1125 Preclinical development of MT-303, a novel LNP-formulated GPC3-specific CAR mRNA, for in vivo programming of monocytes to treat hepatocellular carcinoma

<h3>Background</h3> Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The five-year survival rate for HCC is only 18%, demonstrating the high demand for more effective therapies. Glypican-3 (GPC3) is an oncofetal protein which is highly expressed in HCC but not in healthy adult tissues. Because of its tumor-restricted expression, GPC3 has been the target of bispecific antibodies and T cell therapies. Here, we describe a novel approach using an RNA/lipid nanoparticle formulation to program myeloid cells <i>in vivo</i> to express a GPC3-specific chimeric antigen receptor. <h3>Methods</h3> MT-303 is a lipid nanoparticle formulation (LNP) that encapsulates the mRNA encoding a GPC3-targeted chimeric antigen receptor (CAR). The CAR comprises a GPC3-specific single-chain variable fragment (scFv) and a short flexible linker fused with a truncated human CD89. In support of IND-enabling studies, we conducted preclinical studies to characterize the expression and function of the GPC3 CAR <i>in vitro</i> and in mouse and monkey studies. Expression and pharmacodynamic effects of the anti-GPC3 CAR were evaluated by flow cytometry, and in cytokine release and tumor killing assay. Anti-tumor efficacy of MT-303 was evaluated in a HepG2 HCC xenograft model. <h3>Results</h3> Because the initial mRNA construct resulted in short GPC3 CAR expression, the mRNA was optimized by introducing modifications in the mRNA coding sequence and 3’-UTR. These changes substantially improved the expression level and duration of CAR expression on monocytes. Accordingly, <i>in vitro</i> cytokine production and cytotoxicity were enhanced. The modifications also translated into higher expression <i>in vivo</i> and better anti-tumor efficacy in a HepG2 xenograft model. In line with the natural turnover of monocytes in blood, CAR-positive monocytes were detected up to 48 hours post i.v. injection of MT-303. Anti-tumor activity was associated with increased cytokine and chemokine levels in peripheral blood, an indicator for CAR engagement with GPC3 on tumor cells. In<i> in vitro</i> transfection of human and cynomolgus whole blood cells testing the uptake of MT-303 drug product, GPC3 CAR was selectively expressed in up to 30% of monocytes. Furthermore, dose-dependent expression of the GPC3 CAR was observed in cynomolgus monkeys upon i.v. infusion of MT-303. <h3>Conclusions</h3> Here, we present a novel GPC3-targeting CAR mRNA/LNP that enables myeloid cells to directly kill tumor cells and support the establishment a cytokine-mediated immune response. MT-303 is currently being tested in phase 1 clinical trial for the treatment of HCC. <h3>Trial Registration</h3> Clinical Trial number: NCT06478693.