Abstract Insulin-like growth factor 2 (IGF2) plays an important role both in human and mouse embryonic development and in tumour growth. IGF2 causes IGF1R and insulin receptor isoform A, IR(A), dependent activation of proliferation (MAPK) and survival pathways (AKT-PI3K). IGF2 bioavailability is tightly regulated by six IGF binding proteins and by the IGF2/mannose 6-phosphate receptor (IGF2R). IGF2R is a 15-domain extracellular receptor, frequently mutated in cancer, which clears IGF2 via binding to domain 11 and internalising it for lysosomal degradation. Increased supply of IGF2 occurs in hepatocellular, breast, prostate, colorectal and ovarian tumours. Excessive amounts of IGF2 secreted by some tumours, mainly sarcoma, can lead to activation of IR(A) and cause non-islet cell tumour induced hypoglycaemia. Approaches aiming to counteract the activity of IGF2 by targeting IGF1R often fail due to IGF2-mediated activation of IR(A). A ligand trap that exploits the high specificity and affinity of IGF2R for IGF2 has been designed by the fusion of a mutated form of human domain 11 of IGF2R to a C-terminal human IgG1 Fc domain (Fc-dom11). For the ligand trap to function as a clinically useful IGF2 super-antagonist, the binding affinity of domain 11 to IGF2 was further improved. The binding affinity optimisation strategy consisted of random mutagenesis of domain 11 loops involved in IGF2 binding, followed by yeast surface display in P. Pastoris, FACS and SPR screening, as well as structure-directed mutagenesis, complemented with NMR studies. Several mutants showing enhanced IGF2 binding have been identified, the combination of separately identified mutations improving affinity up to 100 fold. In particular, the mutant domain 113-4D shows an increase in affinity through both on- and off-rate modification attributed to the decrease in flexibility of one of the loops involved in IGF2 binding, as suggested by NMR results. The ability of Fc-dom113-4D to function as an IGF2 antagonist has been tested in vitro, using hepatocellular carcinoma cell lines, and in vivo, where it counteracts IGF2-induced hypoglycaemia in mice. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A246. Citation Format: Susana Frago, Madeleine Strickland, Jennifer Hughes, Christopher Williams, Ryan Nicholls, Lee Garner, Dellel Rezgui, Matthew P. Crump, A. Bassim Hassan. Development of an IGF2 super-antagonist by directed evolution of IGF2R. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A246.