Ex silico engineering of cystine-dense peptides yielding a potent bispecific T cell engager

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 ( K D = 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.

CYpHER: Catalytic extracellular targeted protein degradation with high potency and durable effect

“…1D) as demonstrated in mammalian surface display staining experiments, and also has access to the CNS (30) . Then we further engineered a PD-L1-binding CDP (33) for enhanced pH-dependent release by generating a library of variants that each contained up to two histidine substitutions, and surface-display screened to enrich for variants that conferred strong PD-L1 binding at pH 7.4 but reduced PD-L1 binding to cells after a low-pH rinse (Fig. 1E).…”

“…To construct CYpHER molecules, we began with two cystine-dense peptide (CDP) miniproteins that we’ve recently characterized separately for other in vivo functionalities (30, 33) . We made use of a TfR-binding CDP allelic series (30) that maintains binding at reduced pH (Fig.…”

“…S1) molecule was produced as previously described (33, 78) . All other CYpHER molecules were produced by transient expression in suspension HEK293 cells (ThermoFisher GeneArt) and purified either via Ni-NTA pull-down as previously described (33, 78) or by Protein A columns (Cytiva 28985254 [pre-packed Protein A columns] and 28903059 [buffer kit]) as per manufacturer’s protocol. Proteins were buffer exchanged (Sephadex G25 desalting columns, Cytiva 17085101) into PBS and aliquoted for storage at -80°C.…”

De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery

“…NMR experiments were recorded at 298 K on Bruker AVANCE III 850 MHz spectrometers equipped with a cryogenic triple-resonance probe. Two dimensional (2D) 1 H− 15 N/ 13 C HSQC spectra were recorded to obtain chemical shifts of heavy atoms in backbone and side chains. 2D 1 H− 1 H TOCSY (mixing time, 80 ms) and 2D 1 H− 1 H COSY were acquired for the assignment of NMR signals of peptides.…”

“…In recent years, new strategies have emerged to generate DRPs with novel structures and functions beyond the scope of naturally occurring DRPs. These strategies include computational de novo designs and library-based screenings. − Although these approaches have been powerful and impressive, the diversity in structure and function within both the designed and selected DRPs remains quite limited. This limitation primarily stems from the inherent challenges associated with manipulating the disulfide pairing patterns of DRPs.…”