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

Background

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 in vivo to express a GPC3-specific chimeric antigen receptor.

Methods

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 in vitro 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.

Results

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, in vitro cytokine production and cytotoxicity were enhanced. The modifications also translated into higher expression in vivo 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 in vitro 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.

Conclusions

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.

Trial Registration

Clinical Trial number: NCT06478693.