Abstract
Monoclonal antibodies (mAbs) are widely used in cancer therapy but often show limited efficacy for solid tumors. Enhancing anti-tumor activity by fusing cytokines to tumor-targeting mAbs, which specifically activate immune cells within the tumor microenvironment, represents a promising strategy. However, the optimal design and therapeutic efficacy of antibody-cytokine fusion formats remain unclear. The epithelial cell adhesion molecule (EpCAM), frequently overexpressed in a variety of carcinomas, serves as the target for immunotherapies. In this study, we identified a fully human mAb targeting EpCAM, designated as m801, from a previously constructed phage-displayed fully human antibody library. By fusing m801 with an IL-2 variant (IL-2v) in two configurations, m801.2 (2 anti-EpCAM Fab + 1 IL-2v) and m801.3 (1 anti-EpCAM Fab + 1 IL-2v), we identified m801.2 as the lead candidate due to its superior biophysical properties, including high thermal stability, homogeneity, and low aggregation. Furthermore, m801.2 showed strong binding affinity to EpCAM, with KD values of 0.6 nM, and an EpCAM-expressing tumor cell line, comparable to the original IgG m801. Additionally, m801.2 exhibited IL-2 receptor β subunit (IL-2Rβ)-biased binding activity, with a KD of 27.3 nM, resulting in superior effective T cell activation. In an SW480 xenograft mice model, m801.2 significantly inhibited tumor growth and demonstrated high tolerability. These findings suggest a valuable framework for the future design of immunocytokine therapies.