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- Title
Ex silico engineering of cystine-dense peptides yielding a potent bispecific T cell engager.
- Authors
Crook, Zachary R.; Girard, Emily J.; Sevilla, Gregory P.; Brusniak, Mi-Youn; Rupert, Peter B.; Friend, Della J.; Gewe, Mesfin M.; Clarke, Midori; Lin, Ida; Ruff, Raymond; Pakiam, Fiona; Phi, Tinh-Doan; Bandaranayake, Ashok; Correnti, Colin E.; Mhyre, Andrew J.; Nairn, Natalie W.; Strong, Roland K.; Olson, James M.
- Abstract
Cystine-dense peptides (CDPs) are a miniprotein class that can drug difficult targets with high affinity and low immunogenicity. Tools for their design, however, are not as developed as those for small-molecule and antibody drugs. CDPs have diverse taxonomic origins, but structural characterization is lacking. Here, we adapted Iterative Threading ASSEmbly Refinement (I-TASSER) and Rosetta protein modeling software for structural prediction of 4298 CDP scaffolds and performed in silico prescreening for CDP binders to targets of interest. Mammalian display screening of a library of docking-enriched, methionine and tyrosine scanned (DEMYS) CDPs against PD-L1 yielded binders from four distinct CDP scaffolds. One was affinity-matured, and cocrystallography yielded a high-affinity (KD = 202 pM) PD-L1–binding CDP that competes with PD-1 for PD-L1 binding. Its subsequent incorporation into a CD3-binding bispecific T cell engager produced a molecule with pM-range in vitro T cell killing potency and which substantially extends survival in two different xenograft tumor-bearing mouse models. Both in vitro and in vivo, the CDP-incorporating bispecific molecule outperformed a comparator antibody-based molecule. This CDP modeling and DEMYS technique can accelerate CDP therapeutic development. Maximizing the utility of miniproteins: Miniproteins are an attractive option for targeted therapeutic development. Here, Crook et al. developed a CD3-targeting bispecific T cell engager with a PD-L1–binding cystine-dense miniprotein [also called a cystine-dense peptide (CDP)]. Using CDP sequence modeling and in silico docking, they identified PD-L1–binding candidates and optimized one for testing in vitro and in mice. Co-delivery of activated T cells and the CDP-based molecule extended survival and eliminated tumors more effectively than an anti–PD-L1 antibody in mice with prostate cancer tumors and inhibited tumor growth and extended survival in mice with breast cancer. This modeling and screening technique could improve the development of miniprotein-based therapies.
- Subjects
PEPTIDES; PROSTATE tumors; TUMOR growth; PROTEIN models
- Publication
Science Translational Medicine, 2022, Vol 14, Issue 645, p1
- ISSN
1946-6234
- Publication type
Article
- DOI
10.1126/scitranslmed.abn0402