Yang et al. developed, de novo, an array of concave helical protein scaffolds with diverse shapes and surfaces, high stability, and small sizes, with the goal of enabling them to be combined to target multiple immunomodulatory receptors with convex Ig fold surfaces, and to penetrate tumors. After two rounds of FACS selection against TGFβRII, CTLA-4, and PD-L1 those selected were experimentally optimized by yeast display in regions predicted to be involved in target binding to identify stable high-affinity binders. Optimized binders exhibited robust biological activity. Computational models predicted well the cocrystal structures of the proteins complexed with targets.
Contributed by Paula Hochman
ABSTRACT: Immune receptors have emerged as critical therapeutic targets for cancer immunotherapy. Designed protein binders can have high affinity, modularity, and stability and hence could be attractive components of protein therapeutics directed against these receptors, but traditional Rosetta based protein binder methods using small globular scaffolds have difficulty achieving high affinity on convex targets. Here we describe the development of helical concave scaffolds tailored to the convex target sites typically involved in immune receptor interactions. We employed these scaffolds to design proteins that bind to TGF_RII, CTLA-4, and PD-L1, achieving low nanomolar to picomolar affinities and potent biological activity following experimental optimization. Co-crystal structures of the TGF_RII and CTLA-4 binders in complex with their respective receptors closely match the design models. These designs should have considerable utility for downstream therapeutic applications.
Author Info: (1) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (2) Department of Biochemistry
Author Info: (1) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (2) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (3) Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA. (4) Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. (5) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (6) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (7) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (8) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (9) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (10) Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. (11) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (12) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (13) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (14) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (15) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (16) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (17) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (18) Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA. (19) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (20) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (21) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (22) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (23) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (24) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (25) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (26) Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA. (27) Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. (28) Department of Biochemistry, University of Washington, Seattle, WA, USA. dabaker@uw.edu. Institute for Protein Design, University of Washington, Seattle, WA, USA. dabaker@uw.edu. Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. dabaker@uw.edu.
