Humanization of the Shark V<sub>NAR</sub> Single Domain Antibody Using CDR Grafting
Yifan Zhang, Yaping Sun, Jessica Hong, Mitchell Ho
Abstract
Abstract 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)