Quantifying the Entropic and Energetic Effects of Linker Length and Rigidity within Synthetic HIV-1 Antibodies designed to Bind Bivalently to Env Spikes

Abstract

Due to the low density of envelope (Env) spikes on the surface of HIV-1, neutralizing IgG antibodies rarely bind bivalently using both antigen-binding arms (Fabs) to crosslink between spikes, instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Synthetic antibodies capable of bivalently binding within single Env trimers (intra-spike crosslinking) were previously shown to exhibit increased neutralization potencies. In the initial work, diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA) were considered. Here we investigated whether linkers with different rigidities could enhance diFab potency by synthesizing DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and created a model that predicts their neutralization potencies. Model predictions, verified by experimental data, show that although a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom during bivalent binding. In contrast, the strongest neutralization potencies require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer. These results inform the design of bivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.