Published OnlineFirst February 2, 2010; DOI: 10.1158/1535-7163.MCT-09-0555 Research Article Molecular Cancer Therapeutics A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts Yaolin Wang1, Philip Lipari1, Xiaoying Wang1, Judith Hailey1, Lianzhu Liang1, Robert Ramos1, Ming Liu1, Jonathan A. Pachter2, W. Robert Bishop1, and Yan Wang1 Abstract The insulin-like growth factor-I receptor (IGF-IR) and its ligands (IGF-I and IGF-II) have been implicated in the growth, survival, and metastasis of a broad range of malignancies including pediatric tumors. Blocking the IGF-IR action is a potential cancer treatment. A fully human neutralizing monoclonal antibody, SCH 717454 (19D12, robatumumab), specific to IGF-IR, has shown potent antitumor effects in ovarian cancer in vitro and in vivo. In this study, SCH 717454 was evaluated in several pediatric solid tumors including neuroblastoma, osteosarcoma, and rhabdomyosarcoma. SCH 717454 is shown here to downregulate IGF-IR as well as inhibit IGF-IR and insulin receptor substrate-1 phosphorylation in pediatric tumor cells. IGF-IR and insulin receptor substrate-1 phosphorylation in the tumor cells. In vivo, SCH 717454 exhibits activity as a single agent and sig- nificantly inhibited growth of neuroblastoma, osteosarcoma, and rhabdomyosarcoma tumor xenografts. Combination of SCH 717454 with cisplatin or cyclophosphamide enhanced both the degree and the duration of the in vivo antitumor activity compared with single-agent treatments. Furthermore, SCH 717454 treatment markedly reduced Ki-67 expression and blood vessel formation in tumor xenografts, showing that the in vivo activity is derived from its inhibition of tumor cell proliferation and angiogenesis activity. Mol Cancer Ther; 9(2); 410–8. ©2010 AACR. Introduction The insulin-like growth factor-I receptor (IGF-IR), a transmembrane tyrosine kinase that is selectively acti- In the United States, 12,400 children ages <20 years vated by its ligands IGF-I and IGF-II, has been strongly were diagnosed with cancer and 2,500 died of cancer in implicated in the growth, survival, and metastasis of a 1998 (1). For children between ages 1 and 19 years, cancer wide variety of human tumors, including tumors in chil- ranked fourth as a cause of death. The probability of de- dren and adolescents (4–9). Many pediatric tumors in- veloping cancer before age 20 years is 1 in 300. Neuro- cluding neuroblastoma, Wilms' tumor, osteosarcoma, blastoma, bone tumors including osteosarcoma, and and rhabdomyosarcoma are embryonal tumors of the soft-tissue tumors including rhabdomyosarcoma repre- childhood and exhibit increased expression of IGF-II sent three important groups of pediatric solid tumors and IGF-IR establishing autocrine loops to perpetuate and collectively account for nearly one-fifth of all pediat- growth and survival (10–15). The role of IGF-I in the de- ric malignancies (1). The currently available treatments velopment of Ewing's sarcoma has also been well docu- include surgical resection, chemotherapy, and radiation. mented in addition to the induction of IGF-IR expression Despite recent advances in these therapeutic modalities, by the Ewing's sarcoma fusion protein (16–19). Increased tumors frequently recur, and children with tumor metas- expression of IGF-IR in rhabdomyosarcomas has also tasis have poor prognosis (2, 3). Therefore, there is an un- been linked to chimeric transcription factor PAX3-FKHR met need for new and effective treatment modalities for (20). Highly metastatic neuroblastoma and osteosarcoma pediatric solid tumors. cells possess higher expression of IGF-IR than tumor cells that are less prone to metastasize (21). Furthermore, IGF- Authors' Affiliations: 1Merck Research Laboratory, Kenilworth, New IR activation has been shown to generate strong anti- Jersey and 2OSI Pharmaceutical, Inc., Melville, New York apoptotic signals in various cancer cells including sarcoma Note: Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). and neuroblastoma cells, conferring resistance to the cy- totoxic effects of chemotherapies (6, 21, 22). Epidemio- Corresponding Author: Yaolin Wang, Department of Tumor Biology, Merck Research Laboratory, K15-4-4700, 2015 Galloping Hill Road, logic data have linked elevated plasma levels of IGF-I Kenilworth, NJ 07033. Phone: 908-740-6717; Fax: 908-740-3918. E-mail: and IGF-II with increased risk for major cancers such as [email protected] breast, colon, lung, and prostate cancers, although no doi: 10.1158/1535-7163.MCT-09-0555 such data are currently available for pediatric cancers (6, ©2010 American Association for Cancer Research. 23–25). Importantly, selective impairment of IGF-IR using 410 Mol Cancer Ther; 9(2) February 2010 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst February 2, 2010; DOI: 10.1158/1535-7163.MCT-09-0555 SCH 717454 Inhibits Pediatric Tumor Growth different approaches, including antisense technologies, Schering-Plough Research Institute according to the dominant-negative mutants of IGF-IR, tyrosine kinase in- guidelines of the Association for Assessment and Accred- hibitors, and neutralizing anti–IGF-IR antibodies, inhib- itation of Laboratory Animal Care. Conventional animal ited tumor cell growth, induced tumor cell apoptosis, chow and water were provided ad libitum. Animals were increased tumor sensitivity to cytotoxic drugs, and de- handled according to the protocols approved by the Scher- creased metastasis of cancer cells of both adult and pediat- ing-Plough Institutional Animal Care and Use Commit- ric origins (16, 26–31). These observations provide a strong tee. For all studies, mice were allowed to acclimate for at rationale for targeting IGF-IR against pediatric cancers. least 3 days after receipt, and test animals were random- Monoclonal antibodies and tyrosine kinase inhibitors ized into groups before the start of the treatments. are being vigorously pursued as potential anticancer therapies with antibodies promising to provide greater Measurements of IGF-I and IGF-II specificity and safety. To minimize the potential for immu- Tumor cells were seeded on in RPMI 1640 containing 10% nologic liabilities of non-human antibodies, several fully fetal bovine serum. After cells were attached, medium was human monoclonal antibodies against IGF-1R several changed to serum-free medium. After 24 h, cell medium was fully human monoclonal antibodies against IGF-IR have collected, debris was spun down, and supernatants were recently been generated (32–35). These antibodies exhibit lyophilized. Cells on the plates were trypsinized and potent antitumor effects in breast, colon, prostate, ovarian, counted. Water was added to each lyophilized supernatant and pancreatic cancers in vitro and in vivo and offer great sample (1 mL/2 × 107 cells). IGF-I and IGF-II were mea- potential for success as safe and effective human antican- sured using ELISA kits from Diagnostic System Laboratories. cer therapeutics. We and others have previously described SCH 717454 (19D12, robatumumab), a fully human neu- Western Blot Analysis of IGF-IR and Insulin tralizing anti–IGF-IR antibody that potently inhibits li- Receptor Substrate-1 gand binding and signaling (34, 36, 37). SCH 717454 Cells were treated as described and then lysed in lysis inhibits the growth of various mouse xenograft tumor buffer [50 mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, in vivo models. In the current study, we evaluated the ef- 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, prote- fects of SCH 717454 in mouse xenograft models of neuro- ase inhibitors, and 2 mmol/L sodium vanadate]. The blot blastoma, osteosarcoma, and rhabdomyosarcoma. Our was probed with anti–IGF-IR antibody (Santa Cruz Bio- results show that SCH 717454 alone is efficacious in inhi- technology), anti-phosphorylated IGF-IR (Cell Signaling biting tumor growth in these models and that its combina- Technology), anti-phosphorylated insulin receptor sub- tion with cytotoxic agents (cyclophosphamide and strate-1 (IRS-1) antibody (Cell Signaling Technology), cisplatin) results in a greater antitumor effect than that and anti-total IRS-1 antibody (Santa Cruz Biotechnology) observed for either agent used alone. and visualized by enhanced chemiluminescence. Materials and Methods In vivo Tumor Growth Assessments For tumor implantation, specific cell lines were grown Reagents in vitro, washed once with PBS, and resuspended in SCH 717454 is a fully human neutralizing IgG1 anti- 50% Matrigel (BD Biosciences) to a final concentration of body raised against human IGF-IR using Medarex's Hu- 4×107 to 5 × 107 cells/mL. Nude mice were injected with Mab mouse technology (34). SCH 717454 was prepared, 0.1 mL of this suspension s.c. in the flank region. Tumor stored, and diluted in sodium acetate buffer [20 mmol/L volumes were measured by caliper twice a week. When sodium acetate, 150 mmol/L NaCl (pH 5.0-5.5)]. Control tumor sizes reached 75 to 150 mm3, the animals were ran- human IgG1 was purchased from The Binding Site. Cyclo- domized to appropriate treatment groups, and i.p. treat- phosphamide and cisplatin were obtained from Sigma. ments with either control human IgG1, SCH 717454, Vehicle for these cytotoxic agents was 20% hydroxypro- chemotherapeutic agents, or combinations were initiated. pyl-β-cyclodextran.