Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

Abstract

A central challenge for any therapeutic is targeting diseased over normal cells. Proteolysis is frequently upregulated in disease and can generate proteoforms with unique neo-epitopes. We hypothesize that targeting proteolytic neo-epitopes can enable more effective and safer treatments, reflecting a conditional layer of disease-specific regulation. Here, we characterized the precise proteolytic isoforms of CUB domain containing protein 1 (CDCP1), a protein overexpressed and specifically cleaved in RAS-driven cancers. We validated that the N-terminal and C-terminal fragments of CDCP1 remain associated after proteolysis in vitro and on the surface of pancreatic cancer cells. Using a differential phage display strategy, we generated exquisitely selective recombinant antibodies that target cells harboring cleaved CDCP1 and not the full-length form using antibody-drug conjugates or a bi-specific T-cell engagers. We show tumor-specific localization and anti-tumor activity in a syngeneic pancreatic tumor model having superior safety profiles compared to a pan-CDCP1-targeting antibody. Our studies show proteolytic neo-epitopes can provide an orthogonal AND gate for disease-specific targeting.

A general approach for selection of epitope-directed binders to proteins

“…2a ). 10 This supported that the three binders selectively binds to the proteolytic sites of CDCP1. IgG 0151, from the selection campaign using CDCP1-Fc mutant1 for negative selection, exhibits minimum binding to both mutants, while from selection using CDCP1-Fc mutant2 for negative selection, IgG 0152 recognizes CDCP1-Fc mutant1 with impaired affinity (Kd ∼15 nM) and does not bind to CDCP1-Fc mutant2 , and IgG 0153 partially binds to both mutants (Kd ∼5 nM for CDCP1-Fc mutant1 and 6 nM for CDCP1-Fc mutant1 ).…”

“…2a ). 10 Using CDCP1 as a model system, we aimed to generate antibodies specifically targeting the proteolytic sites (R415-K420) to block its proteolysis.…”

“…8 However, this method is time- and effort-consuming, only works for proteins and expression systems allowing noncanonical amino acid incorporation while not disrupting protein tertiary folding, and often generates non-specific binders. Differential phage selection is a powerful strategy to obtain highly specific antibodies for specific protein conformations 9 , isoforms 10 , and even small molecule complexes 11 . Inspired by this, we developed a more general approach to select and identify epitope-directed antibodies by deploying differential phage selection ( Fig.…”

“…Differential phage selection is a powerful strategy to obtain highly specific antibodies for specific protein conformations 9 , isoforms 10 , and even small molecule complexes 11 . Inspired by this, we developed a more general approach to select and identify epitope-directed antibodies by deploying differential phage selection (Fig.…”

“…1a ). Differential phage selection has been powerfully applied to obtain highly specific antibodies for specific protein conformations 21 , isoforms 22 , and even small molecule complexes 23 . To extend differential selection for EDS we describe a design algorithm to produce a decoy antigen mutated in the EOI to deplete binders that recognize undesired epitopes from a Fab phage library before positive selection against the WT POI.…”