CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics

Abstract

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins—high physical stability, specificity and low production costs—with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…We chose CD4 as target aiming to generate a tool that specifically modifies CD4-positive immune cells that not only play a central role in protecting against infection and cancer, but are also the target of human immunodeficiency virus 15 . To specifically target this cell population, we decided to use DARPin 55_2, which performed best from a series of previously described human CD4-specific DARPins 16 . Like Her2-AAV, also CD4-AAV displaying the CD4-specific DARPin on its capsid surface readily discriminated between CD4-positive and CD4-negative lymphocyte cell lines ( Supplementary Figs 7 and 8).…”

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

“…Antibodies and the N2C (15-kDa) and N3C (18-kDa) types of DARPins employed in the present study cover binding footprints of comparable sizes. DARPins recognize the target by using their surface of ␣-helices and their row of ␤-hairpins, resulting in a groove-like binding surface, and thus, they prefer to bind to the surface of a globular protein domain, or at least structurally welldefined loops (16)(17)(18)(66)(67)(68). Antibodies can do this, too, but they can also bind to an unstructured terminal peptide or long loop, which adapts to a pocket or groove between the antibody variable domains.…”

“…Early rounds of selection (rounds 1 to 3) were kept at low stringency with typically 3 to 5 short washing steps (5 min), while the intensity of washing was increased during subsequent rounds of selection (typically 8 to 10 washing steps of 10 min each). The most important adaptation to the standard ribosome display protocol previously described (18,36,37) was the omission of heparin in the panning buffer. Heparin is usually added during panning to prevent nonspecific RNA binding to the protein or surfaces.…”

“…Here, we employed the designed ankyrin repeat protein (DARPin) technology (15)(16)(17)(18)(19) to derive HIV envelope-specific entry inhibitors. We have previously utilized the technology successfully to develop DARPins specific for CD4, which proved to be highly potent in inhibiting entry of divergent HIV strains (18).…”

“…We have previously utilized the technology successfully to develop DARPins specific for CD4, which proved to be highly potent in inhibiting entry of divergent HIV strains (18). DARPins, like the natural ankyrin repeat proteins from which they are derived (20), have exceptional binding properties and recognize particularly well targets with a defined rigid surface, such as folded proteins.…”