IGF1R Blockade with Ganitumab Results in Systemic Effects on the GH–IGF Axis in Mice

G MOODY and others Effects of ganitumab on IGF1R 221:1 145–155 Research in murine tissues

IGF1R blockade with ganitumab results in systemic effects on the GH–IGF axis in mice

Gordon Moody, Pedro J Beltran, Petia Mitchell, Elaina Cajulis, Young-Ah Chung, David Hwang1, Richard Kendall, Robert Radinsky, Pinchas Cohen2 and Frank J Calzone Correspondence should be addressed Oncology Research Therapeutic Area, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA to G Moody 1David Geffen School of Medicine, Los Angeles, California, USA Email 2USC Davis School of Gerontology, Los Angeles, California, USA [email protected]

Abstract

Ganitumab is a fully human MAB to the human type 1 IGF receptor (IGF1R). Binding Key Words assays showed that ganitumab recognized murine IGF1R with sub-nanomolar afﬁnity " ganitumab

(KDZ0.22 nM) and inhibited the interaction of murine IGF1R with IGF1 and IGF2. Ganitumab " IGF1R inhibited IGF1-induced activation of IGF1R in murine lungs and CT26 murine colon " IGFBP3 carcinoma cells and tumors. Addition of ganitumab to 5-ﬂuorouracil resulted in enhanced " receptor pituitary inhibition of tumor growth in the CT26 model. Pharmacological intervention with " human ganitumab in naı¨ve nude mice resulted in a number of physiological changes described " murine previously in animals with targeted deletions of Igf1 and Igf1r, including inhibition of weight gain, reduced glucose tolerance and signiﬁcant increase in serum levels of GH, IGF1

Journal of Endocrinology and IGFBP3. Flow cytometric analysis identiﬁed GR1/CD11b-positive cells as the highest IGF1R-expressing cells in murine peripheral blood. Administration of ganitumab led to a dose-dependent, reversible decrease in the number of peripheral neutrophils with no effect on erythrocytes or platelets. These ﬁndings indicate that acute IGF availability for its receptor plays a critical role in physiological growth, glucose metabolism and neutrophil physiology and support the presence of a pituitary IGF1R-driven negative feedback loop that tightly

regulates serum IGF1 levels through Gh signaling. Journal of Endocrinology (2014) 221, 145–155

Introduction

The type 1 insulin-like growth factor receptor (IGF1R) is a and IGF2R as well as to the insulin-receptor splice variant tyrosine kinase receptor that mediates the positive effects A (IR-A; Frasca et al. 1999). of its ligands, IGF1 and IGF2, on cell growth and survival The role of the ligands and receptors of the (Baserga et al. 1997). IGF1 functions as a hormone and insulin/IGF signaling pathway in mouse development paracrine growth factor and is a key component of both and adult physiology has been the subject of an elegant the pre- and postnatal physiological mechanisms control- series of targeted genetic analyses. In LID (liver-derived ling organ size and tissue homeostasis (Gallagher & Igf1 gene-deleted mouse model) mice, where IGF1 levels LeRoith 2011, Pollak 2012). IGF2 exerts most of its activity were reduced by 75% (Sjogren et al. 1999, Yakar et al. during embryonic development and binds to IGF1R 1999), postnatal growth and development were

http://joe.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/JOE-13-0306 Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 05:07:39PM via free access Research G MOODY and others Effects of ganitumab on IGF1R 221:1 146 in murine tissues

essentially normal; however, the mice displayed insulin inhibits murine IGF1R, providing a unique opportunity to resistance associated with a fourfold increase in serum examine the effects of ganitumab on the somatotropic growth hormone (GH) levels (Yakar et al. 2001). LID GH–IGF1 axis and tissue homeostasis in mice. mice also had reduced splenic cellularity with a decrease in the number of myeloid progenitors, but bone marrow cellularity and immune parameters (B and T cells) were Materials and methods unaffected, suggesting a role for circulating IGF1 in Animals transition or survival of myeloid progenitors in the K K spleen (Welniak et al. 2004). In homozygous Igf1( / ) CD1 nude mice (Charles River Laboratories, Hollister, CA, mice, birth weight was about 60% of normal mice, and USA) or athymic nude mice (Harlan Laboratories, Inc.) the weight of animals that survived was only 30% of were housed in sterile cages, five per cage. The laboratory their normal adult littermates (Baker et al. 1993). Mice housing the cages met all AAALAC specifications. All with homozygous null mutations in Igf1r had normal experimental procedures were performed in accordance embryonic development but died soon after birth with the IACUC and USDA rules and regulations. (45% normal weight), whereas heterozygous mice had normal growth up to the natural weaning period Cell lines and reagents followed by a significant reduction in weight gain and development of insulin resistance (Holzenberger et al. The murine colon carcinoma cell line, CT26, was 2003). Elevations in pituitary GH and IGF1 levels purchased from the American Type Culture Collection C K have been observed in heterozygous Igf1r( / ) mice (ATCC, Manassas, VA, USA) and maintained in RPMI (Holzenberger et al. 2003), and elevations in GH levels supplemented with 10% FBS. Ganitumab was produced by were evident in mice, where circulating IGF1 levels were Amgen, Inc. (Thousand Oaks, CA, USA). A human IgG1 reduced by whole-animal (Liu & LeRoith 1999)and raised against streptavidin (Amgen, Inc.) was used as a liver-specific Igf1 gene disruption (Sjogren et al.1999, nonspecific control for ganitumab. IGF1 and IGF2 were Naranjo et al.2002), supporting the existence of a obtained from Sigma. Ruthenium-labeled IGF1 and IGF2 negative feedback loop where circulating GH/IGF1 levels were generated at Amgen, Inc. and 5-fluorouracil was regulate their own synthesis through IGF1R signaling in acquired from Sandoz (Basel, Switzerland). the pituitary gland. The mIGF1R extracellular domain (ECD) (amino acid Journal of Endocrinology We have shown previously that ganitumab, a fully residues 31–941) was fused to a mIgG1 Fc-coding region human MAB to IGF1R, inhibits the activation of (Bass et al. 1996) to create mIGF1R(ECD)–mFc. The fusion human IGF1R and IGF1R–insulin receptor (IR) hybrids protein was expressed in CHO cells and purified by without cross-reacting with the IR (Beltran et al. 2009, recombinant Protein A Sepharose (GE Healthcare, Pasan- 2011). In addition, we have demonstrated that targeting dena, CA, USA) affinity chromatography. a specific epitope in the cysteine-rich (CR) domain of IGF1R results in subtle but distinct differences in the Characterization of ganitumab cross-reactivity by molecular profile of ganitumab when compared with Biacore solution equilibrium binding assays IGF1R antibodies targeting different domains (Calzone et al. 2013). A number of clinical trials evaluating The Biacore equilibrium method was used to determine

ganitumab and other therapeutic anti-IGF1R antibodies the equilibrium binding constant (KD) of ganitumab for for the treatment of human cancer have been completed mIGF1R(ECD)–mFc as described previously (Beltran et al.

(Rodon et al. 2008, Yuen & Macaulay 2008, Heidegger et al. 2009). The KD was obtained from nonlinear regression of 2011). Though the optimal context and combination the competition curves using an n-curve one-site homo- strategy for positioning a clinical IGF1R antibody geneous binding model (KinExA Pro software, Sapidyne remain to be elucidated (Guha 2013), data from these Instruments, Inc., Boise, ID, USA). studies continue to reveal new roles for IGF1R in An ORIGEN binding assay was used to determine the physiology and disease; therefore, it is important to effect of ganitumab on human IGF1 and IGF2 binding develop animal models that help us understand the to murine IGF1R(ECD)–mFc using procedures provided by consequences of inhibiting IGF1R in biological systems. the manufacturer (IGEN, Inc., Gaithersburg, MD, USA). In this study, we demonstrate that, in addition to its Dynal M450 paramagnetic beads coated with sheep anti- interaction with human IGF1R, ganitumab reacts with and mouse IgG were used as the solid support phase for the

IGF1R(ECD)–mFc. Each assay contained PBS, 0.05% Tween Tumor growth inhibition was expressed relative to the 20 (Mallinckrodt, St. Louis, MO, USA), 0.1% BSA, 50 ng initial and final mean tumor volumes of the control group. receptor loaded onto 1!106 M450 beads, 0.25 nM ruthe- Lysates of lungs and CT26 tumors (mIGF1R) were nium-labeled IGF1 or 0.5 nM ruthenium-labeled IGF2, analyzed by immunoprecipitation using 5 mg anti-IGF1R K K K 10 6–10 11 M ganitumab or rituximab and 10 11– mAb L2C and Protein G Sepharose (GE Healthcare) and K 10 6 M IGF1 or IGF2 in a volume of 100 ml assay buffer. by western blotting using anti-phospho-IGF1R/ After incubation at room temperature in the dark for 2 h, an phospho-IR antibody (Cell Signaling Technologies, Inc.) M384 instrument (Roche Ltd) was used to remove free and sheep anti-mouse-HRP (GE Healthcare). An anti-b ruthenium-labeled ligand and to determine the amount of tubulin antibody (Sigma) was used to probe the non- receptor-bound ligand. immunoprecipitated lysate to normalize the i.p. signal. Western blots were developed using Pierce Supersignal Pico (Pierce, Rockford, IL, USA) and images were captured Inhibition of murine IGF1R autophosphorylation with the VersaDoc imaging system (Bio-Rad). Densitome- Serum-starved CT26 murine colon carcinoma cells were try was performed on blot images using Quantity One pretreated with increasing concentrations of ganitumab or software (Bio-Rad). hIgG1 (500–0.032 nM) for 10 min before addition of 2 nM IGF1 for 15 min. Murine IGF1R was immunoprecipitated Measurement of IGF1R in murine peripheral blood with 5 mg human anti-mIGF1R MAB LC2 (Amgen, Inc.) and immunoblotted with anti-phosphorylated IGF1R/ Mouse whole blood (0.5 ml) was harvested from naı¨ve phosphorylated IR antibody (Cell Signaling Technologies, female athymic nude mice by cardiac puncture into Inc., Danvers, MA, USA) and anti-IGF1Rb antibody C-20 EDTA/heparin-coated capillary tubes. Red blood cells were (Santa Cruz Biotechnology, Inc.) to detect phosphorylated lysed, and white blood cells were stained for flow cytometry and total mIGF1R levels respectively. The specificity of all with ganitumab followed by staining with goat anti-hIgG- antibodies was confirmed using genetically engineered cell FITC, anti-CD11b-PE and anti-GR-1-APC (allophycocyanin, lines and western blotting. BD Biosciences, San Jose, CA, USA). Events were captured using FACSCalibur and analyzed using CellQuest software (BD Biosciences). In vivo pharmacodynamic and efficacy studies Journal of Endocrinology Naı¨ve and tumor-bearing mice were used to examine the Measurement of peripheral neutrophils, platelets and effects of ganitumab on levels of phospho- and total mouse erythrocytes IGF1R in vivo. One million CT26 cells were injected subcutaneously into female athymic nude mice. For In all experiments, 150 ml of blood were collected from pharmacodynamic assays, when the average tumor size anesthetized animals by retro-orbital bleeding using reached 300–450 mm3, mice were randomly divided into heparin-coated glass capillary tubes. Blood was collected four groups (nZ3 per group). Two groups of mice were into Microtainer tubes with EDTA and diluted 1:1 with pretreated with 1 mg ganitumab and two with 1 mg hIgG1 0.1% BSA in PBS. Peripheral blood cell types were by i.p. injection. After 6 h, one ganitumab group and one quantified using the ADVIA120 Hematology System hIgG1 group were administered with human IGF1 (5 mg) (Siemens Healthcare Diagnostics, Los Angeles, CA, USA). by i.v. injection. Control groups were administered with The effects of ganitumab on peripheral neutrophils 1! PBS. Tumors were collected 15 min post IGF1 challenge were assessed in eight female athymic nude mice dosed and snap frozen in liquid nitrogen. The same procedure intraperitoneally with hIgG1 or ganitumab (300 mg/dose) was followed for collection of lung tissue from naı¨ve mice twice per week for a total of five doses. Peripheral (nZ2). For efficacy studies, 250 mm3 tumors were divided neutrophils were quantified once per week for 7 weeks. into four groups (nZ10). Mice were dosed with either The effects of ganitumab on the number of peripheral ganitumab or hIgG1 twice per week (1 mg for the first dose neutrophils in the presence of mGCSF were also and 300 mg for the following doses), 5-fluorouracil determined in female athymic nude mice. Five mice were (40 mg/kg) for 5 days or the combination of both agents. dosed with 300 mg ganitumab or hIgG1 (day 1) followed by Treatment was continued for the length of the experiment. two doses (once daily, days 2 and 3) of mGCSF (2.5 mg s.c.) Tumor volumes and body weights were measured twice per or PBS (s.c.). The 3-day dosing cycle was repeated six times week using calipers and an analytical scale respectively. for 3 weeks. The number of neutrophils was determined

once per week on days 6, 13 and 20 after the start of dosing 1 ng/ml and intra-assay and inter-assay CV values – !10% using the ADVIA120 Hematology System. (range 10–60 ng/ml). Serum mGH levels were determined using an enzyme immunoassay (EIA) kit (ALPCO, Salem, NH, USA) according to the manufacturer’s instructions Glucose tolerance test (detection limit: 0.5 ng/ml; intra-assay and inter-assay CV Thirty male athymic nude mice were dosed by i.p. values: !10 and !14% respectively). injection with ganitumab or hIgG1 (300 mg/dose) twice weekly for 4 weeks. Twenty-four hours after the last dose, Statistical analysis mice were fasted for 16 h. Twenty-five mice in each group were then challenged with an i.p. injection of 25% glucose Statistical analysis: in the xenograft experiment, the effect (2 g/kg) for 15, 30, 60, 90 or 120 min (five mice per time of the combination treatment was compared with that point); five mice in each group were not challenged with of treatment with either agent using repeated-measures glucose to establish basal blood glucose levels. At each ANOVA (RMANOVA). In the body weight experiment, time point, mice were anesthetized using isoflurane, and RMANOVA with Fisher’s protected least significant blood was drawn by cardiac puncture for serum analysis difference (PLSD) post hoc was used. For the pharmacody- using the AU400 glucose analyzer (Olympus Life and namic assays (mouse lung pharmacodynamic assay, CT26 Material Science Europa, Clare, Ireland). In pilot studies, it tumor pharmacodynamic assay, CBC analysis and glucose was found that the use of different cohorts per time point tolerance test), one-way ANOVA with Scheffe’s post hoc produced the most consistent and least variable results test was used. Power calculations: for the body weight when blood glucose was measured. experiment, ten mice per group were used based on historical body weights to detect differences between 5% Inhibition of weight gain by ganitumab (nZ17) and 10% (nZ5) in mouse body weight in the ganitumab-treated cohort. For the glucose tolerance test, Total body weight gain of naı¨ve, adolescent athymic five mice per time point were chosen based on a pilot female mice was measured during a 28-day ganitumab study to detect w30% change in glucose levels between treatment period. Mice were divided into groups based the ganitumab-treated and control-treated mice at each on the body weight (ten animals/group; average weight, time point. For the in vivo phosphorylation assay using 19.6 g) and treated with either ganitumab (80 or Journal of Endocrinology CT26 tumors, values from in vitro pIGF1R experiment were 300 mg/dose) or hIgG1 (300 mg/dose) twice weekly by i.p. used to predict changes in pIGF1R in vivo.Forthis injection. The weight was measured twice weekly using an analytical scale. experiment, three tumor samples would allow detection of 20% difference in pIGF1R levels in mice.

Measurement of mouse serum markers (IGF1, mIGFBPs and mGH) Results To determine the effect of ganitumab on the serum levels of mIGF1, mIGFBPs and mGH, 7-week old, athymic nude Ganitumab binds mIGF1R and inhibits IGF1- and female mice (15 per group) were dosed by i.p. injection IGF2-mediated activation of mIGF1R with either hIgG1 or ganitumab (300 mg/dose) twice per week for 30 days. Fifteen mice per group were killed on day Ganitumab bound to purified mIGF1R(ECD)–mFc with a G 30 for serum analysis. The levels of mIGF1, mIGFBP1, KD of 0.22 0.05 nM (Fig. 1A) and potently inhibited the mIGFBP2, mIGFBP3 and mALS were measured using binding of hIGF1 and hIGF2 to mIGF1R(ECD)–mFc with Ki G G mouse-specific ELISAs (Anzo et al. 2008, Hwang et al. values of 1.9 0.04 and 1.2 0.19 nM respectively (Fig. 1B). 2008, Lee et al. 2010). The assay specifications were as Ganitumab also inhibited IGF1-induced autophosphoryla- follows: mIGF1 – sensitivity 0.1 ng/ml, no cross-reactivity tion of mIGF1R in CT26 murine colon carcinoma cells in a with mIGF2 and intra-assay and inter-assay coefficient dose-dependent manner (Fig. 1C) at similar concentrations of variation (CV) values – !10% (range 1–10 ng/ml); as those observed in human cell lines (Beltran et al.2009). mIGFBP1, mIGFBP2 and mIGFBP3 – sensitivity 0.2 ng/ml No inhibition of ligand-induced autophosphorylation was and intra-assay and inter-assay CV values – !6 and !8% observed using hIgG1, and ganitumab alone had no respectively (range 1–6 ng/ml) and mALS – sensitivity agonistic activity in CT26 cells (Fig. 1C).

A 110 with IGF1. The IGF1-induced activation of mIGF1R was K 0.22 + 0.05 nM D completely inhibited when mice were pretreated with 90 ganitumab (1 mg/dose), but not when mice were pretreated 70 with hIgG1 (Fig. 2A). The low levels of basal phosphorylated

50 mIGF1R did not allow detection of any further inhibition by ganitumab (Fig. 2A). The effective dose of ganitumab in 30 a single-dose pharmacodynamic study was described 0.5 nM IGF1R previously (Beltran et al.2009). 10 1.5 nM IGF1R

% Free mulGF1R (ECD)–mFc 5.0 nM IGF1R Intravenously administered IGF1 induced an approxi- –10 mate ﬁvefold increase in the levels of phosphorylated 10–13 10–12 10–11 10–10 10–9 10–8 10–7 10–6 10–5 Ganitumab concentration (M) mIGF1R in CT26 tumors (Fig. 2B). Pretreatment with ganitumab (1 mg/dose) completely inhibited this stimu- B 110 lation and led to degradation (O70%) of total mIGF1R both hIGF1 90 hIGF2 in the absence and in the presence of IGF1 (Fig. 2B). The extensive receptor downregulation observed following gani- 70 tumab treatment prevented the accurate characterization 50 of changes in the basal pIGF1R levels after ganitumab

30 treatment. Ganitumab treatment of CT26 established xenografts % RU IGF remaining 10 resulted in consistent tumor growth inhibition of w30%.

–10 However, combining ganitumab with 5-fluorouracil –12 –10 –8 –6 resulted in w80% tumor growth inhibition (Fig. 2C). Log ganitumab concentration (M) Thedegreeoftumorgrowthinhibitionachievedin C IgG1 (nM) – – 500 – – – – – – – combination with 5-fluorouracil was statistically better Ganitumab (nM) – 500 – 500 100 20 4 0.8 0.16 0.032 than 5-fluorouracil alone (P!0.001). IGF1 (2 nM) ––+++ + ++++

pIGF1R Ganitumab treatment reduces peripheral blood

Journal of Endocrinology Total IGF1R neutrophils

Using flow cytometric analysis, ganitumab detected high Figure 1 levels of IGF1R in murine (GR1C/CD11bC) cells (Fig. 3A). Interaction of ganitumab with mIGF1R. (A) Ganitumab KD measurement. We therefore sought to determine whether the loss of Binding affinity of ganitumab to mIGF1R(ECD)–mFc measured using the Biacore equilibrium method. (B) Ganitumab blockade of hIGF1 and hIGF2 IGF1R activity in response to ganitumab treatment could binding. A binding assay with mIGF1R(ECD)–mFc was used to evaluate generate a myeloid lineage phenotype as shown pre- Z ganitumab blockade of ruthenium-labeled hIGF1 and hIGF2 (n 2, viously (Welniak et al. 2004). Mice treated with ganitumab meanGS.D.). (C) Ganitumab inhibition of mIGF1R phosphorylation. Serum-starved CT26 cells were pretreated with increasing concentrations (300 mg/dose) for 2 weeks displayed a marked reduction of ganitumab before challenge with 2 nM IGF1 for 15 min. IGF1R (up to 50%) in the number of peripheral neutrophils when phosphorylation was determined by immunoprecipitation and western compared with hIgG1 controls (Fig. 3B, P!0.01) with a blotting. pIGF1R, phosphorylated IGF1R; RU, relative units. maximal reduction on day 17, after reaching steady-state serum levels based on the previous pharmacokinetic studies using this dosing regimen (Beltran et al. 2009). Ganitumab inhibits IGF1-induced activation of mIGF1R The effect was reversed following a 14-day recovery period in murine lungs and syngeneic CT26 tumors during which ganitumab was cleared (half-lifeZ3–4 days). The pharmacological activity of ganitumab in mice was No changes in erythrocytes or platelets were observed in tested using an IGF1 challenge pharmacodynamic model mice treated with ganitumab (Fig. 3B). described previously (Beltran et al.2009). Treatment of mice To investigate the mechanisms involved in neutrophil with IGF1 led to an increase in the levels of phosphorylated depletion, we determined the effect of ganitumab on mIGF1R in murine lungs (Fig. 2A). Mouse lungs were chosen neutrophil induction with mGCSF. As expected, neutrophil for the PD assay because they displayed the highest level of levels increased (five- to eightfold) after mGCSF adminis- IGF1R activation of any organ in the mouse upon challenge tration, with a moderate decline over a 3-week period with

A CT26 CT26 cells Murine lung cells Murine lung 0.20 No IGF1 hIgG1 – ++ – – –+––+ + IGF1 Ganitumab – –– + + ––++– 0.15 IGF1 (5 µg) + –– – – +++++ 0.10 pIGF1R *

0.05

tIGF1R Phospho/total IGF1R 0.00

lgG1 IgG1

B C Ganitumab Ganitumab hIgG1 ++++++–––––– Ganitumab ––––––++++++ 2500 hIgG1+PBS *78% TGI;P< 0.001 vs IGF1 –––+++––– +++ ganitumab or 5-FU alone Ganitumab+PBS

pIGF1R ) 2000 hIgG1+5-FU 3 Ganitumab+5-FU tIGF1R 1500 β-tubulin (lysate) 1000

5 * Tumor volume (mm volume Tumor 500 Total IGF1R 4 pIGF1R 0 1314 15 16 17 18 19 20 21 3 Time (days)

2 total IGF1R (RU) Phosphorylated or 1 * * * * 0

Journal of Endocrinology hIgG1 hIgG1 Ganitumab hIgG1+IGF1Ganitumab hIgG1+IGF1

Ganitumab+IGF1 Ganitumab+IGF1

Figure 2 Inhibition of mIGF1R activation by ganitumab in vivo. (A) (Left panel) IGF1-induced mIGF1R activation in CT26 colon carcinoma tumors. Each lane Ganitumab inhibited IGF1-induced activation of mIGF1R in murine lungs. of the blot represents an individual mouse treated once with 1 mg/dose of Each lane represents an individual mouse treated once with 1 mg/dose of ganitumab or hIgG1 for 6 h (nZ3 per group). The bar chart shows the ganitumab or hIgG1 for 6 h (nZ2 per group). In vitro cultures of CT26 cells amount of phosphorylated and total IGF1R; data are shown as the were used as positive control for mIGF1R phosphorylation status. (Right meanGS.D.(*P!0.05 ganitumab vs hIgG1). pIGF1R, phosphorylated IGF1R; panel) Densitometric analysis of IGF1R activation from the image on the RU, relative units. (C) Efficacy of ganitumab in combination with left. Bars represent mean of the band intensity GS.D. Values were 5-flurouracil in the CT26 model. CT26 xenografts were randomly divided normalized to total IGF1R levels from CT26-positive controls. Significant into four groups (nZ10): i) hIgG1CPBS, ii) ganitumabCPBS, iii) hIgG1C inhibition of pIGF1R was observed in ganitumab-treated mice receiving 5-Flurouracil and iv) ganitumabC5-flurouracil. Data are shown as mean IGF1 challenge (*P!0.001). (B) Ganitumab significantly inhibited tumor volume GS.E.M.

repeated dosing. Pretreatment with ganitumab inhibited expression of IGF1R was lowered by gene deletion the increase in neutrophils by 75% (P!0.01) (Fig. 3C). (Holzenberger et al. 2003). The effect of ganitumab on glucose uptake in naı¨ve male athymic nude mice was determined after a glucose challenge (2 g/kg) in pretreated Ganitumab causes impaired glucose tolerance in animals. Ganitumab-pretreated mice had signiﬁcantly male mice higher serum glucose levels than hIgG1-pretreated mice Male mice were chosen to perform glucose-uptake (Fig. 4A). The maximum difference was observed at experiments since they showed signiﬁcantly greater 30 min, when ganitumab-treated mice had a 40% higher reductions in glucose uptake than females when blood glucose levels than hIgG1-treated mice (PZ0.004).

A All cells Proportion of GR1+/CD11b+ cells 105 250 200 104 150 103 100 Cell count

GR1 APC area 50 102 0 102 103 104 105 102 103 104 105 CD11b PE area Ganitumab FITC area

BC1.6 Neutrophils Erythrocytes 25 1.4 hIgG1 Platelets mGCSF 1.2 20 mGCSF + ganitumab 1.0 15 0.8 0.6 10 * * 0.4 * 5 *

Ratio (ganitumab:hIgG1) Ganitumab Recovery 0.2 * Neutrophil count (1000/ml) 0.0 0 0 5 10 15 20 25 30 35 40 10 17 24 Days Days

Figure 3 Effects of ganitumab on murine peripheral neutrophils. (A) Ganitumab The ratio of the number of neutrophils in the ganitumab-treated group to detected mIGF1R on the surface of murine (GR1C/CD11bC) peripheral the number of neutrophils in the hIgG1-treated group is shown on the blood cells using FACS analysis. The boxed area (gray) in the left panel indicated days. Significant differences were measured between day 17 or shows the GR1C/CD11bC cells. The right panel shows that most GR1C/ day 24 and day 3 or day 38 (*P!0.01). Mice were dosed with 300 mg CD11bC cells (smaller gray peak) are also ganitumab positive. The orange ganitumab twice per week (nZ8 per group). (C) Ganitumab significantly data points represent negative cells. (B) Ganitumab significantly decreased inhibited the increase in peripheral blood neutrophils induced by mGCSF the number of peripheral neutrophils in naı¨ve female athymic mice. (*P!0.01; nZ5). Data are shown as the meanGS.E.M. Journal of Endocrinology Ganitumab inhibits weight gain in result in elevated levels of serum mIGF1 and mGh. Indeed, athymic female nude mice statistically significant increases in the levels of mGh, mIGF1 and mIGFBP3 (*PZ0.048, †P!0.0001 and The phenotypes of mice that carry Igf1r and Igf1 gene ‡P!0.0001) were observed in ganitumab-treated mice vs disruptions suggest that receptor blockade with ganitumab hIgG1-treated mice on day 30 (Fig. 4C). Statistically should inhibit increases in body weight gain (Yakar et al. significant increases in the levels of mIGFBP3 and mGh 2005). Athymic nude mice were chosen to study the effects (P!0.037, P!0.01) were also observed on day 19 (data not of ganitumab on weight gain to minimize immune shown). No measurable changes in mIGFBP1, mIGFBP2, responses against ganitumab, and females because the effect mALS, glucose or insulin levels were obtained in any of the of IGF1R deficiency in female mice on longevity was more groups (data not shown). A significant direct correlation pronounced than in male mice (Holzenberger et al.2003). (r2Z0.7, P!0.0001) between mIGF1 levels and mIGFBP3 Statistically significant inhibition (w50%, P!0.02) of body levels was observed in ganitumab-treated mice; no weight gain was observed in mice treated with 300 mg/dose correlation was observed in hIgG1-treated mice (Fig. 4D). of ganitumab twice per week by i.p. injection (Fig. 4B). Discussion Ganitumab increases serum levels of mIGF1, Several anti-IGF1R MABs have been described in the mIGFPB3 and mGh scientific literature (Kull et al. 1983, Soos et al. 1992, The genetic analysis of endocrine IGF1 signaling in mice Xiong et al. 1992, Li et al. 1993, Burtrum et al. 2003, indicates that IGF1R regulates the serum IGF1 levels Maloney et al. 2003, Cohen et al. 2005). However, to our through inhibition of pituitary Gh release (Yakar et al. knowledge, no data have been published that convin- 2001). Thus, treatment of mice with ganitumab should cingly demonstrate that these antibodies physically and

AB C D 6 † 112 hIgG1 hIgG1 1600 16 5 * mGH 2500 hIgG1 Ganitumab 110 Ganitumab ‡ 1400 * mIGF1 14 Ganitumab 4 108 2000 1200 mIGFBP3 12 106 ‡ 1000 10 3 ‡ 1500 104 * 800 8 2 102 600 6 1000

100 † mGH (ng/ml)

Blood glucose (g/l) 400 4 1 500 mIGFBP3 (ng/ml) % Initial body weight 98 200 2 0 96 0 0 0

mIGF1 or mIGFBP3 (ng/ml) 0204060 80 100 120 0 5 10 15 20 25 30 0 100 200 300 400 500 Time (min) Time (days) hIgG1 hIgG1 hIgG1 mIGF1 (ng/ml) Ganitumab Ganitumab Ganitumab

Figure 4 Physiological changes induced by ganitumab treatment. (A) Sensitization ganitumab-treated groups compared with the control group (*PZ0.028). of male mice to glucose challenge (2 g/kg) by pretreatment with (C) Ganitumab vs hIgG1 significantly increased serum levels of mGH, ganitumab (300 mg/dose, twice per week, nZ5). Significant differences in mIGF1 and mIGFBP3 on day 30 (*PZ0.048, †P!0.0001 and ‡P!0.0001 blood glucose levels were observed at 15, 30, 60 and 90 min post glucose respectively). Data are from two experiments with a mean of 15 mice per † ‡ 2 challenge (*P!0.05, P!0.004, P!0.02). (B) Effect of ganitumab on body group GS.D. (D) There was a significant, direct correlation (r Z0.7, weight gain in naı¨ve mice. Ganitumab at 300 mg/dose inhibited body P!0.0001) between the levels of mIGF1 and mIGFBP3 in individual weight gain when compared with control hIgG1 in naı¨ve, 7-week-old, ganitumab-treated mice after 30 days of treatment. female athymic mice (nZ10). Weight gain was significantly different in the

functionally interact with mIGF1R. In this study, we have Given the effect of ganitumab on body weight, it was demonstrated that ganitumab recognized mIGF1R and not surprising that ganitumab treatment led to alterations inhibited its interaction with IGF1 and IGF2. Ganitumab in levels of circulating GH and IGF1. Elevation of levels also inhibited the activation of mIGF1R in the normal of pituitary Gh and/or IGF1 has been consistently murine lung and established murine tumors. The observed in response to genetic inhibition of IGF1R C K inhibition of mIGF1R by ganitumab enhanced the signaling in Igf1r( / ) animals (Holzenberger et al. 2003), cytotoxic activity of 5-flurouracil against the murine and in mice where circulating IGF1 levels were reduced by colorectal carcinoma model CT26. The cross-reactivity of whole-animal (Liu & LeRoith 1999) and liver-specific Igf1 ganitumab to mIGF1R is a unique characteristic of this gene disruption (Sjogren et al. 1999, Naranjo et al. 2002). molecule that allowed us to pharmacologically examine Based on these genetic data, it has been hypothesized that the effects of IGF1R inhibition on mouse physiology. there is a negative feedback loop in the pituitary whereby Journal of Endocrinology The phenotypic changes in nude mice treated with Gh release is suppressed through IGF1R signaling driven ganitumab observed in the present study were similar by circulating total IGF1 (Fig. 6). The pituitary Gh excess C K to the changes observed in the genetic manipulation of in Igf1r( / ) mice was associated with a 20–30% increase in the GH–IGF1 axis through Igf1r- and Igf1-targeted gene circulating IGF1 (Holzenberger et al. 2003); we observed a disruption (Holzenberger et al. 2003, Yakar et al. 2005). similar effect when IGF1R was inhibited by ganitumab. The inhibition of weight gain was comparable to the Thus, it is likely that synthesis of IGF1 in the liver was C K phenotype of the Igf1r( / ) mice (Holzenberger et al. 2003) stimulated by the excess Gh produced when pituitary and liver-specific Als (Als knockout (ALSKO)) or Igf1CAls IGF1R signaling was inhibited by ganitumab, thereby (LIDCALSKO) gene disruption (Yakar et al. 2005). Gani- resulting in the serum IGF1 concentration changes tumab appeared to be highly effective at inhibiting weight observed in the treated mice. Importantly, our data show gain (w50% inhibition) during the treatment period in that the pituitary feedback occurs in a rapid, tightly naı¨ve mice. The lack of a more severe effect on body regulated fashion. weight as a result of ganitumab treatment when compared In ganitumab-treated mice, the levels of circulating with targeted homozygous deletion of Igf1 or Igf1r was IGF1 and IGFBP3 produced by the liver appeared to be probably due to the age of the animals at the time when highly correlated, even though these proteins are pro- treatment was started. In contrast to the genetic experi- duced in different cells (Le Roith et al. 2001). The loss of ments where mice were exposed to IGF1 or IGF1R hepatic IGF1 synthesis in LID and LIDCALSKO mice was inhibition early in their development, treatment with associated with a corresponding decrease in serum IGFBP3 ganitumab did not begin until weeks 4 through 7 and (Sjogren et al. 1999, Naranjo et al. 2002). The genetic data proceeded for 30 days. At this age, the average mouse body and our pharmacological data with respect to IGFBP3 are weight is w20 g, and body weight gain is limited to a most easily reconciled if IGF1R signaling has no direct further 10–20%. role in regulating circulating IGFBP3 levels in mice.

We propose that in well-fed mice, accumulation of IGFBP3 (Fig. 1C). Pharmacodynamic assays using murine lung in circulation requires assembly into a complex with IGF1 tissue and murine CT26 colon carcinoma tumors showed (and ALS), while excess IGFBP3 is eliminated by a rapid that ganitumab blocked ligand binding to IGF1R in both clearance mechanism. Thus, in the mouse, circulating tissue types but may downregulate IGF1R more efficiently IGFBP3 levels are dependent on the total IGF1 concen- in tumor cells. In vivo IGF1R downregulation was also tration. This mechanism is consistent with the obser- observed previously in human tumor xenografts vation that no change in Igfbp3 mRNA levels was treated with ganitumab (Beltran et al. 2011). The exact C detected in LID and LID ALSKO mice on normal diets mechanisms causing the differences in receptor down- (Naranjo et al. 2002). regulation between the two tissue types are unknown The modest impairment in glucose uptake and at this time but may involve differences in antibody metabolism that we observed in ganitumab-treated mice exposure, endosomal/lysosomal machinery or local is similar to what was observed in mice in which IGF1R immune infiltrates that drive antibody–receptor cross- activity had been genetically reduced (Holzenberger et al. linking internalization and degradation. 2003). Basal glucose levels were normal in ganitumab- In summary, we have shown that treatment of mice treated mice, but their ability to respond to glucose with ganitumab, a fully human MAB that is active against challenge was significantly compromised when compared both human and murine IGF1R, led to a number of rapid with controls. The underlying mechanism may be related physiological changes predicted for a pharmacological to Gh-mediated insulin resistance that had been charac- inhibitor with IGF1R activity. Treatment not only repro- terized previously in LID and LID ALSKO mice (Yakar et al. duced phenotypic phenomena in mouse models with 2001). However, it is also possible that ganitumab induced targeted IGF axis disruption, but also predicted physio- glucose intolerance through its inhibitory effect on IGF1R logical and pharmacological changes in patients treated in pancreatic b-islet cells (Kulkarni 2002) or on IGF1R–IR with ganitumab monotherapy in the clinic. Further hybrids in peripheral tissues (Pandini et al. 2002). Our clinical and preclinical analyses of these and other results provide further support for the concept that IGF1R changes using ganitumab may identify useful biomarkers signaling contributes to the regulation of blood glucose to optimize the development and utilization of IGF1R- and is consistent with early observations of hyperglycemia antagonistic agents in the clinic. in clinical trials of IGF1R inhibitors (Rodon et al. 2008). The mild and reversible deficiency in myelopoiesis Journal of Endocrinology associated with ganitumab was also seen in LID mice (Welniak et al. 2004) where, similar to the effects of Declaration of interest G M, P J B, E C, P M, Y-A C, R K and R R are Amgen, Inc. employees and own ganitumab, splenic myeloid progenitors were reduced Amgen, Inc. stock. F J C was an employee of Amgen, Inc. and owns Amgen, by about 50%, while all other hematologic parameters Inc. stock. D H and P C received grant support from Amgen, Inc. (including B and T cells) were unaffected. The mechanism by which IGF1R blockade results in neutrophil depletion is not yet clear but may involve blockade of progenitor differen- Funding tiation as described previously in cells that do not This study was sponsored by Amgen, Inc. Additional grant support was provided by the National Institute on Aging (NIA 1P01AG034906). express IRS1 (Valentinis et al.2000). Interestingly, ganitumab inhibited the increase in peripheral neutrophils induced by mGCSF suggesting a requirement for IGF1R Acknowledgements signaling in progenitor differentiation or mobilization. We thank Grace Chung, Larry Daugherty and Keith Kelley for assistance Clinical data also demonstrated a reduction in neutrophil with flow cytometry, Sylvia Copon for assistance with the ADVIA120 counts in some patients with cancer who received ganitu- Hematology System, Robert Ortiz for assistance with the pharmacokinetic mab as monotherapy (Tap et al.2012). Our rodent studies analysis, Barbara Felder for assistance with immunohistochemistry, Renato Baserga for critical review of the manuscript and Kathryn Boorer might provide a suitable model to better understand the role (Amgen, Inc.) for editorial assistance. of IGF1R in neutrophil differentiation and survival. The trough serum ganitumab concentration required to achieve 50% inhibition of BxPC-3 and References MiaPaCa2 pancreatic carcinoma xenograft models Z Anzo M, Cobb LJ, Hwang DL, Mehta H, Said JW, Yakar S, LeRoith D & (EC50 3.05 mg/ml) (Beltran et al. 2009) was tenfold higher Cohen P 2008 Targeted deletion of hepatic Igf1 in TRAMP mice leads to than the IC50 for inhibition of mIGF1R activation in vitro dramatic alterations in the circulating insulin-like growth factor axis

but does not reduce tumor progression. Cancer Research 68 3342–3349. Lee C, Safdie FM, Raffaghello L, Wei M, Madia F, Parrella E, Hwang D, (doi:10.1158/0008-5472.CAN-07-3165) Cohen P, Bianchi G & Longo VD 2010 Reduced levels of IGF-I mediate Baker J, Liu JP, Robertson EJ & Efstratiadis A 1993 Role of insulin-like differential protection of normal and cancer cells in response to fasting growth factors in embryonic and postnatal growth. Cell 75 73–82. and improve chemotherapeutic index. Cancer Research 70 1564–1572. (doi:10.1016/0092-8674(93)90680-O) (doi:10.1158/0008-5472.CAN-09-3228) Baserga R, Hongo A, Rubini M, Prisco M & Valentinis B 1997 The IGF-I Le Roith D, Scavo L & Butler A 2001 What is the role of circulating IGF-I? receptor in cell growth, transformation and apoptosis. Biochimica et Trends in Endocrinology and Metabolism 12 48–52. (doi:10.1016/ Biophysica Acta 1332 F105–F126. S1043-2760(00)00349-0) Bass J, Kurose T, Pashmforoush M & Steiner DF 1996 Fusion of insulin Li SL, Kato J, Paz IB, Kasuya J & Fujita-Yamaguchi Y 1993 Two new receptor ectodomains to immunoglobulin constant domains monoclonal antibodies against the alpha subunit of the reproduces high-affinity insulin binding in vitro. Journal of Biological human insulin-like growth factor-I receptor. Biochemical and Chemistry 271 19367–19375. (doi:10.1074/jbc.271.32.19367) Biophysical Research Communications 196 92–98. (doi:10.1006/bbrc. Beltran PJ, Mitchell P, Chung YA, Cajulis E, Lu J, Belmontes B, Ho J, Tsai MM, 1993.2220) Zhu M, Vonderfecht S et al. 2009 AMG 479, a fully human Liu JL & LeRoith D 1999 Insulin-like growth factor I is essential for anti-insulin-like growth factor receptor type I monoclonal antibody, postnatal growth in response to growth hormone. Endocrinology 140 inhibits the growth and survival of pancreatic carcinoma cells. 5178–5184. (doi:10.1210/endo.140.11.7151) Molecular Cancer Therapeutics 8 1095–1105. (doi:10.1158/1535-7163. Maloney EK, McLaughlin JL, Dagdigian NE, Garrett LM, Connors KM, MCT-08-1171) Zhou XM, Blattler WA, Chittenden T & Singh R 2003 An anti- Beltran PJ, Chung YA, Moody G, Mitchell P, Cajulis E, Vonderfecht S, insulin-like growth factor I receptor antibody that is a potent Kendall R, Radinsky R & Calzone FJ 2011 Efficacy of ganitumab inhibitor of cancer cell proliferation. Cancer Research 63 5073–5083. (AMG 479), alone and in combination with rapamycin, in Naranjo WM, Yakar S, Sanchez-Gomez M, Perez AU, Setser J & LeRoith D Ewing’s and osteogenic sarcoma models. Journal of Pharmacology 2002 Protein calorie restriction affects nonhepatic IGF-I production and Experimental Therapeutics 337 644–654. (doi:10.1124/jpet.110. and the lymphoid system: studies using the liver-specific IGF-I 178400) gene-deleted mouse model. Endocrinology 143 2233–2241. Burtrum D, Zhu Z, Lu D, Anderson DM, Prewett M, Pereira DS, Bassi R, (doi:10.1210/endo.143.6.8852) Abdullah R, Hooper AT, Koo H et al. 2003 A fully human monoclonal Pandini G, Frasca F, Mineo R, Sciacca L, Vigneri R & Belfiore A 2002 antibody to the insulin-like growth factor I receptor blocks ligand- Insulin/insulin-like growth factor I hybrid receptors have different dependent signaling and inhibits human tumor growth in vivo. Cancer biological characteristics depending on the insulin receptor isoform Research 63 8912–8921. involved. Journal of Biological Chemistry 277 39684–39695. Calzone FJ, Cajulis E, Chung YA, Tsai MM, Mitchell P, Lu J, Chen C, Sun J, (doi:10.1074/jbc.M202766200) Radinsky R, Kendall R et al. 2013 Epitope-specific mechanisms of IGF1R Pollak M 2012 The insulin and insulin-like growth factor receptor family in inhibition by ganitumab. PLoS ONE 8 e55135. (doi:10.1371/journal. neoplasia: an update. Nature Reviews. Cancer 12 159–169. pone.0055135) Rodon J, DeSantos V, Ferry RJ Jr & Kurzrock R 2008 Early drug development Cohen BD, Baker DA, Soderstrom C, Tkalcevic G, Rossi AM, Miller PE, of inhibitors of the insulin-like growth factor-I receptor pathway: Tengowski MW, Wang F, Gualberto A, Beebe JS et al. 2005 Combination lessons from the first clinical trials. Molecular Cancer Therapeutics 7 therapy enhances the inhibition of tumor growth with the fully human 2575–2588. (doi:10.1158/1535-7163.MCT-08-0265)

Journal of Endocrinology anti-type 1 insulin-like growth factor receptor monoclonal antibody Sjogren K, Liu JL, Blad K, Skrtic S, Vidal O, Wallenius V, LeRoith D, Tornell J, CP-751,871. Clinical Cancer Research 11 2063–2073. (doi:10.1158/ Isaksson OG, Jansson JO et al. 1999 Liver-derived insulin-like growth 1078-0432.CCR-04-1070) factor I (IGF-I) is the principal source of IGF-I in blood but is not required Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A, Goldfine ID, for postnatal body growth in mice. PNAS 96 7088–7092. (doi:10.1073/ Belfiore A & Vigneri R 1999 Insulin receptor isoform A, a newly pnas.96.12.7088) recognized, high-affinity insulin-like growth factor II receptor in fetal Soos MA, Field CE, Lammers R, Ullrich A, Zhang B, Roth RA, Andersen AS, and cancer cells. Molecular and Cellular Biology 19 3278–3288. Kjeldsen T & Siddle K 1992 A panel of monoclonal antibodies for the Gallagher EJ & LeRoith D 2011 Minireview: IGF, insulin, and cancer. type I insulin-like growth factor receptor. Epitope mapping, effects on Endocrinology 152 2546–2551. (doi:10.1210/en.2011-0231) ligand binding, and biological activity. Journal of Biological Chemistry Guha M 2013 Anticancer IGF1R classes take more knocks. Nature Reviews. 267 12955–12963. Drug Discovery 12 250. (doi:10.1038/nrd3992) Tap WD, Demetri G, Barnette P, Desai J, Kavan P, Tozer R, Benedetto PW, Heidegger I, Pircher A, Klocker H & Massoner P 2011 Targeting the insulin- Friberg G, Deng H, McCaffery I et al. 2012 Phase II study of ganitumab, a like growth factor network in cancer therapy. Cancer Biology & Therapy fully human anti-type-1 insulin-like growth factor receptor antibody, 11 701–707. (doi:10.4161/cbt.11.8.14689) in patients with metastatic ewing family tumors or desmoplastic small Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, round cell tumors. Journal of Clinical Oncology 30 1849–1856. Cervera P & Le Bouc Y 2003 IGF-1 receptor regulates lifespan and (doi:10.1200/JCO.2011.37.2359) resistance to oxidative stress in mice. Nature 421 182–187. Valentinis B, Navarro M, Zanocco-Marani T, Edmonds P, McCormick J, (doi:10.1038/nature01298) Morrione A, Sacchi A, Romano G, Reiss K & Baserga R 2000 Insulin Hwang DL, Lee PD & Cohen P 2008 Quantitative ontogeny of murine receptor substrate-1, p70S6K, and cell size in transformation and insulin-like growth factor (IGF)-I, IGF-binding protein-3 and the differentiation of hemopoietic cells. Journal of Biological Chemistry 275 IGF-related acid-labile subunit. Growth Hormone & IGF Research 18 25451–25459. (doi:10.1074/jbc.M002271200) 65–74. (doi:10.1016/j.ghir.2007.07.007) Welniak LA, Karas M, Yakar S, Anver MR, Murphy WJ & LeRoith D 2004 Kulkarni RN 2002 Receptors for insulin and insulin-like growth factor-1 Effects of organ-specific loss of insulin-like growth factor-I production and insulin receptor substrate-1 mediate pathways that regulate islet on murine hematopoiesis. Biology of Blood and Marrow Transplantation function. Biochemical Society Transactions 30 317–322. (doi:10.1042/ 10 32–39. (doi:10.1016/j.bbmt.2003.09.008) BST0300317) Xiong L, Kasuya J, Li SL, Kato J & Fujita-Yamaguchi Y 1992 Growth- Kull FC Jr, Jacobs S, Su YF, Svoboda ME, Van Wyk JJ & Cuatrecasas P 1983 stimulatory monoclonal antibodies against human insulin-like Monoclonal antibodies to receptors for insulin and somatomedin-C. growth factor I receptor. PNAS 89 5356–5360. (doi:10.1073/pnas.89.12. Journal of Biological Chemistry 258 6561–6566. 5356)

Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B & LeRoith D 1999 Yakar S, Pennisi P, Kim CH, Zhao H, Toyoshima Y, Gavrilova O & LeRoith D Normal growth and development in the absence of hepatic 2005 Studies involving the GH–IGF axis: lessons from IGF-I and IGF-I insulin-like growth factor I. PNAS 96 7324–7329. (doi:10.1073/pnas. receptor gene targeting mouse models. Journal of Endocrinological 96.13.7324) Investigation 28(Suppl 5) 19–22. Yakar S, Liu JL, Fernandez AM, Wu Y, Schally AV, Frystyk J, Chernausek SD, Yuen JS & Macaulay VM 2008 Targeting the type 1 insulin-like Mejia W & Le Roith D 2001 Liver-speciﬁc igf-1 gene deletion leads to growth factor receptor as a treatment for cancer. Expert muscle insulin insensitivity. Diabetes 50 1110–1118. (doi:10.2337/ Opinion on Therapeutic Targets 12 589–603. (doi:10.1517/14728222. diabetes.50.5.1110) 12.5.589)

Received in ﬁnal form 2 February 2014 Accepted 3 February 2014 Accepted Preprint published online 3 February 2014 Journal of Endocrinology