P1435: PRECLINICAL EVALUATION OF ZILOVERTAMAB-BASED ANTI-ROR1 CHIMERIC ANTIGEN RECEPTORS IN NK AND T CELLS

Abstract

Background: The receptor tyrosine kinase-like orphan receptor ROR1 is a promising target for immunotherapies of many cancers, including mantle cell lymphoma, diffuse large B-cell lymphoma, B-cell acute lymphocytic leukemia and chronic lymphocytic leukemia. Normally only expressed during embryonic development and only minimally, if at all, in adult normal tissues, ROR1 is overexpressed on malignant cells. Zilovertamab is a monoclonal antibody binding ROR1 that is currently in Phase 1/2 clinical trials. The antibody blocks survival signals mediated by Wnt5a via the ROR1 receptor. A different approach is the use of the antigen binding domain of the antibody in chimeric antigen receptors (CARs) expressed in NK or T cells to induce cellular cytotoxicity against ROR1-expressing cancer cells. Aims: This study aimed to evaluate different CAR designs in NK cells and T cells for cytotoxicity and cytokine production in vitro and control of cancer growth in a murine in vivo model. Methods: Two CARs using different linkers between the single chain antibody fragment (scFv) and the transmembrane domain were designed and cloned into lentiviral vectors. The two constructs were expressed in the cell line NK92 and the cells’ response against ROR1-positive and negative cancer cell lines was measured by degranulation and interferon-γ production. The construct that showed a stronger activation was tested in an in vivo model. Immune deficient NSG mice were injected with ROR1-positive JEKO-1 cells transgenic for luciferase. The cancer cell burden was monitored by intravital imaging. When cancer cell growth was established, the mice received either 1, 3, or 9 x 106 human T cells expressing the CAR or untransduced T cells. Results: NK92 cells expressing either of the two CARs showed enhanced degranulation and IFN-γ production in response to the ROR1-expressing cancer cell lines RPMI 8226 and JEKO-1. NK cells expressing the CAR containing the shorter linker between the scFv and the transmembrane domain exhibited higher activation levels. Primary human T cells expressing this CAR construct demonstrated degranulation and cytokine production during co-incubation with the ROR1-expressing JEKO-1 cell line but not the ROR1-negative Jurkat cell line. Injection of anti-ROR1 human CAR-T cells into NSG mice with established cancer growth greatly reduced the cell burden of JEKO-1 while untransduced T cells were unable to control cancer cell growth. Summary/Conclusion: The zilovertamab-based CAR tested in this study enhances the NK cell response towards ROR1-expressing cancer cells and enables T cells to clear fast-growing cancer cells in an in vivo model. These findings support the development of anti-ROR1 cell therapies for the potential treatment of ROR1-positive hematological malignancies.