Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers
Shion A. Lim, Jie Zhou, Alexander J. Martinko, Yung-Hua Wang, Ekaterina V. Filippova, Veronica Steri, Donghui Wang, Soumya G. Remesh, Jia Liu, Byron Hann, Anthony A. Kossiakoff, Michael J. Evans, Kevin K. Leung, James A. Wells
Shion A. Lim, Jie Zhou, Alexander J. Martinko, Yung-Hua Wang, Ekaterina V. Filippova, Veronica Steri, Donghui Wang, Soumya G. Remesh, Jia Liu, Byron Hann, Anthony A. Kossiakoff, Michael J. Evans, Kevin K. Leung, James A. Wells
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Research Article

Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers

Abstract

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal “AND” gate for improving the therapeutic index.

Authors

Shion A. Lim, Jie Zhou, Alexander J. Martinko, Yung-Hua Wang, Ekaterina V. Filippova, Veronica Steri, Donghui Wang, Soumya G. Remesh, Jia Liu, Byron Hann, Anthony A. Kossiakoff, Michael J. Evans, Kevin K. Leung, James A. Wells

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Figure 5

IgG CL03 specifically targets c-CDCP1–expressing pancreatic cancer cells.

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IgG CL03 specifically targets c-CDCP1–expressing pancreatic cancer cells...
(A) Differential phage selection strategy to identify a c-CDCP1–specific Ab. Fab-phage was precleared with fl-CDCP1-Fc prior to positive selection with c-CDCP1-Fc. (B) BLI shows specific binding of IgG CL03 to c-CDCP1-Fc, but not to fl-CDCP1-Fc (KD = 150–840 pM, Supplemental Table 1). (C) Negative-stain EM 3D reconstruction of c-CDCP1 with CL03 Fab. Left: 2D class averages of c-CDCP1 (cut 3) plus CL03 Fab in the absence and presence of anti-Fab VHH. Right: 3D EM maps of CDCP1 (cut 3) plus CL03 Fab plus VHH with crystal structure of Fab (green) and VHH (blue) modeled into the density. (D) Immunofluorescence of HPAC, PL5, and HPNE cells with Alexa Fluor 488–labeled IgG CL03 (left panels) and IgG 4A06 (right panels). IgG CL03 specifically stains PL5 cells, while IgG 4A06 stains both HPAC and PL5 cells. No staining is observed for HPNE. Scale bars: 20 μm. (E) Flow cytometry shows that IgG CL03 binds to PL5 and PL45 cells, but not HPAC or HPNE cells. Data are represented as mean ± SEM. n = 3. (F) Top: schematic of ADC cell-killing assay. Bottom: dose-dependent ADC-mediated cell killing with IgG CL03 was only observed against PL5 and PL45 and only in the presence of both the primary and secondary Abs. Data are represented as mean ± SEM. n = 3. ****P < 0.0001, 2-way ANOVA. (G) In vivo PET imaging of 89Zr-labeled IgG CL03 in PL5 mouse xenografts shows tumor localization. Data represent individual values and mean ± SEM. n = 4.