High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules.
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.
Shen
Yue-Chun
YC
Division of Cardiology, Department of Internal Medicine, The University of Michigan Medical School, Ann Arbor, 48109, USA.
Wang
Xue-Hao
XH
Wang
Xiao-Ming
XM
Chen
Zao-Lai
ZL
Shen
Xi-Ping
XP
Zhao
Chao-Chen
CC
Li
Jun
J
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
D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins; D004247:DNA