mRNAs encoding NKG2D ligand-targeted bispecific T-cell engagers confer robust antitumor activity

Abstract

Natural killer group 2, member D (NKG2D) ligands are highly expressed in various tumor cells and therefore represent an attractive target for cancer immunotherapy. A bispecific T-cell engager (BiTE) with a fused extracellular domain of NKG2D and an anti-CD3 single-chain fragment variable (scFv) has shown significant anti-tumor potency. To circumvent the manufacturing challenges and short serum half-life of BiTEs, we developed lipid nanoparticle-encapsulated messenger RNA (mRNA) encoding α-CD3-mNKG2D fusion proteins. In vitro functional analysis revealed that the BiTE-encoded mRNA induced T cell activation and cell cytotoxicity against tumor cells. An in vivo study using an A20 lymphoma mouse model showed that BiTE-encoding mRNA significantly inhibited tumor growth. Notably, BiTE constructs without an antibody Fc domain exhibited markedly higher anti-tumor efficacy than BiTE constructs with an Fc fragment. Additionally, we designed a human version of the BiTE mRNA encoding an NKG2D and an anti-CD3 (OKT3) scFv fusion protein. Human BiTE mRNA treatment significantly upregulated CD69 expression on T cells, promoted cytotoxicity against tumor cells in vitro, and suppressed tumor growth in a peripheral blood mononuclear cell-engrafted, tumor-bearing mouse model. Based on these preclinical findings, NKG2D BiTE mRNA holds promise as a potential anti- tumor treatment meriting further clinical development.

Keywords

Messenger RNA / NKG2D / CD3 / Bispecific T-cell engager / Cancer immunotherapy

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