Geiger, MartinaStubenrauch, Kay-GunnarSam, JohannesRichter, Wolfgang F.Jordan, GregorEckmann, JanHage, CarinaNicolini, ValeriaFreimoser-Grundschober, AnneRitter, MirkoLauer, Matthias E.Stahlberg, HenningRingler, PhilippePatel, JigarSullivan, EricGrau-Richards, SandraEndres, StefanKobold, SebastianUmaña, PabloBrünker, PeterKlein, Christian2020-08-252020-08-252020-08-252020-06-2410.1038/s41467-020-16838-whttps://infoscience.epfl.ch/handle/20.500.14299/171106T-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.text::journal::journal article::research article