A New Disulfide-Linked Dimer of a Single-Chain Antibody Fragment Against Human CD47 Induces Apoptosis in Lymphoid Malignant Cells In Vitro and In Vivo Via the HIF-1α Pathway: A Possible Novel Therapeutic Agent for B-CLL.
Authors: Sagawa Morihiko , Shimizu Takatsune , Xian Mingji , Chizuka Aki , Fukushima Naoshi , Kinoshita Yasuko , Ohizumi Iwao , Ikeda Yasuo , Okabe Hisafumi , Kizaki Masahiro 
Published: October 2019

Abstract
CD47 belongs to the immunoglobulin superfamily, and is expressed as a 50 kDa cell surface antigen in a wide variety of tissues. CD47 is associated with integrins of β1, β2, and β3, and serves as both a receptor for thrombospondin (TSP) and a ligand for the transmembrane signal regulatory protein SIRP-α . CD47 has a number of different functions, including platelet activation, cell motility, leukocyte adhesion, migration and phagocytosis. Recently, it was reported that the ligation of CD47 rapidly induces cell death in T-cells and chronic lymphocytic leukemic B cells (B-CLL) in a caspase-independent manner (Nat Med, 1999;5:1277–1284). These findings indicate that CD47 ligation may have potential as a new therapeutic approach for lymphoid malignancies. B-CLL is the most common hematological malignancy in Western countries. Although new therapeutic agents such as fludarabine and 2-chlorodeoxyadenosine have been introduced in the clinic, B-CLL is not considered curable, and thus there is an immediate need for new, effective drugs. In an attempt to establish a novel therapeutic agent for B-CLL, we first generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Soluble MABL (10μg/ml) with goat anti-mouse IgG (GAM) induced apoptosis of CD47-positive CCRF-CEM and MOLT-4 cells. However, because MABL caused hemoagglutination, we created a disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) to get rid of this adverse effect. The S-S diabody did not cause hemoagglutination. Treatment with the S-S diabody (0–0.1 μg/ml for 0–24 hours) alone was fully active in inducing apoptosis of primary samples from patients with B-CLL and CD47-positive lymphoid cells (MOLT-4 and JOK-1) in a dose- and time-dependent manner. In addition, administration of the S-S diabody (30 mg/kg for 5 days) significantly prolonged the survival of SCID mice inoculated with JOK-1 cells compared with that of the control mice (median survival 21 days vs. more than 30 days, p<0.05). These results demonstrate that the S-S diabody induced the ligation of CD47 more efficiently than original MABL without causing hemoagglutination. Transmission electron microscopic analysis showed that the S-S diabody induced typical morphological changes of apoptosis as well as cell-cell adhesion in MOLT-4 cells. To address the molecular mechanism of apoptosis by the ligation of CD47 with the S-S diabody, we performed gene expression profiling analysis. Gene expression profiling with Affimetrix gene chips of MOLT-4 cells demonstrated 54 genes, including HIF-1a and its down-stream genes (RTP801, EGR-1, BNIP3, and VEGF), that were upregulated by the treatment with S-S diabody. Moreover, knockdown of these molecules by siRNA partially repressed S-S diabody-induced apoptosis in MOLT-4 cells. These results suggest that induction of apoptosis by CD47 ligation may be mediated through various HIF-1α pathways. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies. In addition, the newly established disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) may have potential as a novel therapeutic agent for the treatment of B-CLL and other incurable lymphoid malignancies.