The 2EF Antibody Targets a Unique N-Terminal Epitope of Trop-2 and Enhances the In Vivo Activity of the Cancer-Selective 2G10 Antibody

SIMPLE SUMMARY: We sought recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. The 2EF mAb was developed, and demonstrated efficient access to Trop-2 at cell–cell junctions in breast cancer cells in culture and in prostate tumors that were not accessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of cancer cells in vitro, with the highest activity at high cell density. Correspondingly, 2EF showed the highest anticancer activity on densely packed, Trop-2-expressing tumors. The 2EF mAb enhances the activity of the cancer-only binding 2G10 mAb against tumors in vivo, opening novel avenues for Trop-2-targeted therapy. ABSTRACT: 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.