Fig. 2: mRNA-encoded TCE mediates target-specific cell killing and induces target-dependent T cell activation. | Nature Communications

Fig. 2: mRNA-encoded TCE mediates target-specific cell killing and induces target-dependent T cell activation.

From: Organ-specific delivery of an mRNA-encoded bispecific T cell engager targeting glypican-3 in hepatocellular carcinoma

Fig. 2

A MTS105 is a novel LNP for efficient liver-tropic delivery with a size of 65–95 nm. mRNA encapsulated in MTS105 was optimized for improved intracellular stability and translational efficiency, including UTRs and CDS. The mRNA-encoded TCE was composed of ScFvs targeting GPC3 and CD3, separated by a flexible peptide linker. B T cell-dependent cellular toxicity assay (TDCC). Human PBMCs and Hepa1-6-hGPC3-luc or MC38-luc tumor cell lines were co-cultured for 48 h with supernatants from the MTS105-transfected 293T cell line at a 5:1 E:T ratio. rTCE and ERY974 were used as a positive control. Specific lysis was assessed via bioluminescence. C Cytokines released after MTS105-encoded TCE-induced specific lysis. IFN-γ and TNF were measured with ELISA using the supernatants from the TDCC assay (B). rTCE and ERY974 were used as a positive control. D TDCC assay. Human PBMCs and Hepa1-6-hGPC3-luc or MC38-luc tumor cell lines were co-cultured for 48 h with MTS105-encoded TCE containing mouse serum at a 5:1 E:T ratio. rTCE and ERY974 were used as a positive control. Specific lysis was assessed via bioluminescence. E MTS105-mediated CD8 T cell activation after 24 h of incubation of human PBMCs as effector cells with Hepa1-6-hGPC3-luc or MC38-luc target cells at a 5:1 E:T ratio. The concentration of 1111 pM for MTS105-transfected supernatant, rTCE and ERY974 was shown in the assay. T cell activation was assessed by measuring CD25, CD69 and CD107a levels on CD4 and CD8 T cells by flow cytometry. F MTS105-containing serum mediated cytotoxicity after coincubation of human PBMCs with GPC3+ HepG2 cells and GPC3- HL-60 cells as target cells. Cytotoxicity assays were performed at a 10:1 E:T ratio. Serum of cynomolgus monkey was collected after MTS105 administration, serially diluted, and added to the cocultures, followed by incubation for 24 h. The OD value detected with cell cytotoxicity by LDH was measured as readout. All data are expressed as mean ± SD (n = 3 biological replicates). Differences between the two groups were assessed by a two-sided Student’s t-test, while comparisons across multiple groups were performed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Back to article page