CN117430707A

CN117430707A - Preparation method of CIK cells and application of CIK cells in treatment of cancers

Info

Publication number: CN117430707A
Application number: CN202311385661.6A
Authority: CN
Inventors: 陈锦泽; 张化杰
Original assignee: Beijing Runzhou Biotechnology Co ltd
Current assignee: Chongqing Tcrcure Biological Technology Co ltd
Priority date: 2023-10-25
Filing date: 2023-10-25
Publication date: 2024-01-23
Anticipated expiration: 2043-10-25
Also published as: CN117430707B

Abstract

The invention provides a preparation method of CIK cells and application thereof in treating cancers. Meanwhile, the EGFR is used as a target to prepare and obtain a specific monoclonal antibody, and the monoclonal antibody has higher affinity with EGFR and has extremely strong activity of inhibiting proliferation of nasopharyngeal carcinoma cells. After the CIK cells prepared by the invention are combined with EGFR monoclonal antibodies, the combined type EGFR monoclonal antibody has a synergistic effect of increasing the killing effect of nasopharyngeal carcinoma, and has excellent application prospect.

Description

Preparation method of CIK cells and application of CIK cells in treatment of cancers

Technical Field

The application relates to the field of biology, in particular to a preparation method of CIK cells and application of CIK cells in treating cancers.

Background

CIK cells are heterogeneous cells, a mixed population of cells. Cd3+cd56+ cells and cd3+cd8+ cells are the major components thereof, and the major effector cells thereof are cd3+cd56+ cells. Studies have shown that the induced expansion of CD3+CD56+ cells is derived from CD3+CD56-T cells, but not from CD3-CD56+ natural killer cells, or CD3+CD56+ cells that are originally present in vivo. Although CD3+CD56+ cells only contain 1% -5% of peripheral blood lymphocytes, the cells can be rapidly amplified under certain culture conditions, and the amplification amount can reach more than 1000 times in general about 20d of in vitro culture, and can be further amplified in the presence of IL-2 after reinfusion. In the course of culturing CIK cells, cd3+ cells, i.e., T cells, are predominantly expanded and non-T cells are eliminated. After 2 weeks of culture, mononuclear cells were purified by immunomagnetic bead technique, and it was confirmed that cd3+cd56+cik cells account for 85% of the total number of all cultured cells, and a large number of active CIK cells obtained according to the above method provided a sufficient number for clinical tumor immunotherapy. CIK cells have stronger tumor killing activity than LAK cells and TIL cells.

The tumor killing mechanism of CIK cells may be a cytotoxic effect mediated by NK cell activating receptor NKG2D, i.e. the killing effect is initiated by interaction of activating receptor NKG2D on CIK cells with the corresponding ligand NKG2DL on tumor cells. Cd4+cd25+ regulatory T cells (tregs) can inhibit the anti-tumor activity of CIK cells, whereas transforming growth factor B (TGF-B) and glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR) molecules have a more important role in the immunosuppressive function of cd4+cd25+ cells. The CD4+CD25+ Treg content in CIK cells before and after DC induction was (13+ -5)% and (10+ -4)%, respectively, confirming that the amount of Treg in CIK cells after DC induction was reduced, while secretion amounts of IFN-y and IL-6 in DC+CIK cell groups were observed to be higher than that of CIK cells alone. It is suggested that DCs can enhance the antitumor activity of CIK cells, and that their effect on tregs is likely one of the mechanisms by which DCs activate CIK cells. When blocking TGF-B, IL-10, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and GITR with specific antibodies, respectively, it was found that blocking TGF-B and GITR molecules enhanced the proliferation and killing activity of CIK cells, suggesting that these two molecules play an important role in the inhibitory effect of CD4+CD25+ Treg on CIK cells.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in China, and is common in southern areas, and the survival rate of 5 years is 50-60%. At present, the treatment of NPC is mainly radiotherapy, but the clinical curative effect is limited. The Epidermal Growth Factor Receptor (EGFR) belongs to a human EGFR family member, is highly expressed in various human tumors, is closely related to apoptosis, angiogenesis and tumor metastasis, and can be used as a target for targeted treatment of partial malignant tumors, and the clinical application of the EGFR in non-small cell lung cancer is very mature. The EGFR expression level is also closely related to the occurrence and development of NPC. In recent years, molecular targeted therapies for NPC represented by EGFR have highlighted unique therapeutic advantages in the clinic. Currently, EGFR-targeted drugs are mainly classified into two categories according to their mechanism of action: the first is a small molecule tyrosine kinase inhibitor that reversibly competes with EGFR kinase for ATP binding sites on the receptor and induces the production of inactive homo-or heterodimers to inhibit activity, such as gefitinib and erlotinib. The twentieth monoclonal antibody acting on the extracellular region of the receptor is a mouse and human monoclonal antibody, is an EGFR monoclonal antibody, can block cell signaling, and can slow down cell division by regulating cell cycle checkpoint molecules, such as cetuximab and nituzumab. The two kinds of medicines have different acting sites, but all play roles in blocking EGFR-mediated signal transduction pathway, blocking phosphorylation of related enzymes and reducing generation of corresponding proteins, thereby inhibiting tumor growth, metastasis and angiogenesis, promoting apoptosis and improving sensitivity of tumors to chemo-and radiotherapy. However, at present, the variety of EGFR-homed monoclonal antibodies is still insufficient, and particularly, the research for providing the combined treatment of nasopharyngeal carcinoma with CIK cells is still under further development.

EGFR monoclonal antibody and CIK cells are important tools for tumor biological targeting treatment, but the problems of feasibility of combined application of the EGFR monoclonal antibody and the CIK cells in nasopharyngeal carcinoma treatment, synergistic effect and capability of effectively improving the killing effect on tumor cells are all to be verified.

Disclosure of Invention

The invention provides a CIK cell which can be used for killing nasopharyngeal carcinoma.

Furthermore, the invention provides monoclonal antibodies specific for EGFR targets.

The monoclonal antibody is EGFR monoclonal antibody 3D7, and the sequence of the light chain variable region is shown in SEQ ID NO:1, the heavy chain variable region sequence is shown as SEQ ID NO: 2.

Light chain variable region sequence VL (SEQ ID NO: 1)

NIVFTQASDSVPVTPGTIVSISCRSSNVPTFYKKYMMLYWFLQSPGQSPQRLILHKNLASGVPDDFSGRGSGTDFTLRISMVEAEDVGVYYCLCFGQWTMIFGGGTKLEIK

Heavy chain variable region sequence VH (SEQ ID NO: 2)

EVQFLETGAGLVQPTGSRGLSCEGSYHLNWQEVSWVRQLPGKTLEWIGDGA

LGQHPAKYAPSIKDRFTTIFHMHCIMLTQMSNVRSEDTATYFCIAHYSYGGFA

YWGQGTSVTVSS

Furthermore, the monoclonal antibody has better affinity property and the property of killing nasopharyngeal carcinoma cells.

Specifically, the EGFR monoclonal antibody 3D7 of the present invention may also be modified and still retain antibody activity.

"conservative amino acid substitutions" refers to any amino acid substitution of a given amino acid residue, wherein the substituted residue is chemically similar to the given residue such that the polypeptide function (e.g., enzymatic activity) is not substantially reduced. Conservative amino acid substitutions are well known in the art. In a preferred embodiment, the conservative amino acid substitutions may be any one that occurs in one of the following six groups: 1. small aliphatic, substantially nonpolar residues: ala, gly, pro, ser and Thr;2. large aliphatic, non-polar residues: ile, leu, and Val; met;3. polar negatively charged residues and amides thereof: asp and Glu;4. amides of polar negatively charged residues: asn and Gln; his;5. polar positively charged residues: arg and Lys; his; and 6. Large aromatic residues: trp and Tyr; phe. In a preferred embodiment, the conservative amino acid substitution will be any one of the following, listed as a natural residue (conservative substitution) pair: ala (Ser); arg (Lys); asn (Gln; his); asp (Glu); gln (Asn); glu (Asp); gly (Pro); his (Asn; gln); ile (Leu; val); leu (Ile; val); lys (Arg; gln; glu); met (Leu; ile); phe (Met; leu; tyr); ser (Thr); thr (Ser); trp (Tyr); tyr (Trp; phe) and Val (Ile; leu).

In certain embodiments, an isolated antibody, or antigen-binding portion thereof, comprises a first polypeptide portion comprising a human heavy chain constant region; and a second polypeptide portion comprising a human light chain constant region. An isolated antibody or antigen binding portion thereof described herein can comprise a human IgG1 Fc domain comprising (1) a mutation at Kabat position 238 that reduces binding to an Fc-gamma-receptor (fcγr), wherein proline 238 (P238) is mutated to one of the residues selected from the group consisting of lysine, serine, alanine, arginine, and tryptophan, and wherein the antibody or antigen binding portion thereof has reduced fcγr binding; or (2) an alanine at Kabat position 297.

In some embodiments, a pharmaceutical composition is provided that comprises an effective amount of an anti-EGFR antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In some embodiments, a pharmaceutical composition is provided that comprises an effective amount of an anti-EGFR antibody and an effective amount of at least one additional therapeutic agent, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant. In some embodiments, the at least one additional therapeutic agent is selected from those described above.

In some embodiments, the formulation materials of the pharmaceutical composition are non-toxic to the recipient at the dosages and concentrations employed. In some embodiments, the pharmaceutical composition comprises a formulation material for modifying, maintaining or maintaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, and lysine); an antimicrobial agent; antioxidants (e.g., ascorbic acid, sodium sulfite, and sodium bisulfite); buffers (e.g., borates, bicarbonates, tris-HCl, citrates, phosphates, and other organic acids); bulking agents (e.g., mannitol and glycine); chelating agents (e.g., ethylenediamine tetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, and hydroxypropyl-beta-cyclodextrin); a filler; monosaccharides, disaccharides, and other sugars (e.g., glucose, mannose, and dextrins); proteins (e.g., serum albumin, gelatin, and immunoglobulins); coloring agents, flavoring agents, and diluents; an emulsifying agent; hydrophilic polymers (e.g., polyvinylpyrrolidone); a low molecular weight polypeptide; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, and polyethylene glycol); sugar alcohols (e.g., mannitol and sorbitol); a suspending agent; surfactants or moisturizers (e.g., poloxamers, PEG, sorbitan esters, polysorbates (e.g., polysorbate 20 and polysorbate 80), triton, tromethamine, lecithin, cholesterol, and tyloxapol); stability enhancers (e.g., sucrose and sorbitol); tonicity enhancing agents (e.g., alkali metal halides (e.g., sodium chloride or potassium chloride), mannitol, and sorbitol); a delivery vehicle; a diluent; an excipient; and a pharmaceutical adjuvant.

In some desirable embodiments, the antibody is administered by parenteral injection. For parenteral administration, the anti-EGFR antibody may be formulated as a solution, suspension, emulsion or lyophilized powder with a pharmaceutically acceptable parenteral carrier. For example, the carrier may be a solution of the antibody or mixture thereof dissolved in an acceptable carrier (e.g., an aqueous carrier, such a carrier being water, saline, ringer's solution, dextrose solution, or 5% human serum albumin, 0.4% saline, 0.3% glycine, or the like). Liposomes and non-aqueous carriers (e.g., fixed oils) can also be used. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of antibody in these formulations can vary widely, e.g., from less than about 0.5% by weight, typically or at least about 1% by weight up to 15% by weight or 20% by weight, based primarily on liquid volume, viscosity, etc., and is selected according to the particular mode of administration selected. The carrier or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by conventional techniques.

Furthermore, the invention also provides application of EGFR monoclonal antibody 3D7 and CIK cells in preparing a pharmaceutical composition for treating nasopharyngeal carcinoma.

Furthermore, the invention also provides application of EGFR monoclonal antibody 3D7 and CIK cells in preparing a pharmaceutical composition for treating human nasopharyngeal carcinoma CNE-2 cells.

Further, included within the scope of the invention are kits for practicing the methods. The kit comprises at least one or more antibodies of the invention, nucleic acids encoding the antibodies, or cells containing the antibodies, and CIK cells. In one embodiment, the antibodies of the invention are provided generally in lyophilized form in a container. The antibodies, which may be conjugated to a label or toxin or unconjugated, are typically included in a kit along with buffers (e.g., tris, phosphate, carbonate, etc.), stabilizers, biocides, inert proteins (e.g., serum albumin), or the like. Typically, these materials are present in an amount of less than 5% by weight of the amount of active antibody, which is typically present in a total amount of at least about 0.001% by weight based on the antibody concentration. In general, it is desirable to include inert supplements or excipients to dilute the active ingredient, wherein the excipients may be present in an amount of about 1% to 99% by weight of the total composition.

Drawings

FIG. 1 growth inhibition of EGFR monoclonal antibody 3D7 on poorly differentiated human nasopharyngeal carcinoma CNE-2 cells; FIG. 2 graph of the results of the combined killing rate characteristics of CIK cells and monoclonal antibody 3D7

Detailed Description

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs.

EXAMPLE 1 immunization and fusion screening of EGFR monoclonal antibodies

Egfrvlll recombinant protein, cat No.: CS10718, whankestan Biotechnology Co., ltd.

Male BALB/c mice were immunized for 6-8 weeks for four rounds. The previous three rounds are all injected subcutaneously by multiple points, 1 time every two weeks, and the injection quantity is 80 mug each time; the serum antibody titer of the mice was determined to be 1:200000 by indirect enzyme-linked immunosorbent assay (ELISA) 10d after the end of the third round of immunization. A fourth immunization was performed by intraperitoneal injection, with an immunization dose of 60. Mu.g.

Spleens of mice with four rounds of immunization are taken, fully ground and spleen cells are collected. After cell counting, spleen cells and Sp2/0 cells in logarithmic growth phase were counted at 10:1, centrifuging at 1500r/min for 5min, and discarding the supernatant. Placing the centrifuge tube in 37 deg.C warm water, slowly dripping polyethylene glycol (PEG 1500) solution within 1min, mixing, and standing for 1min. 5mL of preheated DMEM medium is added within 2.5min, and the volume is fixed and centrifuged. Cells were resuspended in HAT medium containing trophoblast cells and plated according to the number of splenocytes. After 10d of incubation, HT medium was replaced. When larger cell clusters appear in the fused cells, EGFR proteins are coated, and positive clones are screened by an indirect ELISA method. And (3) performing amplification culture on the screened positive holes, subcloning by a limiting dilution method after the cell density reaches 70%, and screening to obtain the hybridoma cell strain with the strongest positive reaction, namely 3D7.

8 weeks male BALB/c mice were intraperitoneally injected with paraffin oil 600. Mu.L/mouse. After 14D, 3D7 hybridoma cells in logarithmic growth phase were collected and adjusted to 2X10 ⁶ mL ^-1 In the same wayMice were injected at a dose of 500 μl/mouse. Ascites fluid is collected after 7d, purified by ammonium octoate sulfate precipitation and protein G column affinity chromatography, and the concentration of the monoclonal antibody is adjusted to 1mg/mL for standby by protein quantitative determination.

Antibody titers were detected by indirect ELISA. The purified antibody was diluted to a mass concentration (0.5 mg/mL), then diluted to a gradient of 1:409600, and the OD value was analyzed to evaluate the antibody titer. The result shows that the titer of the EGFR monoclonal antibody 3D7 reaches 1: 204800.

The antibody variable region was analyzed to extract 3D7 hybridoma RNA, which was sent to the golden Biotech Co.Ltd for sequencing. By sequence analysis, the sequence of the light chain variable region is identified as SEQ ID NO:1, the heavy chain variable region sequence is shown as SEQ ID NO: 2.

Example 2 affinity identification of EGFR monoclonal antibody 3D7

The invention uses SurfacePlasmonResonance (SPR) to determine antibody affinity. SPR experiments were performed in HBS-EP buffer (10 mmol/LHEPES, 150mmol/LNaCl, 3mmol/LEDTA, 0.005% polysorbate 20, pH 7.4) using a BIAcore T200 instrument. The egfrvlll protein was immobilized on CM5 chip by amine coupling reaction so that it remained sufficient ligand binding capacity. The subtraction of the body effect is performed using the control flow channel as a reference plane. The immobilized and reference channels were injected with increasing concentrations of the antibodies of the invention and control antibodies, and the chips were then treated with regeneration solution (2 mol/LNaCl,10mmol/L sodium acetate, pH 4.5). And finally, the data are evaluated by using BIAEvalation software, and the equilibrium dissociation constant Kd is calculated according to the fitted saturated binding curve. A specific control antibody is an anti-EGFRVIII [ L8A4] antibody, cat#

/SKUEDK002，The results are shown in Table 1.

TABLE 1 dissociation constant results for antibodies

Antibody name	Dissociation rate constant (Kd)
		EGFR monoclonal antibody 3D7	(6.34±0.21)×10 ^-8 mol/L
Control: anti-EGFRVIII [ L8A4]]Antibodies to	(2.16±0.15)×10 ^-9 mol/L

From Table 1, it can be seen that the EGFR monoclonal antibody 3D7 of the present invention shows extremely strong affinity and stability compared with the control anti-EGFRVIII [ L8A4] antibody, and can be used for the subsequent targeted treatment and detection application of nasopharyngeal carcinoma.

Example 3 Activity detection of EGFR monoclonal antibody 3D7

The poorly differentiated human nasopharyngeal carcinoma CNE-2 cells are cultured by conventional methods. With RPMI-1640 culture solution containing 10% FPS and 100U/ml penicillin and 100U/ml streptomycin at 37deg.C and 5% CO ₂ Saturated humidity, 2-3d liquid exchange and passage for 1 time. The cells in logarithmic growth phase were used in the experiment.

CNE-2 cells were digested and counted at 2X10 per well ³ The cells are evenly inoculated on a 96-well culture plate, and after 12 hours of culture, serum-free culture solution is changed for continuous culture for 12 hours, so that the cells synchronously grow. Then, 100. Mu.l of RPMI-1640 culture medium containing 1% FPS was replaced, and the monoclonal antibodies of the present invention and control monoclonal antibodies were added to each well, with final drug concentrations of 0, 62.5, 125, 250, 500. Mu.g/ml, 4 multiplex wells for each concentration, and blank controls. After further incubation for 24h, 20. Mu.l of MTT solution at 5mg/ml was added to each well, incubated for 4h, centrifuged at 1500r/min for 10min, the supernatant was discarded, the pellet was dissolved in 150. Mu.l of DMSO, absorbance was measured at 570nm, and the experiment was repeated 3 times. The cell growth inhibition was calculated according to the following formula:

cell growth inhibition (%) = [1- (drug-treated group OD value-blank group OD value) (no drug-control group OD value-blank group OD value) ] x100%. The positive control was purchased from beijing bertai biopharmaceutical company limited using nituzumab. The results are shown in FIG. 1.

As can be seen from FIG. 1, the monoclonal antibody of the present invention has a dose-dependent inhibition of proliferation of poorly differentiated human nasopharyngeal carcinoma CNE-2 cells, and at a concentration of 500. Mu.g/ml, the inhibition rate of cell growth reaches (51.37.+ -. 2.87)% higher than that of the control.

Meanwhile, the result of using western bolt to detect the inhibition of the monoclonal antibody on the EGFR protein expression quantity in each experimental group shows that compared with CNE-2 cells without monoclonal antibody, the monoclonal antibody has the protein inhibition rate reaching (93.47+/-2.34)% under the action of 125 mug/ml monoclonal antibody and the protein inhibition rate reaching (98.59+/-3.27)% under the action of 500 mug/ml monoclonal antibody, which shows that the monoclonal antibody has better EGFR expression inhibition property.

EXAMPLE 4 screening identification and Activity verification of CIK cells

PBMC were extracted from 10ml peripheral blood of healthy adults by Ficoll density gradient centrifugation and the RPMl1640 medium was washed 3 times, suspended in RPMl1640 complete medium containing 500U/ml INF-. Gamma.and cultured in a 5% CO2 incubator at 37℃for 24 hours. Adding rhlL-2400U/ml, rhlL-1 alpha 100U/ml and human CD3 monoclonal antibody 100ng/ml for continuous culture. 1-time supplement of rhIL-2 for each 2d liquid change is as before, and the concentration is adjusted to 2X10 ⁶ After 15d of culture, a small amount of cells were taken and examined by a flow cytometer. The proportion of CD3+CD56+ cells reaches 45.31%, the proportion of CD8+CD28+ cells reaches 52.37%, and the proportion of CD3+ cells reaches 93.59%, which indicates that the preparation and the expansion of the CIK cells are better.

Inoculating CNE-2 cells in logarithmic growth phase into 96-well plate with 1×10 per well ⁴ Mu.l, cultured for 24h. CIK cells and CNE-2 cells were added at different target ratios. Each group is provided with 5 compound holes. After incubation for 24h, 30. Mu.l of MTT at 5mg/ml was added to each well and incubated for 4h. The supernatant was aspirated, 100. Mu.l of DMSO was added to each well and the A value was determined by an microplate reader at a wavelength of 590nm with shaking for 30 min. Killing rate = [1 one (experimental well a value-effector cell well a value)/target cell well a value]x100%. The results are shown in Table 2.

TABLE 2 killing Activity of CIK cells on nasopharyngeal carcinoma cells

Effective target ratio	1:10	1:20	1:30
				CNE-2	37.35±3.48(％)	46.87±4.17(％)	69.43±5.24(％)

As can be seen from table 2, the most potent killing activity was obtained at an effective target ratio of 1:30.

Example 5 Combined Activity verification of CIK cells and monoclonal antibodies of the invention

Inoculating CNE-2 cells in logarithmic growth phase into 96-well plate with 1×10 per well ⁴ Mu.l, cultured for 24h. Three experimental groups A, B, C, D, E and control groups of single target cells (CNE-2 cells) and single effector cells (CIK cells) were set up. Group A: nituzumab + CIK cells + CNE-2 cells, group B: CIK cells+CNE-2 cells. Group C: nituzumab +CNE-2 cells, group D: 3D7 mab + CIK cells + CNE-2 cells, group E: 3D7 mab +CNE-2 cells.

Wherein the concentration of the monoclonal antibodies is 1mg/ml, and the effective target ratio of CIK cells is 30: 1. Each group is provided with 5 compound holes. After incubation for 24h, 30. Mu.l of MTT at 5mg/ml was added to each well and incubated for 4h. The supernatant was aspirated, 100. Mu.l of DMSO was added to each well and the A value was determined by an microplate reader at a wavelength of 590nm with shaking for 30 min. Killing rate = [1- (experimental well a value-effector cell well a value)/target cell well a value ] x100%. The results are shown in FIG. 2.

As can be seen from fig. 2, the 3D7 mab of the present invention has an effect of better killing rate of cancer cells with nimuzumab, especially has a stronger killing rate when being used in combination with CIK cells, and achieves (97.31 ±2.01)%, which is higher than the (90.23±1.93)% killing rate of positive control, which fully indicates that the monoclonal antibody of the present invention has a better application prospect when being combined with CIK.

When practicing or testing embodiments of the invention, optional methods and materials similar or equivalent to those described in the specification may be used, with the preferred methods, devices, materials described in the specification. However, before describing the materials and methods of the present invention, it is to be understood that the specific size, shape, dimensions, materials, methods, means, etc. described in this specification may be changed according to conventional experimental methods and optimization purposes, and thus the present invention is not limited thereto. And it is to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the scope of the appended claims.

Claims

1. A monoclonal antibody against the EGFR target for the treatment of cancer, characterized in that the monoclonal antibody is EGFR monoclonal antibody 3D7, the light chain variable region of which has the sequence set forth in SEQ ID NO:1, the heavy chain variable region sequence is shown as SEQ ID NO: 2.

2. Use of the monoclonal antibody of claim 1 in the preparation of a medicament for inhibiting nasopharyngeal carcinoma.

3. The use of the monoclonal antibody and CIK cells according to claim 1 for preparing a pharmaceutical composition for inhibiting nasopharyngeal carcinoma, wherein said CIK cells are prepared by extracting human blood with PBMC by Ficoll density gradient centrifugation, washing with RPMl1640 medium 3 times, suspending in 500U/ml INF-gamma-containing RPMl1640 complete medium, and concentrating at 37deg.C 5% CO ₂ Incubator culture 24h; adding rhlL-2400U/ml, rhlL-1 alpha 100U/ml and human CD3 monoclonal antibody 100ng/ml for continuous culture; changing liquid 1 time every 2d, and adjusting concentration to 2×10 ⁶ And/ml, culturing for 15 days to obtain CIK cells.

4. The use according to claim 3, wherein the nasopharyngeal carcinoma is a human nasopharyngeal carcinoma CNE-2 cell.

5. A pharmaceutical composition for treating nasopharyngeal carcinoma comprising the monoclonal antibody of claim 1 and a CIK cell.