Disparities between Antibody Occupancy, Orientation, and Cytotoxicity in Immunotherapy.

We report fusion proteins designed to bind spatially distinct epitopes on the extracellular portion of HER2, a breast cancer biomarker and established therapeutic target, and recruit IgG (either anti-His6 or serum IgG) to the cell surface. When the proteins were incubated with anti-His6 antibody and various concentrations of a single HER2-binding protein His6 fusion, we observed interference and a decrease in antibody recruitment at HER2-binding protein concentrations exceeding ∼30 nM. In contrast, concomitant treatment with two or three distinct HER2-binding protein His6 fusions, and anti-His6 , results in increased antibody recruitment, even at relatively high HER2-binding protein concentration. In some instances, increased antibody recruitment leads to increased antibody-dependent cellular cytotoxicity (ADCC) activity. While a fusion protein consisting of a HER2-binding nanobody and Sac7d, a protein evolved to recognize the Fc domain of IgG, binds IgG from serum, antibody recruitment does not lead to ADCC activity. Rationales for these disparities are provided. Collectively, our findings have implications for the design of efficacious targeted immunotherapeutic biologics, and ensembles thereof.

Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1.

Drugs that block HIV-1 entry are relatively limited. Enfuvirtide is a 36-residue synthetic peptide that targets gp41 and blocks viral fusion. However, Enfuvirtide-resistant HIV has been reported, and this peptide drug requires daily injection. Previously, we have reported helix-grafted display proteins, consisting of HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these biologics inhibit HIV-1 entry with relatively modest and varied potency (IC50 = 190 nM to >1 µM). Here, we report that gp41 C-peptide helix-grafted Sac7d (Sac7d-Cpep) potently suppresses HIV-1 entry in a live virus assay (IC50 = 1.9-12.4 nM). Yeast display sequence optimization of solvent exposed helix residues led to new biologics with improved expression in E. coli (a common biosimilar expression host), with no appreciable change in entry inhibition. Evolved proteins inhibit the entry of a clinically relevant mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide resistant. Fusion proteins designed for serum stability also potently suppress HIV-1 entry. Collectively, we report several evolved biologics that are functional against an Enfuvirtide-resistant strain and are designed for serum stability.

Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins.

Many therapeutically-relevant protein-protein interactions (PPIs) have been reported that feature a helix and helix-binding cleft at the interface. Given this, different approaches to disrupting such PPIs have been developed. While short peptides (<15 amino acids) typically do not fold into a stable helix, researchers have reported chemical approaches to constraining helix structure. However, these approaches rely on laborious, and often expensive, chemical synthesis and purification. Our premise is that protein-based solutions that stabilize a therapeutically-relevant helix offer a number of advantages. In contrast to chemically constrained helical peptides, or minimal/miniature proteins, which must be synthesized (at great expense and labor), a protein can be expressed in a cellular system (like all current protein therapeutics). If selected properly, the protein scaffold can stabilize the therapeutically-relevant helix. We recently reported a protein engineering strategy, which we call "helix-grafted display", and applied it to the challenge of suppressing HIV entry. We have reported helix-grafted display proteins that inhibit formation of an intramolecular PPI involving HIV gp41 C-peptide helix, and HIV gp41 N-peptide trimer, which contain C-peptide helix-binding clefts. Here, we used yeast display to screen a library of grafted C-peptide helices for N-peptide trimer recognition. Using 'hits' from yeast display library screening, we evaluated the effect helix mutations have on structure, expression, stability, function (target recognition), and suppression of HIV entry.

Helix-Grafted Pleckstrin Homology Domains Suppress HIV-1 Infection of CD4-Positive Cells.

The size, functional group diversity and three-dimensional structure of proteins often allow these biomolecules to bind disease-relevant structures that challenge or evade small-molecule discovery. Additionally, folded proteins are often much more stable in biologically relevant environments compared to their peptide counterparts. We recently showed that helix-grafted display-extensive resurfacing and elongation of an existing solvent-exposed helix in a pleckstrin homology (PH) domain-led to a new protein that binds a surrogate of HIV-1 gp41, a validated target for inhibition of HIV-1 entry. Expanding on this work, we prepared a number of human-derived helix-grafted-display PH domains of varied helix length and measured properties relevant to therapeutic and basic research applications. In particular, we showed that some of these new reagents expressed well as recombinant proteins in Escherichia coli, were relatively stable in human serum, bound a mimic of pre-fusogenic HIV-1 gp41 in vitro and in complex biological environments, and significantly lowered the incidence of HIV-1 infection of CD4-positive cells.

GLUE that sticks to HIV: a helix-grafted GLUE protein that selectively binds the HIV gp41 N-terminal helical region.

Methods for the stabilization of well-defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Here, we report the stable and functional display of an HIV gp41 C-peptide helix mimic on a GRAM-Like Ubiquitin-binding in EAP45 (GLUE) protein. C-peptide helix-grafted GLUE selectively binds a mimic of the N-terminal helical region of gp41, a well-established HIV drug target, in a complex cellular environment. Additionally, the helix-grafted GLUE is folded in solution, stable in human serum, and soluble in aqueous solutions, and thus overcomes challenges faced by a multitude of peptide drugs, including those derived from HIV gp41 C-peptide.