Construction,Expression,in Vitro Activity Detection And Application Of Novel Dimerized Antibody F（ab’）₂ To Different Targets

Background and Objective:With the increasing maturity of monoclonal antibody technology,commercial monoclonal antibody for disease treatment has been studied in depth.However,the gradually revealed deficiencies of full-length antibody limit its clinical application.For example,total antibody has a large molecular weight,it is difficult to penetrate tumor tissue,and Fc end is easy to cause side effects caused by non-specific binding.Therefore,the construction and expressing of active small molecule antibodies may become a new therapeutic direction,such as single chain variable region scfv,antigenbinding fragment Fab,etc.Compared with monovalent small molecule antibodies,the construction of bivalent or polyvalent antibodies can enhance the binding ability to the antigen,increase the molecular weight can prolong the half-life of the drug,slow down the clearance rate in vivo,etc.Common bivalent or polyvalent antibodies such as F(ab’)2,Triabodies,Tetrabodies,etc.Based on the monoclonal antibodies against Anti-CD 19,AntiCD20 and Anti-BCMA previously screened in our laboratory,the combined sequence of cysteine(Cys)and proline(Pro)was introduced into the CH1 terminal of the chimeric human and mouse Fab fragment antibody to form the Fab’.Then the thiol group of cysteine is oxidized to disulfide bond in order to form bivalent or polyvalent antibody,so as to enhance the affinity with the target antigen.Besides,Antibody conjugated drugs(ADC)are an important branch of antibody drugs.Antibodies can reduce toxic side effects due to their targeting,and conjugated toxin drugs can reduce the minimum effective dose.In this paper,this genetically engineered form will also be used to construct dimerized fusion protein,so as to form dimerized antibody conjugated drugs,to detect the binding activity of dimerized antibody conjugated drugs and target cell was significantly enhanced.Methods:Based on the existing prokaryotic expression vector PAYZ-Anti-CD 19Fab,six genetically engineered plasmids(CPP)3、(CPP)2、(CPP)、(CP)3、(CP)2、(CP)were constructed and transformed into Escherichia coli 16C9 by heat shock method.The target proteins were expressed by low phosphorus induced expression medium and purified by Protein G affinity chromatography.Various proteins were identified by SDS-PAGE.The molecular weight and relative content of major components of Anti-CD 19Fab-(CP)3 were identified by mass spectrometry.The binding activity of different modified proteins to target antigen was detected by flow cytometry,and the optimal structure of(CP)3 was determined.In order to verify the generality of this modification method,the CH1 terminal of Anti-CD20 and Anti-BCMA Fab fragment antibody was constructed with(CP)3 structure in the same way as the existing modification technology(CPP)3 structure.The relative content of dimer was determined by electrophoresis and the binding activity to target cells was determined by flow cytometry.In order to further study whether the modified form is suitable for antibody conjugators,a prokaryotic expression plasmid of Anti-CD 19Fab conjugated cobase protein(LDP)was constructed.After the fusion protein was expressed and purified,the relative content of the dimer was determined by electrophoresis and the binding activity of the fusion protein to the target antigen was detected by flow cytometry.Results:Different modified forms of targeting CD 19 proteins were successfully constructed and expressed.The results of gel electrophoresis and flow cytometry showed that the introduction of(CP)3 structure was better for the construction of dimerized antibody and had higher binding activity to target cells than Fab monomer protein and(CPP)3 structure introduced by the existing modification technology.Dimerized antibodies targeting CD20 and BCMA were successfully constructed and expressed.Electrophoresis results showed that the introduced(CP)3 structure could form a higher proportion of dimerers than the introduced(CPP)3 structure,and flow cytometry showed stronger binding activity to target cells.CD 19-targeting Fab antibody conjugations with LDP were successfully constructed and expressed,and the results showed that the introduction of(CP)3 structure could form a higher proportion of dimerized fusion protein,with stronger binding activity to target cells.Conclusions:The Anti-CD 19Fab,Anti-CD20Fab,and Anti-BCMA Fab antibodies formed a higher proportion of dimers at the CH1 terminal by introducing(CP)3 than(CPP)3.Flow cytometry showed that(CP)3 antibodies had higher binding activity to target cells.This conclusion is also applicable for antibody conjugations targeting CD 19.Compared with monovalent antibody Fab,the higher the proportion of dimers formed by the genetically engineered antibody,the stronger the affinity for the target antigen.