title: Selection and characterization of an HIV-1 gp120-binding affibody ligand.
abstract: To evaluate the possibility of generating novel proteins binding to highly glycosylated viral proteins, affibody ligands were selected by bacteriophage display technology to the HIV-1 envelope glycoprotein gp120 (glycoprotein 120), from a combinatorial protein library based on the 58-amino-acid-residue staphylococcal Protein A domain. The predominant variant from the bacteriophage selection was produced in Escherichia coli and characterized by biosensor analyses. Both univalent and bivalent affibody molecules were shown to bind selectively to the gp120 target molecule in a biosensor analysis. The dissociation equilibrium constants (KD) were determined to be approx. 100 nM for the univalent affibody and 10 nM for the bivalent affibody, confirming the stronger gp120 binding of the bivalent affibody ligand. The affibody constructs were further introduced into the Ad5 (adenovirus type 5) fibre gene, and the recombinant fibres were shown to bind selectively to gp120 in a biosensor analysis and to gp160 transiently expressed in African-green-monkey (Cercopithecus aethiops) kidney cells. Neither the affibody ligand nor the Ad5 fibres showed any virus neutralization activity, suggesting that the affibody bound to a non-neutralizing site on gp120. To investigate the binding site for the affibody ligand on gp120, CD4 (cluster of differentiation 4) and a panel of mAbs (monoclonal antibodies) known to bind to gp120 were allowed to compete with the affibody ligand in a biosensor study. Two mAbs, 670-30D and 697-30D, were found to compete with gp120 for overlapping binding sites. Although neutralization effects were not achieved in this initial investigation, the successful selection of a gp120-binding affibody ligand indicates that future affibody-based strategies might evolve to complement antibody-based efforts for HIV-1 therapy. Strategies for directed selection of affibody ligands binding to neutralizing epitopes and the potential of using adenovirus for gene-therapy-mediated efforts are discussed.
authors.affiliation: School of Biotechnology, AlbaNova University Center, Kungl Tekniska Högskolan, SE-106 91 Stockholm, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Wikman
authors.forename: Eric
authors.initials: E
authors.lastname: Rowcliffe
authors.forename: Mikaela
authors.initials: M
authors.lastname: Friedman
authors.forename: Petra
authors.initials: P
authors.lastname: Henning
authors.forename: Leif
authors.initials: L
authors.lastname: Lindholm
authors.forename: Sigvard
authors.initials: S
authors.lastname: Olofsson
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques; D015704:CD4 Antigens / Q000737:chemistry; D002460:Cell Line; D002522:Chlorocebus aethiops; D019281:Dimerization; D018604:Epitope Mapping; D005455:Fluorescent Antibody Technique; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D008024:Ligands; D019151:Peptide Library; D011993:Recombinant Fusion Proteins