Abstract 4995: anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer
Authors: Blankenship John W. , Misher Lynda , Mitchell Danielle , Zhang Nicole , Tan Philip , Hoyos Gabriela H. , Ravikumar Padma , Bader Robert , McMahan Catherine J. , Miller Robert E. , Bannink Jeannette , Fang Hang , Parr Lara , Dasovich Maria , Bienvenue David , Aguilar Megan , Xu Carina , Daugherty Mollie , Woodruff Brian , Gross Jane A. 
Published: July 2016

Abstract
Background: Effective treatment of metastatic, triple-negative breast cancer (TNBC) remains a highly unmet medical need. We have developed ES425, a bispecific ADAPTIR™ (modular protein technology) molecule that redirects T-cell cytotoxicity to tumor cells expressing ROR1 (receptor tyrosine kinase-like orphan receptor 1), an oncofetal antigen expressed on TNBC and other malignancies. Results are presented for studies run to examine in vitro and in vivo activity of ES425 in preclinical models of TNBC.
Materials and Methods: Target-dependent cytotoxic activity was examined in vitro by treating ROR1(+) cell lines and ROR1(−) cell lines with ES425 in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined using chromium release assays. T cells were assessed for activation and proliferation using multi-color flow cytometry. Pharmacokinetics of ES425 in NOD/SCID gamma (NSG) mice was determined using single intravenous dose of approximately 10 mg/kg. Serum concentrations at time points ranging from 15 minutes to 504 hours were used to calculate the terminal elimination half-life of ES425. To assess activity in vivo, NOD/SCID mice were implanted subcutaneously with the ROR1(+) TNBC tumor cell line MDA-MB-231 and purified human T cells and treated with ES425. This model was run twice with T cells from different human donors. Tumor growth was assessed by measuring tumor volume.
Results: ES425 efficiently redirected T cell cytotoxicity against ROR1(+) cell lines at low picomolar concentrations in vitro. Cytotoxic activity was dependent on expression of ROR1 by the target cells. T cells were activated and proliferated in response to ES425 in the presence of ROR1(+) target cells; proliferation was not observed in response to ROR1(−) cells. In vivo, pharmacokinetic analysis showed a serum half-life of approximately 7 days in NSG mice, and ES425 inhibited growth of MDA-MB-231 tumors in mouse xenografts. Repeat experiments showed similar inhibition of tumor growth and an improvement in overall survival.
Conclusions: These studies show that ES425 may be an efficient agent for redirecting T cell cytotoxicity in preclinical TNBC models and merits investigation as a potential therapeutic in TNBC and other malignancies.
Citation Format: John W. Blankenship, Lynda Misher, Danielle Mitchell, Nicole Zhang, Philip Tan, Gabriela H. Hoyos, Padma Ravikumar, Robert Bader, Catherine J. McMahan, Robert E. Miller, Jeannette Bannink, Hang Fang, Lara Parr, Maria Dasovich, David Bienvenue, Megan Aguilar, Carina Xu, Mollie Daugherty, Brian Woodruff, Jane A. Gross. anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4995.