Abstract 2470: Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies
Authors: Pietsch E. Christine , Dong Jianying , Zhang Xiaochun , Chin Diana , Hawkins Rebecca , Dinh Thai , Zhou Mimi , Strake Brandy , Feng Ping-Hua , Rocca Meredith , Dos Santos Cedric , Shan Xiaochuan , Danet-Desnoyers Gwenn , Kaiser Elizabeth , Quinn Hillary , Nemeth Jeffrey A. , Attar Ricardo 
Published: August 2015

Abstract
CD47 is a ubiquitously expressed cell surface Ig superfamily member. CD47 mediates a variety of biological processes, including leukocyte adhesion/migration, T-cell activation, apoptosis, and phagocytosis due to physical interaction with various proteins (integrins, thrombospondin-1, and signal regulatory protein alpha [SIRPα]). The CD47-SIRPα interaction negatively regulates phagocytosis.
Enhanced CD47 expression has been described on hematological and solid cancers, suggesting that CD47 may mediate cancer cell escape from immune surveillance (e.g., by macrophages or neutrophils). Published studies demonstrated that anti-CD47 antibodies induced phagocytosis or apoptosis of cancer cells in vitro and mediated in vivo anti-tumor efficacy.
Hypothesizing that blocking the CD47-SIRPα interaction will lead to phagocytosis and elimination of tumor cells, we generated 23 unique anti-CD47 monoclonal antibodies (mAbs), which potently block the CD47-SIRPα interaction. Upon further functional testing and optimization, three candidates emerged with nanomolar affinity to human and cynomolgus monkey CD47 and lack of hemagglutination and platelet aggregation activity: C47B157, C47B161 and C47B222 (human CD47 KD 3.53, 2.87, and 1.12 nM, respectively). To further characterize these antibodies and to better understand the contribution of merely blocking the CD47-SIRPα interaction to in vitro and in vivo anti-tumor activity, the mAbs were cloned into an effector function silent (IgG2sigma) and competent (IgG1) Fc backbone. In vitro ADCP assays demonstrated that IgG1 C47B157, C47B161, and C47B222 enhanced phagocytosis 4-fold over PBS control, while the IgG2sigma mAbs did not enhance phagocytosis. Subsequently, the anti-tumor activity was characterized in three human acute myeloid leukemia (AML) xenograft models (HL60/NSG, MV4-11/NSG, and Kasumi-3/NSG mice). At 10mg/kg C47B157, C47B161, and C47B222 completely suppressed growth of leukemia cells across all models in peripheral blood as IgG1 (0.74-2.78%) vs. control (23.2-88.5%) at time of control sacrifice. As IgG2sigma, C47B222 caused greatest suppression at 10 mg/kg in all models (1.6-2.2%). Additional in vivo studies with either AML cell lines or AML patient-derived primary cells revealed that although IgG2sigma C47B222 reduced disease outgrowth in peripheral blood and distinct organs (e.g., spleen), merely blocking the CD47-SIRPα interaction did not yield a survival advantage. In non-human primates, a single dose of IgG1 C47B222 caused ∼40% decrease in red blood cell count, hematocrit and hemoglobin at 1 mg/kg. In contrast, IgG2sigma C47B222 had minimal impact on red cell indices at 1 and 10 mg/kg.
In conclusion, the findings suggest that efficacy and tolerability of anti-CD47 mAbs are Fc effector function dependent. The need to balance efficacy and tolerability raise questions on the feasibility of targeting CD47 in the development of cancer therapeutics.
Citation Format: E. Christine Pietsch, Jianying Dong, Xiaochun Zhang, Diana Chin, Rebecca Hawkins, Thai Dinh, Mimi Zhou, Brandy Strake, Ping-Hua Feng, Meredith Rocca, Cedric Dos Santos, Xiaochuan Shan, Gwenn Danet-Desnoyers, Elizabeth Kaiser, Hillary Quinn, Jeffrey A. Nemeth, Ricardo Attar. Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2015-2470