Functional Deimmunization of Botulinum Neurotoxin Protease Domain via Computationally Driven Library Design and Ultrahigh-Throughput Screening.
ACS Synth Biol
; 12(1): 153-163, 2023 01 20.
Article
de En
| MEDLINE
| ID: mdl-36623275
ABSTRACT
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.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Anticorps
Type d'étude:
Diagnostic_studies
/
Screening_studies
Limites:
Animals
Langue:
En
Journal:
ACS Synth Biol
Année:
2023
Type de document:
Article
Pays d'affiliation:
États-Unis d'Amérique