Engineered pH-dependent recycling antibodies enhance elimination of Staphylococcal enterotoxin B superantigen in mice

Abstract

A new modality in antibody engineering has emerged in which the antigen affinity is designed to be pH dependent (PHD). In particular, combining high affinity binding at neutral pH with low affinity binding at acidic pH leads to a novel antibody that can more effectively neutralize the target antigen while avoiding antibody-mediated antigen accumulation. Here, we studied how the in vivo pharmacokinetics of the superantigen, Staphylococcal enterotoxin B (SEB), is affected by an engineered antibody with pH-dependent binding. PHD anti-SEB antibodies were engineered by introducing mutations into a high affinity anti-SEB antibody, 3E2, by rational design and directed evolution. Three antibody mutants engineered in the study have an affinity at pH 6.0 that is up to 68-fold weaker than the control antibody. The pH dependency of each mutant, measured as the pHdependent affinity ratio (PARratio of affinity at pH 7.4 and pH 6.0), ranged from 6.7-11.5 compared to 1.5 for the control antibody. The antibodies were characterized in mice by measuring their effects on the pharmacodynamics and pharmacokinetics (PK) of SEB after co-administration. All antibodies were effective in neutralizing the toxin and reducing the toxin-induced cytokine production. However, engineered PHD antibodies led to significantly faster elimination of the toxin from the circulation than wild type 3E2. The area under the curve computed from the SEB PK profile correlated well with the PAR value of antibody, indicating the importance of fine tuning the pH dependency of binding. These results suggest that a PHD recycling antibody may be useful to treat intoxication from a bacterial toxin by accelerating its clearance. ARTICLE HISTORY

An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH

“…Previous efforts to engineer pH-sensitive protein–protein interactions guided this work. The naturally pH-dependent interaction between human IgG1 Fc and the neonatal Fc receptor (FcRn) has been engineered to adjust antibody in vivo half-lives ( 26 , 27 , 28 ), while novel pH-dependent binding has been introduced into other binding partners via histidine scanning mutagenesis ( 15 , 29 ). In both cases, pH sensitivity relies on the presence of ionizable histidines in the binding interface, whose pK a of ∼6.0 can be modulated by adjacent residues.…”

Monoclonal Antibody Engineering and Design to Modulate FcRn Activities: A Comprehensive Review

“…Furthermore, it is interesting to note that despite the increased affinity for FcRn at neutral pH, these sweeping variants have no effect on endogenous IgG levels contrarily to the ABDEG developed by Argenx discussed later in Section 3 , which has a much stronger affinity for FcRn at neutral pH [ 71 ]. The sweeping antibody technology has shown promising results in neutralizing the staphylococcal enterotoxin B in a murine model, decreasing the toxin levels, and opening a new way for treating some bacterial infections [ 73 ]. Finally, a recent proof of concept has been published by Bogen et al who have generated a bispecific sweeping antibody targeting two tumor antigens, one CEACAM5, in a pH-dependent manner, and the other CEACAM6 in a pH-independent manner.…”

pH-responsive antibodies for therapeutic applications

“…A recent study showed the application of recycling and sweeping antibodies for the removal of toxins. The activity of conventional and pH-responsive variants of an antibody against Staphylococcal enterotoxin B (SEB) superantigen in a mouse model were compared [ 32 ]. Although all analyzed antibodies neutralized the toxin and reduced cytokine production, the pH-responsive molecules eliminated the toxin significantly faster than the conventional molecules.…”

“…In the case of eculizumab, a small library of 66 variants was created by replacing each position within CDRs by histidine [ 27 ]. In other examples, 20 variants of anti-SEB antibody 3E2 were designed as yeast displayed scFvs, but only nine of them were successfully expressed, suggesting that introduced histidine affected folding [ 32 ]. Some properties of mutants can be predicted with reasonable accuracy by computational methods.…”

“…More detailed analyzes based on surface plasmon resonance or biolayer interferometry were done in parallel with ELISA [ 27 , 29 ]. Some authors also did cell-based assays at different pH at a very early stage of biotherapeutic development [ 13 , 32 ]. Then, selected leading variants were further engineered.…”