Recombinant human Fab antibody fragments to HIV-1 Rev and Tat regulatory proteins: Direct selection from a combinatorial phage display library

Abstract

A human Fab phage display library has been produced from peripheral blood lymphocytes of an individual who was asymptomatic after 10 years of infection with human immunodeficiency virus type-1 (HIV-1). The library was panned against the HIV-1 Rev and Tat regulatory proteins and several clones, producing Fab binding to these proteins, were isolated (3 to Rev and 4 to Tat) with binding constants varying from 10 −6M to 10 −8M. DNA sequencing demonstrated two unique anti-Rev Fab clones, but the four anti-Tat Fab comprised only two unique IgG 1 heavy chain Fd fragments, illustrating redundancy of light chains. Peptide mapping of the epitopes recognized by these Fab indicated that three of the anti-Tat Fab were directed to the functional domain between amino acid residues 22–33 of the Tat molecule, and that binding was inhibited by reduction of this cysteine-rich region with dithiothreitol. The anti-Rev Fab were directed to sites adjacent to the Rev basic nucleolar localization sequence (residues 52–64) and to the Rev activation domain (residues 75–88). Binding constants were of a similar order to that of an anti-Rev single-chain Fv fragment (SFv) used successfully for intracellular immunization, and as such intracellular effects with the human anti-Tat and anti-Rev Fab are not precluded. These newly described human antibody fragments to HIV-1 regulatory proteins may be critical moieties for gene therapeutic protocols, to control HIV-1 replication in human cells.