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

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 (alpha IGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (V-L and V-H) from mouse IgG were cloned from a hybridoma producing the 1H7 alpha IGF-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 alpha IGF-IR scFv, including the N-terminal signal peptide, V-L, linker peptide, V-H, and C-terminal DYKD tag. Two types of soluble alpha IGF-IR scFvs, a prototype alpha IGF-IR scFv and its alternative type alpha IGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. alpha IGF-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 alpha IGF-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 alpha IGF-IR scFv-Fc is a dimeric antibody, alpha IGF-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 alpha IGF-IR scFv-Fc (500 mu g/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the alpha IGF-IR scFv-Fc is a first-generation recombinant alpha IGF-IR for the potential development of future alpha IGF-IR therapeutics.