Construction And Anti-Carcinoma Activity Of Anti-GPC3×CD3 Bispecific Antibody On Hepatoma Carcinoma

Hepatocellular Carcinoma(HCC)is a common malignancies that endanger human health in the world.The mortality of liver cancer in China accounts about 55%.Studies have shown that Glypican-3(GPC3)is highly expressed in some malignant tumors,with a positive rate of more than 60%in hepatocellular carcinoma,and not expressed in normal liver tissue and non-hepatoma tissue.Therefore,GPC3 is a potential target in clinical diagnosis and treatment.GC33 is a high-affinity humanized antibody that specifically targets GPC3.It can kill GPC3-positive hepatocellular carcinoma cells through ADCC function.The safety of GC33 has been demonstrated in Phase I clinical trials,but its results in clinical phase II trials is unsatisfactory,GC33 failed to significantly prolong the patient’s progression-free survival(PFS)and overall survival(OS)compared with the control group.Bispecific antibodies can recruit T cells to the tumor site through the CD3 target,specifically killing tumor cells,and the retention of the Fc functional domain can stimulate immune cells in the tumor microenvironment to exert their anti-tumor effects.In this research,two different forms of anti-GPC3×CD3 bispecific antibodies were constructed by genetic engineering methods.One is the fusion of single-chain antibodies to the C-terminus of the light chain,and another is replacement of the variable region with single-chain antibody in natural antibodies.Then the bispecific antibodies were expressed in CHO cells by transient transfection.The results showed that the design of the single-chain antibody linked to the C-terminus had a higher antibody expression level of 13 mg/L,about 40 times of another design.In addition,which antibody attached to the C-terminus of light chain has a greater impact on the binding activity of the bispecific antibody.In the present study,the bispecific antibody with GC3 3 single chain antibody attaching to the C-terminus of the light chain can retained GPC3 and CD3 binding activity as well.However,when the anti-CD3 single chain antibody attached to the C-terminus of the light chain,it significantly reduced the binding activity to the CD3 target.Finally,the CD3K-SCFV-GC33 bispecific antibody binds well to the GPC3 and CD3 and has a high level of expression.The purity of the CD3K-SCFV-GC33 is also above 95%by SDS-PAGE and HPLC analysis.In vitro biological activity verification of CD3K-SCFV-GC33,T cells could be highly stimulated by the bispecific antibody and produced higher levels of IL-2 and IFN-γ.These cytokines can induce the other immune cells(such as NK cells and macrophages)to exert immune function;CD3K-SCFV-GC3 3 antibody could rapidly kill liver cancer cells in 24 hours,and the killing rate was more than 60%at 48 hours,about three times comparing to GC33 in vitro.In the subcutaneous xenograft model of NOD/SCID mice,fluorescence imaging showed that both CD3K-SCFV-GC33 and GC33 had targeting function in vivo,but CD3K-SCFV-GC33 was more stable and showed almost no attenuation of fluorescence intensity after 72 hours.Furthermore,CD3K-SCFV-GC33 was able to inhibit tumor growth same as GC33.Therefore,this study successfully constructed an anti-GPC3×CD3 bispecific antibody with a high expression level,purity and good anti-tumor function both in vitro and in vivo.We provide a pattern for the design of bispecific antibody,and also provide a new tool for the development of advanced therapeutic drug for liver cancer.