Humanization of Camelid Single-Domain Antibodies

Humanization of therapeutic antibodies derived from animal immunizations is often required to minimize immunogenicity risks in humans, which can cause potentially harmful and serious side effects and reduce antibody efficacy. Humanization is typically applied to conventional monoclonal antibodies derived in rodents as well as single-domain antibodies isolated from camelids and sharks (VHHs and VNARs). A streamlined protocol is described here for sequence humanization of camelid VHHs, which represent a promising biotherapeutic format with many desirable attributes. From human framework selection and complementarity-determining region grafting strategies to empirical scoring for prioritization of back-mutations, step-by-step instructions, and templates are provided along with bioinformatics resources to assist each step of the humanization process. Alternative approaches, warnings, and caveats are also presented.