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 variet y 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 bin d with high affinity. To investigate the mitogenic and anti-apoptotic activ ities of IGF-IR and to achieve better inhibition of IGF-IR function, single -chain antibodies against human IGF-IR (alpha IGF-IR scFvs) were constructe d and expressed. IgG cDNA encoding variable regions of light and heavy chai ns (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 ta g. Two types of soluble alpha IGF-IR scFvs, a prototype alpha IGF-IR scFv a nd its alternative type alpha IGF-IR scFv-Fc, were constructed and expresse d 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 syn thase 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 dodec yl sulfate/polyacrylamide gel electrophoresis analysis under reducing and n onreducing conditions indicated that alpha IGF-IR scFv-Fc is a dimeric anti body, alpha IGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was es timated to be approximately 10(8) M-1, which was one-order of magnitude low er 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 f irst-generation recombinant alpha IGF-IR for the potential development of f uture alpha IGF-IR therapeutics.