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 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.
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 (inter-spike crosslinking) , instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Synthetic antibodies designed to bivalently bind a single Env trimer (intra-spike crosslinking) were previously shown to exhibit increased neutralization potencies. In initial work, diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA) were considered. Anticipating future experiments to improve synthetic antibodies, we investigate whether linkers with different rigidities could enhance diFab potency by modeling DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and characterizing their neutralization potential . Model predictions suggest that while a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom when both Fabs are bound . In contrast, the strongest neutralization potencies are predicted to require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer , and avidity can be further boosted by incorporating more Fabs into these constructs . These results inform the design of multivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.
A
(inter-spike crosslinking)
R
capable of bivalently binding within single Env trimers
designed to bivalently bind a single Env trimer
R
Here we investigated
Anticipating future experiments to improve synthetic antibodies, we investigate
R
synthesizing
modeling
R
created a model that predicts their neutralization potencies
characterizing their neutralization potential
R
, verified by experimental data, show that although
suggest that while
R
during bivalent binding
when both Fabs are bound
A
are predicted to
A
, and avidity can be further boosted by incorporating more Fabs into these constructs
R
bivalent
multivalent