Yeast surface display has proven to be an effective tool in the discovery and evolution of ligands with new or improved binding activity. Selections for binding activity are generally carried out using immobilized or fluorescently labeled soluble domains of target molecules such as recombinant ectodomain fragments. While this method typically provides ligands with high affinity and specificity for the soluble molecular target, translation to binding true membrane-bound cellular target is commonly problematic. Direct selections against mammalian cell surfaces can be carried out either exclusively or in combination with soluble target-based selections to further direct towards ligands for genuine cellular target. Using a series of fibronectin domain, affibody, and Gp2 ligands and human cell lines expressing a range of their targets, epidermal growth factor receptor and carcinoembryonic antigen, this study quantitatively identifies the elements that dictate ligand enrichment and yield. Most notably, extended flexible linkers between ligand and yeast enhances enrichment ratios from 1.4±0.8 to 62±57 for a low-affinity (>600 nM) binder on cells with high target expression and from 14±13 to 74±25 for a high-affinity binder (2 nM) on cells with medium valency. Inversion of the yeast display fusion from C-terminal display to N-terminal display still enables enrichment albeit with 40% to 97% reduced efficacy. Collectively, this study further enlightens the conditions – while highlighting new approaches – that yield successful enrichment of yeast-displayed binding ligands via panning on mammalian cells.

Molecularly-targeted microbubbles (MBs) are increasingly being recognized as promising contrast agents for oncological molecular imaging with ultrasound. With the detection and validation of new molecular imaging targets, novel binding ligands are needed that bind to molecular imaging targets with high affinity and specificity. In this study we assessed a novel class of potentially clinically translatable MBs using an engineered 10th type III domain of human-fibronectin (MB-FN3VEGFR2) scaffold-ligand to image VEGFR2 on the neovasculature of cancer. The in vitro binding of MB-FN3VEGFR2 to a soluble VEGFR2 was assessed by flow-cytometry (FACS) and binding to VEGFR2-expressing cells was assessed by flow-chamber cell attachment studies under flow shear stress conditions. In vivo binding of MB-FN3VEGFR2 was tested in a transgenic mouse model (FVB/N Tg(MMTV/PyMT634Mul) of breast cancer and control litter mates with normal mammary glands. In vitro FACS and flow-chamber cell attachment studies showed significantly (P<0.01) higher binding to VEGFR2 using MB-FN3VEGFR2 than control agents. In vivo ultrasound molecular imaging (USMI) studies using MB-FN3VEGFR2 demonstrated specific binding to VEGFR2 and was significantly higher (P<0.01) in breast cancer compared to normal breast tissue. Ex vivo immunofluorescence-analysis showed significantly (P<0.01) increased VEGFR2-expression in breast cancer compared to normal mammary tissue. Our results suggest that MBs coupled to FN3-scaffolds can be designed and used for USMI of breast cancer neoangiogenesis. Due to their small size, stability, solubility, the lack of glycosylation and disulfide bonds, FN3-scaffolds can be recombinantly produced with the advantage of generating small, high affinity ligands in a cost efficient way for USMI.

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The goal of this study was to create an accessible, inexpensive, and engaging experiment to teach high school and undergraduate chemistry or biology students about intermolecular forces and how they contribute to the behavior of biomolecules. We developed an enzyme-linked immunosorbent assay (ELISA) to probe specific structure–function relationships in the context of a protein–protein interaction that can be completed within a week of 45 min daily classes or a single 3–4 h lab using accessible reagents and materials (e.g., micropipettes and camera phones). The assay detected the high-affinity interaction between immunoglobulin G (IgG) and an engineered fibronectin domain protein. To demonstrate the impact of small chemical changes on intermolecular interactions, four mutant fibronectin domains were engineered, each with a single amino acid change, to provide a variety of chemical groups in the hypothesized binding site that resulted in a range of affinities for IgG (equilibrium dissociation constants from 1.5–696 nM). The experiment was implemented with two classes of high school chemistry students. Students effectively differentiated between strong and weak protein–protein interactions (median correlation coefficient between observed and expected results = 0.88) and demonstrated keen interest in the assay and concepts. Students were asked to then design and conduct a variation of the ELISA to test their own hypotheses regarding various experiment parameters to great success. Image acquisition for assay colorimetry was identified as a potential area of improvement. We have shown that this experiment is accessible to high school students both fiscally and academically and can be a fun and effective tool to apply their knowledge of intermolecular forces within the context of proteins. We have shown that the experiment could also be implemented in an undergraduate laboratory setting to allow for advanced inquiry into protein–protein interaction quantification and data analysis. This experience helps students at a variety of academic levels make conceptual connections across the fields of chemistry, physics, and biology.

The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping mutations and sorted them, via yeast display and flow cytometry, for stability and target binding. Deep sequencing of functional variants revealed effective mutations both in clone-dependent contexts and broadly across binders to epidermal growth factor receptor (EGFR), insulin receptor, and immunoglobulin G. Functional mutants averaged 4.3 charge neutralizing mutations per domain while maintaining net negative charge. We evolved an EGFR-targeted Gp2 mutant that reduced charge density by 33%, maintained net charge, and improved charge distribution homogeneity while elevating thermal stability ( T = 87 ± 1 °C), improving binding specificity, and maintaining affinity ( K = 8.8 ± 0.6 nM). This molecule was conjugated with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid for Cu chelation and evaluated for physiological distribution in mice with xenografted A431 (EGFR) and MDA-MB-435 (EGFR) tumors. Excised tissue gamma counting and positron emission tomography/computed tomography imaging revealed good EGFR tumor signal (4.7 ± 0.5%ID/g) at 2 h post-injection and molecular specificity evidenced by low uptake in EGFR tumors (0.6 ± 0.1%ID/g, significantly lower than for non-charge-modified Gp2, p = 0.01). These results provide charge mutations for an improved Gp2 framework, validate an effective approach to charge engineering, and advance performance of physiological EGFR targeting for molecular imaging.