To prevent systemic T cell activation and on-target/off-tumor toxicity, Geiger et al. designed a T cell bispecific antibody approach comprising an inert Fc region, two antigen-binding Fabs (targeting FOLR1, overexpressed in ovarian and lung cancers), and an anti-CD3 Fab fused with a cleavable linker to an anti-idiotypic anti-CD3 scFv that acted as a mask to prevent CD3 ligation. The linker was cleaved by proteases found within the TME, leading to target cell killing in vitro (in human cancer cell lines), activation (in undigested patient-derived tumor samples), and tumor growth inhibition in vivo, while sparing normal lung and kidney cells.
Contributed by Anna Scherer
ABSTRACT: T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.