Recombinant human Fab antibody fragments to HIV-1 Rev and Tat regulatory proteins: direct selection from a combinatorial phage display library.
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(-6)M to 10(-8)M. DNA sequencing demonstrated two unique anti-Rev Fab clones, but the four anti-Tat Fab comprised only two unique IgG1 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.
Pilkington
G R
GR
Intracel Corporation, Cambridge, MA 02139, USA.
Duan
L
L
Zhu
M
M
Keil
W
W
Pomerantz
R J
RJ
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D015723:Gene Library; D015694:Gene Products, rev / Q000276:immunology*; D015696:Gene Products, tat / Q000276:immunology*; D015316:Genetic Therapy; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D013329:Structure-Activity Relationship; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015696:Gene Products, tat; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus