mRNA-Lipid Nanoparticle (LNP) Delivery of Humanized EpCAM-CD3 Bispecific Antibody Significantly Blocks Colorectal Cancer Tumor Growth

Abstract

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.

Lipid Nanoparticle (LNP) Delivery Carrier-Assisted Targeted Controlled Release mRNA Vaccines in Tumor Immunity

“…In the field of tumor therapy, the application of LNP-assisted mRNA vaccines has aroused extensive research interest [ 154 ]. In past studies [ 155 , 156 , 157 , 158 , 159 , 160 ], researchers have achieved a series of encouraging results by using LNP carriers to deliver mRNA vaccines to tumor models (such as liver cancer). Some studies [ 161 , 162 , 163 , 164 ] have shown that LNP carriers can effectively deliver mRNA vaccines and stimulate tumor antigen-specific immune responses in liver tumor mouse models.…”

Informed by Cancer Stem Cells of Solid Tumors: Advances in Treatments Targeting Tumor-Promoting Factors and Pathways

“…Catumaxomab is a similar BiTE antibody construct targeting EpCAM and CD3 that was initially approved in the European Union in 2009 for intraperitoneal treatment of malignant ascites but was withdrawn for commercial reasons in 2017 [101]. Interestingly, a recent paper on mRNA-lipid nanoparticle (LNP) delivery of EpCAM-CD3 BiTEs shows promise in vivo [102]. Intratumoral injection of EpCAM-CD3 hFc mRNA-LNPs (1 µg/mouse) in combination with intravenous injection of T cells significantly decreased OVCAR-5 tumor growth in mice [102].…”

“…Interestingly, a recent paper on mRNA-lipid nanoparticle (LNP) delivery of EpCAM-CD3 BiTEs shows promise in vivo [102]. Intratumoral injection of EpCAM-CD3 hFc mRNA-LNPs (1 µg/mouse) in combination with intravenous injection of T cells significantly decreased OVCAR-5 tumor growth in mice [102].…”

mRNA-based vaccines and therapeutics: an in-depth survey of current and upcoming clinical applications

“…When used with mRNA-LNP technology, these antibodies showed high specificity for killing EpCAM + T cells. The EpCAM scFv-CD3 scFv-human Fc also significantly blocked OVCAR-5 xenograft tumor growth in vivo [ 69 ]. The applications of in vivo therapeutic antibody expression as treatments for specific diseases are summarized in Fig.…”