Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Abstract

Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

“…DARPins that have already been developed against several other targets, such as lactococcal phage TP901-1 (32), HIV gp120 (33), and human IgE (34), are potentially applicable in LAB-mediated therapy. We genetically fused DARPins I_07 and I_19 to the C terminus of the AcmA protein and to the Usp45 secretion signal and expressed the resulting fusion proteins in L. lactis by using nisin-controlled expression (NICE).…”

Human Immunodeficiency Virus Immune Cell Receptors, Coreceptors, and Cofactors: Implications for Prevention and Treatment

“…48,49 A third class of candidate anti-HIV drugs currently being examined utilizes CD4-specific designed ankyrin repeat protein (DARPin) technology, which promises a potent and stable inhibitory strategy with low production costs and high specificity to CD4 that can outcompete viral gp120 binding. 50,51 Cyclotriazadisulfonamides (CADAs) are yet another type of small molecule that target CD4 rather than envelope (Env) proteins, and work by knocking down CD4 in an mRNA-independent manner 52 ; however, long-term adverse effects on the immune system are of concern. Lignosulfonic acid, a low-cost lignin-derived polyanionic macromolecule, has also shown potency in inhibiting HIV entry by interfering with gp120 binding to the cell surface of CD4 + T cells, although the mechanism of action is not yet fully understood.…”

Biobetters

“…From such combinatorial libraries DARPins with high affi nities have been selected, normally via ribosome display, against a variety of proteins, including several medically relevant targets: for example, HIV gp120 (Mann et al 2013 ;Schweizer et al 2008 ), EpCAM (Stefan et al 2011 ), Alzheimer amyloid-β peptide (Aβ) (Hanenberg et al 2014 ) and, as will be described in greater detail below, VEGF-A (Stahl et al 2013 ). Since the advantageous biophysical properties of the parental ankyrin scaffold such as high-level expression, solubility, and stability are often retained in these DARPins, they are also well suited for the generation of multispecifi c constructs (Molecular Partners, http://www.molecularpartners.com ).…”