Immunocompetent mouse models of cancer reveal the superiority of cellular targets over stromal targets for the development of anticancer bispecific antibodies

Abstract Bispecific antibodies are a rapidly advancing class of biopharmaceuticals with substantial potential for cancer immunotherapy. While bispecific antibodies have shown notable success in treating certain hematological malignancies, their application for solid tumors remains limited. The extra domain B of fibronectin (EDB) represents a promising pan-tumoral stromal target, offering an attractive alternative to conventional cellular tumor antigens, which often face limitations with respect to specificity in solid tumors. In this study, we describe the generation and characterization of a T cell engaging bispecific antibody which targets murine CD3 using the 2C11 clone and EDB with the L19 clone. Specifically, the bispecific antibody consists of a Fab fragment (targeting CD3) fused with two single-chain Fv fragments (targeting EDB) at the C-terminus. The bispecific antibody was produced in Chinese Hamster Ovary cells and purified to homogeneity. To compare stromal and cellular targeting, two murine tumor cell lines naturally secreting EDB in the stroma were transduced to express the target on the cell surface. In both cell lines, biodistribution analysis revealed increased tumor uptake in the cellular model compared to the stromal one. Similarly, treating immunocompetent cellular EDB tumor-bearing mice with the bispecific antibody improved anti-cancer activity. By contrast, no significant therapeutic benefit was observed in the stromal model. These findings underscore the importance of direct tumor cell targeting compared to stromal targeting for effective bispecific antibody therapy.