Functional evaluation of novel soluble insulin-like growth factor (IGF)-II-specific ligand traps based on modified domain 11 of the human IGF2 receptor.
Ligands transported by the mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor (IGF2R) include IGF-II- and mannose 6-phosphate-modified proteins. Increased extracellular supply of IGF-II, either secondary to loss of the clearance function of IGF2R, loss of IGF binding protein function, or increased IGF2 gene expression, can lead to embryonic overgrowth and cancer promotion. Reduced supply of IGF-II is detrimental to tumor growth, and this suggests that gain of function of IGF-II is a molecular target for human cancer therapy. Domain 11 of IGF2R binds IGF-II with high specificity and affinity. Mutagenesis studies have shown that substitution of glutamic acid for lysine at residue 1554 results in a 6-fold higher affinity for IGF-II (20.5 nmol/L) than native domain 11 (119 nmol/L). Here, we generate a novel high-affinity IGF-II ligand trap by fusion of mutated human 11(E1554K) to a COOH-terminal human IgG1 Fc domain (11(E1554K)-Fc). The resulting homodimer has a significantly increased affinity for IGF-II (1.79 nmol/L) when measured by surface plasmon resonance. IGF-II signaling via the IGF-I receptor and the proliferative effect of IGF-II were specifically inhibited by 11(E1554K)-Fc in both HaCaT and Igf2(-/-) mouse embryonic fibroblast cells. These data confirm that a novel engineered and soluble IGF2R-11(E1554K)-Fc protein functions as an IGF-II-specific and high-affinity ligand trap in vitro and that this protein has potential application as an IGF-II antagonist for cancer therapy following in vivo experimental evaluation.
Prince
Stuart N
SN
Weatherall Institute for Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom.
Foulstone
Emily J
EJ
Zaccheo
Oliver J
OJ
Williams
Christopher
C
Hassan
Andrew Bassim
AB
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D004331:Drosophila melanogaster / Q000254:growth & development / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D004625:Embryo, Nonmammalian; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000502:physiology*; D015603:Keratinocytes / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D018345:Mice, Knockout; D010843:Pichia / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism*; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013936:Thymidine / Q000378:metabolism
D008024:Ligands; D017527:Receptor, IGF Type 2; D011961:Receptors, Fc; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; D013936:Thymidine