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Constitutive Btk signaling drives several B-cell cancers. Here the authors demonstrate key allosteric intramolecular interactions between the SH2 domain and the kinase domain of Btk, and propose an alternative approach for inhibition of both wild-type and tyrosine kinase inhibitor-resistant Btk.

Precise binding mode identification and subsequent affinity improvement without structure determination remain a challenge in the development of therapeutic proteins. However, relevant experimental techniques are generally quite costly, and purely computational methods have been unreliable. Here, we show that integrated computational and experimental epitope localization followed by full-atom energy minimization can yield an accurate complex model structure which ultimately enables effective affinity improvement and redesign of binding specificity. As proof-of-concept, we used a leucine-rich repeat (LRR) protein binder, called a repebody (Rb), that specifically recognizes human IgG1 (hIgG1). We performed computationally-guided identification of the Rb:hIgG1 binding mode and leveraged the resulting model to reengineer the Rb so as to significantly increase its binding affinity for hIgG1 as well as redesign its specificity toward multiple IgGs from other species. Experimental structure determination verified that our Rb:hIgG1 model closely matched the co-crystal structure. Using a benchmark of other LRR protein complexes, we further demonstrated that the present approach may be broadly applicable to proteins undergoing relatively small conformational changes upon target binding.

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.