title: High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules.
abstract: OBJECTIVE
  To construct and produce a recombinant bispecific humanized single-chain Fv (sFv) /Interleukin-2 (IL-2) fusion protein by using mammalian cells.
  
  
  METHODS
  The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency.
  
  
  RESULTS
  The fusion protein was constructed and high efficiently expressed with yields up to 102 +/- 4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy (V(H)) and light (V(L)) domains of monoclonal antibody (mAb) 520C9 directed against the human HER-2/neu (c-erbB2) proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL-2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells.
  
  
  CONCLUSIONS
  The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.
authors.affiliation: Division of Cardiology, Department of Internal Medicine, The University of Michigan Medical School, Ann Arbor, 48109, USA.
authors.forename: Yue-Chun
authors.initials: YC
authors.lastname: Shen
authors.forename: Xue-Hao
authors.initials: XH
authors.lastname: Wang
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Wang
authors.forename: Zao-Lai
authors.initials: ZL
authors.lastname: Chen
authors.forename: Xi-Ping
authors.initials: XP
authors.lastname: Shen
authors.forename: Chao-Chen
authors.initials: CC
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D004247:DNA / Q000032:analysis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D047468:Immunoprecipitation; D007376:Interleukin-2 / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D014162:Transfection
chemical_list: D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins; D004247:DNA