Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth

Cancer Immunol Immunother. 2000 Jul;49(4-5):243-52. doi: 10.1007/s002620000115.


Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (alphaIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 alphaIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92-98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the alphaIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble alphaIGF-IR scFvs, a prototype alphaIGF-IR scFv and its alternative type alphaIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. alphaIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A--agarose chromatography. Levels of alphaIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that alphaIGF-IR scFv-Fc is a dimeric antibody. alphaIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 10(8) M(-1), which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of alphaIGF-IR scFv-Fc (500 microg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the alphaIGF-IR scFv-Fc is a first-generation recombinant alphaIGF-IR for the potential development of future alphaIGF-IR therapeutics.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / isolation & purification
  • Cell Division / immunology
  • Chromatography
  • Cloning, Molecular
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Immunologic
  • Electrophoresis, Polyacrylamide Gel
  • Glutamate-Ammonia Ligase / metabolism
  • Humans
  • Hybridomas / immunology
  • Hybridomas / metabolism
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Heavy Chains / immunology
  • Immunoglobulin Light Chains / genetics
  • Immunoglobulin Light Chains / immunology
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology*
  • Mice
  • Models, Immunological
  • Protein Structure, Tertiary
  • Receptor, IGF Type 1 / immunology*
  • Surface Plasmon Resonance
  • Time Factors
  • Transfection
  • Tumor Cells, Cultured


  • Antibodies, Monoclonal
  • DNA, Complementary
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Receptor, IGF Type 1
  • Glutamate-Ammonia Ligase