Binding Activity Difference of Anti-CD20 scFv-Fc Fusion Protein Derived from Variable Domain Exchange

Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 single-chain Fv fragment (scFv) to the human IgG1 CH2 (i.e., C(gamma)2) and CH3 (i.e., C(gamma)3) domains with the human IgG1 hinge (i.e. H-gamma). Given the relationship between structure and function of protein, the 3-D structures of the two engineered antibody fragments were modeled using computer-aided homology modeling method. Furthermore, the relationship between 3-D conformation and their binding activity was evaluated theoretically. Due to the change of active pocket formed by CDRs, the HL23 (V-H-Linker-V-L-H-gamma-C(gamma)2- C(gamma)3) remained its activity because of its preserved conformation, while the binding activity of the LH23 (V-L-Linker-V-H-H-gamma-C(gamma)2-C(gamma)3) was impaired severely. Experimental studies by flow cytometry and fluorescence microscopy showed that HL23 possessed significantly superior binding activity to CD20-expressing target cells than LH23. That is to say, the order of variable regions could influence the binding activity of the fusion protein to CD20(+) cell lines, which was in accordance with the theoretical results. The study highlights the potential relationship between the antibody binding activity and their 3-D conformation, which appears to be worthwhile in providing direction for future antibody design of recombinant antibody.