Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments

Abstract

SUMMARY The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes.

Focal accumulation of aromaticity at the CDRH3 loop mitigates 4E10 polyreactivity without altering its HIV neutralization profile

“…To that end, we selected a pyrene-based acetamide compound with four fused aromatic rings that we termed Fus4. This is a highly hydrophobic/aromatic molecule that efficiently interacts with VL membranes ( Rujas et al., 2020 ). Specifically, MD simulations revealed that Fus4 locates within one leaflet of the bilayer, with its center of mass submerged into the upper sections of the acyl chains, close to the interface, and with its longitudinal axis almost parallel to the membrane normal ( Figure 2 A) ( Rujas et al., 2020 ).…”

“…This is a highly hydrophobic/aromatic molecule that efficiently interacts with VL membranes ( Rujas et al., 2020 ). Specifically, MD simulations revealed that Fus4 locates within one leaflet of the bilayer, with its center of mass submerged into the upper sections of the acyl chains, close to the interface, and with its longitudinal axis almost parallel to the membrane normal ( Figure 2 A) ( Rujas et al., 2020 ).

Figure 2 Fus4 engraftment onto the truncated CDRH3 restores antiviral activity of the Fab ΔLoop (A) Depth of insertion and possible orientations adopted in the lipid bilayer by the aromatic compound Fus4.

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“…Hence, the near pan-neutralization breadth of MPER antibodies warrants engineering efforts to improve their potency. In this regard, we have previously shown that site-selective chemical modification of the anti-MPER bnAb 10E8 with aromatic residues dramatically increased its capacity to block viral infection ( Rujas et al., 2020 ). Similarly, here we showed that aromatic grafting in the parental 4E10 resulted in 30-fold improvement of the neutralization potency of this antibody.…”

“…Site-selective chemical conjugation was carried out as previously described ( Rujas et al., 2020 ). In brief, a cysteine-substituted Fab derivative was first generated by site-directed mutagenesis using the KOD-Plus mutagenesis kit (Toyobo, Osaka, Japan).…”

“…Here, we have re-engineered the CDRH3 loop of the human bnAb 4E10 ( 100 WGWL 100c ) to minimize off-target interactions with the lipid membrane while preserving its neutralization potency. Specifically, we have removed the hydrophobic residues at the apex of the CDRH3 and replaced them by a polycyclic aromatic compound (pyrene) previously described to improve antibody binding to native MPER in HIV-1 virions ( Rujas et al., 2020 ). This trimming-and-grafting strategy concentrated the hydrophobicity/aromaticity, otherwise spanned over several residues of the CDRH3, into a single location of the loop resulting in a re-engineered 4E10 antibody variant with favorable functional properties.…”