Evaluation of site-directed mutagenesis on stability and affinity of Bevacizumab antibody

Bevacizumab is an anti-angiogenesis monoclonal antibody which prevents the interaction of VEGF-A withVEGFR and thereby inhibits the activation of VEGF signaling pathways that promote neovascularization.The type of amino acids at the site of antigen and CDR interaction is important and also the amino aciddiversity is accumulated in the three complementarity determining regions (CDRs). Cysteine, proline andmethionine are avoided for their chemical reactivity or constraints in folding. Isoleucine and leucine areunderrepresented to decrease the surface hydrophobicity. Furthermore, arginine, lysine, serine, threonine,tyrosine and asparagine are preferred as these amino acids occur frequently in the paratope, where they areinvolved in antigen interaction. As a result, these substitutions enable us to optimize antibodies’ affinity andstability. VEGF-A amino acid sequence and Bevacizumab amino acid sequence was also extracted fromhttps://www.rcsb.org with ۶BFT PDB code. This web server was also used for CDR prediction. Initialy weused pymol software to visualize antibody-epitope interaction. Some amino acids were selected as significantresidues in Bevacizumab structure by employing the results of different software. These residues located inone of the three CDR regions. Pymol software can predict amino acid substitution influence on antibodies’affinity by measuring bond length and strength. In order to explore the effect of amino acid substitution onantibodies’ stability mCSM web server also was used. Several characteristics of monoclonal antibody shouldbe developed in order to use them as therapeutic agents, including biding affinity, folding stability andpharmacokinetics. Optimization strategies for monoclonal antibody provide the possibility of modificationand improvement of an antibody molecule. By analysis with mCSM some of mutants of antibody improvedstability, especially conversion of tyrosine ۱۰۲ to glutamic acid. Evaluation of optimization for antibodyaffinity by Pymol software indicated that the conversion of tryptophan ۱۰۸ to arginine improved affinity.