Up-regulation of CD47 is an immune evasion mechanism used by different cancers to evade immune surveillance. Through its interaction with signal-regulatory protein alpha (SIRPα) on myeloid cells, CD47 delivers a universal “don’t eat me” signal to phagocytes, which prevents immune cells from efficiently eliminating tumor cells. Blockade of the SIRPα–CD47 innate immune checkpoint has therefore emerged as a new way to treat cancer. Several CD47-targeting molecules are in development with encouraging results obtained with monoclonal antibodies (mAb). However, the pharmacologic properties and the safety profile of molecules indiscriminately blocking CD47 can be improved by selectively inhibiting CD47 only on tumor cells. For this purpose, we generated bispecific antibodies (bsAbs) capable of targeting blockade of CD47 specifically to malignanT-cells through the co-engagement of a tumor-associated antigen (TAA). The bsAb NI-1801 specifically targets mesothelin (MSLN)-positive tumors. NI-1801 was shown to bind to MSLN-positive tumor cells, but not to MSLN-negative cells expressing physiologic levels of CD47 (e.g., leukocytes, erythrocytes, platelets). NI-1801 blocks the CD47-SIRPα interaction in a MSLN-dependent manner and thus minimizes the side effects related to a nonspecific blockade of CD47 on healthy cells. Studying antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) of various MSLN-positive human tumor cell lines revealed that NI-1801 markedly enhanced killing as compared to amatuximab (an anti-MSLN mAb in clinical trials) and to the corresponding anti-MSLN mAb, exemplifying the role of blocking the “don’t eat me” signal to target cancer. NI-1801 also showed efficacy in various xenograft tumor models and analysis of the tumor microenvironment (TME) revealed a significant increase in leukocyte subpopulations (macrophages/monocytes and NK cells) of NI-1801 treated mice, suggesting that NI-1801 mediates the recruitment of monocytes from blood. Additionally, NI-1801 treatment affected the ratio between MHC-II-low and MHC-II-high macrophages in the TME. Finally, nonhuman primate studies with NI-1801 demonstrated a linear elimination profile, minimal target-mediated drug disposition and no hematologic toxicity. Taken together, these results illustrate that this strategy possesses potent anticancer activities both in vitro and in vivo in conjunction with favorable pharmacologic and toxicologic profiles. Citation Format: Stefano Majocchi, Valery Moine, Xavier Chauchet, Lucile Broyer, Laura Cons, Laurence Chatel, Eric Hatterer, Vanessa Buatois, Hasnaa Haddouk, Gerard Didelot, Giovanni Magistrelli, Yves Poitevin, Ulla Ravn, Anne Papaioannou, Francoise Richard, Limin Shang, Marie H. Kosco-Vilbois, Nicolas Fischer, Walter G. Ferlin, Krzysztof Masternak. Selective blockage of the innate immune checkpoint receptor CD47 on mesothelin (MSLN) positive solid tumor cells via dual targeting bispecific antibodies alters the tumor microenvironment to control tumor growth [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A088.