Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity

Abstract

A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

A Novel gp41-Binding Adnectin with Potent Anti-HIV Activity Is Highly Synergistic when Linked to a CD4-Binding Adnectin

“…1). Using techniques described previously (14), the library was translated in vitro, and each unique adnectin protein was coupled with the mRNA encoding it via a puromycin linker ( Fig. 2A).…”

“…These individual adnectins were expressed in bacteria with an attached 6ϫHis tag and isolated with a high-throughput purification system ( Fig. 2C) (14). A simple enzyme-linked immunosorbent assay (ELISA)-based binding assay was devised, where the same IZN17 and IZIZ targets used during selection were bound to a neutravidin-coated plate, followed by addition of the adnectins at concentrations between 50 nM and 1 M. After washing, readout of bound adnectins was accomplished with an anti-6ϫHis antibody coupled with horseradish peroxidase (HRP).…”

“…Inhibition of cell-cell fusion is detected as a decrease in luciferase activity. In this assay, 23 adnectins were capable of inhibiting fusion by at least 50% at concentrations of less than 1 M. These adnectins were further assessed in a replicating virus assay, using a modified NL 4-3 virus (RepRluc) containing a luciferase reporter gene (14,28). Of these, the 2428_G03 adnectin was chosen, as it possessed good and consistent inhibitory activity in both assays ( Table 2).…”

“…Adnectins are small proteins (10 to 12 kDa) derived from the 10th type III fibronectin domain of human fibronectin (10)(11)(12)(13). Selection from libraries based on this scaffold using mRNA display has previously generated adnectins that bind to CD4 and inhibit infection of CD4-expressing target cells by HIV-1 (14). We hypothesized that, due to their small size, adnectins would be able to access the N17 site of gp41 better than MAbs, thereby inhibiting functional six-helix-bundle formation and membrane fusion with high potency.…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…An early example of this development is an Adnectin monobody which was evolved to target CD4, a target receptor for HIV entrance into cells, and thus sterically block HIV internalisation via CD4. The major advantage was that, in contrast to an antibody, the monobody could block the HIV interaction epitope without blocking the natural function of the CD4 receptor [102]. In order to improve upon this mechanism, the anti-CD4 monobody was then fused to an anti-gp41 monobody which binds a surface protein on the HIV capsid ( Figure 3D).…”