Compositions involving cell proliferation inhibition

Abstract

The present invention relates to a composition for inhibiting cell proliferation. The present invention prepares and obtains a specific monoclonal antibody against the G250 protein, which can significantly inhibit the proliferation of cancer cells and the expression of the G250 protein within cancer cells. The antibody is prepared into a drug and is expected to be used to treat the corresponding cancer.

Description

Technical Field

[0001] The present application relates to the biological field, and more particularly to a composition for inhibiting cell proliferation. Background Art

[0002] Renal cell carcinoma (RCC) accounts for over 85% of malignant renal tumors, ranking second among urinary tract tumors. 90% of these are clear cell carcinomas. RCC is cryptic and difficult to detect in its early stages. Despite rapid advances in imaging technology in recent years, early diagnosis remains challenging. Furthermore, RCC is highly malignant and insensitive to both radiotherapy and chemotherapy. The incidence of RCC in my country has been increasing annually. RCC is not a simple disease, with numerous histological types, including clear cell carcinoma, multilocular cystic RCC, papillary RCC, and chromophobe RCC.

[0003] Kidney cancer is a multi-gene disease, and its development and progression are influenced by mutations in multiple genes. Recently, researchers from the Sanger Institute in the UK, the National Cancer Centre Singapore, and other institutions discovered PBRM1, the second most mutated gene in renal cell carcinoma. Using large-scale exome sequencing, the scientists found that the PBRM1 gene is mutated in 41% (92 / 227) of clear cell renal cell carcinomas. This gene, also located on the short arm of chromosome 3 (3p21), encodes the protein BAF180, a subunit of the SWI / SNF chromosome remodeling complex (PBAF). The SWI / SNF complex, first discovered in Saccharomyces cerevisiae, is a highly conserved ATP-dependent chromosome remodeling complex. Its human homolog is called PBAF. It plays a role in replication, transcription, DNA repair, and cell proliferation and differentiation. It is composed of multiple subunits, and the loss of any one subunit results in functional loss of the complex. Loss of SWI / SNF complex function is associated with many malignancies. The PBRM1 protein (BAF180) is composed of 1,689 amino acids, including a DNA-binding domain (HMG), two protein-interacting components (BAH1 and BAH2), and six acetylated histone-binding domains (BD1-BD6). It plays a crucial role in cell mitosis and gene transcription. PBRM1 gene mutations have been found in breast cancer, where it functions as a tumor suppressor gene. Researchers using RNA interference (RNAi) have demonstrated that PBRM1 is a tumor suppressor gene in renal cell carcinoma (RCC), at least in part, involved in the recognition and modification of histone H3. As a newly discovered RCC gene, in-depth research on PBRM1 will provide new insights into the diagnosis and treatment of RCC.

[0004] In recent years, the development of molecular biology experimental techniques, such as microarray (TMA) technologies such as gene sequencing and high-throughput tissue sequencing, has enabled the detection and analysis of the expression of hundreds of different biomolecules, including DNA, RNA, and proteins. However, inactivation of the VHL gene remains the predominant genetic alteration in renal cancer, particularly in clear cell renal cell carcinoma (ccRCC). Nickerson et al. found that 91% of patients with pathologically confirmed clear cell renal cell carcinoma had mutations or hypermethylation of the VHL gene. The VHL gene is located on the short arm of chromosome 3 (3p25.3), and inactivation occurs through mutation, loss of heterozygosity (LOH), and hypermethylation. VHL gene inactivation results in the loss of normal function of the VHL protein it encodes. This, in turn, prevents the effective degradation of hypoxia-inducible factor (HIF), resulting in activation of hypoxia-inducible genes.

[0005] In 1986, Dutch scientists discovered a new renal cancer-associated antigen, G250, and conducted systematic and in-depth studies, finding that G250 exhibits excellent renal cancer specificity. The high specificity and sensitivity of G250McAb binding to renal cancer cells makes it suitable for targeted tumor therapy. Chemically conjugating G250McAb to a vector enabled targeted transfection of the vector-carried reporter gene, CFP, into renal cancer cells, while also doubling the transfection efficiency of GFP. Researchers also found that when using MAbG250 for targeted immunotherapy of renal cancer, efficacy ceased to improve with increasing antibody concentration beyond a certain level. Analysis suggests that because the efficacy of G250-targeted therapy is positively correlated with the amount of MAbG250 bound to the G250 antigen, once a certain amount of G250 antibody is injected into the body, all G250 antigenic determinants present on renal cancer cells are targeted, resulting in saturation. Therefore, increasing the dose and concentration of the injected antibody does not improve the efficacy of MAbG250 in treating renal cancer. Therefore, to further enhance the efficacy of targeted immunotherapy for renal cancer, particularly high-grade renal cancer, upregulating G250 antigen expression is an effective approach. Studies have shown that IL-2 and IFNα cytokines upregulate G250 antigen expression in renal cancer cell lines 786-0 and OS-RC-2, with a clear dose- and time-dependent effect. Furthermore, an additive effect was observed when the drugs were used in combination. Therefore, IL-2 and IFNα were used in combination to enhance G250 antigen expression in renal cancer cells. Specifically, a complex formed by ligating G250McAb and a plasmid encoding interleukin (IL)-2 was transfected into renal cancer cells. The complex was found to be able to transfer the IL-2 gene into renal cancer cells through antibody-mediated endocytosis, where it remained in the perinuclear zone for over 20 hours. IL-2 secretion by transfected renal cancer cells persisted for 15 days. These findings suggest that G250McAb and the various antibodies derived from it hold great significance in targeted immunotherapy for renal cancer. Summary of the Invention

[0006] The present invention overcomes the defects of the prior art and provides a specific G250 monoclonal antibody.

[0007] Specifically, the light chain variable region sequence of the G250 antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0008] Furthermore, the present invention also provides use of the G250 monoclonal antibody in preparing a pharmaceutical composition for treating renal cancer.

[0009] Specifically, the pharmaceutical composition further contains a pharmaceutically acceptable carrier or a second therapeutic agent.

[0010] Examples of suitable pharmaceutically acceptable excipients include one or more polymers, wetting agents or surfactants, pH adjusters, isotonicity adjusters, preservatives, buffers, and chelating agents, or any combination thereof.

[0011] Examples of suitable pharmaceutically acceptable pH adjusting agents include, but are not limited to, sodium hydroxide, citric acid, hydrochloric acid, boric acid, acetic acid, phosphoric acid, succinic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium oxide, calcium carbonate, magnesium carbonate, magnesium aluminum silicate, malic acid, potassium citrate, sodium citrate, sodium phosphate, lactic acid, gluconic acid, tartaric acid, 1,2,3,4-butanetetracarboxylic acid, fumaric acid, diethanolamine, monoethanolamine, sodium carbonate, sodium bicarbonate, triethanolamine, or any combination thereof. In one embodiment, the pharmaceutically acceptable pH adjusting agent is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0012] Examples of suitable pharmaceutically acceptable preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride, benzyl bromide, benzyl alcohol, disodium EDTA, phenylmercuric nitrate, phenylmercuric acetate, ethylmercuric sodium thiosalicylate, thimerosal, acetate and phenylmercuric borate, polymyxin B sulfate, chlorhexidine, methyl and propyl parabens, phenylethyl alcohol, quaternary ammonium chloride, sodium benzoate, sodium propionate, stabilized chlorine oxygen complex (stabilized oxychloro complex), sorbic acid or a mixture thereof. Preferred pharmaceutically acceptable preservatives include disodium EDTA (disodium ethylenediaminetetraacetic acid) and benzalkonium chloride or a mixture thereof. In one embodiment, the pharmaceutically acceptable preservative is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume). Examples of suitable pharmaceutically acceptable buffers include, but are not limited to, sodium chloride, glucose, lactose and phosphate buffered saline (PBS) or any combination thereof. Other suitable pharmaceutically acceptable buffers include, but are not limited to, disodium succinate hexahydrate, borate, citrate, phosphate, acetate, saline, tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), HEPES (N-2-hydroxyethyl piperazine-N1-2-ethane sulfonic acid), sodium phosphate, sodium borate, saline, citrate, carbonate, phosphate, and / or mixtures thereof to achieve the desired osmolarity. In one embodiment, the pharmaceutically acceptable buffer is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0013] Examples of suitable pharmaceutically acceptable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), disodium EDTA and its derivatives, citric acid and its derivatives, nicotinamide and its derivatives, sodium deoxycholate and its derivatives, or mixtures of these chelating agents. In one embodiment, the pharmaceutically acceptable chelating agent is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0014] Examples of suitable pharmaceutically acceptable wetting agents or surfactants include, but are not limited to, amphoteric, nonionic, cationic or anionic molecules. Suitable surfactants include, but are not limited to, polysorbates, sodium lauryl sulfate (sodium lauryl sulfate), dodecyldimethylamine oxide, sodium docusate, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, N,N-dimethyldodecylamine-N-oxide, cetyltrimethylammonium bromide, polyoxyethylene (10) lauryl ether, surfactants (polyoxyethylene fatty alcohol polyoxyethylene ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol), bile salts (e.g., sodium deoxycholate and sodium cholate), polyoxyethylene castor oil, nonylphenol ethoxylate, cyclodextrin, lecithin, methylbenzethonium chloride, carboxylates, sulfonates, petroleum Sulfonates, alkylbenzenesulfonates, naphthalenesulfonates, olefinsulfonates, alkyl sulfates, sulfates, sulfated natural oils and fats, sulfated esters, sulfated alkanolamides, alkylphenols (ethoxylated and sulfated), ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylates, polyethylene glycol esters, sorbitan esters and their ethoxylated derivatives, fatty acid glycol esters, carboxamides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkylamines and polyoxyethylene alicyclic amines, N,N,N,N-tetrasubstituted ethylenediamines, 2-alkyl-1-hydroxyethyl-2-imidazolines, N-coco-3-aminopropionic acid / sodium salt (N-coco 3-aminopropionic acid / sodium salt), N-tallow 3-iminodipropionate disodium salt, N-carboxymethyl n dimethyl n-9octadecenyl ammonium hydroxide, N-cocoamidoethyl-N-hydroxyethylglycine sodium salt, etc., polyoxyethylene, sorbitan monolaurate and stearate, (polyethoxylated castor oil), (ethylene oxide / 12-hydroxystearic acid), polysorbate, tyloxapol, and any combination thereof. Preferred pharmaceutically acceptable surfactants include tyloxapol and (sorbitan monooleate) or a mixture thereof.

[0015] Examples of suitable pharmaceutically acceptable polymers include, but are not limited to, cellulose derivatives (e.g., hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose polymers, hydroxyethyl cellulose, sodium carboxymethyl cellulose, carboxymethylene hydroxyethyl cellulose, and carboxymethyl hydroxyethyl cellulose, or any combination thereof), acrylates (e.g., acrylic acid, acrylamide, and maleic anhydride polymers, copolymers, or mixtures thereof), and mixtures thereof. Polymer blends may also be used. A preferred pharmaceutically acceptable polymer is hydroxyethyl cellulose. In one embodiment, the pharmaceutically acceptable polymer is present in an amount of about 0.01% to about 5.0% (weight / volume), preferably about 0.05% to about 2% (weight / volume), more preferably about 0.1% to about 1.0% (weight / volume), for example, about 0.1%, 0.2%, 0.5%, or 1.0% (weight / volume).

[0016] In one embodiment, the pharmaceutically acceptable wetting agent or surfactant is present in an amount of about 0.01% to about 5.0% (weight / volume), preferably about 0.05% to about 2.0% (weight / volume), more preferably about 0.1% to about 1.0% (weight / volume), for example about 0.1%, 0.2%, 0.5%, 1.0% (weight / volume).

[0017] Examples of suitable pharmaceutically acceptable isotonicity adjusting agents include, but are not limited to, D-mannitol, glucose, glycerol, sodium chloride, potassium chloride, calcium chloride, and magnesium chloride, or any combination thereof. Various nitrates, citrates, acetates, or mixtures thereof may also be used. In one embodiment, the pharmaceutically acceptable isotonicity adjusting agent is present in an amount of about 0.1% to about 5.0% (weight / volume), preferably about 1% to about 3% (weight / volume).

[0018] Furthermore, the present invention provides a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition contains the monoclonal antibody of the present invention.

[0019] Specifically, the pharmaceutical compositions of the present invention can be used to treat cancer or as a cancer vaccine. The cancer may be a cancer type selected from uterine cancer, upper gastrointestinal squamous cell carcinoma, all other upper gastrointestinal cancers, thyroid cancer, sarcoma, urothelial renal carcinoma, all other renal cancers, prostate cancer, pancreatic cancer, ovarian cancer, neuroendocrine cancer, multiple myeloma, melanoma, lymphoma, small cell lung cancer, lung adenocarcinoma, all other lung cancers, leukemia, hepatobiliary cancer, hepatobiliary duct cancer, head and neck cancer, colorectal cancer, cervical cancer, breast cancer, bladder cancer, and anorectal cancer. In some embodiments, the cancer type is selected from anal cancer, bladder cancer, colorectal cancer, esophageal cancer, head and neck cancer, liver / bile duct cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, plasma cell neoplasms, and gastric cancer. In some embodiments, the cancer type is selected from thyroid cancer, melanoma, sarcoma, myeloid neoplasms, kidney cancer, prostate cancer, breast cancer, uterine cancer, ovarian cancer, bladder cancer, urothelial cancer, cervical cancer, anorectal cancer, head and neck cancer, colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, gallbladder cancer, upper gastrointestinal cancer, multiple myeloma, lymphoma, and lung cancer. Preferably, the cancer is kidney cancer.

[0020] The pharmaceutical compositions of the present invention are suitable for administration of the peptides by any acceptable route, such as oral (enteral), intranasal, intraocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal administration. Subcutaneous administration is preferred, intradermal administration is most preferred, and administration via an infusion pump is also possible.

[0021] It will be appreciated that the features of the invention disclosed and described herein can be used not only in combination but also individually within the scope of the intended use of the invention.

[0022] The pharmaceutical composition of the present invention further contains a second therapeutic agent, which may be a chemotherapeutic agent.

[0023] The chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, antitumor antibiotics, cytoskeletal disruptors (taxans), topoisomerase inhibitors, mitotic inhibitors, corticosteroids, kinase inhibitors, nucleotide analogs, and platinum agents.

[0024] In some embodiments, the anticancer agent is a chemotherapeutic agent selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, antitumor antibiotics, taxans, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, kinase inhibitors, nucleotide analogs, and platinum agents.

[0025] Also provided herein are methods for treating a cancer patient, the methods comprising: administering the pharmaceutical composition of the present invention to a subject identified as a cancer patient.

[0026] Beneficial effects

[0027] The present invention prepares and obtains a specific monoclonal antibody against the G250 protein, which can significantly inhibit the proliferation of cancer cells and the expression of the G250 protein in cancer cells. The antibody is prepared into a drug and is expected to be used to treat the corresponding cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Effects of monoclonal antibodies on G250 protein expression in cancer cells DETAILED DESCRIPTION

[0029] Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although 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. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0030] Example 1 Preparation of G250 Monoclonal Antibody

[0031] (I) Preparation of G250 antigen protein

[0032] Recombinant G250 protein was prepared according to conventional methods in the art. Specifically, primers were designed based on the G250 cDNA sequence (Genbank accession number BC014950). The upstream primer was 5'-AGAAGCTTTTCCAATGCACGTACAGCC-3', plus two protective bases and a HindIII restriction site; the downstream primer was 5'-TACTCGAGGCATAATGAGCAGGACAGG-3', plus two protective bases and an XhoI restriction site. The PCR product should be 1520 bp in length (the G250 coding region sequence is 1380 bp and is included therein). The primers were synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. 1.5 μl of cDNA from human renal carcinoma 786-0 cells was used as a template, and a 50 μl reaction system contained 1.0 U of DNA polymerase, 5 μl of PCR buffer, 0.2 mmol / L dNTPs, 2 mmol / L MgSO4, and 0.2 μmol / L primers. Denature at 94°C for 2 minutes, perform 35 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 68°C for 2 minutes, and finally 68°C for 5 minutes. PCR products were purified using a PCR purification kit. The purified PCR product and plasmid pcDNA3.0 were double-digested with HindI and XhoI, respectively. The digested fragments were recovered using a DNA gel recovery kit (Shanghai Sangon Biotechnology Service Co., Ltd.) and ligated with T4 DNA ligase. Competent Escherichia coli DH5α was prepared using the calcium chloride method. 9 μl of the ligation product was transformed and plated on SOB plates containing ampicillin and cultured overnight. PCR amplification and sequencing were performed, and the successfully transformed pcDNA3.0-G250 plasmid was extracted and identified. The recombinant plasmid was then introduced into CHO cells using liposomes as a medium. After selection with G418 at a final concentration of 400 μg / mL, transfection-positive cell clones were obtained and expanded in RPMI1640 medium. A large amount of culture supernatant was collected and protein purified using agarose gel coupled with G250 antibody. Specifically, 6 mg of G250 monoclonal antibody was coupled to cyanogen bromide-activated Sepharose 4B. 100 mL of RPMI1640 medium containing G250 was collected and centrifuged at high speed to remove cell debris. The supernatant was passed through an anion exchange Q column, and the target protein was eluted with 25 mmol / L CaCl2. The eluted fraction was passed through an agarose gel affinity chromatography column coupled with G250 antibody, and the G250 protein was eluted with 0.1 mol / L sodium citrate (pH 3.5). The eluate was quantified for protein and freeze-dried for later use.

[0033] (II) Preparation of G250 protein monoclonal antibody

[0034] BALB / c mice were immunized three times with recombinant G250 protein, mixed with an equal volume of Freund's complete adjuvant, via subcutaneous injection at multiple sites. Each immunization was two weeks apart, with a dose of 120 μg per mouse. Seven days after the third immunization, mice were pulsed with unadjuvanted G250 protein. Four days later, spleen cells from the mice with the highest titer were fused.

[0035] Cell fusion and positive cell screening were performed according to conventional methods. SP2 / 0 cells were fused with spleen cells in the presence of PEG1500. After fusion, DMEM medium was replaced with HAT and HT. After hybridoma cells grew to approximately one-third of the basal area, the supernatant was aspirated and the antibody titer was determined by indirect ELISA. Wells with strong positive results were selected for subcloning and screening, resulting in the identification of the hybridoma cell line G4H9 that secretes anti-G250 protein antibodies.

[0036] Example 2 Identification of G4H9 Monoclonal Antibody Subtypes

[0037] Inject 1×10 6 After incubating the mouse with G4H9 hybridoma cells, a noticeable abdominal bulge was observed. The supernatant of the ascites was collected after centrifugation and titered by indirect ELISA, resulting in a titer of 1:256,000. The ascites monoclonal antibody was crudely extracted by ammonium sulfate precipitation and further purified using Protein G. SDS-PAGE analysis revealed two distinct bands at approximately 25 kD and 56 kD for the G4H9 monoclonal antibody, representing the light and heavy chains, respectively. This confirmed the purity of the monoclonal antibody, and the antibody concentration was adjusted to 2 mg / mL for later use.

[0038] The subtype of the purified antibody was identified using a mouse Ig class / monoclonal antibody subclass detection kit. The results showed that the G4H9 monoclonal antibody of the present invention was of IgG1 type, and the light chain was a κ chain.

[0039] Example 3 Affinity and specificity of G4H9 monoclonal antibody and sequence identification

[0040] Specificity identification: Protein G250, renal cell carcinoma 786-0 cell lysate, human serum albumin, whey protein, mouse serum, and E. coli lysate were diluted 1:10 and coated onto an ELISA plate at a 100 μg / mL concentration. The plate was incubated at 4°C overnight, blocked, and washed. Afterwards, the monoclonal antibody G4H9 of the present invention was added dropwise at a 1:1000 dilution. The plate was incubated at room temperature for 1 hour. The plate was washed three times with PBST, and goat anti-mouse IgG alkaline phosphatase (1:5000) was added dropwise. The plate was incubated at room temperature for 1 hour. After washing as above, PNPP was added and the plate was developed at 37°C for 40 minutes. The results were recorded using an ELISA reader.

[0041] Table 1 Specificity of monoclonal antibodies

[0042] Sample type Identification results (- for negative, + for positive) G250 protein + Renal cell carcinoma 786-0 cell lysate + Human albumin - whey protein - Mouse serum - E. coli lysate -

[0043] From the results in Table 1, it can be seen that the monoclonal antibody of the present invention can specifically react with G250 protein and renal cancer 786-0 cell lysate, but not with other proteins, indicating that the monoclonal antibody of the present invention has good specificity.

[0044] The affinity constant of the human G4H9 monoclonal antibody was determined using a Biacore T200 instrument. Anti-mouse Fc antibodies were covalently coupled to a CM5 biosensor chip via amino group binding. The anti-mouse Fc antibody on the chip captured the monoclonal antibody. Various concentrations of G250 recombinant protein were flowed over the antibody on the chip at a flow rate of 20 μL / min. Human G250 recombinant protein bound to the candidate antibody with an association time of 120 s and a dissociation time of 300 s. Kinetic fitting using BIAevaluation software yielded an affinity constant, indicating a KD of (4.52 ± 0.17) nM for the monoclonal antibody of the present invention.

[0045] RNA was extracted from the hybridoma cell line G4H9 and cDNA was synthesized. The first-strand cDNA product obtained was used as a template for PCR amplification. The light chain gene was amplified using Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and mIg-kR primers, while the heavy chain gene was amplified using Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and mIg-HR primers. The light and heavy chain genes were sent to Shanghai Bioengineering for sequencing. The results showed that the light chain variable region sequence of the G4H9 monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0046] Example 4 Analysis of the effect of monoclonal antibodies on cancer cells

[0047] Human renal cancer 786-0 cell line was purchased from Hongshun Biotechnology. Renal cancer 786-0 cells were cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin, and cultured and passaged in a cell culture incubator at 37°C and 5% CO2.

[0048] Equal amounts of cells in the logarithmic phase were plated in 96-well plates. Low, medium, and high dose experimental groups, as well as negative and positive control groups, were divided into experimental groups treated with 10, 100, and 200 μg / mL monoclonal antibodies, negative control groups with equal amounts of normal saline, and positive control groups with 100 μM c-Myc small molecule inhibitor 10058-F4. Each group was intervened for 72 hours. 20 μL of MTT (5 mg / mL) solution was added to each well and incubated for 4 hours. The optical density (OD) value of each well was detected at 570 nm, and the proliferation inhibition rate of human renal cancer 786-0 cells was calculated as (1- OD570nm of experimental group / OD570nm of control group) × 100%. Five replicate wells were set up for control, and each group was repeated 3 times. The results are shown in Table 2.

[0049] Table 2 Effects of each group on the inhibition rate of cancer cells

[0050] Group Inhibition rate (%) High-dose experimental group 74.58±1.53* Medium dose experimental group 62.47±0.74* Low-dose experimental group 53.59±0.62* Negative control group 0 Positive control group 55.42±0.83*

[0051] As can be seen from the results in Table 2, the monoclonal antibodies of the present invention have a dose-dependent effect in treating cancer. Like the positive control group, the difference relative to the negative control group is highly significant (P < 0.01). Under high concentrations of monoclonal antibodies, the inhibitory effect is even better than that of the positive control group.

[0052] Human renal cancer 786-0 cell line was purchased from Hongshun Biotechnology. Renal cancer 786-0 cells were cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin, and cultured and passaged in a cell culture incubator at 37°C and 5% CO2.

[0053] Equal amounts of cells in the logarithmic phase were taken and seeded in 96-well plates. The medium-dose and high-dose experimental groups and the control group were treated with 100 and 200 μg / mL monoclonal antibodies and equal amounts of normal saline, respectively, and each group was intervened for 48 hours. The cells in each group were lysed, the protein was extracted, and 12% SDS-PAGE gel electrophoresis was performed at the same time, and then transferred to a nitrocellulose membrane (NC membrane). The transferred NC membrane was then blocked with 5% skim milk at room temperature for 2 hours and washed three times with PBST. The prepared monoclonal antibody (1:5000 dilution) was added as the primary antibody, incubated at room temperature for 1 hour, and washed three times with PBST after the incubation was completed. Then, the IRDye700RD-labeled goat anti-mouse IgG secondary antibody was added and incubated at room temperature for 40 minutes. After the incubation was completed, it was washed three times with PBST. The labeled NC membrane was scanned and analyzed using Infrared Imaging Systems. The results are shown in the figure. Figure 1 shown.

[0054] from Figure 1It can be seen that lane A is the G250 protein expression band of the control group, lane B is the G250 protein expression band after treatment with a medium dose of monoclonal antibody, and lane C is the G250 protein expression band after treatment with a high dose of monoclonal antibody. After treatment with the monoclonal antibody of the present invention under high concentration conditions, the expression of G250 protein is basically inhibited, and the medium dose of monoclonal antibody also has a significant inhibitory effect compared with the control group, and the difference is significant (P<0.05).

[0055] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in an appropriate manner in any one or more embodiments or examples. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A monoclonal antibody that specifically inhibits G250 expression, characterized in that The sequence of its light chain variable region is shown in SEQ ID NO: 1, and the sequence of its heavy chain variable region is shown in SEQ ID NO:

2.

2. Use of a monoclonal antibody that specifically inhibits G250 expression in the preparation of a pharmaceutical composition for inhibiting the proliferation of renal cancer 786-0 cells, wherein: 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.

3. The use according to claim 2, characterized in that The pharmaceutical composition further contains a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that Suitable pharmaceutically acceptable carriers include wetting agents or surfactants, pH adjusters, isotonicity adjusters, preservatives, buffers or chelating agents.

5. A pharmaceutical composition for inhibiting G250 expression, characterized in that The invention contains a monoclonal antibody that specifically inhibits G250 expression. The light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO:

2.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further contains a pharmaceutically acceptable carrier.

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