329 Pre-clinical efficacy of a novel anti-GPC3 in vivo CAR-M for hepatocellular carcinoma

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Background

Chimeric antigen receptor macrophage (CAR-M) cell therapies have the potential to mediate robust anti-tumor immunity via phagocytosis, cytokine/chemokine release, antigen presentation, activation of the tumor microenvironment (TME) and T cell recruitment. Phase I data have demonstrated that autologous ex vivo CAR-M are well-tolerated, induce reprogramming of the solid TME, promote epitope spreading, and mediate anti-tumor activity.¹ Here, we have developed a novel off-the-shelf approach to directly reprogram endogenous myeloid cells in vivo by systemically delivering lipid nanoparticles (LNP) encapsulating mRNA encoding CARs targeting glypican-3 (GPC3). GPC3 is a tumor-associated surface antigen that is overexpressed in hepatocellular carcinoma (HCC) with minimal expression on normal tissues. The CAR architecture was optimized to maximize antigen-dependent myeloid activation.

Methods

Primary human monocytes and macrophages were used to assess binding, phagocytosis, killing, and cytokine release using flow cytometry, Incucyte^®-based cytotoxicity assays, and Meso Scale Discovery (MSD) or Nomic Bio based cytokine release assays. The efficacy and tolerability of CAR-encoding mRNA/LNP were evaluated in vivo in CD34+ humanized mice harboring GPC3+ solid tumor xenografts.

Results

In vitro analysis showed that anti-GPC3 CAR-M had a high binding affinity to GPC3, and lack of binding to related glypican proteins. A CAR comprising a hinge, transmembrane, and signaling domain optimized for myeloid cells demonstrated enhanced antigen-dependent macrophage activation without tonic signaling. Functional evaluation demonstrated CAR expression on human macrophages for more than 7 days in vitro leading to dose-dependent cytotoxicity against multiple GPC3+ tumor lines. At physiologic levels, soluble GPC3 did not interfere with the cytotoxic activity of anti-GPC3 CAR-M. In humanized immune system mice with disseminated GPC3+ solid tumors, systemic administration of anti-GPC3 CAR LNP/mRNA led to robust anti-tumor activity and suppressed tumor lesions in the liver. Myeloid cells were the primary CAR+ cells after systemic LNP administration. Treated animals tolerated repeat mRNA/LNP administration with no significant elevation in plasma cytokine levels or other signs of toxicity.

Conclusions

These data demonstrated that anti-GPC3 CAR-M can be directly produced in vivo utilizing mRNA/LNP, reducing tumor burden in translationally relevant humanized solid tumor models. In vivo CAR-M represents a novel off-the-shelf cell therapy strategy for patients with GPC3+ solid tumors, including HCC.

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