Design and assembly of anti-CD16 ScFv antibody with two different linker peptides

Several studies have focused on the effect of different lengths of linker peptides on the properties of single-chain Fv (ScFv). The expressing level and stability of anti-CD16 ScFv with common linker peptide (Gly(4)Ser)(3) were very poor. Considering 3-D structures of heavy and light chain variable region gene of anti-CD16 antibody, a novel linker peptide PT7 (i.e. Gly(3)SerAla(3)) was designed. As a comparison, the linker PT5 (Gly(4)Ser) was chosen to construct anti-CD16 ScFv. A molecular modeling of anti-CD16 ScFv antibody with the two different linker peptides was designed using computer-assisted modeling techniques and molecular dynamics method. Based on the crystal structure of human IgG1 Fc fragment-Fc gamma receptor III (Fc gamma RIII) complex, putative interactions between anti-CD16 ScFv antibody with two different linker peptide and Fc gamma receptor III fragment were predicted with Docking method. Using molecular graphic techniques, the structure-function relationship of anti-CD16 ScFv antibody with two different linker peptides was analyzed and the combining ability was predicted. The binding activity to Daudi cells by FACS showed that anti-CD16 ScFv antibody with two different linker peptides possessed similar ability and the experimental result was consistent with the theoretical prediction.