Eliminating Autoreactivity of Variants of Antibody VRC07 by Glycan Masking

Background: Antibody VRC07, a clonal relative of VRC01 identified from next-generation-sequencing analysis of PBMC's from Donor 45, shows improved HIV-1 neutralization breadth and potency compared to VRC01. Efforts are being made to further improve the potency of VRC07, and a number of mutations improve the potency of VRC07. Some of the more potent mutations, however, induce autoreactivity, which in turn negatively impacts effectiveness and safety. Here we investigate the ability of introduced N-linked glycans to shield potency-enhancing mutations from auto-antigen and to eliminate the induced autoreactivity. Methods: For three different autoreactive VRC07 antibody variants, we designed a total of five single glycan antibody mutants by computationally designing N-linked glycosylation sequons proximal to the antibody residues responsible for autoreactivity. The NGlycPred algorithm, a Random Forest-based classifier utilizing structural properties and patterns as attributes, was used to predict the glycan occupancy of the N-linked glycosylation sequons. Three autoreactivity assays, Anti-cardiolipin ELISA, Luminex AtheNA Multi-Lyte ANA test, and HEp-2 ANA test were used to assess the autoreactivity of these glycan mutants.