A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH

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 significantly 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 cytotoxic 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

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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. Dosing volume was 0.2 mL/injection. All in vivo efficacy studies were done with 10 mice per treatment group unless specifically indicated. At various Cell Lines intervals of treatment (usually twice per week), tumor Neuroblastoma cell lines (SK-N-AS and SK-N-FI), rhab- volumes were measured, and antitumor activity was ex- domyosarcoma cell lines (SJCRH30 and RD), and the oste- pressed as the percent inhibition of tumor size. Body osarcoma cell line SJSA-1 were obtained from the weight was recorded at the time of tumor measurement. American Type Culture Collection. These cells were main- Animals were euthanized according to the Institutional tained in the RPMI 1640 supplemented with 10% heat- Animal Care and Use Committee guidelines. Statistical inactivated fetal bovine serum (Invitrogen/Life Technologies). analysis by Student's t test was used to compare treatments.

Animals Ex vivo Immunohistochemical Analysis of Animals used for tumor xenografts in the study were Tumor Ki-67 6- to 15-week-old female nude mice (nu/nu) weighing 18 Xenograft tumor samples were collected after treat- to 24 g (Charles River Laboratories). Mice were housed at ment with SCH 717454. Tumor samples were fixed in

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10% buffered formalin, embedded in paraffin, and cut in- 2 min and rinsed with distilled water. Slides were evalu- to 4 to 6 μm sections with Microtome onto slides. After ated under an optical light microscope, and photographs deparaffinization and rehydration, antigens were re- were obtained showing brown staining of Ki-67 in nuclei trieved with 1× Cytomation target retrieval solution from of cells. Ki-67 signal was analyzed and quantified using Dako in a decloaker chamber at 95°C for 20 min and then ImagePro Plus 5.0 (Media Cybernetics). at room temperature for 20 min followed by sequential rinsing with distilled water and PBS at room tempera- Analysis of Tumor Blood Vessel Formation ture. Slides were incubated with hydrogen peroxide for To visualize blood vessel within the tumors, 5 min to quench endogenous hydrogen peroxidase activ- xenografted mice were treated with either control IgG1 ity. After rinsing twice with TBS-0.1% Tween 20, slides or SCH 717454 when tumor size reached ∼150 to were incubated with mouse anti–Ki-67 antibody (BD 200 mm3. Xenograft mice were treated twice (on days 0 Pharmingen) and mouse control IgG (Vector Laborato- and 3). On day 5 after the initiation of SCH 717454 treat- ries), each at a concentration of 5 μg/mL, for 15 min ment (day 16 post-inoculation), mice were anesthetized, and then rinsed again with TBS-0.1% Tween 20. Slides and FITC-labeled Lycopersicon esculentum (tomato lectin; were incubated sequentially with biotinylated goat anti- FITC-lectin), an endothelial cell selective reagent (Vector mouse IgG for 15 min, peroxidase-labeled streptavidin Laboratories), was injected slowly via tail vein (0.1 mL of (Dako) for 15 min, and diaminobenzidine/hydrogen per- 1 mg/mL FITC-lectin solution). Cardiac perfusion was oxide chromogen substrate (Vector Laboratories) for done with 10 mL of 4% paraformaldehyde followed by 5 min. All incubation steps were done at room tempera- 10 mL PBS. Tumor was dissected and immersed sequen- ture. Slides were counterstained with hematoxylin for tially in 12%, 15%, and 18% sucrose for 1 h each and then

Figure 1. SCH 717454 downregulates IGF-IR and inhibits both basal and IGF-I–induced phosphorylation of IGF-IR and IRS-1 in pediatric tumor lines. A, SK-N-FI cells either untreated or treated with control IgG1 (lane 2; 80 nmol/L) or various concentrations of SCH 717454 (0.2-80 nmol/L) for 0.5 or 4 h before stimulation with IGF-I (50 ng/mL) for 10 min. Cell lysates were subjected to Western analysis and probed with phosphorylated IGF-IR–specific antibody (top). The filter was then reprobed with an antibody that recognizes total IGF-IR. For the 4 h time point, the same blot was stripped and reprobed with anti-actin antibody as loading control. B, SK-N-FI cells were either untreated or treated with control IgG1 (lane 2; 80 nmol/L) or various concentrations of SCH 717454 (0.2-80 nmol/L) for 0.5 h before stimulation with IGF-I (50 ng/mL) for 10 min. Cell lysates were probed with anti-phosphorylated IRS-1 antibody (pY612). C, various pediatric tumor cell lines were either untreated (control; lane 1) or treated with SCH 717454 (20 nmol/L, 4 h; lane 2) or IGF-I (100 ng/mL, 20 min; lane 3). Cell lysates were then probed with anti-phosphorylated IRS-1 antibody (pY612). The blots were stripped and reprobed with anti-total IRS-1 antibody.

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30% sucrose overnight at 4°C. Tumor samples were embedded in Tissue-Tek OCT from Sakura, and 20 μm tissue Table 1. Secretion of IGF-I and IGF-II by pediatric sections were prepared. Tissue sections were analyzed tumor lines using a Leica confocal microscope. The fluorescent lectin signal intensity and total area (pixels) were analyzed and Cell IGF-I IGF-II quantified using ImagePro Plus 5.0. SK-N-AS + ++++ Results SK-N-FI + ++++ SJSA-1 ++ ++++ SJCRH30 ++++ ++++ In vitro Characterization of Tumor Cell Lines RD + ++++ Upon stimulation by its ligands (IGF-I and IGF-II), the activated (phosphorylated) IGF-IR phosphorylates the NOTE: Cells were cultured in serum-free medium for 24 h. IRS-1 (the immediate downstream signaling molecule), Medium was collected, and each medium sample was as- which results in further downstream events leading to in- sayed for IGF-I and IGF-II by ELISA. +, 0.1 to 1 ng/106 creased cell proliferation and cell survival. To study the cells; ++, 1 to 10 ng/106 cells; +++, 10 to 50 ng/106 cells; IGF-IR signaling pathway in the neuroblastoma, osteo- ++++, >50 ng/106 cells. sarcoma, and rhabdomyosarcoma, we looked at the status of total IGF-IR as well as IGF-IR phosphorylation in response to SCH 717454 in these tumor cell lines. We dis- covered that, shortly after treatment with SCH 717454 cyclophosphamide on the same dosing schedules and (0.5 h), the IGF-I–stimulated phosphorylation of IGF-IR frequency. At day 21 after treatment initiation, the SK- was significantly inhibited in the neuroblastoma SK-N- N-FI xenograft tumor was inhibited by 96% in the FI cells, whereas the level of total IGF-IR protein remains 0.04 mg SCH 717454 dose group and resulted in 11% tu- unchanged (Fig. 1A, top). Extended treatment with SCH mor regression in the 0.1 mg dose group (Fig. 2A). In con- 717454 (4 h) resulted in both inhibition of IGF-IR phos- trast, treatment with cisplatin or cyclophosphamide phorylation and receptor downregulation (Fig. 1A, bot- resulted in only 54% or 39% tumor growth inhibition, tom). Similar responses were observed in the other respectively, in this neuroblastoma xenograft model. pediatric tumor cell lines used in this study (data not The effect of combined administration of SCH 717454 shown). Next, we looked at signaling molecule down- and cisplatin on SK-N-FI tumor xenografts was eval- stream of the receptor by analyzing the phosphorylation uated. The combined administration of 0.04 mg SCH status of IRS-1 in these tumor cells. The SK-N-FI neuro- 717454 and cisplatin was significantly more effective blastoma cells exhibited high basal expression of phos- (34%tumorregression)thaneitheragentalone phorylated IRS-1, and this signal is further enhanced by (Fig. 2A). The combination of 0.1 mg SCH 717454 with IGF-I. Treatment with SCH 717454 results in a dose- cisplatin (38% tumor regression) was not better com- dependent inhibition of the IGF-I–stimulated IRS-1 phos- pared with 0.1 mg SCH 717454 used alone (11% tumor phorylation (Fig. 1B), confirming that the IGF-I signaling regression) based on statistical analysis. This is probably pathway is intact in this pediatric tumor line and that this due to the fact that treatment with 0.1 mg SCH 717454 pathway can be effectively inhibited by anti–IGF-IR anti- had already resulted in significant tumor regression by body SCH 717454. Phosphorylated IRS-1 was also de- itself, that suggesting maximal in vivo response was tected in other unstimulated pediatric tumor cell lines, reached at 0.1 mg SCH 717454 in this xenograft model. and this basal phosphorylation was inhibited by SCH Similar results were observed when SCH 717454 was 717454 (Fig. 1C). These pediatric tumor cell lines pro- combined with cyclophosphamide (Fig. 2B). No signifi- duced IGF-II in large amounts, with SJCRH30 cells re- cant body weight loss was observed in both single-agent leasing IGF-I in substantial quantities (Table 1). The and combination groups during the course of study (Sup- data shown here show that pediatric tumor cells used plementary Fig. S1). in the present study possess the functional IGF-IR and have the capacity to establish autocrine loops in promot- Inhibition of Osteosarcoma Growth In vivo ing tumor growth in vivo. Subcutaneous inoculation of the SJSA-1 osteosarcoma line into nude mice resulted in a time-dependent growth Inhibition of Neuroblastoma Growth In vivo of tumors. This tumor growth was effectively suppressed After SK-N-FI tumor cells were inoculated s.c. to nude by both SCH 717454 and cyclophosphamide as single mice, treatment was initiated when the average tumor agents (Fig. 2C). At day 14 after treatment initiation, tu- size reached 150 mm3 (∼21 days post-inoculation). mor growth was inhibited by 71%, 82%, and 88% at 0.02, Mice in various groups were received either control 0.1, and 0.5 mg SCH 717454, respectively, and by 97% at human IgG1 (0.1 mg/mouse), SCH 717454 (0.04 or 100 mg/kg cyclophosphamide. There was no discernible 0.1 mg/mouse), cisplatin (2 mg/kg i.p. twice per week), tumor regression by single-agent treatments. However, cyclophosphamide (100 mg/kg i.p. once per week), or combinations of 100 mg/kg cyclophosphamide with combination of SCH 717454 with either cisplatin or SCH 717454 at doses of 0.1 and 0.5 mg regressed tumors

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by 41% and 53%, respectively (P < 0.05), on day 10 after phosphamide and SCH 717454 combination groups the treatment initiation. To determine if combination where SCH 717454 treatment continued, tumor growth treatments could affect the duration of the antitumor re- was substantially delayed with the mean tumor size sponse, after the first week of treatment, SCH 717454 reaching 1,000 mm3 by day 38 (Fig. 2C). Quantitation dosing (but not cyclophosphamide dosing) was contin- by the log tumor cell kill method (value ≥ 2.0; Supplemen- ued until the mean tumor size reached 1,000 mm3.In tary Table S1) showed the combination treatment elicited groups receiving SCH 717454 as a single agent, the tu- a further and substantial tumor response in this osteo- mors reached 1,000 mm3 by day 21. In contrast, in cyclo- sarcoma model. No significant body weight loss was

Figure 2. SCH 717454 inhibits pediatric tumor growth in xenograft models as a single agent and in combination with chemotherapeutic agents. A and B, nude mice were inoculated s.c. with SK-N-FI neuroblastoma cells (5 × 106 per mouse). When the average tumor size reached 150 mm3 (on day 21 post-inoculation), tumor-bearing mice were randomized to indicated treatment groups (10 mice per group) and treatments (i.p. route) with either 0.1 mg control IgG1 antibody, 0.04 mg or 0.1 mg SCH 717454 (twice weekly), 100 mg/kg cyclophosphamide (once weekly), 2 mg/kg cisplatin (twice weekly), or both SCH 717454 and cyclophosphamide were initiated. C, nude mice were inoculated s.c. with SJSA-1 osteosarcoma cells (7 × 106 per mouse). When tumor size reached 96 mm3, mice randomized to indicated treatment groups (10 per group) were treated i.p. (twice weekly) with either control IgG1 antibody, SCH 717454, 100 mg/kg cyclophosphamide, or both SCH 717454 and cyclophosphamide. Note that cyclophosphamide dosing ceased after three doses, whereas SCH 717454 dosing continued until the tumor volume reached 1,000 mm3. D, effects of SCH 717454 on RD rhabdomyosarcoma xenograft growth as a single agent and in combination with cyclophosphamide. Nude mice were inoculated s.c. with RD rhabdomyosarcoma cells (7 × 106 per mouse). Treatment (i.p. twice weekly for 0.5 mg control IgG1, 0.1 and 0.5 mg SCH 717454, and 100 mg/kg cyclophosphamide) was initiated when the average tumor size reached 125 mm3. On day 18 after the treatment initiation, when control group tumor volume reached 660 mm3, the study was terminated and tumor growth inhibition or regression in various groups was calculated. Bars, SE. *, P < 0.05, compared with IgG1 control; #, P < 0.05, compared with single treatments.

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observed in both single-agent and combination groups during the course of study (Supplementary Fig. S2).

Inhibition of Rhabdomyosarcoma Growth In vivo The efficacy of SCH 717454 as a single agent and in combination with cyclophosphamide was examined in s.c xenografts derived from SJCRH30 and RD rhabdomyosarcoma cell lines. In the RD rhabdomyosarcoma model (Fig. 2D), SCH 7171454 alone inhibited tumor growth by 39% and 58% at 0.1 and 0.5 mg dose (twice per week), respectively, compared with control IgG1 group (0.5 mg twice per week). In this model, treatment with cyclophosphamide (100 mg/kg twice per week) alone inhibited tumor growth completely and caused a 15% tumor regression. This tumor regression was significantly enhanced to 65%, when 0.5 mg SCH 717454 was combined with 100 mg/kg cyclophosphamide. In the SJCRH30 model (Supplementary Fig. S3), SCH 717454 alone inhibited tumor growth by 37% and 53% at 0.1 and 1 mg dose (twice per week), respectively. Cyclophos- phamide (100 mg/kg once per week) by itself was only inhibited by 37% of tumor growth compared with the 1mg control IgG1 group (twice per week). However, combination of 1 mg SCH 717454 with cyclophospha- Figure 3. SCH 717454 inhibits tumor expression of Ki-67 in vivo. A, SK-N-FI neuroblastoma xenograft tumors were collected and analyzed mide increased the tumor growth inhibition to 61%, bet- P by Ki-67 immunostaining and H&E staining on twice weekly treatment ter than either single agent used alone ( < 0.05). with 0.1 mg SCH 717454 for 2 wk. B, SJSA-1 osteosarcoma xenograft tumors were collected 3 d after a single treatment of SCH 717454. Inhibition of In vivo Tumor Cell Proliferation Average tumor size at the time of treatment was 155 and 130 mm3 for To further elucidate the mechanism of action of SCH SK-N-FI and SJSA-1 xenografts, respectively. Tumor proliferation status was assessed with anti–Ki-67 antibody by immunohistochemistry. 717454, we set to determine if reduction of tumor size is Tissue sections were counterstained with hematoxylin. associated with inhibition of tumor cell proliferation. The nuclear proliferation marker Ki-67 was immunohisto- 3 chemically evaluated ex vivo using tumor xenograft tis- when the tumor size reached 150 to 200 mm on day 11 sues collected after treatment. In SK-N-FI neuroblastoma post-inoculation. Compared with control IgG1, 0.5 mg xenograft tumor obtained after twice weekly treatment SCH 717454 treatment (two doses) reduced in the inten- with 0.1 mg SCH 717454 for 2 weeks, the tumor Ki-67 sity of the fluorescent lectin staining by 74%, showing staining was reduced by 38% and along with significant thinner blood vessels and reduced branches (Fig. 4B). change in xenograft tumor morphology (shown in This result suggests that SCH 717454 has antiangiogen- H&E staining; Fig. 3A). In the SJSA-1 osteosarcoma xeno- esis effect and results in the reduction of blood vessel for- graft samples obtained 3 days after a single dose of SCH mation during tumor growth, thereby contributing to its in vivo 717454, the staining of Ki-67 was reduced in a dose- antitumor effect . Taken together, SCH 717454 can dependent manner by 37% and 51%, respectively, after both directly inhibit the IGF-I signaling pathway and 0.1 and 0.5 mg SCH 717454 treatment (Fig. 3B). These data modulate the blood vessel formation via its antiangio- show that SCH 717454 can effectively block pediatric tu- genesis effect. mor cell proliferation in vivo. Moreover, Ki-67 and related proliferation biomarkers could be used as potential biomar- Discussion kers in the clinical development of SCH 717454 and other anti–IGF-IR therapies (biologics and small molecules). There is strong evidence supporting a critical role of IGF-IR in tumorigenesis, tumor survival, and metastasis Inhibition of In vivo Tumor Angiogenesis in a wide variety of human cancers including pediatric In addition to dysregulated growth signal, new blood tumors (3, 6). Our present study showed that SCH vessel needs to be formed to feed the accelerated growth 717454, a fully human neutralizing monoclonal antibody of tumor. To evaluate the effect of SCH 717454 on specific for IGF-IR, inhibits tumor growth as a single angiogenesis within the tumor tissue, blood vessel forma- agent. In addition, its combination with conventional tion was studied in the SJSA-1 tumor xenograft mice chemotherapeutic agents (cyclophosphamide and cisplat- (n = 5; Fig. 4A) using FITC-lectin, which specifically recog- in) results in a greater antitumor effect than that observed nizes the vascular endothelial cells. Treatment (Fig. 4A, for either single agent alone in neuroblastoma, osteosar- arrow) was initiated in the SJSA-1 tumor-bearing mice coma, and rhabdomyosarcoma tumor xenograft models.

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SCH 717454 inhibits rhabdomyosarcoma xenograft correlates with the antibody plasma level (33). These growth only partially while inhibiting the growth of neu- findings are consistent with our observations that a roblastoma and osteosarcoma tumors almost completely. near-maximal downregulation of Ki-67 in neuroblastoma This finding is consistent with the reported existence in xenografts occurs within 1 day of SCH 717454 adminis- rhabdomyosarcoma cells of redundant growth factor tration and persists for at least 3 days and suggest that autocrine loops (14) and suggests that the IGF/IGF-IR the prolonged antiproliferative activity of SCH 717454 loop represents a dominant mechanism in the growth against neuroblastoma and osteosarcoma may be due, of neuroblastoma and osteosarcoma xenografts. The data at least in part, to the sustained receptor downregulation. presented here are also consistent with the results re- SCH 717454 was also shown here to suppress the forma- ported by the investigators of the Pediatric Preclinical tion of new blood vessels in the SJSA-1 tumor xenograft. Testing Program (36). In their study, SCH 717454 showed These findings are consistent with reports showing that antitumor activity against a panel of pediatric tumor IGF-IR activation regulates vascular endothelial growth models with the most potent activities in Ewing's sarco- factor production by tumor cells (42, 43) and that inhibi- ma, osteosarcoma, and neuroblastoma models. Recently, tion of IGF-IR action by antisense, antibody, or tyrosine ki- several anti–IGF-IR monoclonal antibodies including nase inhibitor reduces both neovascularization and SCH 717454 have shown single-agent activity in Ewing's vascular endothelial growth factor expression in neuro- sarcoma in phase II clinical trials providing clinical proof blastoma and Ewing's sarcoma tumors (28, 44). Impor- of concept supporting importance of the IGF-IR pathway tantly, vascular endothelial growth factor levels are in Ewing's sarcoma (37–40). elevated in sera from children with neuroblastoma, osteo- Downregulation of the proliferation marker Ki-67 and sarcoma, Ewing's sarcoma, and rhabdomyosarcoma (45). histopathologic evidence for reduced numbers of cancer Thus, inhibition of angiogenesis appears to be a contribut- cells in SCH 717454–treated mice (Fig. 3) confirm inhibi- ing factor to the antitumor activity of SCH 717454. tion by SCH 717454 of pediatric cancer cell proliferation IGFs are known to protect a variety of cancer cells in vivo. These observations are consistent with the dem- from the cytotoxic effects of radiation and chemothera- onstrated ability of SCH 717454 to downregulate IGF-IR pies (22, 46, 47). Recent evidence suggests that IGF-IR and to inhibit both basal and IGF-I–induced IGF phos- activation can confer resistance to anti-HER2 antibody phorylation of IRS-1 in vitro (Fig. 1). Studies using fully trastuzumab in breast cancer and to anti–epidermal human monoclonal antibodies including SCH 717454 growth factor receptor antibody in human glioblastoma have clearly shown that these antibodies inhibit the pro- cells (48, 49). Consistent with these findings are reports liferation of adult tumor cells by inhibiting IGF-I binding showing that sensitivity of various cancer cells (in- (thereby preventing IGF-IR activation) and by inducing cluding some pediatric tumors) to chemotherapy and IGF-IR downregulation (32, 34, 36, 41). Notably, IGF-IR radiation can be enhanced by various approaches to downregulation in the tumor and peripheral blood cells block IGF-IR action (7, 47). Inhibition of IGF-IR signaling in vivo by CP-751,871 is evident on its administration and by kinase inhibitors increases the susceptibility of

Figure 4. SCH 717454 inhibits vascular endothelial growth factor production and blood vessel formation in tumor xenograft. A, growth of SJSA-1 osteosarcoma xenografts. Nude mice bearing SJSA-1 tumor received i.p. 0.5 mg of either control IgG1 antibody or SCH 717454 (arrow; n = 5 per treatment group). B, tumor samples collected on day 5 post-initiation of treatment, and frozen sections were prepared for immunohistochemical study. Blood vessels within the tumor samples were visualized by FITC-lectin, and staining signal intensity was analyzed by ImagePro software.

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SCH 717454 Inhibits Pediatric Tumor Growth

rhabdomyosarcoma, osteosarcoma, and Ewing's sarcoma liferation is dependent on the activated IGF-IR pathway. to several different chemotherapeutic agents, including We conclude that anti–IGF-IR monoclonal antibody SCH cisplatin, doxorubicin, and vincristine (29, 32), and ex- 717454 is a promising therapeutic candidate for treating pression of a dominant-negative mutant of IGF-IR in- pediatric solid tumors as a single agent and in combina- duces apoptosis of Ewing's sarcoma cells and enhances tion with chemotherapeutic agents. their sensitivity to the cytotoxicity of doxorubicin (49). Ef- ficacy of other anti–IGF-IR antibodies in combination Disclosure of Potential Conflicts of Interest with various anticancer agents in preclinical mouse xenograft models was reported recently (33, 41, 50). We Y. Wang, P. Lipari, X. Wang, J. Hailey, L. Liang, R. Ramos, M. Liu, W. R. showed here that combination of SCH 717454 with either Bishop, and Y. Wang are employees of Merck & Co. J.A. Pachter is an cyclophosphamide or cisplatin in the neuroblastoma, os- employee of OSI Pharmaceutical. teosarcoma, and rhabdomyosarcoma models results in a greater antitumor effect than that observed for either sin- Acknowledgments gle agent used alone. We found that administration of SCH 717454 does not cause body weight loss or elicit We thank Truyen Nguyen for technical support on the blood vessel microscopic imaging study, Abdul-Samad Ponery for quantitation of Ki- overt toxicity during the entire course of treatment. More 67 and fluorescent lectin signal, and Cynthia Seidel-Dugan and Dan importantly, combination of SCH 717454 with various cy- Hicklin for critical reading of the article. The costs of publication of this article were defrayed in part by the totoxics does not result in further body weight loss. This payment of page charges. This article must therefore be hereby marked finding suggests that this anti–IGF-IR antibody can be advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate used in the clinic with a full dose of standard chemother- this fact. apy regimen. Our studies provided mechanistic insight to Received 3/6/09; revised 12/1/09; accepted 12/6/09; published use an anti–IGF-IR antibody in settings where tumor pro- OnlineFirst 2/2/10.

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418 Mol Cancer Ther; 9(2) February 2010 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 Wang, Philip Lipari, Xiaoying Wang, et al.

Mol Cancer Ther 2010;9:410-418. Published OnlineFirst February 2, 2010.

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