Functional Deimmunization of Botulinum Neurotoxin Protease Domain via Computationally Driven Library Design and Ultrahigh-Throughput Screening

Botulinum neurotoxin serotype A (BoNT/A) is a widely
used cosmetic
agent that also has diverse therapeutic applications; however, adverse
antidrug immune responses and associated loss of efficacy have been
reported in clinical uses. Here, we describe computational design
and ultrahigh-throughput screening of a massive BoNT/A light-chain
(BoNT/A-LC) library optimized for reduced T cell epitope content and
thereby dampened immunogenicity. We developed a functional assay based
on bacterial co-expression of BoNT/A-LC library members with a Förster
resonance energy transfer (FRET) sensor for BoNT/A-LC enzymatic activity,
and we employed high-speed fluorescence-activated cell sorting (FACS)
to identify numerous computationally designed variants having wild-type-like
enzyme kinetics. Many of these variants exhibited decreased immunogenicity
in humanized HLA transgenic mice and manifested in vivo paralytic activity when incorporated into full-length toxin. One
variant achieved near-wild-type paralytic potency and a 300% reduction
in antidrug antibody response in vivo. Thus, we have
achieved a striking level of BoNT/A-LC functional deimmunization by
combining computational library design and ultrahigh-throughput screening.
This strategy holds promise for deimmunizing other biologics with
complex superstructures and mechanisms of action.