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.

Generation of affibody molecules specific for HPV16 E7 recognition

“…Since the first HER2 binding affibody molecule discovered [23, 24], a serial of affibody molecules targeting different proteins including EGFR, IGF-1R and HIV-1-gp120 have been reported [20, 25, 26]. The affibody molecules are small size proteins.…”

A 2-Helix Small Protein Labeled with 68Ga for PET Imaging of HER2 Expression

“…1A). Thus, Affibody binders with high affinity and specificity against a wide variety of desired targets, such as human epidermal growth factor receptor type 2 ( HER2 ) (3,4), epidermal growth factor receptor (5,6), human serum albumin (7), interleukin-2 receptor alpha/CD25 (8), transcription factor c-Jun (9), Alzheimer amyloid β-peptides (10), and HIV-1 gp120 (11), have been quickly identified and selected using phage-display library technology and affinity maturation.…”

A Gradient of Sitewise Diversity Promotes Evolutionary Fitness for Binder Discovery in a Three-Helix Bundle Protein Scaffold

“…Numerous sites were constrained in their amino acid diversity (details in Supporting Information). Amino acid diversity at likely hot spot affibody positions was guided by amino acid prevalence in natural antibody interfaces (Abysis database; CDR-H3 diversity, Kabat sites 95–102) and previously evolved affibodies found in literature 5–7,9,11–15,18,55–78 as well as those generated in-house with the first generation library. The prevalence of each amino acid, except Gly and Arg, were calculated based on equally weighted CDR-H3 diversity via Abysis and previously evolved binder data, yielding the codon design which we call B* for the rest of the manuscript (Figure S1).…”

“…345 unique evolved sequences were used from numerous references. 5–7,9,11–15,18,55–59,61–78,96 (B) Computed destabilization upon mutation in the context of diverse paratopes. The median change in folding free energy (ΔΔG f ) upon mutation to the indicated amino acid at the indicated site was computed using FoldX.…”