Agonist Redirected Checkpoint (ARC), SIRPα-Fc-CD40L, for Cancer Immunotherapy

Abstract Combination immunotherapy with bispecific abs, linked scFv’s or T cell engagers have not shown that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule, likely due to a loss of target avidity. Fusion proteins incorporating the extracellular domain of type I membrane proteins (eg. Enbrel) or type II membrane proteins (eg. SIRPα-Fc), linked via an antibody Fc domain, are both functional despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of SIRPα (CD172a) and the ECD of CD40L, adjoined by a central Fc domain. The SIRPα end of the ARC binds CD47 at 3.59 nM affinity, but does not cause hemolysis, compared to CD47 mAbs. The CD40L end binds CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates NFκB-functional activity (independent of Fc receptor cross-linking) and IL2 and TNFα secretion in an ex vivo super-antigen (SEB) assay. Furthermore, live cell imaging shows that SIRPα-Fc-CD40L enhanced phagocytosis of tumor cells by primary macrophages. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to either CD47 blocking antibody, CD40 agonist antibody or their combination. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed. These data demonstrate feasibility of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which may uniquely poise macrophages in the TME for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis.