Targeting B-Cell Malignancies with Anti-ROR1 CAR T-Cell Therapy

Abstract

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy has been shown to induce highcomplete response rates in the majority of B-cell lymphoma patients. However, over 50% ofpatients relapse within one year. A major cause of failure appears to be due to loss of CD19expression on the tumor cell surface. This indicates that novel therapeutic strategies are stillneeded in clinic. Receptor tyrosine kinase like orphan receptor 1(ROR1) is an oncofetal receptorfor Wnt5α that is expressed in multiple embryotic tissues but absent in virtually all adult tissues.However, ROR1 is expressed at high levels in chronic lymphocytic leukemia (CLL), mantle celllymphoma (MCL), diffuse Large B-Cell Lymphoma (DLBCL), and several solid tumors. The highexpression level of ROR1 in tumor cells is associated with adverse clinical prognosis. Inaddition, a small percentage of cancer cells with characteristics of undifferentiated leukemiacells or cancer stem cells were found to overexpress ROR1. Using hybridoma technology, wegenerated an anti-ROR1 monoclonal antibody that specifically recognized ROR1 protein. Theantibody bound to ROR1-expressing MCL cell lines but not to normal donor T cells, B cells, ormonocytes, indicating the specificity of the antibody. Lentiviral transduction of a CAR moleculederived from the anti-ROR1 antibody into primary human T cells redirected their specificityagainst B-cell lymphoma cell lines. We found the anti-ROR1 CAR T cells specifically lysed B-cell lymphoma tumor cells at high efficiency but not normal B cells. At an effector: target ratio of1:1, the anti-ROR1 CAR T cells lysed over 95% of B-cell lymphoma tumor cells during 3-days ofcoculture. To determine the optimal CAR construct, we evaluated various hingetransmembrane, and costimulatory domains in the CAR molecule. We found that incorporationof the CD28 hinge and transmembrane domain in the CAR construct dramatically enhanced thein vitro lysis efficiency of anti-ROR1 CAR-T cells. In conclusion, we developed a novel andpotent anti-ROR1 CAR T-cell therapy product that may be used for treatment of various B-cellmalignancies and ROR1-expressing solid tumors.