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 (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.
Li
S L
SL
Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Liang
S J
SJ
Guo
N
N
Wu
A M
AM
Fujita-Yamaguchi
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D002455:Cell Division / Q000276:immunology; D002845:Chromatography; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D018448:Models, Immunological; D017434:Protein Structure, Tertiary; D017526:Receptor, IGF Type 1 / Q000276:immunology*; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D017526:Receptor, IGF Type 1; D005974:Glutamate-Ammonia Ligase