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.
Highlights
T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing
We recently described 2 + 1 TCBs consisting of an inert Fc region, two tumor antigen-binding Fab fragments and one Fab fragment binding to CD3 on the T-cell receptor[2,3]
In non-human primates, on-target toxicity has been observed when a highly potent FOLR1-TCB with EC50 values in the single-digit pM range was evaluated in tolerability experiments at single doses as low as 10 μg/kg 24
Summary
T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While FOLR1-TCB was efficacious in vitro and in xenograft models, severe on-target toxicity in the lung of non-human primates was observed[24] Based on this experience, we chose FOLR1-TCB as a relevant model to show proof-of-concept for masking the anti-CD3 moiety with an anti-idiotypic antibody scFv fused via a protease cleavable linker to the TCB. We chose FOLR1-TCB as a relevant model to show proof-of-concept for masking the anti-CD3 moiety with an anti-idiotypic antibody scFv fused via a protease cleavable linker to the TCB For this purpose, we fused a specific anti-idiotypic anti-CD3 scFv Nterminally to the anti-CD3 variable heavy chain connected by a protease cleavable linker and demonstrated that active proteases located in the tumor microenvironment lead to cleavage and subsequent unmasking of the anti-CD3 targeting moiety. Unmasking results in efficient killing of FOLR1-positive tumor cells in vitro and in vivo while sparing normal cells with low FOLR1 expression
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