Development of a Novel, Allogeneic GPRC5D-Directed CAR for Treatment of Multiple Myeloma Patients

Abstract

Despite the success of BCMA-targeted therapy in advanced multiple myeloma, the disease is still considered incurable. Relapse following BCMA-directed cellular and antibody-based therapies is commonly associated with decreased surface expression of BCMA on malignant plasma cells. The G protein-coupled receptor, class C group 5 member D (GPRC5D) represents an additional target whose expression is limited to bone marrow and hair follicles, with selectively increased expression on malignant plasma cells. Thus far, the use of GPRC5D-targeted CAR T cells has provided a strong clinical benefit in multiple myeloma with limited on-target, off-tumor effects. To this end, we have validated novel, fully human GPRC5D-specific CARs for use with our hypoimmune (HIP), allogeneic CAR T cell platform. This will allow rapid delivery of allogeneic GPRC5D CAR T cells from healthy donors that simultaneously circumvent “non-self” recognition through TCR, B2M and CIITA gene disruption and promote persistence through innate immune evasion by leveraging CD47 overexpression. For early selection of candidate GPRC5D binders, T cells from healthy donors were transduced with VSV-G pseudotyped lentivirus containing second-generation, fully-human GPRC5D CARs. A clinically validated GPRC5D CAR was produced in similar fashion and used as benchmark control for initial characterization experiments. Transduction efficiency of GPRC5D CARs was analyzed via flow cytometry. GPRC5D CAR efficacy was analyzed in vitro via 24-hour luciferase or long-term Incucyte cytotoxicity assays against RPMI-8226 and MM.1S multiple myeloma target cells. Systemic MM.1S in vivo tumor models were performed in NSG mice to further assess efficacy of lead CAR candidates. Here we show our candidate GPRC5D CARs demonstrate in vitro cytotoxicity and effector cytokine production that is comparable to clinically validated benchmark controls. In 5-day Incucyte cytotoxicity assays, GPRC5D CAR T cells expand up to 10-fold and reduce tumor cell counts at starting E:T ratios of 1:8. Furthermore, in MM.1S in vivo tumor challenge models, these GPRC5D CARs significantly reduce flux and prolonged survival relative to tumor only controls with tumor clearance that was in line with clinical benchmark controls. Using ex vivo VSV-G production, our GPRC5D CAR T cells control multiple myeloma tumor cells both in vitro and in vivo, demonstrating efficacy that is on par with GPRC5D clinical benchmark CAR T cells. Studies to further define the efficacy of HIP-modified GPRC5D CAR T cells are ongoing. Moving forward, further interrogating GPRC5D CAR activity against multiple myeloma tumor cells with varying levels of target antigen expression will be critical to understanding the benefit of deploying GPRC5D CAR T cells within the multiple myeloma clinical landscape.