LAUSR

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. Description A PD-L1–binding cystine-dense peptide produced using in silico and in vitro screening outperforms an antibody in a bispecific T cell engager. 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.