title: A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway.
abstract: CD47 belongs to the immunoglobulin superfamily and is associated with β-integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B-chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47-mediated apoptosis in human lymphoid malignant cells. Treatment with S-S diabody alone induced apoptosis of CD47-positive primary B-CLL and leukemic cells (MOLT-4 and JOK-1). In addition, administration of S-S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK-1 cells. In gene expression profiling of the S-S diabody-treated MOLT-4 cells, hypoxia inducible factor (HIF)-1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF-1α, RTP801 and BNIP3 were increased. Knockdown of HIF-1α by siRNA repressed S-S diabody-induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S-S diabody might have potential as a novel therapeutic agent for B-CLL.
authors.affiliation: Division of Hematology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Morihiko
authors.initials: M
authors.lastname: Sagawa
authors.forename: Takatsune
authors.initials: T
authors.lastname: Shimizu
authors.forename: Naoshi
authors.initials: N
authors.lastname: Fukushima
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Kinoshita
authors.forename: Iwao
authors.initials: I
authors.lastname: Ohizumi
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Uno
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Kikuchi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Hisafumi
authors.initials: H
authors.lastname: Yamada-Okabe
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kizaki
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000627:therapeutic use; D017209:Apoptosis; D051928:CD47 Antigen / Q000276:immunology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D008854:Microscopy, Electron; D024101:Mitochondrial Proteins / Q000235:genetics; D055503:Protein Multimerization; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000627:therapeutic use*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; C508686:BNip3 protein, mouse; D051928:CD47 Antigen; D004268:DNA-Binding Proteins; C450979:Ddit4l protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D057127:Single-Chain Antibodies; D014157:Transcription Factors