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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.

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease-relevant macromolecules that present a challenge to small-molecule reagents. Additionally, high-throughput screening and evolution-based methods often make the discovery of new protein binders simpler than the analogous small-molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease-relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies-a small and stable protein that can be evolved to recognize virtually any disease-relevant receptor-are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP-binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell-penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.

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 ...

We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti-dinitrophenyl (anti-DNP) antibodies to the surface of HER2-positive breast cancer cells, resulting in their targeted destruction by antibody-dependent cellular cytotoxicity. Since nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease-relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.

PURPOSE: Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. PROCEDURES: sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[(18)F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([(18)F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[(125)I]iodobenzoate ([(125)I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [(18)F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [(18)F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[(18)F]RL-I conjugates. RESULTS: [(18)F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [(125)I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [(18)F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [(125)I]SGMIB-2Rs15d.Uptake of [(18)F]RL-I-2Rs15d in subcutaneous xenografts was 16-20 %ID/g over 1-3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [(18)F]RL-I-2Rs15d but not with [(18)F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [(18)F]RL-I-2Rs15d and [(18)F]RL-I-5F7. CONCLUSIONS: Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blot are common bioanalytical techniques. Successful execution traditionally requires the use of one or more commercially available antibody-small-molecule dye, or antibody-reporter protein conjugates that recognize relatively short peptide tags (

Increased cellular levels of protein-protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein-protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin's putative binding face and large surface areas involved in gankyrin-dependent protein-protein interactions present a dramatic challenge to small molecule discovery. The size, high folding energies, and well-defined surfaces present in many proteins overcome some of the challenges faced by small molecule discovery. We used split-superpositive Green Fluorescent Protein (split-spGFP) reassembly to screen a 5×10(9) library of resurfaced proteins that are shape complementary to the putative binding face of gankyrin and identified mutants that potently and selectively bind this oncoprotein in vitro and in living cells. Collectively, our findings represent the first synthetic proteins that bind gankyrin and may represent a general strategy for developing protein basic research tools and drug leads that bind disease-relevant ankyrin repeats.

Alanine scanning mutagenesis of a recently reported prostate cancer cell-selective Protein Transduction Domain (PTD) was used to assess the specific contribution each residue plays in cell uptake efficiency and cell-selectivity. These studies resulted in the identification of two key residues. Extensive mutagenesis at these key residues generated multiple mutants with significantly improved uptake efficiency and cell-selectivity profiles for targeted cells. The best mutant exhibits ~19-fold better uptake efficiency and ~4-fold improved cell-selectivity for a human prostate cancer cell line. In addition, while the native PTD sequence was capable of delivering functional fluorescent protein to the interior of a prostate cancer cells, only modest functional enzyme delivery was achieved. In contrast, the most potent mutant was able to deliver large quantities of a functional enzyme to the interior of human prostate cancer cells. Taken together, the research described herein has significantly improved the efficiency, cell-selectivity, and functional utility of a prostate cancer PTD.

A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein–protein interaction involving the oncoprotein gankyrin. However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly. We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin–S6 ATPase binding interaction by isothermal titration calorimetry.

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is an RNA-mediated disease. Dramatically expanded (CUG) repeats accumulate in nuclei and sequester RNA-binding proteins such as the splicing regulator MBNL1. We have employed resin-bound dynamic combinatorial chemistry (RBDCC) to identify the first examples of compounds able to inhibit MBNL1 binding to (CUG) repeat RNA. Screening an RBDCL with a theoretical diversity of 11 325 members yielded several molecules with significant selectivity for binding to (CUG) repeat RNA over other sequences. These compounds were also able to inhibit the interaction of GGG-(CUG)(109)-GGG RNA with MBNL1 in vitro, with K(i) values in the low micromolar range.

The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.

2-Nitrobenzaldhyde Methyl propionylacetate Zinc(II) chloride Tin(II) chloride 2-Ethyl-3-quinolinecarboxylic acid, methyl ester 2-Ethyl-3-quinolinecarboxylic acid 2-Ethyl-3-quinolinecarboxylic acid hydrochloride Keywords: quinoline skeleton; quinine; chloroquine; lewis acid; one-pot synthesis; anti-malarial drugs

Gaining control over cell adhesion and growth is a critical step in microscale tissue engineering, as well as biosensor fabrication, applied cell biology, and high-throughput cell-based screening. Control over cell adhesion and growth is typically achieved by patterning small molecule or macromolecule reagents with affinity for a cell surface receptor onto a non-adhesive surface. These reagents are often susceptible to environmental and/or enzymatic degradation and their preparation and purification increase the overall cost and complexity of surface fabrication. Surface topology can influence cell adhesion and growth; however, engineering a surface with well-defined topology typically requires expensive and/or specialized equipment and/or multi-step processes such as microcontact printing. In this Paper we show that cell-imprinted features on the surface of a polyacrylamide hydrogel act as surface contact cues that promote cell adhesion and growth. In some cases the shape of cell-imprints dramatically affect cell adhesion. Collectively, we demonstrate that cell-imprinting polyacrylamide hydrogels is an inexpensive and straightforward method for programming cell adhesion and growth.