Studies On Construction Of Anti-B7-H4-scFv-C_H3 Antibody And Its Functional Analysis

The aim of this study was to modify the B7-H4 single chain antibody by human anti-B7-H4 single chain antibody with human IgG1 CH3 to obtain a fusion protein that inhibits tumor growth. Our previous studies have constructed the anti-B7-H4 single chain antibody library and a murine specific anti-B7-H4 specific single chain antibody was screened by ribosome display technology. However, absence of Fc fragments in this single chain antibody resulted in shortness of half-life, easily clearance from blood and increased complexity in identification. Therefore, we connected the anti-B7-H4-scFv with human IgG1 CH3 gene to humanize the anti-B7-H4 antibody. Results were as follows:1 Generation of humanized anti-B7-H4-scFv-CH3 and construction of expression vector of pET43.1a/scFv-CH3The total RNA was insolate from mononuclear cells of human peripheral blood.The IgG1 CH3 gene was amplified by reverse RT-PCR. To construct recombinant gene,the anti-B7-H4-scFv gene was ligated with human IgG1 CH3 gene by SOE-PCR.The results showed that the correct anti-B7-H4-CH3 gene sequence was obtained as evidenced by agarose gel electrophoresis and gene sequencing.2 Expression, purification and identification of the anti-B7-H4-scFv-CH3 proteinThe recombinant fusion gene was inserted into vector pET43.1a ,then transferred to E. coli BL21 (DE3) and induced by IPTG to express protein. The protein was purified by Ni-NTA and further identified by Western blot. And we identified its antigen specificity and affinity binding by ELISA and Western blot. The results showed that we successfully built the anti-B7-H4-scFv expression vector and obtained soluble protein that could combine for hunman B7-H4 high affinity and specificity.3 Biological function detection of anti-B7-H4-scFv-CH3 proteinTo detect the biological functions of the anti-B7-H4-scFv-CH3 protein,we generated a Lewis lung cancer subcutaneous transplant model by using C57BL/6 mice.The tumor-bearing mice were randomly divided into two groups. In the control group,mice were injected with saline, and mice in the experimental group were treated with anti-B7-H4-scFv-CH3 protein. Tumor volume and body weight were measured in each group after injection. Finally the mouse the were killed and the tumor were stripped,pathological histology of tumor was observed with HE staining.The results showed that the tumor volume and weight of the control group were significantly higher than that of the experimental group ,the density of tumor cells in the experimental group was lower than that in the control group and tumor necrosis can be seen in the experimental group ,the antibody could inhibit the tumor growth of Lewis tumor bearing mice and showed good biological activity. Anti-B7-H4-scFv-CH3 fusion protein provides the basis for the study of targeted therapy of cancer.