| Hepatocellular carcinoma（HCC）is the fifth most common tumor in the world and the third leading cause of cancer-related death worldwide.Its morbidity/mortality rate is close to 1.0,and nearly 600,000 deaths occur each year.At present,significant progress has been made in the treatment of HCC and drug development.However,because of its high degree of malignancy and relapse,the prognosis of HCC patients is still poor,and the five-year survival rate is only 30%to 40%.In addition to traditional methods such as radiotherapy,chemotherapy,and surgical treatment,biological therapy has gradually highlighted its advantages in the field of malignant tumor treatment.Biological therapy is currently a new type of cancer treatment,including molecular gene therapy,immunotherapy,cell therapy,and targeted therapy.Among them,molecular targeted therapy utilizes a specific structural molecule possessed by a tumor tissue or a cell as a target,and uses a type of therapy such as an antibody or a ligand that specifically binds to a target molecule to achieve direct treatment or targeted therapy.As one of the members of the molecular-targeted drug family,based on specific antibodies and Antibody-drug conjugates（ADCs）,it reduces the release of small molecule toxic drugs due to its specific binding to tumor targeting markers.The advantages of non-specific killing effect on surrounding normal tissues have become a research hotspot in targeted therapy of liver cancer.Antibody drug conjugates ADCs include specific antibodies,linkers,and small molecule toxic drugs that kill tumor cells by specifically binding to tumor tissue surface antigens to release small molecule toxic drugs.As the primary condition in ADCs technology,the selection of highly expressed antigens in tumor tissues is the key link.c-Met is a product encoded by the oncogene Met and has tyrosine kinase activity as a member of the growth factor receptor family.c-Met has low or no expression in normal tissues,but it has obvious up-regulated expression in many tumor tissues,such as liver cancer,lung cancer,and breast cancer.Studies have demonstrated that c-Met is involved in several key steps such as the occurrence,development,metastasis,and abnormal vascular proliferation of hepatocellular carcinoma.Therefore,c-Met has also become a potential target for targeted antibody therapy.Platinum drugs have become an important component of cancer chemotherapy drugs since its discovery.As a third-generation platinum drug,oxaliplatin has shown its broad spectrum of in vitro cytotoxicity and in vivo anti-tumor activity in various tumors.The treatment plays a significant advantage,but due to its obvious neurotoxicity and side effects of gastrointestinal toxicity,it limits its further application in the treatment of clinical tumors.The aim of this study was to couple anti-c-Met antibodies with oxaliplatin by chemical coupling.The targeting of the combined antibodies and the cytotoxicity of oxaliplatin exert a specific effect on the killing of tumor cells.While maintaining the dose of oxaliplatin at the same time to improve the effect of chemotherapy,and reduce the toxic side effects of oxaliplatin on normal tissues,and further lay the foundation for the exploration of c-Met as a target for molecular targeted therapy of hepatocellular carcinoma.Objective:1.The human full-molecule anti-c-Met antibody was constructed,expressed and purified by a full human immunotype Fab phage antibody library,and its immunological activity was assayed and its affinity detected.2.Coupling of human anti-c-Met antibody with oxaliplatin and analysis of conjugate properties by chemical coupling3.To observe the cytotoxicity of human anti-c-Met IgG-oxaliplatin on hepatoma cells at the cellular level;4.Analysis of in vivo anti-tumor efficacy and toxicity of human anti-c-Met IgG-oxaliplatin in vivo in nude mice bearing hepatocellular carcinoma.Methods:1.Amplify heavy light chain variable region of human anti-c-Met Fab antibody,construct human eukaryotic anti-c-Met antibody eukaryotic expression vector by Infusion PCR,and use E.coli DH5αto amplify and send detection sequence.2.The eukaryotic recombinant expression vector was transfected into 293Freestyle（293F）cells.After 6 days,cell culture supernatants were collected and purified by Protein A affinity column in AKTA protein purifier.3.To verify the immunological properties of human whole-body antibodies by immunoprecipitation,mass spectrometry analysis and BLItz affinity detection.4.To construct cMet-shocked cell line shMet-HepG2,and detect the expression of c-Met in shMet-HepG2 cell line by RT-PCR,qRT-PCR and Western Blot.5.Coupling of human whole molecule anti-c-Met antibody and oxaliplatin by means of chemical synthesis of aminocarboxyl coupling to obtain the coupled product anti-c-Met IgG-OXA,and HPLC and GB/T 9723-88 Flame Atomic Absorption Spectrometry to identify the drug properties of the coupled product.The stability of the conjugated drug was tested under different pH conditions,and the binding ability of c-Met IgG-OXA to c-Met at the protein and cell level was analyzed by ELISA and FACS.6.The internalization capacity of anti-c-Met IgG-OXA in cells with different c-Met expression levels was detected by FACS and cell immunofluorescence assays.7.Anti-c-Met IgG-OXA and free oxaliplatin were applied to c-Met high expression hepatoma cells HepG2 and c-Met knockout hepatoma cells shMet-HepG2,through CCK8,cell scratch healing experiment,Transwell Invasion assays,Annexin V/PE apoptosis assays,and cell cycle assays examined the cytotoxic effects of anti-c-Met IgG-OXA and free oxaliplatin on cells.At the same time,they were compared and analyzed to observe the difference of cytotoxic effects of oxaliplatin on different c-Met expression levels in HepG2 cells.8.HepG2-luc cell line was used to construct a HepG2-luc tumor-bearing nude mouse model,and treated with different concentrations of anti-c-Met IgG-OXA and free oxaliplatin,through in vivo imaging,tumor volume weight monitoring and tumor tissue pathological changes to compare the anti-tumor effect of different drugs.At the same time,comparison of survival,body weight changes,heart,lung,spleen,kidney,and jejunum lesions,and diarrhea after drug administration were performed to verify the toxic side effects of the two drugs on nude mice.Results:1.A human-derived anti-c-Met Fab antibody was screened by PCR using the full human immunotype Fab antibody library.The length of the VH gene and Vκgene was 348 bp and 324 bp,respectively.The human recombinant anti c-Met heavy light chain eukaryotic recombinant expression vector containing the human heavy and light chain constant region and variable region was successfully constructed by Infusion PCR.The recombinant E.coli DH5αwas transformed into a large number of recombinant expression vectors and sequenced correctly.2.The human anti-c-Met IgG was synthesised by transfected the recombinant expression vector into 293F cells,and the optimized productivity was about4mg/100ml.After purification using AKTA protein purification system,the prepared antibodies were shown at 55kD and 25kD by SDS-PAGE.The bands were obvious,indicating that the purity of the antibodies obtained after purification was high.3.The antigen-antibody complexes were obtained by immunoprecipitation assay.Western blot results showed that using the anti-c-Met antibody as a primary antibody,the antigen-antibody complexes had obvious bands at 170kD and 150kD,which demonstrated the antibodies prepared in this experiment.Can specifically bind to c-Met protein.Using BLltz to detect the affinity of the antibody,the KD value was5.56×10-10.It was verified at the kinetic level that the anti-c-Met IgG could be stable with the c-Met protein with higher affinity.4.Coupling of human anti-c-Met IgG antibody molecule with oxaliplatin using aminocarboxyl chemical bond coupling,separation and purification to obtain conjugate anti-c-Met IgG-OXA,HPLC and GB/T 9723-88 Flame atomic absorption spectrometry and other methods confirmed the successful coupling of antibody and chemotherapeutic drug.The ratio of drug to antibody DAR was 4.35:1.In vitro release experiments showed that the anti-c-Met IgG-OXA was stable under neutral conditions of pH 7.2,with a cumulative release rate of approximately 14.6%at 256 h;at pH 4.0 acidic conditions,anti-c-Met IgG-OXA was able to The free oxaliplatin was released quickly.The cumulative release rate at 72 h was 86.3%,much higher than the cumulative release rate of 8.1%at 72 h after pH 7.2.5.Construction of c-Met knockout hepatoma cell line shMet-HepG2,RT-PCR,qRT-PCR and Western Blot results showed that c-Met in the shMet-HepG2 cell line was significantly down-regulated in both nucleic acid and protein levels.6.ELISA showed that anti-c-Met IgG-OXA can specifically bind to recombinant c-Met protein with a significant dose-effect relationship;FACS results showed that anti-c-Met IgG-OXA can express high-expression hepatoma cells with c-Met HepG2binds,but has no obvious binding ability with c-Met knockout hepatoma cells shMet HepG2 cells.This shows that anti-c-Met IgG-OXA has better targeting ability in protein and cell levels.7.FACS analysis showed that the anti-c-Met IgG-OXA could specifically bind to and integrate into c-Met-expressing hepatoma HepG2 cells,and it has obvious time-dependence,while c-Met knockout hepatocellular carcinoma The cells shMet-HepG2 cells had no obvious binding and drug internalization.It was confirmed by fluorescence invert microscope that the anti-c-Met IgG-OXA had the same characteristics as described above,and could specifically bind to the hepatoma cells expressing c-Met and internalize into the tumor cells.8.The CCK-8 assay results showed that compared with the c-Met knockout cell line shMet-HepG2 cells,the inhibition rate of anti-c-Met IgG-OXA on the proliferation of HepG2 cells with high expression of c-Met protein was significantly higher than that of the c-Met knockout cell line shMet-HepG2.The cell line shMet-HepG2 was knocked out by c-Met and the difference was statistically significant（P<0.05）.The results of cell scratch healing experiments showed that the migration of HepG2 cells after treatment with conjugated drugs was significantly lower than that of shMet-HepG2 cells（P<0.05）;the results of Transwell invasion assay showed high expression of c-Met protein.HepG2 cells in the control group had significantly more membrane cells than the experimental group,and the difference was statistically significant（P<0.05）.The invasive ability of the c-Met knockout cell line shMet-HepG2 cells treated with conjugated drugs was similar to that of the control group.The difference was not statistically significant（P>0.05）.9.The results of Annexin V/PE apoptosis assay showed that the apoptosis rate of c-Met cell line HepG2 cells treated with anti-c-Met IgG-OXA was significantly higher than that of c-Met knockout cell line shMet-HepG2 cells.The difference was statistically significant（P<0.05）.Cell cycle experiments confirmed that compared with shMet HepG2,anti-c-Met IgG-OXA could significantly promote the S phase arrest of c-Met high-expression HepG2 cells,and the difference was statistically significant（P>0.05）.10.HepG2-luc tumor bearing nude mice were treated with different doses of anti-c-Met IgG-OXA and free oxaliplatin,respectively.It was observed that both anti-c-Met IgG-OXA and free oxaliplatin had significant inhibition on tumor growth.Anti-c-Met IgG-OXA（containing oxaliplatin 5mg/kg）and free oxaliplatin（10mg/kg）have similar mean tumor inhibition rate,but the anti-tumor effect was significantly lower than anti c-Met IgG-OXA（containing oxaliplatin 10 mg/kg）treatment group.Compared with the low-dose oxaliplatin low-dose group（1 mg/kg）,the anti-c-Met IgG-OXA low-dose group（containing oxaliplatin 1 mg/kg）had a significant anti-tumor effect.11.The survival of tumor-bearing nude mice at different doses of anti-c-Met IgG-OXA and free oxaliplatin was recorded within 80 days.We observed that anti c-Met IgG-OXA can significantly improve the survival rate of tumor-bearing nude mice.Body weight of nude mice with different treatment confirmed that anti-c-Met IgG-OXA significantly reduced the weight loss caused by the administration of free oxaliplatin alone.Comparison of diarrhea,spleen size,and H&E staining of the jejunum,heart,lung,kidney,and spleen tissues of nude mice after administration of tumor-bearing nude mice showed that anti-c-Met IgG-OXA significantly reduced the damage to normal tissues and organs caused by free oxaliplatin.Conclusion:Our group successfully prepared fully human anti-c-Met IgG and successfully coupled anti-c-Met IgG with oxaliplatin by chemical coupling of aminocarboxyl groups to prepare anti-c-Met IgG-OXA.Anti-c-Met IgG-OXA has an obvious targeted killing effect in vitro,and can also specifically target killer tumor cells in vivo experiments.While increasing the anti-tumor effect of oxaliplatin,it can significantly reduce Orssa.Lipoplatin has toxic side effects on normal tissue organs.The results of the study are rich in the treatment of hepatocellular carcinoma,and provide the basis for clinical application of A-anti-c-Met IgG-OXA. |
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