mRNA-Lipid Nanoparticle (LNP) Delivery of Humanized EpCAM-CD3 Bispecific Antibody Significantly Blocks Colorectal Cancer Tumor Growth
The epithelial cell adhesion molecule (EpCAM) is often overexpressed in many types of tumors, including colorectal cancer. We sequenced and humanized an EpCAM mouse antibody and used it to develop bispecific EpCAM-CD3 antibodies. Three different designs were used to generate bispecific antibodies such as EpCAM-CD3 CrossMab knob-in-hole, EpCAM ScFv-CD3 ScFv (BITE), and EpCAM ScFv-CD3 ScFv-human Fc designs. These antibody designs showed strong and specific binding to the EpCAM-positive Lovo cell line and T cells, specifically killed EpCAM-positive Lovo cells and not EpCAM-negative Colo741 cells in the presence of T cells, and increased T cells’ IFN-gamma secretion in a dose-dependent manner. In addition, transfection of HEK-293 cells with EpCAM ScFv-CD3 ScFv human Fc mRNA-LNPs resulted in antibody secretion that killed Lovo cells and did not kill EpCAM-negative Colo741 cells. The antibody increased IFN-gamma secretion against Lovo target cells and did not increase it against Colo741 target cells. EpCAM-CD3 hFc mRNA-LNP transfection of several cancer cell lines (A1847, C30, OVCAR-5) also demonstrated functional bispecific antibody secretion. In addition, intratumoral delivery of the EpCAM-CD3 human Fc mRNA-LNPs into OVCAR-5 tumor xenografts combined with intravenous injection of T cells significantly blocked xenograft tumor growth. Thus, EpCAM-CD3 hFc mRNA-LNP delivery to tumor cells shows strong potential for future clinical studies.
Fig. EpCAM-CD3 hFc mRNA-LNPs delivered to OVCAR-5 tumors with intravenous injection of T cells significantly decreased OVCAR-5 xenograft tumor growth. (A) The schedule for mRNA-LNP intratumoral (i.t.) injections and three T cell intravenous (i.v.) injections to OVCAR-5 tumor groups. Note: c. marks cellular delivery of EpCAM-CD3-hFc mRNA-LNP pre-mixed with OVCAR-5 cancer cells; i.t. marks intratumoral delivery; i.v. marks intravenous delivery of T cells. (B) EpCAM-CD3 hFc mRNA-LNPs combined with T cells significantly decreased OVCAR-5 xenograft tumor growth. (C) The schedule for mRNA-LNP intratumoral injection and a single injection of T cells i.v. delivered to OVCAR-5 xenograft tumor model. (D) EpCAM-CD3 hFc mRNA-LNPs significantly decreased OVCAR-5 tumor growth combined with a single injection of T cells. (E) Tumor size and weight significantly decreased with EpCAM-CD3-hFc mRNA-LNP and T cell treatment. Images of tumors (left panel) are shown at the end of experiment. Bars with averages of tumor weight are shown in the right panel. For tumor weights: * p < 0.05, EpCAM-CD3 hFc mRNA-LNP group versus PBS and GFP mRNA-LNP groups, Student’s t-test. (F) There was no increase in ALT, AST, amylase, or LDH levels in mouse serum collected at the end of treatment for the EpCAM-CD3-hFc mRNA-LNP group versus the GFP mRNA-LNP group for the OVCAR-5 xenograft tumor model.
Cancers 2023, 15 (10), 2860