Abstract
Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancers by potentiating antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing the phagocytosis of tumor cells by macrophages and enhancing the cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; both of these processes are potentiated by CD40 signaling. Here we generated a novel, two-sided fusion protein incorporating the extracellular domains of SIRPα and CD40L, adjoined by a central Fc domain, termed SIRPα-Fc-CD40L. SIRPα-Fc-CD40L bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor cross-linking. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Murine SIRPα-Fc-CD40L outperformed CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and synergized with immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated a type I interferon response in macrophages and potentiated the activity of ADCP-competent targeted antibodies both in vitro and in vivo These data illustrated that whereas CD47/SIRPα inhibition could potentiate tumor cell phagocytosis, CD40-mediated activation of a type I interferon response provided a bridge between macrophage- and T-cell-mediated immunity that significantly enhanced durable tumor control and rejection.
Highlights
Tumor immunogenicity results from the processing, and subsequent presentation of tumor antigens released from dead or dying tumor cells on MHC molecules expressed on antigen-presenting cells (APC)
Purified SIRPa-Fc-CD40 ligand (CD40L) was analyzed for the presence of each individual domain by Western blotting using anti-SIRPa, anti-Fc, and anti-CD40L (Fig. 1B), revealing a glycosylated protein that formed a dimer under nonreducing conditions at the predicted monomeric molecular weight of 88.1 kDa
Calreticulin on tumor cells serves as a prophagocytic signal, facilitating tumor cell phagocytosis following blockade of the CD47/SIRPa pathway [36]. We demonstrated that both calreticulin and Fc receptor engagement was required for efficient phagocytosis of CD20þ B-cell lymphoma cells by the combination of SIRPa-Fc-CD40L and rituximab using a calreticulin-blocking peptide, confirming the importance of Fc interactions for Fc-competent targeting antibodies and providing evidence that the initiation of phagocytosis by SIRPa-Fc
Summary
Tumor immunogenicity results from the processing, and subsequent presentation of tumor antigens released from dead or dying tumor cells on MHC molecules expressed on antigen-presenting cells (APC). CD47 is expressed by many somatic and hematopoietic tissues and prevents red blood cell (RBC) and platelet destruction by macrophages and splenic CD4þ dendritic cells Studies hypothesized that the role of CD47 as a macrophage “do not eat me” signal might explain the observed antitumor benefit following treatment with CD47 blocking antibodies or SIRPa-Fc fusion proteins [8,9,10]. Other studies extended these observations to show that SIRPa signaling in CD8aþ DCs enhances cGAS/STINGmediated sensing of phagocytosed tumor mitochondrial DNA, pro-
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