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 111
In-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.
Results: Scatchard analysis showed high and specific binding affinity ( In-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.
Introduction
CS-1008 (tigatuzumab) is a humanized monoclonal immunoglobulin G (IgG 1 ) with agonistic activity upon binding to human death receptor 5 (DR5). It was developed by Daiichi Sankyo Company Ltd. from a murine anti-DR5 monoclonal antibody, TRA-8 (mTRA-8) by complementarity determining region grafting (1, 2) . DR5 is a member of the TNF receptor superfamily, which contains a cytoplasmic death domain that is capable of initiating an apoptotic signal upon binding with its endogenous ligand TRAIL (3) . Apoptosis is thought to be deregulated in cancer cells 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 described: Lexatumumab (human IgG 1 ; Human Genome Sciences), Apomab (human IgG 1 ; Genentech), AMG-655 (human IgG 1 ; Amgen), LBY135 (chimeric IgG1; Novartis), and TRA-8/CS-1008 (murine IgG1/humanized IgG1; Daiichi Sankyo Company; ref. 4) .
Both mTRA-8 and CS-1008 antibodies have shown specific in vitro cytotoxicity and antitumor efficacy against solid tumor xenografts in vivo. mTRA was selected for its low hepatocellular toxicity and ability to trigger apoptosis in vitro in a variety of human tumor cell lines without the use of cross-linking agents (1) . In vivo antitumor efficacy was shown in different mouse models bearing human xenografts (1, 5) and can be enhanced in combination with chemotherapeutic agents and/or radiation (5-7).
CS-1008 has similar in vitro and in vivo antitumor activity as mTRA-8 (2) .
Phase I trials in humans have shown linear pharmacokinetics in doses ranging from 1 to 8 mg/kg/week with a half-life of 8to 16 days. There were no partial responses to treatment but stable disease was noted in 7 of 17 treated patients with the duration of response ranging from 81 to 798 days. CS-1008 dose-limiting toxicity was not reached, and no evidence of immunogenicity to the antibody was detected (8) .
CS-1008 induces cell death in a variety of DR5-expressing human cancer cell lines in vitro. DR5 expression is required to induce cell death but expression alone is not sufficient. The development of a bioimaging technique for assessing DR5-positive tumors in vivo could help in selecting patients that benefit from CS-1008 treatment. Tumor concentration of CS-1008 in vivo and the impact of dose on tumor saturation of DR5 remain key questions in the determination of the optimal dosing schedule for therapeutic benefit. We therefore aimed to establish and validate a molecular imaging technique for measuring DR5 occupancy and saturation in vivo, and to determine the relationship of antibody:DR5 interaction with therapeutic response.
Materials and Methods

Monoclonal antibodies and cell lines
The murine anti-DR5 monoclonal antibody mTRA-8 (IgG 1 ) and CS-1008, a humanized anti-DR5 monoclonal antibody (IgG 1 ), were provided by Daiichi Sankyo Company Ltd. Hu3S193, a humanized anti-LewisY (Le Y ), was provided by the Ludwig Institute for Cancer Research (Melbourne, Australia) and was used as an IgG 1 isotype control (9) . COLO 205, a DR5-expressing human colorectal adenocarcinoma cell line (10); CT26, a DR5-negative mouse colorectal adenocarcinoma cell line (11) have been tested by single-point Lindmo assays as part of each study using radiolabeled CS-1008 or hu3S193. CT26 is a mouse colon cancer cell line and was tested for its mouse thompsonjames@example.com. A431 cells were grown in DMEM/F-12 with 10% v/v FBS, 1% v/v penicillin-streptomycin (10,000 U/mL in 0.85% NaCl), and 1% v/v L-glutamine (200 mM/mL in 0.85% NaCl; Invitrogen). COLO 205, WiDr, and CT26 cells were grown in RPMI-1640 with 10% v/v FBS and 1% v/v penicillin-streptomycin (10,000 U/mL in 0.85% NaCl).
Flow cytometry
Adherent COLO 205, WiDr, and CT26 cells were trypsinized and resuspended in fresh culture medium. Cells were aliquoted into 1.5 mL eppendorf tubes at a density of 1 Â 10 6 cells per tube. Cells were centrifuged at 3,000 Âg for 3 minutes, and the pellets were resuspended in 100 mL of an antihuman CD262/DR5-PE antibody (1:5 concentration; Affymetrix eBioscience Inc.). A cells-only control and a negative control (1:100 Goat antihuman IgG-g chain specific F(ab 0 ) 2 PE; Sigma-Aldrich) were included. All samples were incubated on ice for 30 minutes. Cells were then centrifuged at 3,000 Âg for 3 minutes and the supernatant was discarded. Cells were washed three times with 1 mL Dulbecco PBS (LifeTechnologies). After the final wash, cells were resuspended in 100 mL FACS-FIX (D-glucose 3.2% w/v, formaldehyde 2.5% v/v in DPBS). Samples were transferred to a Corning Costar 96-well v-bottom plate (Corning Life Sciences). Samples were analyzed on the Guava EasyCyte Mini System, GuavaExpress Pro, CytoSoft v 5.3 (Merck Millipore).
COLO 205 and WiDr cells were positive for DR5 expression, with a median fluorescence for COLO 205 cells of 44.5 and WiDr cells of 29.9. CT26 cells were negative (similar to controls) with a median fluoresence of 4.6.
TRAIL sensitivity testing
Cells were seeded in 96-well microtiter plates at 2,500 to 5,000 cells per well. TRAIL (Life Technologies) or mTRA-8 (0-1,000 ng/mL) were added the following day and one plate was harvested for a time 0 (T ¼ 0) measurement. Remaining cell plates were incubated for 72 hours. Cell viability was assessed using the MTS colorimetric viability assay with MTS as a substrate (Promega). Absorbance was assessed at 490 nm using a VersaMax Microplate Reader (Molecular Devices) and SoftMax Pro 5.4.1 software (Molecular Devices). Absorbance at 630 nm was also determined as background, and the value was subtracted from the 490 nm reading. Experiments were performed in triplicates and repeated for two independent runs. All data were normalized to signal at the time of compound addition (T ¼ 0). Dose-response
Translational Relevance
CS-1008 (tigatuzumab) is an antihuman death receptor (DR5) agonist that induces apoptosis and has cytotoxic activity against human cancer cell lines. Tumor concentration of CS-1008 in vivo and the impact of dose on tumor saturation of DR5 remain key questions in the determination of the optimal dosing schedule for therapeutic benefit. This study reports on the exploration of targeting of anti-DR5 humanized antibody CS-1008 to DR5-expressing tumors in vivo, and quantitative analysis of antibody:DR5 binding and occupancy. The relationship of in vivo imaging of CS-1008 in tumor to therapeutic response was also established. Our results showed that in vivo saturation of DR5 was predictive of therapeutic dose-response threshold. This novel and important information is highly relevant to DR5 modulation as a therapeutic approach and informs the design of clinical trials of anti-DR5 antibodies in patients with cancer.
curves were analyzed using GraphPad Prism 4.03 (Graphpad Software Inc.).
Radiolabeling and quality assurance
Analytic grade reagents, sterile technique, and pyrogenfree plasticware were used in all labeling steps. mTRA-8, CS-1008, and hu3S193 were radiolabeled with Indium-111 ( 111 In; MDS Nordion) via the bifunctional metal ion chelating agent CHX-A 00 -DTPA as previously described (12, 13) . Radiolabeling was performed on the day of injection into mice. Before injection, the percentage of unbound radionuclide content was determined by instant thin-layer chromatography (ITLC) as previously described (14) . Serum stability was assessed by incubating 5.0 mg of radiolabeled antibody in 200 mL of healthy donor human serum at 37 C for an 11-day period. Radiochemical purity and singlepoint immunoreactivity assays at 0 (no incubation), 2, and 11 days of incubation were determined.
In vitro binding of 111 In-CHX-A 00 -DTPA-CS-1008 Lindmo assay. Immunoreactivity of radiolabeled CS-1008 for COLO 205 cells was determined by nonlinear extrapolation to binding at infinite antigen excess using a "Lindmo" assay (15) . Briefly, 20 ng of radiolabeled CS-1008 was added to a range of COLO 205 cell concentrations (0, 3.13, 6.25, 12.5, 25, 50, 100, and 200 Â 10 6 cells/mL) and incubated for 45 minutes at room temperature with continuous mixing throughout to keep the cells in suspension. Cells were washed three times to remove unbound antibody, and pellets were measured in a gamma counter (Wizard 2 ; PerkinElmer). Three radioactive antibody standards of radiolabeled CS-1008 at the same concentration as that initially added to the cells were measured at the same time as cell pellets. The percentage of binding of CS-1008 to COLO 205 cells was calculated by the formula (cpm cell pellet/mean cpm radioactive antibody standards) Â 100. The percentage of binding was graphed against COLO 205 cell concentration using Graphpad Prism 4.03, and immunoreactivity was calculated as the 1/Y-intercept of the inverse plot of both values.
Competitive binding assay. Unlabeled CS-1008 at concentrations ranging from 5 to 200 mg/mL was added to 200 Â 10 6 /mL COLO 205 cells and mixed. Samples were done in duplicate. Subsequently, 20 ng of radiolabeled mTRA was added. After 45-minute incubation at room temperature, the cells were washed and counted as described earlier for the Lindmo assay.
Saturation binding and Scatchard.
111 In-labeled CS-1008 at concentrations ranging from 0.1 to 5 mg/mL in duplicate were added to 20 Â 10 6 per mL COLO 205 cells and mixed. To estimate nonspecific binding, duplicate samples of 111 In-labeled CS-1008 at concentrations ranging from 0.1 to 5 mg/mL were added to 20 Â 10 6 per mL COLO 205 cells in the presence of 140 mg/mL of cold CS-1008 and mixed. After 45-minute incubation at room temperature, the cells were washed and total bound antibody samples and nonspecifc bound antibody samples were counted. The immunoreactive fraction was taken into account in calculating the amount of free, reactive antibody [(immunoreactive fraction Â total bound antibody) À specific bound antibody], and specific binding (total bound antibody À nonspecific bound antibody) was graphed against reactive free (nmol/L). The apparent dissociation constant (K d ) and total receptor number per cell (B max ) at equilibrium were determined by nonlinear regression
using GraphPad Prism version 4.03. To show the Scatchard plot, specific bound antibody/reactive free versus specific bound (nmol/L) was plotted. To convert specific bound in cpm/mL to specific bound in nmol/L, the following formula was used: specific bound (nmol/L) ¼ specific bound
À6 g/mg) Â (1/150,000 mol/g) Â 10 9 nmol/mol. ELISA. To further assess the impact of radiolabeling on CS-1008 binding, ELISA of samples of CS-1008, 111 Inlabeled CS-1008, and a control IgG 1 was performed. NUNC MaxiSorp F96 plates were coated with TRAIL R2 antigen (R&D Systems) and blocked with 3% FCS-PBS. All samples (in triplicate) were diluted in half-log series (10 mg/mL to 0.01 mg/mL). Goat antihuman IgG-AP (Sigma-Aldrich) was used as a secondary antibody. Plates were washed with 0.05% Tween20-PBS between each step. After adding substrate of rNPP (r-Nitrophenyl Phosphate; Sigma-Aldrich), the absorbance was measured at 405 nm after 10 minutes. In-IgG 1 (5.0 mg, 8.8 mCi)] at 0.2 mg/kg plus unlabeled antibody in a total volume of 0.1 mL 0.9% w/v of NaCl containing a total protein dose of 10 mg/kg CS-1008 and IgG 1 respectively. Blood, tumor, and normal tissues were collected at designated time points from euthanized animals (n ¼ 5/time point). Mice injected with radiolabeled control IgG 1 were euthanized at 3, 72, 168, and 336 hours post injection (n ¼ 4/time point) and blood, tumor, and tissues were collected.
Gamma camera imaging and computerized tomography imaging. Planar gamma camera images of 111 In-labeled CS-1008 (0.2 mg/kg) and 111 In-labeled IgG 1 control antibody (0.2 mg/kg) distributions in COLO 205 tumor-bearing mice, and computerized tomography (CT) images, were obtained for 2 mice per antibody preparation on days 0, 2, 3, 7, and 10 post injection. Imaging procedures involved anesthesia of mice by intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (20 mg/kg) mixture. Mice were placed supine on a Philips Skylight gamma camera with energy windows set for 111 In (20% windows at 171 and 245 keV). Images were acquired in a 128 Â 128 or 256 Â 256 matrix and a standard containing a known amount of 111 In activity was included in the field of view. Twenty-minute images were collected. CT scanning, to generate cross-sectional imaging of anatomic structures, was also undertaken (Philips Gemini 2 slice PET-CT scanner; slice thickness, 0.6 mm; pixel size, 0.097 mm).
Imaging analysis involved whole body, liver, and tumor regions of interest (ROI) being defined for each time point. Counts of whole body and liver were compared between groups as an internal control to guarantee that equal amounts of radioactivity were administered in each dose-level group. Counts of the tumors were converted to %ID/g using a standard of known activity that was scanned at the same time. Tumor volumes (mL) were determined on the basis of markup of tumors in crosssectional CT images. To convert tumor volumes (mL) to tumor mass (g), animals were sacrificed after CT imaging, and tumors were removed and weighed (n ¼ 12). The conversion factor was determined as the slope of the linear regression curve.
Receptor occupancy study. A further series of biodistribution and planar gamma camera imaging studies explored the saturation of the DR5 receptor in vivo. Details of the experimental procedures and calculation of receptor occupancy are provided in Supplementary Data. (9) . The therapeutic effects of CS-1008 in COLO 205 tumorbearing mice have been previously described (2) .
Statistical analysis. Data were analyzed using one-way ANOVA when comparing multiple groups. Data were analyzed using an unpaired two-tailed Student t test when comparing two groups (treated to control). Differences were considered significant at a P value of less than 0.05. All analyses were conducted using Graphpad Prism version 4.03.
Results

TRAIL sensitivity
COLO 205 cells showed a 50% proliferation thompsonjames@example.com ng/mL of mTRA-8 and 14 ng/ml of TRAIL, and WiDr cells at 54 ng/mL of TRAIL and 208 ng/mL of mTRA-8. CT26 cells showed minimal responsiveness to TRAIL or mTRA-8.
Antibody radiolabeling
Radiochemical purity assessment of radiolabeled mTRA-8, CS-1008, and control IgG 1 by ITLC confirmed more than 98% bound 111 In-CHX-A 00 DTPA. Figure 1 shows the immunoreactivity-binding curve for radiolabeled CS-1008 (Fig.  1A) . The immunoreactivity of 111 In-radiolabeled CS-1008 was 13.3%. Because of the low immunoreactivity, a competition binding assay was performed to demonstrate specific binding of CS-1008 (Fig. 1B) . At 10 mg/mL of CS-1008, 75% of 111 In-labeled mTRA binding could be blocked with CS-1008, indicating that CS-1008 and mTRA are competing for the same binding sites. Saturation binding analysis of 111 In-labeled CS-1008 showed specific binding with highaffinity binding sites on COLO 205 cells (Fig. 1C) 
37
C was high, with retention of immunoreactivity, radiochemical purity, and construct integrity for up to 11 days.
ELISA
The labeling of CS-1008 with 111 In showed minimal impact on binding to DR5 (TRAIL R2; Fig. 1D ).
Biodistribution
In an initial biodistribution study, tumor uptake and biodistribution of 111 In-labeled CS-1008 at a dose of 0.2 mg/kg was studied in mice bearing human COLO 205 tumors and mice bearing murine CT26 tumors (Table 1  and Supplementary Table S1 ). CS-1008 does not cross-react to the rodent death receptor and therefore a murine colon cancer cell line was chosen as negative control. The %ID/g of In-labeled CS-1008 in CT26 was observed at 72 hours post injection (%ID/g, 11.79 AE 1.68; Fig. 2A) .
The specific uptake in vivo was confirmed by an additional biodistribution study comparing 111 In-labeled CS-1008 at a dose level of 10 mg/kg with 111 In-labeled IgG 1 isotype control hu3S193 at a dose level of 10 mg/kg in COLO 205 tumor-bearing mice at different time points after injection. At the 10 mg/kg dose level, 111 In-CS-1008 tumor uptake was lower at all time points compared with the 0.2 mg/kg dose level, and peaked at 72 hours post injection (%ID/g, 11.64 AE 1.59, n ¼ 4). The isotype control antibody showed an initial tumor uptake of 5.55 AE 0.97%ID/g at 3 hours post injection, declining progressively over time.
A further biodistribution study of 111 In-labeled CS-1008 (0.2 mg/kg) in WiDr and CT26 tumor-bearing mice was performed and showed specific localization of 111 In-labeled CS-1008 in WiDr tumors, but at a lower level than in COLO 205 tumors (Fig. 2C and Supplementary Table S2 ).
Gamma camera imaging and DR5 occupancy of In-labeled CS-1008 (0.2 mg/kg) in BALB/c nu/nu mice bearing DR5-positive COLO 205 or DR5-negative CT26 tumors was performed. Tumor uptake of 111 In-labeled CS-1008 was evident as of day 2 post injection and continued for more than 7 days (Supplementary Fig. S1 ). Initial blood pool activity was seen on day 0 and decreased over time. In a further series of imaging studies, tumor uptake of 111 In-labeled CS-1008 at different antibody dose levels was conducted to explore the saturation of DR5 in vivo in tumors with different levels of DR5 expression, high (COLO 205) and low (WiDr). Six groups of mice (n ¼ 4) were injected with 111
In-labeled CS-1008 (5 mg, 8.5 mCi) at different dose levels of unlabeled CS-1008 (0.2, 0.5, 1, 3, 10, and 30 mg/kg). Planar gamma camera imaging and CT imaging were conducted at 48 hours post injection when the effect of CS-1008 on tumor size was minimal ( Fig. 2B and D) . Figure 3 shows representative planar gamma camera images and CT scans at 48 hours post injection for 0.2 mg/kg (Fig. 3A) , 0.5 mg/kg (Fig. 3B) , and 10 mg/kg (Fig. 3C ) CS-1008 in COLO 205 tumor-bearing mice. After imaging, mice were sacrificed; tumors were weighed and counted for radioactivity. A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay ( Supplementary Fig. S2 ) of COLO 205 tumor sections collected from mice treated with 0.2 mg/kg (Fig. 3A) and 10 mg/kg (Fig. 3C) CS-1008 showed an increase in apoptosis in tumors from animals treated with 10 mg/kg compared with animals treated with 0.2 mg/kg. Supplementary Fig. S3 shows representative images of WiDr tumor-bearing mice at 48 hours post injection for 0.2 mg/kg ( Supplementary Fig. S3A ), 0.5 mg/kg ( Supplementary Fig.  S3B ), and 10 mg/kg ( Supplementary Fig. S3C ) CS-1008. Figure 3D shows %ID/g in the COLO 205 tumors at different doses (n ¼ 4). At 48 hours post injection at a 0.2 mg/kg dose level, tumor uptake of 111 In-labeled CS-1008 was highest (28.15 AE 2.02%ID/g; n ¼ 4), declining progressively with increased doses of cold CS-1008. DR5 saturation starts at 1 mg/kg dose level and is complete at 3 mg/kg. Figure 3E shows the %ID/cm 3 curve generated from region of interest (ROI) analysis of tumor images. To correct for the difference between tumor volumes (cm 3 ) measured by CT analysis and tumor volumes (g) weighed, a correlation curve was established and conversion tumor volumes (cm 3 ) into tumor volumes (g) required a correlation factor of 0.69 AE 0.01. Figure 3F shows the %ID/g generated from image analysis after correcting the tumor volumes and this curve is consistent with the curve generated from the biodistribution data (Fig. 3D) . The results for WiDr tumor analysis are shown in Fig. 3G .
Using a baseline value of 5.07%ID/g at 48 hours post injection of 0.2 mg/kg 111 In-labeled isotype control, a receptor occupancy curve was generated. Figure 4A shows the occupancy of COLO 205 DR5 antigen binding sites by 111 In-CS-1008 with increasing protein dose at 48 hours post injection. To compare receptor occupancy at different dose levels with in vivo tumor growth inhibition (TGI) at different dose levels, TGI values (%) measured by Yada and colleagues were plotted against the imaged CS-1008 antibody dose levels ( Fig. 4B; ref. 2). The curves in Fig. 4A and B show the same saturation trend, with DR5 saturation occurring between 1 and 3 mg/kg dose levels. WiDr tumors showed a similar trend for receptor saturation between 1 and 3 mg/kg dose levels (Fig. 4C ), and this receptor saturation level also predicted the dose of maximal therapeutic response (Fig.  4D) . The level of TGI was less with WiDr tumors than with COLO 205 tumors, in keeping with the reduced DR5 expression and lower concentration of CS-1008 achieved in tumors in vivo.
Pharmacokinetics
No differences in serum clearance were observed between different antibody dose levels (0.2, 1 mg/kg vs. 10 mg/kg) with mean values (n ¼ 15; SD) for a half-life (t 1/2a ) of 5.71 AE 0.91 hours, b half-life (t 1/2b ) of 141.78 AE 20.69 hours, total clearance (C L ) of 0.02 AE 0.1 mL/h, and volume of distribution at steady state (V ss ) of 4.47 AE 0.25 mL. The pharmacokinetic parameters for each dose level are shown in Table 2 .
Discussion
We have shown that CS-1008, a humanized IgG 1 anti-DR5 antibody, demonstrates specific uptake in TRAIL-sensitive, DR5-positive tumors that is dose dependent and in proportion to DR5 expression in tumor cells. Moreover, CS-1008 has tumor inhibitory effects that are dependent on CS-1008 tumor concentration and reach maximal efficacy when antibody:DR5 binding is saturated, which can be quantitatively measured by molecular imaging of receptor occupancy in vivo with 111 In-CHX-A 00 -DTPA-CS-1008. In-CHX-A 00 -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 AE 0.01 (n ¼ 12). G, tumor uptake (%ID/g) of 111 In-CHX-A 00 -DTPA-CS-1008 in WiDr tumors at different dose levels from gamma well counting. bars, SD.
splenic uptake was seen with 111 In-labeled CS-1008. Radioiodinated recombinant human TRAIL has also been explored as a biomarker for DR4 and DR5 targeting (22) .
Potent antitumor activity of CS-1008 has been previously demonstrated in vitro and in vivo against the colorectal cell line COLO 205, with tumor growth inhibition observed in a dose-dependent manner up to 1 mg/kg dose level (2) . We have demonstrated that receptor occupancy and saturation can be accurately measured by molecular imaging of 111 In-CHX-A 0 -DTPA-CS-1008 imaging in COLO 205 tumors, and that receptor occupancy and saturation correlate precisely with the dosage required for maximal tumor growth inhibition. We have also demonstrated that in tumors with lower levels of DR5 expression (WiDr), similar correlations can be made through molecular imaging of DR5 occupancy and saturation. Interestingly, DR5 expression levels in tumor (assessed by peak uptake of Calculation of antigen-binding site occupancy from the imaging data showed that DR5 binding is saturable at protein doses above 1 mg/kg. Assuming a cell diameter of 10 mm for COLO 205 and WiDr cells, measured CT tumor volumes and an immunoreactive fraction of 10%, approximately three antibodies per receptor at 1 mg/kg, were enough to saturate the tumor and elicit maximum therapeutic effect in vivo. This finding indicates the importance of dose selection on achieving optimal concentration of CS-1008 in tumor, and the ability of molecular imaging approaches to quantitate receptor occupancy in vivo.
There are several factors that might influence the success of imaging DR5 occupancy in patients and might cause patient variability in therapy outcome when using 111 Inlabeled anti-DR5 antibodies in the clinic. First, when calculating %ID/g tumor in the mouse models, 1% of the total injected 111 In-CS-1008 was typically measured in the tumors of which 60% was specific. When translating these findings into the clinic, many solid tumors in patients only show values around 0.01% of the injected dose when using radiolabeled antibodies. Using 111 In-labeled CS-1008 might help to select the patients that show sufficient tumor uptake of 111 In-CS-1008 at tracer levels of cold CS-1008 (e.g., 0.2 mg/kg). Second, Fcg receptors have been proposed as an Fc cross-linking interface in vivo promoting DR5-mediated agonistic cell killing by anti-DR5 antibodies (23) . In patients, these Fcg receptors might be available in the tumor via infiltrating immune cells, but the population and the amount might vary among patients. Third, the data presented here show DR5 occupancy and therapeutic response to CS-1008 in COLO 205 and WiDr tumor models; however, in cells with similar or higher DR5 expression levels, but with slower induction of the apoptotic pathway, receptor turnover, receptor internalization, and antibody pharmacokinetics might play a much larger role. Although the results here have shown proof-of-principle that 111 In-CHX-A 00 -DTPA-CS-1008 can aid in patient selection and estimate optimal therapeutic dosing, further studies are warranted comparing resistant versus sensitive tumor models, and exploring the role of apoptosis pathway induction on effectiveness of treatment (24) . Such studies are currently being performed by our laboratory.
In conclusion, these results suggest that molecular imaging of DR5 occupancy and saturation can play a major role for clinical development, optimal dosing, and patient selection for trials of DR5-targeting antibodies. 

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