A Competition‐Based Strategy for the Isolation of an Anti‐Idiotypic Blocking Module and Fine‐Tuning for Conditional Activation of a Therapeutic Antibody

The masking of therapeutic antibodies by the installation of a blocking module that can be removed under certain physiological conditions, is becoming increasingly important to improve their safety and toxicity profile. To gain access to such masking units, we used chicken immunization in combination with yeast surface display and a competition‐based FACS screening campaign to obtain anti‐idiotypic single‐chain Fv (scFv) fragments. This approach promotes the identification of functional masking units, since specificity and high affinity do not necessarily guarantee a paratope blocking effect. This strategy was used to isolate a scFv masking unit for the therapeutic antibody 6G11 (BI‐1206), which is currently in clinical trials for the treatment of B‐cell lymphoma to block the inhibitory Fcγ receptor IIB (CD32b). N‐terminal fusion of the anti‐idiotypic scFv to the 6G11 light chain successfully abolished binding to FcγRIIB in vitro. For conditional activation, a cleavable linker for the tumor‐associated protease MMP‐9 was implemented. To improve demasking efficiency, the affinity of the scFv mask was attenuated through rational design. The substitution of one key amino acid in the masking scFv reduced the affinity toward the 6G11 paratope by factor 10 but still mediated 9800‐fold blocking of receptor binding. Proteolytic demasking allowed full recovery of therapeutic antibody function in vitro, supporting the concept of conditional antibody activation using this anti‐idiotypic binding module.