Targeted CD47 checkpoint blockade using a mesothelin-directed antibody construct for enhanced solid tumor-specific immunotherapy.

The immune checkpoint CD47 is highly upregulated in several cancers as an innate immune escape mechanism. CD47 delivers a "don't eat me" signal to its co-receptor signal regulatory protein α (SIRPα), thereby inhibiting phagocytosis. Blocking the CD47-SIRPα axis is a promising immunotherapeutic strategy against cancer. However, early trial data has demonstrated on-target off-leukemia toxicity. In addition, the ubiquitous expression pattern of CD47 might contribute to an antigen sink. In this study, we combined low-affinity CD47 checkpoint blockade and specific tumor targeting in a multivalent and multifunctional antibody construct to prevent CD47-related toxicities. First, we established a local inhibitory checkpoint monoclonal antibody (LicMAb) by fusing two N-terminal extracellular domains of SIRPα to a full-length anti-human mesothelin (MSLN)-IgG1 antibody, a well-described tumor-associated antigen in epithelial ovarian cancer (EOC) and pancreatic ductal adenocarcinoma (PDAC). Next, we evaluated the SIRPα-αMSLN LicMAb for mediating a tumor-restricted immune response as observed by antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Our data validates CD47 and MSLN as highly upregulated targets expressed on various solid cancer entities, particularly EOC. We show tumor-specific binding and CD47 blocking by the SIRPα-αMSLN LicMAb even in the presence of healthy CD47-expressing cells. Furthermore, the LicMAb induces NK-cell-mediated cytotoxicity and improves phagocytosis of EOC and PDAC tumor cells. Moreover, cell death in EOC-derived organoids was specifically LicMAb-driven. Hence, the SIRPα-αMSLN LicMAb combines a tumor-restricted blockade of the CD47-SIRPα axis with a specific antitumor response while preventing on-target off-tumor toxicities. Our data supports the multifunctional SIRPα-αMSLN LicMAb as a promising approach to treating solid tumors.