The antibody access to conserved structures on the envelope glycoprotein (Env) is restricted. However, HIV may not have developed protective mechanisms against antibody fragments of smaller size that are not generated by the immune system. Such small fragments targeting sterically restricted regions on the Env could exhibit neutralization activity superior to larger antibodies as has been previously demonstrated for Fab and scFv X5. We have hypothesized that further decreasing the size of the antibody fragments to single domains could lead to exceptionally potent and broadly cross-reactive neutralizers that could access hidden conserved epitopes not accessible by larger antibodies.

 

 A large (size 2x1010) highly diversified library of human antibody variable domains (domain antibodies, dAbs), was constructed and used for selection of binders to conserved Env structures by panning sequentially against Envs from two different isolates one of them complexed with CD4.

 

 The highest affinity binder, m36, neutralized all tested HIV-1 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. The m36 epitope is CD4-induced and close to the CD4 binding site.

 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 and evasion of immune responses.