Fig 1: Expression and functional validation of mRNA-encoded bispecific antibodies in vitro. (A–C) ELISA quantification of (A) EpCAM×CD3, (B) Claudin18.2×41BB, and (C) both BsAbs following transfection of Expi293F cells with increasing doses of the respective mRNAs. (D–F) Functional analysis of EpCAM×CD3 BsAb: (D) T cell-dependent cytotoxicity (TDCC) against NCI-N87 cells, (E) binding to NCI-N87 tumor cells, and (F) binding to Jurkat T cells. (G–I) Functional analysis of Claudin18.2×41BB BsAb: (G) activation of a 4–1BB reporter Jurkat cell line, (H) binding to NCI-N87 cells, and (I) binding to pre-activated primary human T cells. (J) Schematic illustrating the proposed mechanism of synergistic T cell co-activation. (K) Synergistic cytotoxicity assay where a fixed concentration of EpCAM×CD3 conditioned media was combined with increasing concentrations of Claudin18.2×41BB conditioned media. Data are mean ± SD (N=3).
Fig 2: E3C4 enables sustained in vivo production of functional bispecific antibodies. (A) Schematic of the single-dose E3C4 administration and blood sampling timeline. (B and C) Serum concentration-time profiles of (B) EpCAM×CD3 and (C) Claudin18.2×41BB bispecific antibodies following a single intraperitoneal dose of E3C4 (3 or 10 μg). (D and E) Functional activity of serum-derived (D) EpCAM×CD3 (T cell-dependent cytotoxicity) and (E) Claudin18.2×41BB (4–1BB reporter activation) compared to recombinant protein standards. (F and G) Pharmacokinetic profiles of both bispecific antibodies over three weeks of repeated E3C4 administration (3 μg, weekly). Data are mean ± SD (N=5). The dotted lines denote the baseline.
Fig 3: E3C4 achieves potent antitumor efficacy by enhancing T-cell immunity with a favorable safety profile in PBMC-humanized mice. (A) Schematic of the treatment schedule. (B) Tumor growth curves and (C) final tumor weights of subcutaneous NCI-N87 xenografts. (D) Quantification of tumor-infiltrating human CD45+ (hCD45+) T cells. Proportions of Granzyme B+ (E) and Ki67+ (F) cells among hCD45+CD8+ T cells. Serum levels of IL-6 (G), IFN-γ (H), and TNF-α (I) measured 24 h after the first dose. Data are presented as mean ± SD. In Figure 4C–I #P < 0.05, ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01 vs EpCAM×CD3 group; ns, not significant (P > 0.05). N = 5 for tumor volume, weight and immune cell and cytokine analysis.
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