Molecular Imaging of Death Receptor 5 Occupancy and Saturation Kinetics in Vivo by Humanized Monoclonal Antibody CS-1008

Published OnlineFirst September 17, 2013; DOI: 10.1158/1078-0432.CCR-12-3104

Clinical Cancer Imaging, Diagnosis, Prognosis Research

Molecular Imaging of Death Receptor 5 Occupancy and Saturation Kinetics In Vivo by Humanized Monoclonal Antibody CS-1008

Ingrid J.G. Burvenich1, Fook T. Lee1, Glenn A. Cartwright1, Graeme J. O'Keefe3, Dahna Makris1, Diana Cao1, Sylvia Gong3, Anderly C. Chueh2, John M. Mariadason2, Martin W. Brechbiel4, Robert A. Beckman5, Kosaku Fujiwara6, Reinhard von Roemeling5, and Andrew M. Scott1,3

Abstract Purpose: CS-1008 (tigatuzumab; phase I/II), an antihuman death receptor 5 (DR5) agonist, induces apoptosis and has cytotoxic activity against human cancer cell lines. This study reports on the preclinical validation of 111In-labeled anti-DR5 humanized antibody CS-1008 as a diagnostic tool to study the DR5 occupancy in patients with cancer and establish dose ranges for receptor saturation kinetics in vivo. Experimental Design: CS-1008 was radiolabeled and characterized for DR5 binding and labeling efficiency on TRAIL-sensitive DR5–positive colorectal cancer cells (COLO 205 and WiDr). Pharmacokinetic and biodistribution studies were conducted in BALB/c nu/nu mice bearing COLO 205, WiDr, or DR5- negative CT26 colon tumors. Planar gamma camera imaging and computerized tomography (CT) images were obtained to study receptor occupancy in vivo. 111 Results: Scatchard analysis showed high and specific binding affinity (Kd, 1.05 0.12 nmol/L) of In- labeled CS-1008. 111In-labeled CS-1008 was specifically taken up in mice bearing COLO 205 and WiDr tumors with prolonged tumor retention (26.25 2.85%ID/g vs. 12.20 2.24 at 168 hours post injection; n ¼ 5, SD), and uptake correlated both with DR5 expression on tumor cells and antitumor activity. DR5 saturation was shown in vivo via both biodistribution studies and planar gamma camera imaging/CT imaging of 111In-labeled CS-1008. Saturation of DR5 corresponded to maximal in vivo antitumor efficacy. Conclusions: Imaging of DR5 receptor occupancy in vivo correlates with tumor concentration and in vivo efficacy, and is a novel molecular imaging technique that can be used to determine receptor occupancy and effective dose levels of DR5 agonist antibodies in the clinic. Clin Cancer Res; 19(21); 5984–93. 2013 AACR.

Introduction ity determining region grafting (1, 2). DR5 is a member of CS-1008 (tigatuzumab) is a humanized monoclonal the TNF receptor superfamily, which contains a cytoplasmic death domain that is capable of initiating an apoptotic immunoglobulin G (IgG1) with agonistic activity upon binding to human death receptor 5 (DR5). It was developed signal upon binding with its endogenous ligand TRAIL by Daiichi Sankyo Company Ltd. from a murine anti-DR5 (3). Apoptosis is thought to be deregulated in cancer cells monoclonal antibody, TRA-8 (mTRA-8) by complementar- and activating proapoptotic pathways is being explored as a mechanism to render cancer cells more susceptible to death. Several anti-DR5 specific antibodies have been Authors' Affiliations: 1Tumour Targeting Laboratory and 2Oncogenic described: Lexatumumab (human IgG1; Human Genome 3 Transcription Laboratory, Ludwig Institute for Cancer Research; Depart- Sciences), Apomab (human IgG1; Genentech), AMG-655 ment of Nuclear Medicine and Centre for PET, Austin Health, Melbourne, (human IgG ; Amgen), LBY135 (chimeric IgG1; Novartis), Australia; 4Radioimmune & Inorganic Chemistry Section, Radiation Oncol- 1 ogy Branch, Cancer Research Centre, Bethesda, Maryland; 5Clinical Devel- and TRA-8/CS-1008 (murine IgG1/humanized IgG1; Daii- opment-Oncology, Daiichi Sankyo Pharmaceutical Development, Edison, chi Sankyo Company; ref. 4). New Jersey; and 6Global Project Management Department, Daiichi Sankyo Co. Ltd., Tokyo, Japan Both mTRA-8 and CS-1008 antibodies have shown specific in vitro cytotoxicity and antitumor efficacy against Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). solid tumor xenografts in vivo.mTRAwasselectedforits low hepatocellular toxicity and ability to trigger apoptosis Corresponding Author: Andrew M. Scott, Tumour Targeting Laboratory, Ludwig Institute for Cancer Research, 145-163 Studley Road, Heidelberg, in vitro in a variety of human tumor cell lines without the Victoria 3084, Australia. Phone: 61-39496-5876; Fax: 61-39496-5334; use of cross-linking agents (1). In vivo antitumor efficacy E-mail: [email protected] was shown in different mouse models bearing human doi: 10.1158/1078-0432.CCR-12-3104 xenografts (1, 5) and can be enhanced in combination 2013 American Association for Cancer Research. with chemotherapeutic agents and/or radiation (5–7).

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colorectal adenocarcinoma cell line (11); and A431, a LeY Translational Relevance -expressing human epidermoid carcinoma cell line used to CS-1008 (tigatuzumab) is an antihuman death recep- assess LeY antigen binding of hu3S193 were obtained from tor (DR5) agonist that induces apoptosis and has cyto- American Type Culture Collection (ATCC). WiDr, a low- toxic activity against human cancer cell lines. Tumor level DR5-expressing colorectal cancer cell line, was concentration of CS-1008 in vivo and the impact of dose obtained from J.M. Mariadason, Ludwig Institute for Cancer on tumor saturation of DR5 remain key questions in the Research (Melbourne, Australia). Cell lines were authenti- determination of the optimal dosing schedule for ther- cated at the DNA Sequencing and Fragment Analysis Facility apeutic beneﬁt. This study reports on the exploration of at QIMR (Queensland Institute for Medical Research, targeting of anti-DR5 humanized antibody CS-1008 to Queensland, Australia) and expression levels of target recep- DR5-expressing tumors in vivo, and quantitative analysis tors (DR5 or LeY) have been tested by single-point Lindmo of antibody:DR5 binding and occupancy. The relation- assays as part of each study using radiolabeled CS-1008 or ship of in vivo imaging of CS-1008 in tumor to thera- hu3S193. CT26 is a mouse colon cancer cell line and was peutic response was also established. Our results showed tested for its mouse origin at QIMR. A431 cells were grown that in vivo saturation of DR5 was predictive of thera- in DMEM/F-12 with 10% v/v FBS, 1% v/v penicillin–strep- peutic dose–response threshold. This novel and impor- tomycin (10,000 U/mL in 0.85% NaCl), and 1% v/v tant information is highly relevant to DR5 modulation L-glutamine (200 mM/mL in 0.85% NaCl; Invitrogen). as a therapeutic approach and informs the design of COLO 205, WiDr, and CT26 cells were grown in RPMI- clinical trials of anti-DR5 antibodies in patients with 1640 with 10% v/v FBS and 1% v/v penicillin–streptomycin cancer. (10,000 U/mL in 0.85% NaCl).

Flow cytometry Adherent COLO 205, WiDr, and CT26 cells were trypsi- CS-1008 has similar in vitro and in vivo antitumor activity nized and resuspended in fresh culture medium. Cells were as mTRA-8 (2). aliquoted into 1.5 mL eppendorf tubes at a density of 1 Phase I trials in humans have shown linear pharmaco- 106 cells per tube. Cells were centrifuged at 3,000 g for 3 kinetics in doses ranging from 1 to 8 mg/kg/week with a minutes, and the pellets were resuspended in 100 mLofan half-life of 8to 16 days. There were no partial responses to antihuman CD262/DR5-PE antibody (1:5 concentration; treatment but stable disease was noted in 7 of 17 treated Affymetrix eBioscience Inc.). A cells-only control and a patients with the duration of response ranging from 81 to negative control (1:100 Goat antihuman IgG-g chain spe- 0 798 days. CS-1008 dose–limiting toxicity was not reached, cific F(ab )2 PE; Sigma-Aldrich) were included. All samples and no evidence of immunogenicity to the antibody was were incubated on ice for 30 minutes. Cells were then detected (8). centrifuged at 3,000 g for 3 minutes and the supernatant CS-1008 induces cell death in a variety of DR5-expres- was discarded. Cells were washed three times with 1 mL sing human cancer cell lines in vitro. DR5 expression is Dulbecco PBS (LifeTechnologies). After the final wash, cells required to induce cell death but expression alone is not were resuspended in 100 mL FACS-FIX (D-glucose 3.2% w/v, sufficient. The development of a bioimaging technique formaldehyde 2.5% v/v in DPBS). Samples were transferred for assessing DR5-positive tumors in vivo could help in to a Corning Costar 96-well v-bottom plate (Corning Life selecting patients that benefit from CS-1008 treatment. Sciences). Samples were analyzed on the Guava EasyCyte Tumor concentration of CS-1008 in vivo and the impact of Mini System, GuavaExpress Pro, CytoSoft v 5.3 (Merck dose on tumor saturation of DR5 remain key questions in Millipore). the determination of the optimal dosing schedule for therapeutic benefit. We therefore aimed to establish and TRAIL sensitivity testing validate a molecular imaging technique for measuring Cells were seeded in 96-well microtiter plates at 2,500 DR5 occupancy and saturation in vivo, and to determine to 5,000 cells per well. TRAIL (Life Technologies) or the relationship of antibody:DR5 interaction with thera- mTRA-8 (0–1,000 ng/mL) were added the following day peutic response. and one plate was harvested for a time 0 (T ¼ 0) measurement. Remaining cell plates were incubated for 72 Materials and Methods hours. Cell viability was assessed using the MTS colori- Monoclonal antibodies and cell lines metric viability assay with MTS as a substrate (Promega). The murine anti-DR5 monoclonal antibody mTRA-8 Absorbance was assessed at 490 nm using a VersaMax (IgG1) and CS-1008, a humanized anti-DR5 monoclonal Microplate Reader (Molecular Devices) and SoftMax Pro antibody (IgG1), were provided by Daiichi Sankyo Com- 5.4.1 software (Molecular Devices). Absorbance at 630 pany Ltd. Hu3S193, a humanized anti-LewisY (LeY), was nmwasalsodeterminedasbackground,andthevalue provided by the Ludwig Institute for Cancer Research (Mel- was subtracted from the 490 nm reading. Experiments bourne, Australia) and was used as an IgG1 isotype control were performed in triplicates and repeated for two inde- (9). COLO 205, a DR5-expressing human colorectal ade- pendent runs. All data were normalized to signal at the nocarcinoma cell line (10); CT26, a DR5-negative mouse time of compound addition (T ¼ 0). Dose–response

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curves were analyzed using GraphPad Prism 4.03 (Graph- lating the amount of free, reactive antibody [(immunore- pad Software Inc.). active fraction total bound antibody) specific bound antibody], and specific binding (total bound antibody Radiolabeling and quality assurance nonspecific bound antibody) was graphed against reactive Analytic grade reagents, sterile technique, and pyrogen- free (nmol/L). The apparent dissociation constant (Kd) and free plasticware were used in all labeling steps. mTRA-8, CS- total receptor number per cell (Bmax) at equilibrium were 1008, and hu3S193 were radiolabeled with Indium-111 determined by nonlinear regression [Y ¼ Bmax X/(Kd þ X)] (111In; MDS Nordion) via the bifunctional metal ion che- using GraphPad Prism version 4.03. To show the Scatchard lating agent CHX-A00-DTPA as previously described (12, 13). plot, specific bound antibody/reactive free versus specific Radiolabeling was performed on the day of injection into bound (nmol/L) was plotted. To convert specific bound in mice. Before injection, the percentage of unbound radio- cpm/mL to specific bound in nmol/L, the following for- nuclide content was determined by instant thin-layer chro- mula was used: specific bound (nmol/L) ¼ specific bound matography (ITLC) as previously described (14). Serum (cpm/mL) [1/specific activity (cpm/mg)] 1,000 mL/L stability was assessed by incubating 5.0 mg of radiolabeled (L 10 6 g/mg) (1/150,000 mol/g) 109 nmol/mol. antibody in 200 mL of healthy donor human serum at 37C ELISA. To further assess the impact of radiolabeling on for an 11-day period. Radiochemical purity and single- CS-1008 binding, ELISA of samples of CS-1008, 111In- point immunoreactivity assays at 0 (no incubation), 2, and labeled CS-1008, and a control IgG1 was performed. NUNC 11 days of incubation were determined. MaxiSorp F96 plates were coated with TRAIL R2 antigen (R&D Systems) and blocked with 3% FCS–PBS. All samples In vitro binding of 111In-CHX-A00-DTPA-CS-1008 (in triplicate) were diluted in half-log series (10 mg/mL to Lindmo assay. Immunoreactivity of radiolabeled CS- 0.01 mg/mL). Goat antihuman IgG-AP (Sigma-Aldrich) was 1008 for COLO 205 cells was determined by nonlinear used as a secondary antibody. Plates were washed with extrapolation to binding at infinite antigen excess using a 0.05% Tween20–PBS between each step. After adding sub- "Lindmo" assay (15). Briefly, 20 ng of radiolabeled CS-1008 strate of rNPP (r-Nitrophenyl Phosphate; Sigma-Aldrich), was added to a range of COLO 205 cell concentrations (0, the absorbance was measured at 405 nm after 10 minutes. 3.13, 6.25, 12.5, 25, 50, 100, and 200 106 cells/mL) and incubated for 45 minutes at room temperature with con- In vivo binding of 111In-CHX-A00-DTPA-CS-1008 tinuous mixing throughout to keep the cells in suspension. Animal model. In vivo studies were conducted in athy- Cells were washed three times to remove unbound anti- mic BALB/c nu/nu mice (female, 4–6 week-old) obtained body, and pellets were measured in a gamma counter from the Animal Resources Centre (Western Australia, (Wizard2; PerkinElmer). Three radioactive antibody stan- Australia). All animal studies were approved by the Austin dards of radiolabeled CS-1008 at the same concentration as Hospital Animal Ethics Committee and were conducted in that initially added to the cells were measured at the same compliance with the National Health and Medical Research time as cell pellets. The percentage of binding of CS-1008 to Council (NHMRC) Australian Code of Practice for the Care COLO 205 cells was calculated by the formula (cpm cell and Use of Animals for Scientific Purposes. pellet/mean cpm radioactive antibody standards) 100. To establish tumors, mice were injected subcutaneously The percentage of binding was graphed against COLO 205 into the left underside flank with COLO 205 cells (2–5 cell concentration using Graphpad Prism 4.03, and immu- 106 cells), WiDr cells (5 106 cells with Matrigel), or CT26 noreactivity was calculated as the 1/Y-intercept of the cells (1 106 cells) in a total volume of 0.1 mL PBS. Tumor inverse plot of both values. volume was calculated by the formula [(length width2)/2], Competitive binding assay. Unlabeled CS-1008 at con- in which length was the longest axis and width was the centrations ranging from 5 to 200 mg/mL was added to measurement at right angles to length. In general, in vivo 200 106/mL COLO 205 cells and mixed. Samples were studies commenced when tumor volume was 100 to 200 done in duplicate. Subsequently, 20 ng of radiolabeled mm3 (11–12 days post injection). mTRA was added. After 45-minute incubation at room Biodistribution study. In the initial studies, the in vivo temperature, the cells were washed and counted as biodistribution of 111In-labeled CS-1008 at a dose level of described earlier for the Lindmo assay. 0.2 mg/kg was assessed in mice bearing COLO 205, WiDr, Saturation binding and Scatchard. 111In-labeled CS- or CT26 tumors. On day 0, mice received a single intrave- 1008 at concentrations ranging from 0.1 to 5 mg/mL in nous tail vein injection of 111In-labeled CS-1008 (5.0 mg, duplicate were added to 20 106 per mL COLO 205 cells 8.5 mCi) in a total volume of 0.1 mL 0.9% w/v of NaCl. and mixed. To estimate nonspecific binding, duplicate At designated time points after injection of the radio- samples of 111In-labeled CS-1008 at concentrations ranging conjugates (COLO 205: 3, 24, 72, 168, 240, and 336 hours; from 0.1 to 5 mg/mL were added to 20 106 per mL COLO WiDr: 4, 24, 72, and 168 hours; CT26: 3, 24, 72, and 168 205 cells in the presence of 140 mg/mL of cold CS-1008 and hours), groups of 5 mice were sacrificed by overinhalation mixed. After 45-minute incubation at room temperature, of isoflurane followed by cervical dislocation. Blood, the cells were washed and total bound antibody samples tumor, and normal tissues [brain, heart, lung, stomach, and nonspecifc bound antibody samples were counted. The spleen, liver, kidney, small intestine, colon, muscle, bone immunoreactive fraction was taken into account in calcu- (femur) and skin] in triplicate were collected for analysis

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and counted for radioactivity (Wizard2 gamma counter). Pharmacokinetics. Mice (n ¼ 5) were injected with The tissue distribution data were calculated as the mean 111In-labeled CS-1008 (0.2 mg/kg) at three different protein SD percentage injected dose per gram tissue [%ID/g; {radio- dose levels. Unlabeled CS-1008 was added to 111In-labeled activity tissue (counts per minute)/radioactivity standard CS-1008 (0.2 mg/kg) to obtain total protein doses of 0.2, 1, (counts per minute)} 100/tissue weight (g)] for each and 10 mg/kg. At 0.5, 1, 3, 5, 7, 24, 48, 72, 120, 168, 240, radiolabeled construct per time point. and 336 hours post injection, blood samples (10 mL) were The in vivo biodistribution of 111In-labeled CS-1008 and collected and counted in a Wizard2 gamma counter. 111 In-labeled IgG1 control at a dose level of 10 mg/kg was A two-compartment IV bolus model with macropara- also assessed in mice bearing COLO 205 tumors. On day 0, meters, no lag time, and first-order elimination (WNL mice with established xenografts received a single intrave- Model 8) was fitted to serum data for each animal using nous tail injection of CS-1008 or control IgG1 containing unweighted nonlinear, least squares with WinNonLin ver- trace labeled 111In radioconjugates [111In-CS-1008 (6.2 mg, sion 5.2 (Pharsight Corp.). 111 17.8 mCi), In-IgG1 (5.0 mg, 8.8 mCi)] at 0.2 mg/kg plus Therapeutic studies. Mice bearing WiDr tumors (100– unlabeled antibody in a total volume of 0.1 mL 0.9% w/v of 200 mm3) were treated with CS-1008, control IgG, or NaCl containing a total protein dose of 10 mg/kg CS-1008 vehicle (on day 0, 7, and 14) at 0.1, 0.3, 1.0, 3.0, 10.0, or and IgG1 respectively. Blood, tumor, and normal tissues 30.0 mg/kg dose levels (n ¼ 5/dose level), and monitored were collected at designated time points from euthanized for tumor growth using methods previously reported (9). animals (n ¼ 5/time point). Mice injected with radiolabeled The therapeutic effects of CS-1008 in COLO 205 tumor- control IgG1 were euthanized at 3, 72, 168, and 336 hours bearing mice have been previously described (2). post injection (n ¼ 4/time point) and blood, tumor, and Statistical analysis. Data were analyzed using one-way tissues were collected. ANOVA when comparing multiple groups. Data were ana- Gamma camera imaging and computerized tomography lyzed using an unpaired two-tailed Student t test when imaging. Planar gamma camera images of 111In-labeled comparing two groups (treated to control). Differences 111 CS-1008 (0.2 mg/kg) and In-labeled IgG1 control anti- were considered significant at a P value of less than 0.05. body (0.2 mg/kg) distributions in COLO 205 tumor-bear- All analyses were conducted using Graphpad Prism version ing mice, and computerized tomography (CT) images, were 4.03. obtained for 2 mice per antibody preparation on days 0, 2, 3, 7, and 10 post injection. Imaging procedures involved Results anesthesia of mice by intraperitoneal injection of a keta- Flow cytometry mine (100 mg/kg) and xylazine (20 mg/kg) mixture. Mice COLO 205 and WiDr cells were positive for DR5 expres- were placed supine on a Philips Skylight gamma camera sion, with a median fluorescence for COLO 205 cells of 44.5 with energy windows set for 111In (20% windows at 171 and and WiDr cells of 29.9. CT26 cells were negative (similar to 245 keV). Images were acquired in a 128 128 or 256 256 controls) with a median fluoresence of 4.6. matrix and a standard containing a known amount of 111In activity was included in the field of view. Twenty-minute TRAIL sensitivity images were collected. CT scanning, to generate cross-sec- COLO 205 cells showed a 50% proliferation inhibition at tional imaging of anatomic structures, was also undertaken 3.9 ng/mL of mTRA-8 and 14 ng/ml of TRAIL, and WiDr cells (Philips Gemini 2 slice PET-CT scanner; slice thickness, 0.6 at 54 ng/mL of TRAIL and 208 ng/mL of mTRA-8. CT26 cells mm; pixel size, 0.097 mm). showed minimal responsiveness to TRAIL or mTRA-8. Imaging analysis involved whole body, liver, and tumor regions of interest (ROI) being defined for each Antibody radiolabeling time point. Counts of whole body and liver were com- Radiochemical purity assessment of radiolabeled mTRA- pared between groups as an internal control to guarantee 8, CS-1008, and control IgG1 by ITLC confirmed more than that equal amounts of radioactivity were administered in 98% bound 111In-CHX-A00DTPA. Figure 1 shows the immu- each dose-level group. Counts of the tumors were con- noreactivity-binding curve for radiolabeled CS-1008 (Fig. verted to %ID/g using a standard of known activity that 1A). The immunoreactivity of 111In-radiolabeled CS-1008 wasscannedatthesametime.Tumorvolumes(mL)were was 13.3%. Because of the low immunoreactivity, a com- determined on the basis of markup of tumors in cross- petition binding assay was performed to demonstrate spe- sectional CT images. To convert tumor volumes (mL) to cific binding of CS-1008 (Fig. 1B). At 10 mg/mL of CS-1008, tumor mass (g), animals were sacrificed after CT imaging, 75% of 111In-labeled mTRA binding could be blocked with and tumors were removed and weighed (n ¼ 12). The CS-1008, indicating that CS-1008 and mTRA are competing conversion factor was determined as the slope of the for the same binding sites. Saturation binding analysis of linear regression curve. 111In-labeled CS-1008 showed specific binding with high- Receptor occupancy study. A further series of biodistri- affinity binding sites on COLO 205 cells (Fig. 1C). The 111 bution and planar gamma camera imaging studies explored apparent Kd value for In-labeled CS-1008 was 1.05 the saturation of the DR5 receptor in vivo. Details of the 0.12 10 9 mol/L. The number of binding sites per cell was experimental procedures and calculation of receptor occu- 6,578 284 binding sites per cell. The stability of the pancy are provided in Supplementary Data. radiolabeled 111In-CS-1008 in normal human serum at

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Figure 1. A, binding assay for the determination of the immunoreactive fraction of 111In- CHX-A00-DTPA-CS-1008 to COLO 205 cells. B, competition binding of 111In-mTRA-8 in the presence of increasing amounts of unlabeled CS-1008. C, saturation binding assay to determine Kd and Bmax of 111In-CHX-A00-DTPA-CS-1008 antibody to COLO 205 cells. D, ELISA of CS-1008, 111In-labeled CS-1008, and control IgG binding DR5 (TRAIL R2).

37C was high, with retention of immunoreactivity, radio- mg/kg was studied in mice bearing human COLO 205 chemical purity, and construct integrity for up to 11 days. tumors and mice bearing murine CT26 tumors (Table 1 and Supplementary Table S1). CS-1008 does not cross-react ELISA to the rodent death receptor and therefore a murine colon The labeling of CS-1008 with 111In showed minimal cancer cell line was chosen as negative control. The %ID/g of impact on binding to DR5 (TRAIL R2; Fig. 1D). 111In-labeled CS-1008 (0.2 mg/kg) in COLO 205 tumors peaked at 168 hours post injection (%ID/g, 26.25 2.85). Biodistribution The tumor:blood ratio for 111In-labeled CS-1008 increased In an initial biodistribution study, tumor uptake and over time and peaked at 2.92 0.53 at 240 hours post biodistribution of 111In-labeled CS-1008 at a dose of 0.2 injection. The tumor uptake of 111In-labeled CS-1008 in

Table 1. Biodistribution of 111In-CHX-A00-DTPA-CS-1008 in BALB/c nu/nu COLO 205 tumor-bearing mice

Time (h)

Tissues 3 24 72 168 240 336 Tumor 6.08 1.72a 11.18 1.41 20.16 5.48 26.25 2.85 23.84 2.83 21.05 2.00 Blood 30.68 4.84 21.50 1.32 17.45 1.28 13.36 3.99 10.74 3.99 6.03 0.76 Brain 0.54 0.11 0.44 0.04 0.51 0.04 0.46 0.11 0.35 0.05 0.28 0.03 Heart 7.69 0.61 5.43 0.57 5.47 1.26 4.03 0.41 3.28 0.35 2.17 0.16 Lung 12.79 1.73 8.10 1.06 8.77 2.23 8.30 2.49 6.89 0.61 4.41 0.31 Stomach 3.47 0.84 3.03 0.79 3.71 0.83 2.16 0.48 2.12 0.74 1.51 0.22 Spleen 8.75 2.04 7.00 1.79 8.54 3.02 8.78 2.01 7.24 1.13 6.99 0.53 Liver 8.79 1.55 6.48 0.86 8.43 1.63 7.82 0.64 5.78 0.69 4.42 0.38 Kidney 11.81 1.97 9.21 0.95 9.93 0.71 7.74 1.91 6.93 0.84 5.16 0.56 Small intestine 5.48 1.45 4.42 2.46 3.28 1.37 3.30 0.89 3.58 0.86 2.03 0.64 Colon 4.29 1.26 2.57 0.67 2.94 0.54 2.30 0.69 1.99 0.38 1.53 0.16 Muscle 1.73 0.22 1.96 0.36 2.21 0.51 1.97 0.45 1.63 0.23 1.08 1.15 Skin 3.46 1.23 5.33 0.90 8.41 1.91 4.21 1.84 4.92 0.87 3.34 0.66 Bone 4.54 1.09 3.14 0.53 3.02 2.02 3.66 0.65 4.06 0.58 3.03 1.02

aData presented as %ID/g tissue SD (n ¼ 5).

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Figure 2. In vivo specificity of 111In- CHX-A00-DTPA-CS-1008. A, specific tumor uptake of 111In-CS- 1008 (0.2 mg/kg) in DR5-positive COLO 205 xenografts versus DR5- negative CT26 xenografts. B, COLO 205 tumor growth in the presence of 111In-CHX-A00-DTPA- CS-1008 (10 mg/kg; filled squares) and control (open squares); n ¼ 5; bars, SD. C, specific tumor uptake of 111In-CHX-A00-DTPA-CS-1008 (0.2 mg/kg) in DR5-positive WiDr xenografts versus DR5-negative CT26 xenografts. D, WiDr tumor growth in the presence of 111In-CHX-A00-DTPA-CS-1008 (10 mg/kg; filled squares) and control (open squares); n ¼ 5; bars, SD. , P < 0.05, compared with control.

CT26 DR5-negative tumor–bearing mice was significantly studies, tumor uptake of 111In-labeled CS-1008 at different less than that seen in COLO 205 DR5-positive tumor– antibody dose levels was conducted to explore the satura- bearing mice at 72 and 168 hours post injection. Maximum tion of DR5 in vivo in tumors with different levels of DR5 uptake of 111In-labeled CS-1008 in CT26 was observed at 72 expression, high (COLO 205) and low (WiDr). Six groups hours post injection (%ID/g, 11.79 1.68; Fig. 2A). of mice (n ¼ 4) were injected with 111In-labeled CS-1008 (5 The specific uptake in vivo was confirmed by an additional mg, 8.5 mCi) at different dose levels of unlabeled CS-1008 biodistribution study comparing 111In-labeled CS-1008 at a (0.2, 0.5, 1, 3, 10, and 30 mg/kg). Planar gamma camera 111 dose level of 10 mg/kg with In-labeled IgG1 isotype imaging and CT imaging were conducted at 48 hours post control hu3S193 at a dose level of 10 mg/kg in COLO injection when the effect of CS-1008 on tumor size was 205 tumor-bearing mice at different time points after injec- minimal (Fig. 2B and D). Figure 3 shows representative tion. At the 10 mg/kg dose level, 111In-CS-1008 tumor planar gamma camera images and CT scans at 48 hours post uptake was lower at all time points compared with the injection for 0.2 mg/kg (Fig. 3A), 0.5 mg/kg (Fig. 3B), and 0.2 mg/kg dose level, and peaked at 72 hours post injection 10 mg/kg (Fig. 3C) CS-1008 in COLO 205 tumor-bearing (%ID/g, 11.64 1.59, n ¼ 4). The isotype control antibody mice. After imaging, mice were sacrificed; tumors were showed an initial tumor uptake of 5.55 0.97%ID/g at 3 weighed and counted for radioactivity. A terminal deoxy- hours post injection, declining progressively over time. nucleotidyl transferase–mediated dUTP nick end labeling A further biodistribution study of 111In-labeled CS-1008 (TUNEL) assay (Supplementary Fig. S2) of COLO 205 (0.2 mg/kg) in WiDr and CT26 tumor-bearing mice was tumor sections collected from mice treated with 0.2 mg/kg performed and showed specific localization of 111In-labeled (Fig. 3A) and 10 mg/kg (Fig. 3C) CS-1008 showed an CS-1008 in WiDr tumors, but at a lower level than in COLO increase in apoptosis in tumors from animals treated with 205 tumors (Fig. 2C and Supplementary Table S2). 10 mg/kg compared with animals treated with 0.2 mg/kg. Supplementary Fig. S3 shows representative images of WiDr Gamma camera imaging and DR5 occupancy of 111In- tumor-bearing mice at 48 hours post injection for 0.2 mg/kg CHX-A00-DTPA-CS-1008 in vivo (Supplementary Fig. S3A), 0.5 mg/kg (Supplementary Fig. Whole-body imaging of 111In-labeled CS-1008 (0.2 S3B), and 10 mg/kg (Supplementary Fig. S3C) CS-1008. mg/kg) in BALB/c nu/nu mice bearing DR5-positive COLO Figure 3D shows %ID/g in the COLO 205 tumors at 205 or DR5-negative CT26 tumors was performed. Tumor different doses (n ¼ 4). At 48 hours post injection at a 0.2 uptake of 111In-labeled CS-1008 was evident as of day 2 post mg/kg dose level, tumor uptake of 111In-labeled CS-1008 injection and continued for more than 7 days (Supplemen- was highest (28.15 2.02%ID/g; n ¼ 4), declining tary Fig. S1). Initial blood pool activity was seen on day 0 progressively with increased doses of cold CS-1008. DR5 and decreased over time. In a further series of imaging saturation starts at 1 mg/kg dose level and is complete at

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Figure 3. In vivo saturation of 111In-CHX-A00-DTPA-CS-1008 demonstrated by planar gamma camera imaging (A, 0.2 mg/kg; B, 0.5 mg/kg; and C, 10 mg/kg). Representative whole-body images (left), surface-rendered CT (middle), and fused images (right) are shown. D, tumor uptake (%ID/g) of 111In-CHX-A00-DTPA- CS-1008 in COLO 205 tumors at different dose levels from gamma well counting. E, tumor uptake (%ID/cm3)of111In-CHX-A00-DTPA-CS-1008 at different dose levels calculated by ROI analysis of planar gamma camera images and tumor volume (cm3) estimation by CT. F, tumor uptake (%ID/g) of 111In-CHX-A00-DTPA-CS-1008 at different dose levels calculated by ROI analysis of planar gamma camera images and tumor volume (g) estimation by CT using a correlation factor of 0.69 0.01 (n ¼ 12). G, tumor uptake (%ID/g) of 111In-CHX-A00-DTPA-CS-1008 in WiDr tumors at different dose levels from gamma well counting. bars, SD.

3mg/kg.Figure3Eshowsthe%ID/cm3 curve generated predicted the dose of maximal therapeutic response (Fig. from region of interest (ROI) analysis of tumor images. 4D). The level of TGI was less with WiDr tumors than with To correct for the difference between tumor volumes COLO 205 tumors, in keeping with the reduced DR5 (cm3) measured by CT analysis and tumor volumes (g) expression and lower concentration of CS-1008 achieved weighed, a correlation curve was established and conver- in tumors in vivo. sion tumor volumes (cm3) into tumor volumes (g) required a correlation factor of 0.69 0.01. Figure 3F Pharmacokinetics shows the %ID/g generated from image analysis after No differences in serum clearance were observed between correcting the tumor volumes and this curve is consistent different antibody dose levels (0.2, 1 mg/kg vs. 10 mg/kg) with the curve generated from the biodistribution data with mean values (n ¼ 15; SD) for a half-life (t1/2a) of 5.71 (Fig. 3D). The results for WiDr tumor analysis are shown 0.91 hours, b half-life (t1/2b) of 141.78 20.69 hours, in Fig. 3G. total clearance (CL) of 0.02 0.1 mL/h, and volume of Using a baseline value of 5.07%ID/g at 48 hours post distribution at steady state (Vss) of 4.47 0.25 mL. The injection of 0.2 mg/kg 111In-labeled isotype control, a pharmacokinetic parameters for each dose level are shown receptor occupancy curve was generated. Figure 4A shows in Table 2. the occupancy of COLO 205 DR5 antigen binding sites by 111In-CS-1008 with increasing protein dose at 48 hours post Discussion injection. To compare receptor occupancy at different dose We have shown that CS-1008, a humanized IgG1 anti- levels with in vivo tumor growth inhibition (TGI) at different DR5 antibody, demonstrates speciﬁc uptake in TRAIL-sen- dose levels, TGI values (%) measured by Yada and collea- sitive, DR5-positive tumors that is dose dependent and in gues were plotted against the imaged CS-1008 antibody proportion to DR5 expression in tumor cells. Moreover, CS- dose levels (Fig. 4B; ref. 2). The curves in Fig. 4A and B show 1008 has tumor inhibitory effects that are dependent on CS- the same saturation trend, with DR5 saturation occurring 1008 tumor concentration and reach maximal efﬁcacy between 1 and 3 mg/kg dose levels. WiDr tumors showed a when antibody:DR5 binding is saturated, which can be similar trend for receptor saturation between 1 and 3 mg/kg quantitatively measured by molecular imaging of receptor dose levels (Fig. 4C), and this receptor saturation level also occupancy in vivo with 111In-CHX-A00-DTPA-CS-1008.

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Molecular Imaging of DR5 with Tigatuzumab

Figure 4. A, DR5 occupancy curve of 111In-CHX-A00-DTPA-CS-1008 in BALB/c nu/nu COLO 205 tumor- bearing mice at 48 hours post injection. B, tumor growth inhibition of different dose levels of CS-1008 in a COLO 205 tumor model shown as percentage tumor volume of vehicle control group at 24 days post therapy (adapted from Yada and colleagues, ref. 2). C, DR5 occupancy curve of 111In-CHX-A00-DTPA-CS-1008 in BALB/c nu/nu WiDr tumor-bearing mice at 48 hours post injection. D, tumor growth inhibition of different dose levels of CS-1008 in a WiDr tumor model shown as percentage tumor volume of vehicle control group at 24 days post therapy.

Anti-DR5 antibodies were successfully chelated and radi- Endocytosed 111In-DTPA antibodies are delivered to olabeled with 111In-CHX-A00DTPA, with both 111In-labeled lysosomes and hydrolyzed by lysosomal enzymes into mTRA and CS-1008 showing high-affinity binding for DR5 small molecular weight metabolites that are retained within expressing COLO 205 cells. Retention of binding affinity of tumor lysosomes (17, 18). Consistent with a low receptor 111In-CHX-A00-DTPA-CS-1008 following radiolabeling was number expressed on the cell membrane, accumulation of confirmed by ELISA. The in vivo biodistribution and imag- 111In-CHX-A0-DTPA-CS-1008 in DR5-positive tumors was ing studies showed specific DR5-positive tumor localiza- gradual with high tumor uptake by 72 hours, and main- tion, with prolonged retention in COLO 205 tumors despite taining or slightly increasing uptake over a 1-week period. In the low receptor number available for CS-1008 binding contrast to the COLO 205 and WiDr xenografts, no specific observed in saturation binding analysis (6,578 receptors per uptake of radiolabeled 111In-CS-1008 was observed in DR5- cell). The number of binding sites on COLO 205 cells is negative CT26 tumors. Pharmacokinetic analysis showed consistent with the number of DR5 found on other cell no differences between increasing dose levels of unlabeled lines: the number of DR5 per MIA PaCa–2 cell was 8,620 CS-1008. The uptake of 111In-labeled CS-1008 in all normal 1,920, whereas that per 2LMP cell was 11,170 4,170, tissues was consistent with blood pool activity. Other DR5 without significant difference between the two cell lines imaging antibodies reported on are 99mTc-HYNIC-mTRA (P ¼ 0.6141; ref. 16). WiDr tumors, which have lower DR5 (19, 20) and 64Cu-DOTA-conatumumab (21). Comparable expression, also demonstrated specific uptake but at lower tumor uptake (%ID/g) was observed with 111In-labeled CS- levels than seen in COLO 205 tumors. 1008, but in contrast with 64Cu-DOTA-conatumumab, no

Table 2. Pharmacokinetic parameters of 111In-CHX-A00-DTPA-CS-1008 at different dose levels in BALB/c nu/nu COLO 205 tumor-bearing mice

CS-1008 (mg/kg) Cmax mg/mL AUC0-¥ (mg/mL h) t1/2a (h) t1/2b (h) CL (mL/h) Vss (mL) 0.2 1.7 0.1 124.5 11.5 6.1 0.9 120.1 18.7 0.03 0.01 4.3 0.3 1 7.7 0.4 746.2 101.4 4.7 1.1 143.9 29.4 0.02 0.01 4.4 0.4 10 60.7 2.6 7,784.4 2,101.7 6.4 2.1 161.3 61.7 0.02 0.01 4.8 0.6

Abbreviations: Cmax, maximum plasma–serum concentration; AUC, area under the curve; t1/2a, serum distribution half-life; t1/2b, serum

elimination half-life; CL, clearance; Vss, volume of distribution at steady state.

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Burvenich et al.

splenic uptake was seen with 111In-labeled CS-1008. Radio- els; however, in cells with similar or higher DR5 expression iodinated recombinant human TRAIL has also been levels, but with slower induction of the apoptotic pathway, explored as a biomarker for DR4 and DR5 targeting (22). receptor turnover, receptor internalization, and antibody Potent antitumor activity of CS-1008 has been previously pharmacokinetics might play a much larger role. Although demonstrated in vitro and in vivo against the colorectal cell the results here have shown proof-of-principle that 111In- line COLO 205, with tumor growth inhibition observed in a CHX-A00-DTPA-CS-1008 can aid in patient selection and dose-dependent manner up to 1 mg/kg dose level (2). We estimate optimal therapeutic dosing, further studies are have demonstrated that receptor occupancy and saturation warranted comparing resistant versus sensitive tumor mod- can be accurately measured by molecular imaging of 111In- els, and exploring the role of apoptosis pathway induction CHX-A0-DTPA-CS-1008 imaging in COLO 205 tumors, and on effectiveness of treatment (24). Such studies are current- that receptor occupancy and saturation correlate precisely ly being performed by our laboratory. with the dosage required for maximal tumor growth inhibi- In conclusion, these results suggest that molecular imag- tion. We have also demonstrated that in tumors with lower ing of DR5 occupancy and saturation can play a major role levels of DR5 expression (WiDr), similar correlations can be for clinical development, optimal dosing, and patient selec- made through molecular imaging of DR5 occupancy and tion for trials of DR5-targeting antibodies. saturation. Interestingly, DR5 expression levels in tumor (assessed by peak uptake of 111In-CHX-A0-DTPA-CS-1008 Disclosure of Potential Conflicts of Interest in tumor) also correlated with the maximal tumor growth R.A. Beckman is founder and chief scientific officer at Onco-Mind LLC. No potential conflicts of interest were disclosed by the other authors. inhibition achievable, with COLO 205 tumors showing higher uptake and tumor inhibition than WiDr tumors. Authors' Contributions Calculation of antigen-binding site occupancy from the Conception and design: I.J.G. Burvenich, F.T. Lee, R.A. Beckman, K. imaging data showed that DR5 binding is saturable at Fujiwara, A.M. Scott Development of methodology: I.J.G. Burvenich, F.T. Lee, G.J. O’Keefe, K. protein doses above 1 mg/kg. Assuming a cell diameter Fujiwara of 10 mm for COLO 205 and WiDr cells, measured CT tumor Acquisition of data (provided animals, acquired and managed patients, volumes and an immunoreactive fraction of 10%, approx- provided facilities, etc.): I.J.G. Burvenich, F.T. Lee, G.A. Cartwright, G.J. O’Keefe, D. Makris, D. Cao, A.C. Chueh, M.W. Brechbiel, K. Fujiwara imately three antibodies per receptor at 1 mg/kg, were Analysis and interpretation of data (e.g., statistical analysis, biosta- enough to saturate the tumor and elicit maximum thera- tistics, computational analysis): I.J.G. Burvenich, F.T. Lee, G.A. Cart- wright, G.J. O’Keefe, D. Cao, S. Gong, A.C. Chueh, R. von Roemeling, peutic effect in vivo. This finding indicates the importance A.M. Scott of dose selection on achieving optimal concentration of CS- Writing, review, and/or revision of the manuscript: I.J.G. Burvenich, F.T. 1008 in tumor, and the ability of molecular imaging Lee, G.A. Cartwright, G.J. O’Keefe, D. Makris, A.C. Chueh, J.M. Mariadason, M.W. Brechbiel, R.A. Beckman, A.M. Scott approaches to quantitate receptor occupancy in vivo. Administrative, technical, or material support (i.e., reporting or orga- There are several factors that might influence the success nizing data, constructing databases): F.T. Lee, D. Cao, J.M. Mariadason of imaging DR5 occupancy in patients and might cause Study supervision: F.T. Lee, R. von Roemeling, A.M. Scott patient variability in therapy outcome when using 111In- Acknowledgments labeled anti-DR5 antibodies in the clinic. First, when cal- The authors thank Malarmathy Ramachandran, Angela Rigopoulos, and culating %ID/g tumor in the mouse models, 1% of the total Marzena Walkiewicz for their help with animal experiments and quality injected 111In-CS-1008 was typically measured in the control of reagents; Department of Nuclear Medicine and Centre for PET, Austin Health for the use of the Philips Skylight and Philips Gemini 2 PET-CT tumors of which 60% was specific. When translating these scanner; Bridget Chappell for planar gamma camera image acquisition; findings into the clinic, many solid tumors in patients only Sherylene Desmond for CT image acquisition; and Fiona Scott for assistance show values around 0.01% of the injected dose when using with project management. radiolabeled antibodies. Using 111In-labeled CS-1008 might help to select the patients that show sufficient tumor Grant Support 111 This work was financially supported by Daiichi Sankyo Co. Ltd. Opera- uptake of In-CS-1008 at tracer levels of cold CS-1008 tional Infrastructure Support Program funding was also provided by the (e.g., 0.2 mg/kg). Second, Fcg receptors have been proposed Victorian Government. This research was also supported in part by the as an Fc cross-linking interface in vivo promoting DR5- Intramural Research Program of the NIH, National Cancer Institute, Centre for Cancer Research. mediated agonistic cell killing by anti-DR5 antibodies The costs of publication of this article were defrayed in part by the (23). In patients, these Fcg receptors might be available in payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate the tumor via infiltrating immune cells, but the population this fact. and the amount might vary among patients. Third, the data presented here show DR5 occupancy and therapeutic Received October 1, 2012; revised July 30, 2013; accepted August 13, 2013; response to CS-1008 in COLO 205 and WiDr tumor mod- published OnlineFirst September 17, 2013.

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Molecular Imaging of Death Receptor 5 Occupancy and Saturation Kinetics In Vivo by Humanized Monoclonal Antibody CS-1008

Ingrid J.G. Burvenich, Fook T. Lee, Glenn A. Cartwright, et al.

Clin Cancer Res 2013;19:5984-5993. Published OnlineFirst September 17, 2013.

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