Humanization of the Shark V_NAR Single Domain Antibody Using CDR Grafting

The variable domain of the new antigen receptor (V_NAR ) of shark single domain antibodies is evolutionarily distant from the variable regions (V_H ) of mammalian immunoglobulins, yet it still has complementarity-determining regions (CDRs) that are involved in antigen recognition, therefore making it possible to humanize by grafting these CDRs to the framework of human V_H homologs. Here, we show the V_NAR CDR based on an analysis of currently available V_NAR -antigen structure complexes in the global Protein Data Bank archive of 3D structure data, and describe the detailed protocol to humanize V_NAR by CDR grafting, using B6 (an anti-Pseudomonas exotoxin V_NAR ), the most common type (Type II) of shark V_NAR s, as an example. Ongoing efforts will further optimize the protocol for moving shark V_NAR s to the clinic for treating cancer and other human diseases. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Humanize shark V_NAR sequence by CDR grafting Support Protocol 1: V_NAR structure prediction and comparison Support Protocol 2: Measure binding kinetics of humanized V_NAR using bio-layer interferometry (BLI).