Research Article A Human Monoclonal Antibody against Insulin-Like Growth Factor-II Blocks the Growth of Human Hepatocellular Carcinoma Cell Lines In vitro and In vivo Daniel T. Dransfield 1 , Edward H. Cohen 1 , Qing Chang 1 , Lindsay G. Sparrow 2 , John D. Bentley 2 , Olan Dolezal 2 , Xiaowen Xiao 2 , Thomas S. Peat 2 , Janet Newman 2 , Patricia A. Pilling 2 , Tram Phan 2 , Ilka Priebe 3 , Gemma V. Brierley 3 , Niksa Kastrapeli 1 , Kris Kopacz 1 , Diana Martik 1 , Dina Wassaf 1 , Douglas Rank 1 , Greg Conley 1 , Yan Huang 1 , Timothy E. Adams 2 , and Leah Cosgrove 3 Abstract Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR–directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1–104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K D value of 49 and 10 pmol/L, respectively) compared with IGF-I (∼10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor–binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 Å. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I–induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage- dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A. Mol Cancer Ther; 9(6); 1809–19. ©2010 AACR. Introduction Hepatocellular carcinomas (HCC) account for the majority of primary liver cancers, ranking third as a cause of cancer mortality (1). The major etiologic fac- tors are well established and include viral hepatitis (B and C), primary liver disease with hereditary origins (e.g., hemochromatosis), alcohol use, and mycotoxin ex- posure. Therapeutic intervention is dictated by the stage of the disease at diagnosis and may include tu- mor resection and liver transplantation, percutaneous and transarterial intervention, radiation, and other ther- apies. For advanced HCC, no standard therapy exists, although a phase III clinical trial of the multikinase inhibitor sorafenib did result in significantly improved survival (2). There is compelling clinical and experimental evidence that insulin-like growth factor-II (IGF-II) plays a key role in the pathogenesis of HCC (3). IGF-II is a maternally imprinted embryonic growth factor that can elicit a spec- trum of cellular responses, including proliferation and protection from apoptosis, through activation of IGF- IR, an alternatively spliced form of the insulin receptor (IR-A), and the mannose-6-phosphate receptor (IGF-IIR; ref. 4). The mature form of IGF-II (67 amino acids) arises following posttranslational processing, including O-glycosylation and endoproteolysis, of a pro-IGF-II precursor (5). Elevated expression of IGF-II, in part the result of loss of imprinting, is observed in a variety of human malignancies, including cancers of the breast, co- lon, and liver (reviewed in ref. 4). This may be accompa- nied by the secretion of aberrantly processed pro-IGF-II isoforms with novel properties by some tumor types). With respect to HCC, the reactivation of Igf-II transcrip- tion from fetal-specific promoter elements is observed in Authors' Affiliations: 1 Dyax Corp., Cambridge, Massachusetts; 2 CSIRO Molecular and Health Technologies, Parkville, Victoria, Australia; and 3 CSIRO Molecular and Health Technologies, Adelaide, South Australia, Australia Note: Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). The crystal structure has been deposited in the Protein Data Bank with accession code 3KR3. Corresponding Author: Daniel T. Dransfield, Discovery Research, Dyax Corp., 8th Floor, 300 Technology Square, Cambridge, MA 02139. Phone: 617-250-5729; Fax: 617-225-2501. E-mail: ddransfield@dyax.com doi: 10.1158/1535-7163.MCT-09-1134 ©2010 American Association for Cancer Research. 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