Abstract 5567: Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy
Authors: Fromm George , Silva Suresh de , Johannes Kellsey , Patel Arpita , Hornblower Josiah C. , Schreiber Taylor H. 
Published: August 2018

Abstract
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. This is likely because these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. SIRPα-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies 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 immobilized CD47 at 3.59 nM affinity and binds CD47 on the surface of human tumor cells both in vitro and in vivo, but does not bind human platelets or RBCs. Further, no hemolytic activity was observed with the human SIRPα-Fc-CD40L ARC 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 NFκB-luciferase reporter cells (CD40 driven activation of NFκB). 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, time-lapse high content live cell imaging has shown that when activated human macrophages were co-cultured with CD47 positive human tumor cells, SIRPα -Fc-CD40L was shown to enhance phagocytosis. 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 significant 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.
Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5567.