Abstract
Recently, nanobody-based therapeutics have emerged as a highly effective strategy for COVID-19 treatment. However, camelid-derived nanobodies often require humanization engineering to reduce immunogenicity in clinical applications while simultaneously preserving their target-binding affinities. Here, we employed a computational and engineering approach to optimize the binding affinities of complementarity-determining region (CDR)-grafted humanized variants of the camelid-derived nanobody Nb2-67, which exhibits potent SARS-CoV-2 neutralization. By grafting the three CDR loops of Nb2-67 onto the humanized scaffold of the approved therapeutic nanobody Caplacizumab and refining the target-binding interface, we generated five nanobody variants with improved computational humanness scores. Three of these variants (Nb491, Nb273, and Nb1052) retained neutralizing activity. To further enhance their potency, we fused these variants to a self-assembling scaffold, generating three multivalent constructs with higher humanness scores. Pseudovirus assays showed that all the trivalent nanobodies exhibited picomolar neutralizing potency comparable to the original trivalent Nb2-67. Our study presents a novel computational and multivalent engineering strategy that effectively restores the antiviral efficacy of humanized CDR-grafted nanobody variants, offering a valuable approach for developing nanobody-based therapeutics against COVID-19 and other diseases.