Abstract PR06: Targeting CD47 as a novel immunotherapy for multiple myeloma
Authors: Sun Jennifer , Muz Barbara , Alhallak Kinan , Egbulefu Christopher , King Justin , Kohnen Danny , Fiala Mark , Cappocia Benjamin , Manning Pamela , Achilefu Samuel , Vij Ravi , Azab Abdel Kareem 
Published: June 2021

Abstract
Introduction: CD47 is an immune checkpoint highly expressed on the surface of various cancer cells, allowing cells to escape innate immune response by sending inhibitory signals to macrophages, which further hinders the antitumor response by the adaptive immune system. This CD47 “don’t-eat-me signal” has been identified as a novel therapeutic target in cancers. Targeted reprogramming of this interaction using anti-CD47 monoclonal antibodies (mAbs) represents a new class of checkpoint inhibitors that attack tumors via coordinating the innate and adaptive immune systems. Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow (BM). The objective of this study is to investigate the inhibition of CD47 using anti-CD47 mAbs as a novel checkpoint immunotherapy for the treatment of MM.
Methods: We performed analysis of CD47 mRNA expression in healthy, MGUS (pre-condition of MM), and MM subjects. We measured CD47 protein expression on MM cell lines by flow cytometry, labeling MM cells with Vx1000R (anti-human CD47 mAb). We also investigated the effect of hypoxia (1% O2), stromal cells, and 3D tissue engineered bone marrow (3DTEBM) cultures on CD47 expression in MM cells. Next, we tested the cytotoxic activity of Vx1000R on MM cells alone by MTT assay. Additionally, we investigated the effect of Vx1000R on phagocytosis and killing of MM cells (GFP+) by primary macrophages (labeled with DiD dye) at 4 and 24 hours with or without Vx1000R, in 2D cultures and in 3DTEBM cultures. Phagocytosis was determined as GFP+ DiD+ cells and MM survival was analyzed by counting GFP+ by flow cytometry. Moreover, we performed real-time live confocal imaging on 3DTEBM cultures of GFP-MM cells and DiD-macrophages, with or without treatment of Vx1000R, to further visualize the effect of Vx1000R.
Results: CD47 gene expression was increased with MM progression (healthy&lt;MGUS&lt;MM). CD47 protein was highly expressed in MM cells lines (MM.1S, H929, and U266) and primary MM cells. The expression of CD47 was not affected by hypoxia or co-culture with stromal cells in 2D cultures; however, 3DTEBM significantly increased CD47 expression. Vx1000R alone (without macrophages) did not result in direct killing of MM cells in 2D culture or in the 3DTEBM. Vx1000R treatment in presence of macrophages in 2D resulted in mild effect on killing of MM cells, while cultures in 3DTEBM resulted in extensive killing of MM cells (50% killing at 4 hours and 95% killing at 24 hours).
Conclusions: Myeloma cells express high levels of CD47, and this expression is not affected by hypoxia or TME in 2D, but significantly increases in 3DTEBM cultures. Blocking CD47 on MM cells with anti-CD47 mAbs enhanced MM phagocytosis and killing by macrophages, especially in 3DTEBM. These results suggest that anti-CD47 mAbs are applicable for MM treatment, and further studies are warranted to examine the effect of anti-CD47 mAbs as a novel checkpoint immunotherapy to target MM in vivo and in patients.
This abstract is also being presented as Poster A88.
Citation Format: Jennifer Sun, Barbara Muz, Kinan Alhallak, Christopher Egbulefu, Justin King, Danny Kohnen, Mark Fiala, Benjamin Cappocia, Pamela Manning, Samuel Achilefu, Ravi Vij, Abdel Kareem Azab. Targeting CD47 as a novel immunotherapy for multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR06.