Designing of a Functional Chimeric Protein for Production of Nanobodies Against Human CD20: Molecular Dynamics Simulation and In Vitro Verification

Abstract

One major issue in cancer immunotherapy is the large size of mAbs (150 kDa) which usually causes limitation in tumor penetration. Camel nanobodies (< 15 kDa) could be used as an alternative for this problem. Here in, using molecular modeling we designed a novel recombinant chimeric protein containing the extra membrane loop of human CD20 which was fused to the FC region of camel with proper potential for production of camel derived nanobodies. Molecular dynamics simulation showed that the recombinant chimera can interact specifically with therapeutic antibody Rituximab Fab and the interaction site was the residue 164IYNCEPANPSE174. The recombinant chimeric protein was successfully expressed in HEK293-T cells and evaluated in vitro using western blotting analysis. In agreement with simulation results, western blotting analysis also confirmed the interactions between Rituximab and hCD20. The results of this paper show that protein engineering with the help of molecular modelling tools can be promising and efficient approach in developing new nanobodies for caner immunotherapy.