Cancer Therapy: Preclinical Receptor Affinity and Extracellular Domain Modifications Affect Tumor Recognition by ROR1-Specific Chimeric Antigen Receptor T Cells

Purpose: The adoptive transfer of T cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single-chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module, has activity in advanced malignancies. The receptor tyrosine kinase–like orphan receptor 1 (ROR1) is a tumor-associated molecule expressed in prevalent B-lymphoid and epithelial cancers and is absent onnormalmature B cells and vital tissues,making it a candidate for CAR T-cell therapy. Experimental Design: We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T cells expressing each CAR to recognize ROR1þ hematopoietic and epithelial tumors in vitro, and to eliminate humanmantle cell lymphoma (MCL) engrafted into immunodeficient mice. Results: ROR1-CARs containing a short "Hinge-only" extracellular spacer conferred superior lysis of ROR1þ tumor cells and induction of T-cell effector functions compared with CARs with long "Hinge-CH2CH3" spacers. CARs derived from a higher affinity scFV conferredmaximum T-cell effector function against primary CLL and ROR1þ epithelial cancer lines in vitrowithout inducing activation-induced T-cell death. T cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR–modified T cells in mediating regression of JeKo-1 MCL in immunodeficient mice. Conclusions: Our results show that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1þ tumors. T cells modified with an optimized ROR1-CARhave significant antitumor efficacy in a preclinicalmodel in vivo, suggesting theymaybeuseful to treat ROR1þ tumors in clinical applications. Clin Cancer Res; 19(12); 1–12. 2013 AACR.