Abstract 1489: Development of a humanized ROR1 x CD3 bispecific DART® molecule for the treatment of solid and liquid tumors
Authors: Barat Bhaswati , Chichili Gurunadh , Ciccarone Valentina , Tamura James , Gorlatov Sergey , Spliedt Michael , Chen Francine , Koenig Scott , Moore Paul , Bonvini Ezio , Alderson Ralph , Johnson Syd 
Published: July 2016

Abstract
Introduction: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in chronic lymphocytic leukemia and a subset of solid tumors, including lung, breast, ovarian, colon, and pancreatic cancers, as well as sarcoma. Limited adult tissue expression and its absence in normal leukocytes makes ROR1 a promising cancer therapeutic target. We have developed a Dual-Affinity Re-Targeting (DART®) protein for redirecting T lymphocytes to lyse tumor cells via monovalent recognition of ROR1 on tumors and CD3 on T cells. ROR1 x CD3 DART protein was engineered for improved half-life with the incorporation of a modified Fc domain, lacking effector function.
Methods: The ROR1 x CD3 DART protein was stably expressed in CHO cells and purified to homogeneity by a standard antibody platform. Bispecific binding was evaluated by ELISA and SPR analysis. In vitro functional studies were performed with lymphoma and solid tumor cell lines in the presence of primary human T cells. Tumor growth inhibition was evaluated in NOD/SCID/IL-2 gamma chain KO (NOG) mice coimplanted with human T cells and either mantle cell lymphoma (MCL) or lung cancer cell lines (5:1 effector : target cell ratio) followed by treatment with ROR1 x CD3 DART protein by intravenous (IV) administration. In vivo activity was also evaluated in human PBMC-reconstituted NOG/B2m deficient mice bearing established intradermal tumor xenografts following IV treatment with ROR1 x CD3 DART molecule. Pharmacokinetic analysis of the DART molecule was performed in human neonatal Fc receptor (hFcRn) transgenic mice.
Results: The ROR1 x CD3 DART protein displayed the expected bispecific binding for ROR1 and CD3 antigens and retained the affinity and specificity of the parent mAbs. The DART molecule mediated dose-dependent lysis of ROR1-positive MCL and solid tumor (breast, lung, and osteosarcoma) cell lines through recruitment of human T cells. DART molecule-mediated killing of ROR1-expressing target cells was accompanied by target-dependent T-cell activation and cytokine release; however, no activity was observed in the absence of target cells and no cytokine release was observed with human PBMCs alone. The ROR1 x CD3 DART protein displayed extended circulating half-life after administration to hFcRn-transgenic mice. In mouse efficacy studies, the growth of HBL-2 (MCL), HOP-92 (lung cancer), or NIH-1975 (a lung cancer line resistant to erlotinib) cells co-implanted with human T cells in NOG mice was inhibited by treatment with the ROR1 x CD3 DART protein at doses in the mcg/kg range. The ROR1 x CD3 DART molecule also demonstrated antitumor activity with high complete response rates in human PBMC-reconstituted mice bearing established HBL-2 cell xenografts.
Conclusion: The promising in vitro and in vivo activity of the Fc-bearing ROR1 x CD3 DART molecule supports further investigation as a potential candidate for the cancer treatment.
Citation Format: Bhaswati Barat, Gurunadh Chichili, Valentina Ciccarone, James Tamura, Sergey Gorlatov, Michael Spliedt, Francine Chen, Scott Koenig, Paul Moore, Ezio Bonvini, Ralph Alderson, Syd Johnson. Development of a humanized ROR1 x CD3 bispecific DART® molecule for the treatment of solid and liquid tumors. [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 1489.