Agonist Redirected Checkpoint (ARC), SIRPα-Fc-CD40L, for Cancer Immunotherapy Suresh de Silva, George Fromm, Arpita Patel, Kellsey Johannes, Josiah C. Hornblower and Taylor H. Schreiber Shattuck Labs, Inc. Austin, TX & Durham, NC
Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule, which is likely due to a loss of target avidity. Fusion proteins incorporating the extracellular domain (ECD) 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 both in vitro and in vivo, but does not cause hemolysis or hemagluttination in vitro as compared to CD47 mAbs. The CD40L end of the ARC binds immobilized CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates functional activity (independent of Fc receptor cross-linking) in CD40-dependent NFκB-luciferase reporter cells. Addition of SIRPα-Fc-CD40L to an ex vivo super-antigen (SEB) cytokine release assay stimulated secretion of IL2 and TNFα from human PBMC. Furthermore, live cell imaging demonstrated 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 combination antibody therapy. Notably, the superior anti-tumor immunity observed with SIRPα-Fc-CD40L was not accompanied by mucosal inflammation and weight loss stimulated by murine CD40 agonist antibodies. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule. Signal replacement of CD47 by CD40L may uniquely poise macrophages in the tumor microenvironment for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis.