title: Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris.
abstract: Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60-70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders.
authors.affiliation: Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, People's Republic of China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wan
authors.forename: Shengyun
authors.initials: S
authors.lastname: Zhu
authors.forename: Jian
authors.initials: J
authors.lastname: Zhu
authors.forename: Hao
authors.initials: H
authors.lastname: Yang
authors.forename: Shengfu
authors.initials: S
authors.lastname: Li
authors.forename: Youping
authors.initials: Y
authors.lastname: Li
authors.forename: Jingqiu
authors.initials: J
authors.lastname: Cheng
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lu
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D053645:Interleukin-2 Receptor alpha Subunit; D057127:Single-Chain Antibodies