Enhancing affinity of neutralizing SARS-CoV-2 nanobody through facile structure-guided mutations in CDRs

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for development of the next generation therapeutics. This study delves into the refinement and engineering of CDRs through in silico affinity maturation followed by binding validation using ITC and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of RBD of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Subsequent biophysical studies using ITC provided insights into binding affinity and key thermodynamic parameters. Consistent with in silico findings, seven single mutations resulted in enhanced affinity. The mutants were further tested for neutralization activity against SARS-CoV-2 pseudovirus. L106T, L106Q, S107R, and S107Q generated mutants were more effective in virus-neutralizing with IC50 values of [~]0.03 {micro}M, [~]0.13 {micro}M, [~]0.14 {micro}M, and [~]0.14 {micro}M, respectively as compared to the native nanobody (IC50 [~]0.77 {micro}M). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.