Engineered proteins empower biotechnologies in industrial, medical, and agricultural settings. Specifically, molecular recognition binding ligands are integral for targeted therapy and diagnostic applications.1 A common approach to engineer high-affinity, specific binding entails scaffold proteins,2, 3 which consist of a conserved framework to provide structural integrity (for stability and reduced entropic cost of binding4) while accommodating a variable active site which can be engineered to provide new binding functions (a property called evolvability or innovability5). To be effective solutions within these applications, proteins must also exhibit “developability”,6, 7, 8 i.e., remain stable and soluble in complex environments as well as be efficiently produced, often in multifunctional conjugates. Yet, mutations required to change protein function are generally destabilizing resulting in a trade-off between developability and evolvability.9, 10, 11, 12, 13 To complicate matters further, proteins do not come with ideal properties because they evolved to fit environmental pressures rather than for our specific needs.10 Despite extensive engineering of binding ligands across various topologies,2, 14, 15 the factors that dictate performance remain incompletely understood,5, 16 thereby motivating fundamental elucidation of the impact of protein topology, parental framework sequence, paratope structure, and paratope sequence on scaffold developability and evolvability. Moreover, current scaffolds all present various liabilities, inspiring identification of ligand scaffolds with an improved balance of evolvability and developability.

Nature’s primary solution for evolvable binding proteins, the antibody, is the dominant scaffold for clinical and biotechnological applications.17, 18 Yet large size inhibits effective tissue penetration which can decrease therapeutic efficacy,19, 20 multi-domain structures hinder modularity for multifunctional applications,21 and variable developability hinders utility.2, 7, 22 To overcome these limitations, small, single domain scaffolds have been developed23 – including affibodies,24, 25 fibronectin domains,26 knottins,27, 28 and DARPins29 – in a wide variety of topologies with varied binding paratope structures,22 including loops (both flexible21, 26 and constrained30, 31, 32), α helices,24 and β strands.33 Yet, the evolvability and developability of these scaffolds vary significantly, which renders the ideal scaffold uncertain. Maintaining developability is non-trivial because introducing new functions requires varying an appreciable fraction of these small domains.34 Yet, decoupling the engineered paratope from the framework – a beneficial approach to evolvability5, 11 – is challenging in small domains. Stability of the parental molecule promotes evolvability thereby motivating selection of a highly stable framework sequence.10, 12, 13 Rocklin and colleagues computationally designed and experimentally validated a collection of proteins that fold into an array of small topologies with very high stability.35 Yet, the parental sequence must also tolerate a chemically diverse set of paratope sequences to enable introduction of new function. Though these sequences were designed for stability and topology without evolvability as a design criterion, these proteins are compelling ligand scaffolds because of their small size, hyperstability, and variety of potential paratope structures. We seek to answer several core questions: Can synthetic proteins, designed for wild-type protease stability,35 behave as ligand scaffolds (i.e., be robust enough to tolerate mutations to exhibit evolvability while maintaining high developability)?. Which combinations of topology, paratope, and framework are most effective for overall scaffold developability and evolvability?

To answer these questions, we developed a full-factorial experimental design by constructing 45 combinatorial libraries systematically varying in topology, framework sequence, and paratope locations. We measured evolvability via binder discovery to four diverse targets and developability via four high-throughput assays. Through deep sequencing analysis validated by statistical testing, we measured an array of evolvability and developability across design space to identify drivers of performance. Although a hyperstable framework and localized diversity are insufficient criteria for an effective scaffold, select synthetic miniproteins, originally designed solely for foldedness, indeed serve as developable, evolvable scaffolds. Furthermore, a single round of affinity maturation of VEGF binders yielded well-folded, highly stable clones with single-digit nanomolar affinity.