Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

Abstract

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

De novo developed protein binders mimicking Interferon lambda signaling

“…(a) We utilize the unique feature of the ProBi scaffold – two mutable patches (PatchN and PatchC) on one protein molecule (Fig. 2A, reported previously in [26]). This approach imitates the natural signaling molecules such as interleukins and interferons.…”

“…1). They were initially established for ribosome display, so each Patch contains 10 mutable amino acid positions [26]. For yeast display, we used six positions to reduce the complexity of library.…”

“…The library of ProBi scaffold (PatchC) was synthetized by the GENEWIZ company using NNK codons technology [26]. The DNA library of ProBi scaffold (PatchN) was constructed by the restriction-free cloning technique described by Peleg et al [42].…”

“…We started with five protein scaffolds (Table 1, Fig. 1) to increase the odds for identification of high-affinity [26] 4psf [31] Q9NWS0 Q17-I144 15 6 IL-10R2 Sso7d [27] 1sso [32] P61991 A1-K62 7 7 IL-28R1 Kan-Nfr [24] 4h05 [33] Q9F9M5 H(À5)-P90 10 10 IL-10R2 GP2 [28] 2wnm [34] P03704 K35-P79 5 9 < 7% Knottin [29] 1cbh [35] P62694 T1-L36 5 8 < 7%…”

Protein scaffolds: antibody alternatives for cancer diagnosis and therapy

“…Finally, it is possible to select designed scaffold binders against targets via experiments to evaluate their performance. 193,194…”