an LD column, and a MidiMACS sorter. NK cells were then positively selected using anti-DX5 magnetic beads, an MS column, and a MiniMACS sorter (Miltenyi Biotec). Sorting purity was assessed using an EPICS XL flow cytometer. Sorted NK cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (HyClone), 10 ng / mIL-2, and 100 ng / ml IL-15 (Beijing Kexin Jiayuan Biotechnology Co., Ltd., catalog number kx20H-15) for 10 days to obtain expanded and activated NK cells for later use.

[0045] The activated NK cells were made into 2×10 6 / ml cell suspension, and prepared human colon cancer cell line SW620 into 2×10 4 / ml cell suspension; conduct experiments according to the following cell grouping guidelines.

[0046] Group A: 1 ml NK cells (2 × 10⁻⁶) 6 / ml) and 1ml SW620 cells (2×10 4 Mix ( / ml);

[0047] Group B: 1 ml NK cells (4 × 10⁻⁶) 6 / ml) and 1ml SW620 cells (2×10 4 Mix ( / ml);

[0048] Group C: 1 ml HepG2 cells and 1 ml culture medium (target cell natural release group);

[0049] Group D: 1 ml HepG2 cells and 1 ml 1% NP40 (target cell maximum release group)

[0050] The cells were inoculated in 6-well plates and placed in a cell incubator for 6 h; the cell liquid was collected into a centrifuge tube and centrifuged at 1500 rpm / min for 10 min, and the supernatant was collected; the killing activity of the NK cells in groups A and B was determined according to the lactate dehydrogenase instruction. The results are shown in Table 2.

[0051] Table 2 Results of the killing activity of NK cells in each group

[0052] Group Cell-killing activity (%) Group A 48.56±2.85 Group B 54.43±4.17

[0053] As can be seen from the results in Table 2, the activated NK cells can effectively promote the killing activity of the NK cells on the SW620 colon cancer cells.

[0054] Example 4 Animal anti-tumor experiment of NK cells combined with 3F-15 monoclonal antibody

[0055] The human colon cancer cell line SW620 cells in vigorous growth were digested to prepare a cell suspension with a concentration of 10 7 ml of the cell suspension was inoculated subcutaneously in the right armpit of C57BL6 mice under sterile conditions, and 50 nude mice were randomly divided into 4 groups, namely:

[0056] Group A: the test drug EGFR antibody 3F-15 was dissolved in PBS with pH 6.0, and the dose was 0.5 mg / dose;

[0057] Group B: the dose of the positive control drug Cetuximab was 0.5 mg / dose;

[0058] Group C: the high-activity NK cells prepared in Example 3 were 2×10 6 / ml, 0.2 ml / dose;

[0059] Group D: the high-activity NK cells prepared in Example 3 were 2×10 6 / ml, 0.2 ml / dose; 6 h later, the EGFR antibody 3F-15 was administered at a dose of 0.5 mg / dose;

[0060] Each group was administered intramuscularly, once a week, for a total of 4 weeks, and the tumor diameter was measured twice a week during the administration period. The tumor volume (TV) was calculated according to the formula: TV = (ab 2 ) / 2, where a and b represent the length and width, respectively. On the 28th day, the mice were killed, the tumors were weighed, and the tumor inhibition percentage of each administration group was calculated, and the results are shown in Table 3.

[0061] Table 3 Results of the treatment of each group on the transplanted tumors of nude mice

[0062] Group Tumor inhibition percentage (%) Group A 66.58±0.39 Group B 64.39±0.31 Group C 45.63±0.28 Group D 78.56±0.52

[0063] As can be seen from Table 3, the EGFR antibody 3F-15 of the present application has a better tumor inhibition effect, and especially has a synergistic therapeutic effect after the antibody is combined with NK cells, and has a better application prospect.

[0064] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations of these embodiments can be made without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents.

[0065] Although the embodiments of the present application are described above in combination with the drawings, the present application is not limited to the above-described specific embodiments and application fields, and the above-described specific embodiments are merely illustrative and instructive, but are not restrictive. A person of ordinary skill in the art can make many forms under the inspiration of the present specification and without departing from the scope protected by the claims of the present application, and these all belong to the protection of the present application.

Claims

1. A monoclonal antibody 3F-15 specifically targeting EGFR, characterized in that The sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO:

2.

2. A kit comprising immune cells, characterized in that The sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO:

2.

3. Use of NK cells and monoclonal antibody 3F-15 specifically targeting EGFR for the preparation of a kit for the treatment of colorectal cancer, wherein, The sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO:

2.

4. Use according to claim 3, characterized in that The kit further contains a pharmaceutically acceptable carrier. The sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 2.

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