Pre-Clinical Combination of AO-176, a Highly Differentiated Clinical Stage CD47 Antibody, with Either Azacitidine or Venetoclax Significantly Enhances DAMP Induction and Phagocytosis of Acute Myeloid Leukemia

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.9-10
• Main Authors: Donio, Mike J, Wilson, W. Casey, Darwech, Isra, Andrejeva, Gabriela, Capoccia, Benjamin J, Puro, Robyn J, Kashyap, Arun K, Pereira, Daniel S
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-140528

While T cell checkpoint inhibitors are mainstays of cancer immunotherapy, therapies that direct innate immune responses against cancer are lacking. CD47, a “don’t eat me” signal, is an innate immune cell checkpoint which binds SIRPα on macrophages and dendritic cells to limit phagocytosis and its upregulation on tumor cells leads to evasion of immune detection and clearance. Therapeutic antibodies have previously been developed to block CD47 and induce phagocytosis of tumor cells, thus validating the pathway. AO-176, a next generation humanized IgG2 anti-CD47 antibody, was developed to block the CD47/SIRPα interaction and induce tumor cell phagocytosis. Moreover, AO-176 directly kills tumor cells through a non-ADCC-dependent mechanism via induction of programmed cell death type III. In addition to these tumor eliminating properties, AO-176 has the potential for a strong safety profile as a result of its preferential binding to tumor versus normal cells, lack of RBC binding, and enhanced binding to tumor cells at acidic pH. CD47 has previously been shown to be upregulated on acute myeloid leukemia (AML) leukemic stem cells (LSC), enabling their expansion through evasion from phagocytic clearance. As a result, patients with increased CD47 on AML LSCs have worse overall survival. In this study, AO-176 efficacy was evaluated in AML cell lines as a single agent and in combination with azacitidine and venetoclax which are approved therapies for AML. Azacitidine is a cytosine analogue which acts to inhibit DNA methylation, and venetoclax is a potent Bcl-2 inhibitor. Previous studies have shown that azacitidine induces apoptosis of tumor cells and increases cell surface exposure of calreticulin, a DAMP (Damage Associate Molecular Pattern) which provides a strong pro-phagocytic signal. From these findings, it was hypothesized that azacitidine would enable increased tumor cell phagocytosis when combined with AO-176. The potential for a similar enhancement with a combination of AO-176 and venetoclax was also explored. The ability of AO-176, with or without azacitidine or venetoclax, to induce DAMPs on the surface of AML cells was assessed. Cell surface expression of DAMPs, calreticulin and PDIA3, were measured by flow cytometry. AO-176, azacitidine, and venetoclax as single agents potently increased both calreticulin and PDIA3 in a dose-dependent manner on AML cell lines such as HL60 This is the first time, to our knowledge, that venetoclax has been shown to induce D