Abstract 2655: A novel Trop-2/CD3 trivalent bispecific antibody effectively redirects T cells to kill target human pancreatic and gastric cancer cells
Authors: Rossi Diane L. , Cardillo Thomas M. , Rossi Edmund A. , Zalath Maria , Goldenberg David M. , Chang Chien-Hsing 
Published: May 2015

Background: Trop-2 is a type I transmembrane protein that is highly expressed in diverse epithelial cancers, including lung, gastric, colorectal, pancreatic, bladder, mammary, ovarian, uterine, and prostate carcinomas, with limited presence on normal human tissues. Whereas various formats of bispecific antibodies (bsAbs) for redirecting T cells to cancers have shown promise in both pre-clinical and clinical studies, efforts to prolong the circulating half-life of the scFv-based platforms and to prevent the commonly observed cytokine release syndrome are continuing. Herein, we describe a novel T-cell redirecting trivalent bsAb, designated (E1)-3s, which comprises an anti-CD3 scFv covalently conjugated to a stabilized dimer of a Trop-2-targeting Fab. Potential advantages of (E1)-3s include high-level surface expression of Trop-2 on various solid cancers, bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance, and potent T-cell mediated cytotoxicity. Methods: DOCK-AND-LOCKTM was employed to site-specifically link an anti-CD3 (Okt3) scFv to a stabilized Fab dimer of the humanized anti-Trop-2 mAb, hRS7. LC-MS confirmed the mass of the conjugate, which resolved as &gt;90% of the desired monomeric peak by size-exclusion HPLC. Purity was demonstrated by reducing SDS-PAGE, which resolved only the three constituent polypeptide bands comprising (E1)-3s. Results: Fluorescence microscopy showed that (E1)-3s, but not a control conjugate [(19)-3s, anti-CD19/CD3], induced synapse formation between Jurkat (T) and Capan-1 (human pancreatic cancer) cells. Using a 3-fold excess of stimulated T cells in vitro, (E1)-3s induced a potent and specific T-cell-mediated lysis in Capan-1 (115,000 Trop-2 copies/cell) pancreatic cancer (IC50 = 29 pM, Lysismax = 60%), and NCI-N87 (383,000 Trop-2 copies/cell) human gastric cancer cells (IC50 = 0.85 pM, Lysismax&gt; 90%). In vivo efficacy was demonstrated with Capan-1 and NCI-N87 xenografts in NOD-SCID mice by co-injection of purified human T cells and tumor cells at an effector-to-target ratio of 2, followed by five daily injections of (E1)-3s. With Capan-1, the control group reached the endpoint of disease progression (tumors exceeding 1 cm3) on day 24, at which time, the (E1)-3s group had significantly smaller tumors (0.257 ± 0.102 cm3 versus 1.011 ± 0.532 cm3; P=0.0092). For NCI-N87, the (E1)-3s group had significantly smaller tumors than the control group (0.147 ± 0.062 cm3 versus 0.824 ± 0.342 cm3, respectively; P=0.0017) when the latter reached the disease-progression endpoint on day 31. Conclusions: (E1)-3s effectively induced T-cell-mediated killing of Trop-2-expressing pancreatic and gastric cancer cells in vitro and in vivo. Additional cell lines of different solid cancer types, and ones with higher and lower Trop-2 antigen density, are under evaluation.

Citation Format: Diane L. Rossi, Thomas M. Cardillo, Edmund A. Rossi, Maria Zalath, David M. Goldenberg, Chien-Hsing Chang. A novel Trop-2/CD3 trivalent bispecific antibody effectively redirects T cells to kill target human pancreatic and gastric cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2655. doi:10.1158/1538-7445.AM2014-2655