Elimination of plasma soluble antigen in cynomolgus monkeys by combining pH-dependent antigen binding and novel Fc engineering

Abstract

A conventional antibody targeting a soluble antigen in circulation typically requires a huge dosage and frequent intravenous administration to neutralize the antigen. This is because antigen degradation is reduced by the formation of antigen–antibody immune complexes, which escape from lysosomal degradation using neonatal Fc receptor (FcRn)-mediated recycling. To address this, we developed an antigen-sweeping antibody that combines pH-dependent antigen binding and Fc engineering to enhance Fc receptor binding. The sweeping antibody actively eliminates the plasma antigens by increasing the cellular uptake of the immune complex and dissociating the antigens in the acidic endosome for degradation. Strong antigen sweeping can reduce the dosage, potentially achieve higher efficacy, and expand the scope of antigen space available for targeting by antibodies. In this study, to further improve the sweeping efficacy, we developed a novel antibody Fc variant by enhancing Fcγ receptor IIb (FcγRIIb) binding and modulating charge characteristics for increased cellular uptake of the immune complex, together with enhancing FcRn binding for efficient salvage of the antigen-free antibodies. Our Fc variant achieved strong antigen sweeping in cynomolgus monkeys with antibody pharmacokinetics comparable to a wild-type human IgG 1 antibody. The positive-charge substitutions enhanced uptake of the immune complex by FcγRIIb-expressing cells in vitro, which was completely inhibited by an anti-FcγRIIb antibody. This suggests that the strong in vivo sweeping efficacy improved by the charge engineering is more likely achieved by FcγRIIb-dependent uptake of the immune complex rather than nonspecific uptake. We expect this novel Fc engineering can maximize the antigen sweeping efficacy even in humans and create novel therapeutic antibodies that meet unmet medical needs for patients.

Impact of structural modifications of IgG antibodies on effector functions

“…This mutation (P238D) in combination with five additional amino acid substitutions E233D/G237D/H268D/P271G/A330R (termed V12) enhanced binding to FcγRIIb even more (~217-fold) ( 178 , 211 , 244 ). Other sets of substitutions described to enhance IgG1 binding to FcγRIIb encompass G236N/H268D and G236N/H268D/A330K, which are abbreviated V2 and V3, respectively ( 195 ). IgG1 with V12-, V2- or V3- bearing Fcs were found to engage FcγRIIb for its rather recently discovered recycling function ( 244 ), and demonstrated efficient soluble target clearance in vivo when combined with antigen-sweeping Fabs ( 195 , 244 , 245 ).…”

“…Other sets of substitutions described to enhance IgG1 binding to FcγRIIb encompass G236N/H268D and G236N/H268D/A330K, which are abbreviated V2 and V3, respectively ( 195 ). IgG1 with V12-, V2- or V3- bearing Fcs were found to engage FcγRIIb for its rather recently discovered recycling function ( 244 ), and demonstrated efficient soluble target clearance in vivo when combined with antigen-sweeping Fabs ( 195 , 244 , 245 ). Strongly reduced binding to FcγRIIa-131R, but also to both FcγRIIb and FcγRIIa-158F, can be achieved by a single mutation D270A.…”

Preclinical <i>in vitro</i> evaluation of immune suppression induced by GYM329, Fc-engineered sweeping antibody

“…GYM329 inhibits the activation of latent myostatin via the Fab domain and sweeps myostatin in the muscle and plasma via the Fc domain (Muramatsu et al, 2021). IC composed of GYM329 and latent myostatin is readily internalized into LSEC and/ or immune cells by FcγRIIb, and then latent myostatin is effectively degraded (Muramatsu et al, 2021;Hori et al, 2022). GYM329 is expected to have better efficacy than conventional anti-myostatin agents.…”