Human Domain Antibodies Against Hiv-1 As Exceptionally Potent Cross-Reactive Neutralizers

HIV-1 has evolved to escape neutralization by antibodies generated by the immune system but not by antibody fragments of smaller size that could be able to gain access to the highly guarded conserved structures on the envelope glycoprotein (Env). Such small fragments targeting sterically restricted regions on the Env could exhibit neutralization activity superior to larger antibodies as has been demonstrated for Fab and scFv X5 which are significantly more potent than the full-size antibody (IgG1 X5). We have hypothesized that further decreasing the size of the antibody fragments to the smallest independently folded fragments, the antibody domains (about 10-fold smaller than an IgG, but maintaining high binding affinity) could lead to exceptionally potent and broadly cross-reactive neutralizers. To identify such fragments, we constructed a large (size 2.5 × 1010) highly diversified library of human antibody variable domains (domain antibodies, dAbs), and used it for selection of binders to conserved Env structures by panning sequentially against Envs from two different isolates, one of which was complexed with CD4. The highest affinity binder, m36, neutralized all tested HIV-1 primary isolates from clades A, B, C, and D with an activity on average higher than that of C34, a peptide similar to the fusion inhibitor T20 which is in clinical use, and that of m9 which is an improved derivative of scFv X5. Increasing the size by joining m36 to Fc with hinge regions of varying length or to Ch3 domains diminished its neutralizing activity likely due to the sterically restricted nature of its epitope. M36 is the first representative of a novel class of potent and broadly cross-reactive HIV-1 inhibitors based on human dAbs. It has potential as a candidate therapeutic, and as an agent for exploration of the highly protected conserved Env structures with implications for the design of small molecule inhibitors and elucidation of the mechanisms of virus entry into cells.