C1q-targeting bispecific antibodies bypass Fc to trigger complement activation and receptor clustering agonism

Abstract

Complement-dependent cytotoxicity (CDC) and receptor clustering that triggers outside-in signaling are effector functions employed by various therapeutic antibodies to kill their target cells. However, not all antibodies, even of the same subclass, efficiently engage these modes of action, because of constraints like antigen density, clustering, and orientation. To overcome these limitations, we developed bispecific antibodies (bsAbs), binding both the classical pathway initiator C1q and cellular targets, aiming to enhance complement activation and enable C1q-driven receptor clustering. Human IgG1 bsAbs were produced, combining four C1q-binding clones with antibodies binding CD20, CD37, HER2, EGFR, GITR and DR5. Antigen binding, complement activation, CDC and receptor clustering were evaluated on various cancer cell lines, including experiments to uncouple Fab-C1q interaction from Fc-C1q interaction. The bsAbs provided more C1q binding and often more complement C3 deposition and CDC than the corresponding monovalent antibody without C1q-binding Fab-arm. Experiments using bsAbs with inactive Fc domain (LALAPG) and bsAb Fab2 fragments indicate that complement activation by these bsAbs do not require the Fc domain. BsAbs with different C1q-targeting Fab-arms all increase C1q binding, but only some enhance complement activation, likely due to the epitope location on C1q. Furthermore, bsAbs strongly induced receptor clustering of GITR and DR5 in presence of C1q, thereby triggering outside-in signaling. We conclude that receptor clustering and complement activation by therapeutic bsAbs through Fab-C1q binding is feasible. The latter can improve complement activation over traditional Fc-mediated activation for mAbs that naturally do not activate complement well.