Khouloud Boujnah

Movie S1 from 3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability

Movie S1 from 3D-informed targeting of the Trop-2 signal-activation site drives selective cancer vulnerability

Next-generation Trop-2-targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti-Trop-2 monoclonal antibodies (mAb) recognize a single immunodominant epitope. Hence, we removed it by deletion-mutagenesis. Cancer-specific, glycosylation-prone mAb were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation-site-mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2-expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art CDR grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd <10-12 M, and uncleaved/wtTrop-2 in normal cells with Kd 3.16x10-8 M, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2-expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2-targeted therapy.

Movie S1 from 3D-informed targeting of the Trop-2 signal-activation site drives selective cancer vulnerability

Trop-2 proteolytic processing in cancer cells exposes epitopes that were specifically targeted by the 2G10 antibody. We sought additional recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. Trop-2 deletion mutants were employed in immunization and screening procedures, and these led to the recognition of a novel epitope in the N-terminal region of Trop-2, by the 2EF antibody. The 2EF mAb was shown to bind Trop-2 at cell–cell junctions in MCF-7 breast cancer cells, and in deeply seated sites in prostate cancer, that were inaccessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of HT29 colon tumor cells in vitro, with the highest activity at high cell density. In vivo, 2EF showed anticancer activity against SKOv3 ovarian, Colo205, HT29, HCT116 colon and DU-145 prostate tumors, with the highest impact on densely packed tumor sites, whereby 2EF outcompeted benchmark anti-Trop-2 antibodies. Given the different recognition modes of Trop-2 by 2EF and 2G10, we hypothesized the effective interaction of the two mAb in vivo. The 2EF mAb was indeed demonstrated to enhance the activity of 2G10 against tumor xenotransplants, opening novel avenues for Trop-2-targeted therapy. We humanized 2EF by state-of-the-art CDR grafting/re-modeling, yielding the Hu2EF for therapy of Trop-2-expressing tumors in patients.

Movie S1 from 3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability

Movie S1 from 3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability