AD______________ Award Number: W81XWH-07-1-(cid:19)(cid:24)(cid:23)(cid:26) TITLE: (cid:44)(cid:80)(cid:68)(cid:74)(cid:76)(cid:81)(cid:74)(cid:3)(cid:82)(cid:73)(cid:3)(cid:40)(cid:83)(cid:16)(cid:38)(cid:36)(cid:48)(cid:3)(cid:51)(cid:82)(cid:86)(cid:76)(cid:87)(cid:76)(cid:89)(cid:72)(cid:3)(cid:48)(cid:72)(cid:87)(cid:68)(cid:86)(cid:87)(cid:68)(cid:87)(cid:76)(cid:70)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:76)(cid:81)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:47)(cid:92)(cid:80)(cid:83)(cid:75)(cid:3)(cid:54)(cid:92)(cid:86)(cid:87)(cid:72)(cid:80) PRINCIPAL INVESTIGATOR: (cid:39)(cid:85)(cid:17)(cid:3)(cid:46)(cid:85)(cid:76)(cid:86)(cid:87)(cid:72)(cid:81)(cid:3)(cid:36)(cid:71)(cid:68)(cid:80)(cid:86) CONTRACTING ORGANIZATION: University of (cid:55)(cid:72)(cid:91)(cid:68)(cid:86)(cid:3)(cid:43)(cid:72)(cid:68)(cid:79)(cid:87)(cid:75)(cid:3)(cid:54)(cid:70)(cid:76)(cid:72)(cid:81)(cid:70)(cid:72)(cid:3)(cid:38)(cid:72)(cid:81)(cid:87)(cid:72)(cid:85) (cid:3)(cid:43)(cid:82)(cid:88)(cid:86)(cid:87)(cid:82)(cid:81)(cid:15)(cid:3)(cid:55)(cid:59)(cid:3)(cid:3)(cid:26)(cid:26)(cid:19)(cid:22)(cid:19)(cid:3) REPORT DATE: (cid:3)(cid:45)(cid:68)(cid:81)(cid:88)(cid:68)(cid:85)(cid:92)(cid:3)(cid:21)(cid:19)(cid:20)(cid:20) TYPE OF REPORT: (cid:3)(cid:41)(cid:76)(cid:81)(cid:68)(cid:79) PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for public release; distribution unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation. 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Z39.18 Table of Contents Introduction 3 Body 3 Key Research Accomplishments 35 Reportable Outcomes 35 Conclusion 35 References 36 Appendices 37 Page | 2 Introduction The majority of cancer mortalities occur not from the primary tumor but rather from distant metastases. Since the lymphatic system provides a route for the metastatic spread of cancer, it is not surprising that lymph node status serves as the primary prognostic indicator in most cancers. Currently, occult lymph node staging requires surgical removal of lymph nodes for subsequent biopsy, which in itself has significant morbidity. Specifically, in the case of breast cancer, axillary node resection is associated with an elevated risk of breast cancer-related lymphedema. This research plan aims to develop a unique imaging agent to identify metastatic tumor cells within the lymph nodes of cancer patients, initially with breast cancer patients. Development of a non-invasive methodology for nodal staging could eventually enable surgeons to intra-operatively differentiate cancer positive and negative lymph nodes, allowing for the specific resection of positive nodes and retention of cancer negative nodes. The project includes development of a new imaging agent based on anti-Ep-CAM antibody and preclinical testing of the binding and pharmacokinetics with the eventual goal of clinical translation. In achieving these goals, I will (i) modify an established humanized antibody against the epithelial cell adhesion molecule (Ep- CAM) by dual-labeling with a near infrared (NIR) fluorescent dye and a radiotracer to image epithelial cell based cancers in the lymph compartment with optical and nuclear imaging modalities; (ii) deliver low doses of the imaging agent directly into the lymphatic space; and (iii) develop a custom gain-modulated intensified camera for intraoperative optical lymph imaging. Body Preclinical Ep-Cam Imaging Studies Phase I of the preclinical Ep-CAM imaging studies included binding affinity studies, antibody labeling, HPLC validation, stability studies, testing of the affinity of dual-labeled antibody, and specificity (blocking) assays. An antibody against the human epithelial cell adhesion molecule (Ep-CAM) and the control immunoglobulin gamma were purchased from RnD Biosystems (Minneapolis, MN). Antibodies were covalently coupled to IRDye 800CW-NHS Ester from Licor, Inc, (Lincoln, NE). The conjugation ratio was 1.5 to 2.8 IRDye molecules per antibody. The retention of specific binding ability of anti-Ep-CAM-IR800 was tested using two human breast cancer cell lines, MT3 and SKBr3. SKBr3 cells (ATCC, Manassas, VA) express low levels of Ep-CAM while MT-3 cells (German Collection of Microorganisms and Cell Cultures, (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)), Braunschweig, Germany) express high levels of Ep-CAM, as published by Prang, et al, British Journal of Cancer, 2005. Cells were maintained in Dulbecco’s Modified Eagle Medium – Nutrient Mixtures F12 (DMEM/F12) with 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO at 37oC. Cell culture reagents were purchased 2 from Gibco/Invitrogen (Carlsbad, CA) and supplies, including sterile tubes, cell culture flasks and pipettes were purchased from ISCBioexpress (Kaysville, UT) or VWR (Radnor, PA). On the day of experimentation, cells were rinsed in phosphate buffered saline (PBS) and incubated in trypsin for 10-20 minutes. After cells were sufficiently detached from the cell culture flasks, they were removed from the flasks and spun down. For cell binding, the cells were split into tubes each with one million cells, and washed in hanks based salt solution (HBSS) with bovine serum albumin (BSA). Each tube of one million cells was incubated with 1 ng of anti-Ep-CAM-IR800 for 30 minutes to one hour a humidified incubator with 5% CO at 37oC. Unbound 2 antibody was washed off the cells with HBSS-BSA and antibody binding was determined using flow cytometry (FACSAria, BDBiosciences, San Jose, CA), microscopy (Lieca, Bannockburn, IL), or the Odyssey (Licor, Lincoln, NE). Cell nuclei were stained using Sytox Green (Invitrogen, Carlsbad, CA). Page | 3 A B C D Figure 1: Two human breast cancer cell lines SKBr3 (A,C) and MT-3 (B,D) were stained with an anti-EpCAM antibody (red in C,D) and cell nuclei were stained with sytox green (green in all panels). More anti-EpCAM antibody binding is seen on the high Ep-CAM expressing MT-3 cells than on the low Ep-CAM expressing SKBr3 cells. Stability and pH sensitivity of anti-Ep-CAM agent, moving towards dual-labeled in vivo imaging, the anti-Ep- CAM antibody mAb 9601 was labeled with IRDye, Alexafluor 680 or IRDye and Alexafluor 680 in different buffers at different pH levels to test the binding ability of the antibody after conjugations. Labeled antibodies were measured for dye to protein ratios and protein concentration, as described in Table 1. Page | 4 Buffer mAb9601-IRDye 800CW mAb9601-AF680 D:P [] mAb D:P [] mAb mg/ml mg/ml Sodium Bicarbonate 2.4 0.12 2.5 0.13 pH 7.4 Sodium Bicarbonate 1.2 0.28 1.5 0.20 pH 8.0 Sodium Bicarbonate 2.5 0.17 2.3 0.26 pH 8.2 Sodium Phosphate 2.7 0.26 0.62 0.05 pH 8.3 Table 1: Comparison of conjugation efficiency of dye to antibodies in reaction buffers. The samples were then incubated on breast cancer cells to determine their ability to bind to high Ep-CAM expressing cells (MCF-7 and MT-3) and low expressing cells (SKBr3). The images shown are from the Alexafluor 680 labeled antibody. Unfortunately there was too much overlap between the AF680 and IRDye 800 absorbance measurements, so I was unable to quantify the amount of each dye that labeled the antibody in the dual conjugation. HPLC traces were run of each antibody conjugate to determine if there was free dye present after the purification processes and the traces did not show free dye contamination (data not shown). The HPLC tubes were imaged on the Odyssey imager to determine if the fluorescent signal was sufficient for a cell binding assay as shown in figure 2. Figure 2: Fluorescent images of labeled antibodies for Alexafluor 680 (em 710 nm) and IRDye 800CW (em 830 nm). The sample numbers from figure 2 are explained in detail in table 2. The first column is sample number, then buffer, pH, labeling ratio of IRDye to antibody and finally labeling ratio of Alexafluor 680 to antibody. The labeling ratios were controlled so that each antibody received a maximum of 5-fold excess dye to ensure retention of binding ability. Page | 5 Sample Buffer pH IRDye:Ab AF680:Ab 1 NaHCO 7.4 5 0 3 2 NaHCO 7.4 0 5 3 3 NaHCO 7.4 2.5 2.5 3 4 NaHCO 8.0 5 0 3 5 NaHCO 8.0 0 5 3 6 NaHCO 8.0 2.5 2.5 3 7 NaHCO 8.2 5 0 3 8 NaHCO 8.2 0 5 3 9 NaHCO 8.2 2.5 2.5 3 10 Na HPO 8.3 5 0 2 4 11 Na HPO 8.3 0 5 2 4 12 Na HPO 8.3 2.5 2.5 2 4 13 10 mg Ab in 100 ml PBS 7.4 14 5 mg IRDye in 100 ml PBS 7.4 15 5 mg AF680 in 100 ml PBS 7.4 Table 2: Summary of labeled antibody samples in different buffers, pHs and with differing amounts of IRDye and Alexafluor 680. The fluorescent intensities of the different antibody conjugates were measured and the results summarized in figure 3. Page | 6 Figure 3: Fluorescent intensity of the labeled antibodies, with each bar of the final data set representing a control, the first bar is IRDye800 only, the second bar is AF680 only, and the third bar is unlabeled antibody only. There is a contribution at 800nm from the AF680, but no contribution at 700 from IRDye800. In both charts the final data set is IRDye (sample 13), AF680 (sample 14), and antibody with no dye (sample 15). The antibody conjugates were then diluted in a phosphate buffered saline at pH 7.4 for cell binding studies. Three cell lines were used, MCF-7 and MT-3, high Ep-CAM expressing cells, and SKBr3, low Ep-CAM expressing cells. In previous experiments (data not shown) the binding of anti-Ep-CAM to the cells is very dependent upon the confluence of the cells at the time of harvest. The MT-3 and SKBr3 cells shown in figure 4 were grown to 100% confluence, the media changed, and then left to grow for another 3 days. The MCF-7 cells were grown to 70% confluence, the media changed, and then left to grow for another 3 days. In the binding results below, the high Ep-CAM cells that were grown well past confluence displayed the most binding. This is probably explained by the fact that Ep-CAM is an adhesion molecule, so in the presence of more cells for binding interactions, each cell might produce or display more Ep-CAM for binding. Page | 7 MCF-7 MT-3 SKBr-3 Blank mAb- 9601- AF680 NaHCO 3 pH 7.4 mAb- 9601- AF680 NaHCO 3 pH 8.0 mAb- 9601- AF680 NaHCO 3 pH 8.2 mAb- 9601- AF680 Na HPO 2 4 pH 8.3 Figure 4: Binding of labeled anti-Ep-CAM antibody (9601) to high Ep-CAM expressing MCF-7 and MT-3 cells and low Ep-CAM expressing SKBr3 cells. The antibody conjugates retained their ability to bind Ep-CAM positive breast cancer cells in pH up to 8.3, which will be important for the chelating agent DOTA. The DOTA-NHS-ester is best conjugated between pH Page | 8 8.0 and 8.5, so the ability of the antibody conjugate and dyes to withstand a pH above 8 will allow the dual- labeling of anti-Ep-CAM for animal studies. For dual-labeled in vivo imaging, the antibody must be conjugated with the fluorophore (IRDye 800CW) and a chelating agent (DOTA), which sequesters the radio-metal completing the dual-label. Each conjugation step requires an incubation of 4 hours to overnight, so to determine the best order of conjugation or if the conjugations could be done simultaneously, anti-Ep-CAM (9601) was conjugated with 5-fold excess IRDye, 5- fold excess DOTA or 500-fold excess DOTA overnight. After size exclusion and centrifugation to remove non- conjugated IRDye or DOTA, the 9601-IR samples were conjugated with 5- or 500-fold excess DOTA, the 9601-DOTA samples were conjugated with IRDye, and a fresh batch of 9601 was conjugated with 5-fold excess IRDye and DOTA (at either 5- or 500-fold excess) overnight at 4 degrees. Two samples of 9601 were kept unlabeled for testing purposes, one was unprocessed and the other went through all processing steps but without any dye or DOTA for conjugation. Sample Conjugation 1 Purification 1 Conjugation 2 Purification 2 9601-unp NO NO NO NO 9601-p Buffer YES Buffer YES 9601-IR,D 5xIRDye YES 5xDOTA YES 9601-D,IR 5xDOTA YES 5xIR YES 9601-D5,IR 500xDOTA YES 5xIR YES 9601-IR-D Buffer YES 5xIR+5xDOTA YES 9601-IR-D5 Buffer YES 5xIR+500xDOTA YES IT-IR-D Buffer YES 5xIR+5xDOTA YES Table 3: Conjugation ratios and conditions for anti-Ep-CAM (9601), IRDye 800CW (IR), and the chelating agent, DOTA. The ability of these different conjugates to bind to Ep-CAM positive cells was then tested using flow cytometry. Ep-CAM positive MT-3 cells were grown to confluence, trypsinized, washed and 1x106 cells were placed into tubes for antibody binding. The different conjugates and controls of IRDye in DMSO, IRDye in PBS, and buffer with no agent were incubated with cells for 1 hour at 37 degrees. After 1 hour the cells were washed to remove unbound antibody and then secondary antibody, goat anti-mouse-Alexafluor488 (GaM-488) was incubated with the cells to detect presence of mouse antibody. The flow cytometer measuredAlexafluor488 (figure 5) and IRDye 800CW (figure 6) fluorescence in each sample through mean fluorescent intensity and percent positive. Page | 9