Epidermal growth factor receptor downregulation by small heterodimeric binding proteins

No single engineered protein has been shown previously to robustly downregulate epidermal growth factor receptor (EGFR), a validated cancer target. A panel of fibronectin-based domains was engineered to bind with picomolar to nanomolar affinity to multiple epitopes of EGFR. Monovalent and homo- and hetero-bivalent dimers of these domains were tested for EGFR downregulation. Selected orientations of non-competitive heterodimers decrease EGFR levels by up to 80% in multiple cell types, without activating receptor signaling. These heterodimers inhibit autophosphorylation, proliferation and migration, and are synergistic with the monoclonal antibody cetuximab in these activities. These small (25 kDa) heterodimers represent a novel modality for modulating surface receptor levels.National Institutes of Health (U.S.) (NIH grant CA96504)National Institutes of Health (U.S.) (NIH grant CA118705)National Science Foundation (U.S.) (Graduate Research Fellowship Program

Making Connections: A Handbook for Effective Formal Mentoring Programs in Academia

This book, Making Connections: A Handbook for Effective Formal Mentoring Programs in Academia, makes a unique and needed contribution to the mentoring field as it focuses solely on mentoring in academia. This handbook is a collaborative institutional effort between Utah State University’s (USU) Empowering Teaching Open Access Book Series and the Mentoring Institute at the University of New Mexico (UNM). This book is available through (a) an e-book through Pressbooks, (b) a downloadable PDF version on USU’s Open Access Book Series website), and (c) a print version available for purchase on the USU Empower Teaching Open Access page, and on Amazon

Deep Antimicrobial Activity and Stability Analysis Inform Lysin Sequence–Function Mapping

Antibiotic-resistant infectious disease is a critical challenge to human health. Antimicrobial proteins offer a compelling solution if engineered for potency, selectivity, and physiological stability. Lysins, which lyse cells via degradation of cell wall peptidoglycans, have significant potential to fill this role. Yet, the functional complexity of antimicrobial activity has hindered high-throughput characterization for discovery and design. To dramatically expand knowledge of the sequence–function landscape of lysins, we developed a depletion-based assay for library-scale measurement of lysin inhibitory activity. We coupled this platform with a high-throughput proteolytic stability assay to assess the activity and stability of ∼5 × 104 lysin catalytic domain variants, resulting in the discovery of a variant with increased activity (70 ± 20%) and stability (7.2 ± 0.4 °C increased midpoint of thermal denaturation). Ridge regression of the resulting data set demonstrated that libraries with a higher average Hamming distance better informed pairwise models and that coupling activity and stability assays enabled better prediction of catalytically active lysins. The best models achieved Pearson’s correlation coefficients of 0.87 ± 0.01 and 0.61 ± 0.04 for predicting catalytic domain stability and activity, respectively. Our work provides an efficient strategy for constructing protein sequence–function landscapes, drastically increases screening throughput for engineering lysins, and yields promising lysins for further development

Epidermal growth factor receptor downregulation by small heterodimeric binding proteins

No single engineered protein has been shown previously to robustly downregulate epidermal growth factor receptor (EGFR), a validated cancer target. A panel of fibronectin-based domains was engineered to bind with picomolar to nanomolar affinity to multiple epitopes of EGFR. Monovalent and homo-and hetero-bivalent dimers of these domains were tested for EGFR downregulation. Selected orientations of non-competitive heterodimers decrease EGFR levels by up to 80% in multiple cell types, without activating receptor signaling. These heterodimers inhibit autophosphorylation, proliferation and migration, and are synergistic with the monoclonal antibody cetuximab in these activities. These small (25 kDa) heterodimers represent a novel modality for modulating surface receptor levels

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold (<i>p</i> < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold (<i>p</i> = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residuesguided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysisdid not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity (<i>K</i>_d = 6–9 nM)

Effect of Linker Length on Protein Evolution Using Yeast Surface Display

Faculty Advisor: Benjamin HackelThis research was supported by the Undergraduate Research Opportunities Program (UROP)

Hyperstable Synthetic Mini-Proteins as Effective Ligand Scaffolds

Small, single-domain protein scaffolds are compelling sources of molecular binding ligands with the potential for efficient physiological transport, modularity, and manufacturing. Yet, mini-proteins require a balance between biophysical robustness and diversity to enable new functions. We tested the developability and evolvability of millions of variants of 43 designed libraries of synthetic 40-amino acid βαββ proteins with diversified sheet, loop, or helix paratopes. We discovered a scaffold library that yielded hundreds of binders to seven targets while exhibiting high stability and soluble expression. Binder discovery yielded 6–122 nM affinities without affinity maturation and Tms averaging ≥78 °C. Broader βαββ libraries exhibited varied developability and evolvability. Sheet paratopes were the most consistently developable, and framework 1 was the most evolvable. Paratope evolvability was dependent on target, though several libraries were evolvable across many targets while exhibiting high stability and soluble expression. Select βαββ proteins are strong starting points for engineering performant binders

High-Throughput Ligand Discovery Reveals a Sitewise Gradient of Diversity in Broadly Evolved Hydrophilic Fibronectin Domains

<div><p>Discovering new binding function via a combinatorial library in small protein scaffolds requires balance between appropriate mutations to introduce favorable intermolecular interactions while maintaining intramolecular integrity. Sitewise constraints exist in a non-spatial gradient from diverse to conserved in evolved antibody repertoires; yet non-antibody scaffolds generally do not implement this strategy in combinatorial libraries. Despite the fact that biased amino acid distributions, typically elevated in tyrosine, serine, and glycine, have gained wider use in synthetic scaffolds, these distributions are still predominantly applied uniformly to diversified sites. While select sites in fibronectin domains and DARPins have shown benefit from sitewise designs, they have not been deeply evaluated. Inspired by this disparity between diversity distributions in natural libraries and synthetic scaffold libraries, we hypothesized that binders resulting from discovery and evolution would exhibit a non-spatial, sitewise gradient of amino acid diversity. To identify sitewise diversities consistent with efficient evolution in the context of a hydrophilic fibronectin domain, >10⁵ binders to six targets were evolved and sequenced. Evolutionarily favorable amino acid distributions at 25 sites reveal Shannon entropies (range: 0.3–3.9; median: 2.1; standard deviation: 1.1) supporting the diversity gradient hypothesis. Sitewise constraints in evolved sequences are consistent with complementarity, stability, and consensus biases. Implementation of sitewise constrained diversity enables direct selection of nanomolar affinity binders validating an efficient strategy to balance inter- and intra-molecular interaction demands at each site.</p></div

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF