Preparation method of immune cell CIK and its application in cancer treatment

Abstract

The present invention provides a method for preparing immune cells CIK and application thereof in treating cancer, and more specifically provides a CIK cell combined with EGFR monoclonal antibody for treating cancer. The composition has good synergistic therapeutic effect, can be used to prepare a composition for treating cancer, and has good application prospects.

Description

Technical Field

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

[0002] Cancer has always been a major public health problem worldwide. For men, the top ten most common tumors are prostate cancer, lung cancer, colorectal cancer, bladder cancer, skin melanoma, kidney cancer, non-Hodgkin's lymphoma, oral cancer, leukemia and pancreatic cancer, of which prostate cancer accounts for 29%. For women, the top ten most common tumors are breast cancer, lung cancer, colorectal cancer, cervical cancer, skin melanoma, non-Hodgkin's lymphoma, thyroid tumors, pancreatic cancer, kidney cancer, and leukemia, of which breast cancer accounts for 31%. With the rapid development of medical technology, the survival rate of cancer has also been significantly improved, and there are many ways to treat cancer.

[0003] CIK cell therapy, or cytokine-induced killer (CIK), is an adoptive cell therapy that refers to a group of heterogeneous cells obtained by stimulating and culturing mononuclear cells from peripheral blood / bone marrow / cord blood in vitro with multiple cytokines (such as anti-CD3 monoclonal antibodies, IL-2, and IFN-γ, etc.). The main effector cells of CIK cell therapy are CD3+ / CD56+ cells. Since they express two membrane protein molecules, CD3 and CD56, they are also called NK cell-like T lymphocytes, which have the powerful anti-tumor activity of T lymphocytes and the non-MHC-restricted tumor killing advantages of NK cells. Therefore, the application of CIK cells is considered to be the first choice for the new generation of anti-tumor adoptive cell immunotherapy.

[0004] Many research reports have confirmed that CIK cells have strong anti-tumor activity, but their specific tumor killing and regulatory mechanisms have not been fully understood. Possible mechanisms include: CIK cells express fasL and can induce apoptosis of fas+ tumor cells through the fas-fasL pathway. At the same time, CIK cells also express fas, but unlike previous effector cells (such as TIL), CIK cells can not only resist apoptosis induced by fasL+ tumor cells, but also kill fasL+ (ie, fas- cells) and their killing activity is equivalent to killing fas+ tumor cells. Adhesion molecules and co-stimulatory molecules also play a certain role in the process of effector cells killing target cells. Whether these adhesion molecules are also involved in the process of CIK cells killing tumor cells needs further study, but it is certain that LFA / CD11a and ICAM-1 / CD54 play an important role in mediating the cytotoxic effect of CIK cells. Experiments have confirmed that CIK cells kill target cells by releasing cytoplasmic granules and producing BLT esterase, mainly through two pathways. One is that the interaction between CIK cell surface adhesion molecule LFA-1 and ICAM-1 stimulates CIK cell degranulation and releases BLT esterase. This pathway is regulated by the second messenger cAMP (dPcAMP inhibits the tumoricidal activity of CIK cells), but is not affected by immunosuppressive drugs CsA and FK506; the other is that CIK cells stimulate CIK cell degranulation and release BLT esterase through the interaction between their surface CD3 or CD3-like surface receptors and CD3-McAb. Unlike the first pathway, this pathway is regulated by the second messenger cAMP and affected by immunosuppressive drugs CsA and FK506. CIK cell culture supernatant contains biologically active soluble FasL. CIK cells can secrete TRAIL (TRAIL), and TRAIL shows an up-regulation trend in CIK cell culture. TRAIL can induce apoptosis of Jurkat cells. At the same time, CIK cells themselves can also secrete cytokines such as IL-2, IL-6, TNF-α, etc., to enhance cytotoxicity.

[0005] Studies have shown that compared with CIK cell therapy alone, CIK cell combination therapy can achieve better anti-tumor response and OS rate. For example, in the study of CIK combined with cetuximab to treat gastric cancer, the results showed that EGFR monoclonal antibody failed to significantly increase the binding rate of CIK cells and SGC7901, but the killing rate of gastric cancer cells and the inhibition rate of nude mouse transplanted tumors by EGFR monoclonal antibody combined with CIK cells were significantly higher than those of either alone. In the study conducted on the ability of anti-EGFR / anti-CD3 bifunctional antibodies to mediate CIK cells to kill gastric cancer cells in nude mice, the results showed that its killing activity was good and stronger than other cell immunotherapy. CIK cell therapy combined with molecular targeted drugs can enhance the killing of gastric cancer and inhibit tumor growth, providing feasibility for clinical comprehensive treatment of gastric cancer. As a promising treatment method, CIK cell-based immunotherapy is widely used in the field of malignant tumors. However, at present, the development of combined treatment methods with better efficacy needs to be further improved. Summary of the invention

[0006] The present invention first provides a method for preparing immune cell CIK.

[0007] Specifically, the method for preparing CIK cells is a conventional method in the art.

[0008] The present invention further provides a pharmaceutical composition for treating cancer, wherein the composition contains the CIK cells of the present invention and a monoclonal antibody specific to EGFR.

[0009] Furthermore, the cancer of the present invention is skin cancer.

[0010] Specifically, the monoclonal antibody against EGFR of the present invention is the monoclonal antibody against EGFR 3F3, the light chain variable region sequence is shown in SEQ ID NO:1, and the heavy chain variable region sequence is shown in SEQ ID NO:2.

[0011] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous applications may include the following components: sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; 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 adjusting tension, such as sodium chloride or glucose. pH can be adjusted with acid or alkali, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be loaded into ampoules, disposable syringes or multi-dose bottles made of glass or plastic. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersants and sterile powders for the temporary preparation of sterile injectable solutions or dispersants. For intravenous administration, suitable carriers include physiological saline, antibacterial water, TMCremophor EL or phosphate buffered saline (PBS). In all cases, the composition can be sterile and should be easy to inject. It can be stable under production and storage conditions and must be preserved from microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing (for example) water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a mixture thereof. For example, it can be by using a coating (for example lecithin), by maintaining the desired particle size under dispersion and by using a surfactant to maintain appropriate fluidity. The prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents can be preferably included in the composition, such as sugars, polyols (for example mannitol, sorbitol), sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, lead monostearate and gelatin.

[0012] Further, the pharmaceutical composition may include one or more pharmaceutical excipients. Any suitable pharmaceutical excipient can be used, and those of ordinary skill in the art can select suitable pharmaceutical excipients. The non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. Whether a specific excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on various factors well known in the art, including but not limited to the specific antibodies in the mode and dosage form of the dosage form to the subject. If desired, the composition or single unit dosage form may also contain a small amount of wetting agent or emulsifier, or pH buffer. Therefore, the pharmaceutical excipients provided below are intended to be illustrative, rather than non-restrictive.

[0013] In certain embodiments, the pharmaceutical composition comprises a defoamer. Any suitable defoamer can be used. In some aspects, the defoamer is selected from alcohol, ether, oil, wax, silicone, surfactant and a combination thereof. In some aspects, the defoamer is selected from mineral oil, vegetable oil, ethylenebisstearamide, paraffin, ester wax, fatty alcohol wax, long-chain fatty alcohol, fatty acid soap, fatty acid ester, silanol, fluorosilicone, polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octanol, sorbitan trioleate, ethanol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone and a combination thereof.

[0014] In certain embodiments, the pharmaceutical composition comprises a cosolvent. Illustrative examples of cosolvents include ethanol, poly (ethylene) glycol, butylene glycol, dimethylacetamide, glycerol and propylene glycol. In certain embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum and monosodium glutamate. In certain embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum and guar gum. In certain embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimonium bromide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid, polyethylene glycol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, polyoxyglycerol esters, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene glycol succinate. In certain embodiments, the pharmaceutical composition includes an anti-caking agent. Illustrative examples of anti-caking agents include tricalcium phosphate, hydroxymethylcellulose, hydroxypropylcellulose, and magnesium oxide. Other excipients that can be used in pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersants, dissolution enhancers, emulsifiers, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizers, solvents, stabilizers, and sugars.

[0015] Any suitable cancer can be treated with the combination of antibodies and CIK cells provided herein. Illustrative suitable cancers include, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, primary unknown cancer, heart tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibro ... Histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma Pigmented tumors, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous neck cancer, midline cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, Pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, colorectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer and Wilms' tumor.

[0016] Also provided herein are freeze-dried powders containing monoclonal antibodies of the present invention, which can be reconstituted into solutions, emulsions and other mixtures for administration. These preparations can also be reconstituted and formulated as solids or gels. Sterile freeze-dried powders can be prepared by dissolving the solid part containing the monoclonal antibody in a suitable solvent or mixing a small aliquot of a solution containing the monoclonal antibody with a suitable solvent. The solvent can contain excipients or other pharmacological components that improve the stability of the powder or the reconstituted solution prepared by the powder. Excipients that can be used include but are not limited to dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, lactose or other suitable agents. The solvent can also contain a buffer, such as citrate, sodium phosphate or potassium phosphate or other such buffers, which are known to those skilled in the art, usually about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to obtain a freeze-dried preparation. Typically, the solution obtained by sterile filtration is dispensed into a vial for freeze drying. Each vial may contain a single dose of the compound, such as 10-1000 mg or 100-500 mg, or multiple doses of the compound. Briefly, the lyophilized powder may be prepared by dissolving dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, lactose or other suitable agent in an amount of about 1-20% in a suitable buffer such as citrate, sodium phosphate or potassium phosphate or other such buffer known to those skilled in the art to a pH of about neutral. The resulting mixture is sterile filtered or treated to remove particulates and ensure sterility, and dispensed into vials for lyophilization. The lyophilized powder may be stored under suitable conditions, such as at about 4° C. to room temperature.

[0017] Further, the present invention can also provide a monoclonal antibody for detecting EGFR and a kit for detecting EGFR. Of course, the antibody of the present invention can be detected by appropriate detection, such as conventional type of immunoassay. For example, a sandwich assay can be performed, in which the full-length EGFR pro-, EGFR or its fragment is attached to a solid phase. Keep incubating for a long enough time to allow the antibody in the sample to bind to the fixed polypeptide on the solid phase. After the first incubation, the solid phase and the sample are separated. The solid phase is washed to remove unbound materials and interfering substances that may also be present in the sample, such as non-specific proteins. Subsequently, the solid phase containing the target antibody bound to the fixed polypeptide is incubated with a second labeled antibody or an antibody bound to a coupling agent (such as biotin or avidin). This second labeled antibody can be another anti-EGFR antibody. The labeling of antibodies is well known in the art and includes radionuclides, enzymes (such as maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluorite (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, etc. The labeled antibody is incubated with the solid and the labeling combined with the solid is measured. These and other immunoassays can be easily performed by those of ordinary skill in the art. An exemplary method for detecting the presence or absence of EGFR in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample with a labeled monoclonal antibody according to the present invention so that the presence of EGFR in the biological sample is detected. The term "labeling" as used herein with respect to a probe or antibody is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance and the probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent directly labeled. Examples of indirect labeling include detecting a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin so that it can be detected by fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids separated from a subject, as well as tissues, cells, and fluids present in a subject. That is, the detection method of the present invention can be used to detect EGFR in biological samples in vitro and in vivo. For example, techniques for detecting EGFR in vitro include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation, and immunofluorescence. Moreover, techniques for detecting EGFR in vivo include introducing labeled anti-EGFR antibodies into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0018] Further, the pharmaceutically acceptable composition comprising CIK cells can include a variety of carriers and excipients. A variety of aqueous carriers can be used, such as buffered saline, etc. These solutions are sterile and generally free of undesirable substances. Suitable carriers and excipients and formulations thereof are described in the prior art. A pharmaceutically acceptable carrier refers to a biologically or otherwise non-adverse material, that is, the material is applied to a subject without causing adverse biological effects or interacting with other components of the pharmaceutical composition contained therein in a harmful manner. If applied to a subject, the carrier is optionally selected to minimize the degradation of the active ingredient and minimize the adverse side effects in the subject. As used herein, the term pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmaceutically acceptable. The pharmaceutical composition will generally include reagents for buffering and preservation in storage and may include buffers and carriers for appropriate transportation, depending on the route of administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Western blot detection of EGFR protein expression in each group

[0020] Figure 2 The results of the effects of cancer cell apoptosis in each group

[0021] Beneficial Effects

[0022] The present invention provides a preparation method of immune cell CIK and its application in cancer treatment, and more specifically provides a CIK cell combined with EGFR monoclonal antibody for cancer treatment, the composition has good synergistic therapeutic effect, can be used to prepare a composition for cancer treatment, and has good application prospects. Moreover, the antibody provided by the present invention has strong ability to inhibit the activity of EGFR and good affinity characteristics. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0024] Example 1 Proliferation and activation of CIK cells

[0025] 50 mL of peripheral venous blood from healthy volunteers was collected in heparin anticoagulation tubes, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation. The separated PBMCs were inoculated into T25 cell culture flasks, and the cell density was adjusted to 1×10 with Corning 88-551-CM KBM551DC / CIK lymphocyte serum-free medium. 8 The cell culture flask was placed in a 37°C, 5% CO2 incubator and 1000U / mL IFN-γ was added for 24 hours; IL-1α, IL-2 and CD3 monoclonal antibody were then added to make the final concentrations of IL-1α, IL-2 and CD3 monoclonal antibody in the culture medium 300U / ml, 1000U / ml and 600U / ml respectively; then the culture was continued in a constant temperature incubator at 37°C, 5% CO2 concentration and 100% humidity, and the culture medium was added every 3 days to keep the cell concentration at 1×10 6 U / ml, the concentrations of IL-1α, IL-2 and CD3 monoclonal antibodies were maintained at 400U / ml, 1000U / ml and 500U / ml respectively. After 14 days of culture, the activated CIK cells were taken for cell performance analysis, and the results showed that CD3+ was 96.5%, CD3+CD4+ was 15.39%, CD3+CD8+ was 84.21%, and CD3+CD56+ was 20.17%, indicating that good CIK cells were isolated.

[0026] Example 2 Preparation of EGFR monoclonal antibody

[0027] The purchased EGFR recombinant protein ab208473 and abcam were fully emulsified with Freund's complete adjuvant at a ratio of 1:1, and then injected subcutaneously at multiple points on the back of the mice to immunize 6-week-old BALB / c mice, with an immunization dose of 70μg / mouse. The mice were boosted two weeks and four weeks after the first immunization. The mice were tail-cut and blood was collected 3 days after the third immunization, and the immune titer of the mice was detected by indirect ELISA after the serum was separated. The No. 2 mouse with the highest titer was selected for impact immunization 3 days before cell fusion, and the protein dose of 60μg / mouse was injected intraperitoneally. The spleen of the immunized mouse was taken and cell fusion was performed with SP2 / 0 cells in the logarithmic growth phase according to the hybridoma cell fusion technology, and the HAT selection medium was used for culture. Half of the medium was changed on the 4th day after fusion, and the medium was changed completely on the 6th day. The supernatant was collected 24 hours after the medium was changed for indirect ELISA detection and screening, and the positive 38 hybridoma cell wells were expanded and cultured and subcloned and purified three times. After three subclonings, three monoclonal antibody cell lines (2G1, 4D8 and 3F3) were obtained with the highest activity.

[0028] Healthy BALB / c mice were selected and intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Ten days later, three types of hybridoma cells were injected into the peritoneal cavity of different mice separately, with each mouse injected with about 10 6 cells. When the abdominal circumference of the mouse was significantly enlarged and the mouse had difficulty in moving, ascites was extracted aseptically. Protein A (GE Healthcare 17-5079-01) affinity column purification method was used for purification. After SDS-PAGE identification, the concentrations of the three monoclonal antibodies 2G1, 4D8 and 3F3 were 3.8 mg / mL, 3.3 mg / mL and 4.5 mg / mL, respectively.

[0029] Example 3 Activity Identification of EGFR Monoclonal Antibodies 2G1, 4D8 and 3F3

[0030] When SCL-1 human skin squamous cell carcinoma cells (Cat. No.: BES-2496HC, Bolson), grew to cover 80% of the area of ​​the culture flask, 25 cm 2 The culture medium in the culture flask was used to wash the cells once with PBS; about 1 ml of 0.25% trypsin digestion solution was added to the culture flask and observed under an inverted microscope. After the cells shrank and became round, complete culture medium was added to terminate the digestion, and the cells were gently blown to make them fall off, and then the suspension was transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min; the supernatant was discarded, and the precipitated cells were resuspended in 12 ml of complete culture medium, and then divided into bottles and passaged at a ratio of 1:2, and cultured in a 37°C, 5% CO2 cell culture incubator; SCL-1 human skin squamous cell carcinoma cells were cultured at 8×10 3 / well of cells were inoculated in a 96-well plate and cultured for 12 hours. The experimental group treated cells with EGFR monoclonal antibodies 2G1, 4D8 and 3F3 at a concentration of 200μg / mL for 48 hours, the control group added an equal volume of PBS solution, and the positive control group was cetuximab with the same dosage and usage as the above antibodies. 10μL of MTT (5mg / mL) was added to each well, and the cells were cultured for 4 hours. The supernatant was discarded, and 200μL of DMSO solution was added to each well. The cells were shaken at a low speed for 10 minutes at a constant temperature of 37°C to fully dissolve the formazan crystals. The absorbance values ​​of each well were measured at 490nm and 570nm wavelengths using an enzyme marker, and the cell survival rate at each drug concentration was calculated. Cell survival rate = [(experimental well-control well) / (control well-blank well)]×100%. The results are shown in Table 1.

[0031] Table 1 Effect of each antibody on the survival rate of cancer cells (%)

[0032] Group Cell survival rate (%) PBS 109.53±1.37 EGFR monoclonal antibody 2G1 85.38±0.64 EGFR monoclonal antibody 4D8 92.45±0.84 EGFR monoclonal antibody 3F3 37.24±0.31# Cetuximab 44.03±0.28#

[0033] As can be seen from Table 1, the EGFR monoclonal antibody 3F3 and cetuximab have similar effects, and are significantly different from the other two monoclonal antibodies (P<0.05). It can be seen that the EGFR monoclonal antibody 3F3 of the present invention has the strongest property of inhibiting cell survival.

[0034] Western blot was used to detect the expression of EGFR protein in each group: an equal amount of cells from each group were washed three times with PBS, centrifuged at 5000r / min, RIPA lysis buffer was added, and the cells were lysed on ice for 30min. The cells were centrifuged at 12000r / min for 15min to extract cell proteins, and the protein concentration was detected using the BCA protein detection kit. Equal amounts of proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane using a gel electrophoresis instrument. The PVDF membrane was blocked with a TBS-T solution containing 5% skim milk for 1h, followed by incubation with the EGFR primary antibody and the GADPH primary antibody overnight. Finally, the PVDF membrane was washed three times and incubated with a horseradish peroxidase-conjugated antibody at room temperature for 2h. Western blot images were obtained using the Gel imaging system, and 200μL of ECL chemiluminescent reagent was added to the top of the membrane to detect the target protein bands. The results are shown in the figure. Figure 1 shown.

[0035] from Figure 1 It can be seen that the EGFR monoclonal antibody 3F3 and the positive control group of cetuximab have similar EGFR protein expression inhibition effects. Therefore, 3F3 monoclonal antibody was selected for subsequent experiments.

[0036] Example 4 Affinity and sequence identification of EGFR monoclonal antibody 3F3

[0037] The recombinant EGFR protein was coated on the biosensor chip, and the affinity constant of the 3G3 monoclonal antibody was determined using the BiacoreX100 protein interaction system. Sodium acetate at pH 4.8 was selected as the coupling dilution buffer. The coupling level was 1800RU. The 3F3 monoclonal antibody was diluted with a pH 7.4 running buffer to a series of concentrations of 0nM, 9.375nM, 18.75nM, 37.5nM, 75nM, and 150nM. The binding time was set to 180s, the dissociation time was 15min, and the regeneration buffer was 50mM Gly-HCl (pH 1.7). The on-machine test was performed according to the protocol of BiacoreX100controlsoft. The results showed that the affinity constant of the 3F3 antibody was 2.6×10 -9 M.

[0038] cDNA was prepared from 3F3 hybridoma cells and entrusted to Suzhou Genewise Biotechnology Co., Ltd. for variable region determination and analysis. After identification, the light chain variable region sequence of EGFR monoclonal antibody 3F3 was obtained as shown in SEQ ID NO: 1, and the heavy chain variable region sequence was shown in SEQ ID NO: 2.

[0039] Example 5 Combined activity of EGFR monoclonal antibody 3F3 and CIK cells

[0040] SCL-1 human skin squamous cell carcinoma cells were seeded in 12-well plates (3×10 5 After incubation for 12 h, PBS (blank control group), EGFR monoclonal antibody 3F3 (100 μg / mL) (antibody group), EGFR monoclonal antibody 3F3 (100 μg / mL) + CIK cells prepared in Example 1 (3×10 6 / well) (antibody combined with CIK cell group), CIK cells prepared in Example 1 (3×10 6 / well) (CIK cell group), cetuximab (10μg / mL) (positive control group), treat cells for 24h. Digest and collect cells in each group, remove supernatant by centrifugation, wash cells with PBS, resuspend cells with 1× binding buffer, add Annexin V-FITC and PI successively, and incubate at 37℃ in the dark for 15min. Flow cytometry was used to detect cell apoptosis immediately. The results are shown in Figure 2 shown.

[0041] We analyzed the cell apoptosis of different groups in SCL-1 human skin squamous cell carcinoma cells by double staining with Annexin V-FITC and propidium iodide (PI). The results showed that at a concentration of 100 μg / mL, the cell apoptosis rate of the antibody group reached (43.12±1.92)%, while the cell apoptosis rate of the blank control group was (11.13±0.96)% (P<0.01). After the monoclonal antibody was used in combination with CIK, the cell apoptosis rate reached (62.85±1.13)%. The experimental results show that the monoclonal antibody of the present invention can synergistically increase the apoptosis of cancer cells when used in combination with CIK, thereby achieving a tumor-killing effect.

[0042] The present disclosure set forth above may encompass a plurality of different inventions with independent utility. Although each of these inventions is disclosed in its preferred form, its specific embodiments as disclosed and described herein should not be considered restrictive, because multiple changes are possible. The subject matter of the present invention includes all novel and non-obvious combinations and sub-combinations of various elements, features, functions and / or characteristics disclosed herein. The following claims specifically point out certain combinations and sub-combinations that are considered novel and non-obvious. Inventions embodied in other combinations and sub-combinations of features, functions, elements and / or characteristics may be claimed in this application, an application claiming priority to this application, or a related application. These claims (whether or not for different inventions or the same invention and whether or not the scope is wider, narrower, equal or different than the original claims) are also deemed to be included in the subject matter of the invention disclosed herein. Without departing from the scope of the present invention, one or more features from any embodiment or drawings described herein may be combined with one or more features in any other embodiment or drawings described herein. All publications, patents and patent applications cited in this specification are incorporated herein by reference, as if explicitly and individually indicating that each individual publication or patent application is incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made to the invention without departing from the spirit or scope of the appended claims.

Claims

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

2.

2. Use of the monoclonal antibody according to claim 1 in the preparation of a pharmaceutical composition for inhibiting human skin squamous cell carcinoma.

3. Use of the monoclonal antibody according to claim 1 in the preparation of a pharmaceutical composition for inhibiting human skin squamous cell carcinoma cell SCL-1.

4. Use of the monoclonal antibody and CIK cells according to claim 1 in preparing a pharmaceutical composition for inhibiting human skin squamous cell carcinoma, wherein the preparation process of the CIK cells comprises: The isolated PBMCs were inoculated into T25 cell culture flasks, which were placed in an incubator at 37°C and 5% CO2 and cultured with 1000U / mL IFN-γ for 24 hours. IL-1α, IL-2, and CD3 monoclonal antibodies were then added to make the final concentrations of IL-1α, IL-2, and CD3 monoclonal antibodies in the culture medium 300U / ml, 1000U / ml, and 600U / ml, respectively. The cells were then cultured in a constant temperature incubator at 37°C, 5% CO2, and 100% humidity, and the culture medium was added every 3 days to keep the cell concentration at 1×10 6 U / ml, the concentrations of IL-1α, IL-2 and CD3 monoclonal antibodies were maintained at 400U / ml, 1000U / ml and 500U / ml, respectively, and activated CIK cells were obtained after 14 days of culture.

5. The use according to claim 4, characterized in that The pharmaceutical composition is in the form of an injection.

Images