Development of a Red Fluorescent Labeled Agent for Assessing HER2 Expression

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1 APPLICATION NOTE Development of a Red Fluorescent Labeled Agent for Assessing HER2 Expression HER2Sense 645 Author Jeffrey D. Peterson, Ph.D. PerkinElmer, Inc. Waltham, MA USA Abstract Despite the clinical importance of HER2 expression in breast, ovarian, and stomach cancer, until now there have been no established research agents available for non-invasive optical imaging in pre-clinical models of cancer. The ability to image HER2 can provide an important tool for cancer detection and quantification as well as for the assessment of target coverage for HER2-specific therapies. We developed a red fluorescent imaging agent specific for tumor HER2 by conjugating a red fluorescent dye (VivoTag 645) to Trastuzumab, a HER2-specific monoclonal antibody used clinically to treat breast cancer. This application note provides the in vitro and in vivo datasets that characterize this red fluorescent imaging agent and provides detailed protocols and examples that illustrate its utility in pre-clinical imaging. The pairing of HER2Sense with PerkinElmer imaging systems, and other novel red and near infrared (NIR) imaging agents, allows the detection and quantification of multiple important biological processes in the context of tumor HER2 expression.

2 Materials And Methods Fluorescent Agents The HER2Sense 645 fluorescent agent (specific for the HER2 biomarker expressed in certain types of cancer) was used to image tumors implanted in nu/nu mice. The imaging dose for these agents was as recommended in the product insert (4 µg/25 g mouse). Table 1. Basic properties of HER2Sense fluorescent tumor imaging agent. Agent Type HER2Sense 645 HER2-targeted agent Molecuar Weight or Size ~16, g mol -1 Ex/Em Blood Half-life Tissue Half-life 643/661 nm Distribution t 1/ h Terminal t 1/2 2 h h Agent Summary. Characteristics of the agent (MW/size, excitation/emission [Ex/Em], and blood/tissue pharmacokinetics) were determined in multiple indipendent studies. Blood life was measured by blood collection from mice at different times post-intravenous injection. Blood samples were measured for fluorescence levels in a fluorescence microplate reader. Tissue half-lives were determined by time course FMT 4 imaging of tumors. Flow Cytometry and Microscopy Human ovarian carcinoma SKOV-3 cells (HER2/neu positive) and human colon carcinoma COLO 25 (HER2/neu negative), were obtained from American Type Culture Collection (ATCC, Manassas, VA). SKOV-3-luc-D3 cells were obtained from PerkinElmer (Waltham, MA). Cells were routinely cultured in McCoy s 5A medium and RPMI, respectively, with 1% fetal bovine serum and 1% penicillin-streptomycin in 75 cm 2 flasks. Exponentially growing cells between passages 1-1 were used for all experiments. Cells were incubated with HER2Sense (.25 μm), VivoTag 645-labeled control IgG or VivoTag 645 (not shown) for 1 h. Cells were analyzed by flow cytometry (Becton Dickinson LSRII) and fluorescence microscopy using appropriate lasers and filters appropriate for detection of 645 nm wavelength. Cell nuclei were stained with DAPI (blue). SKOV-3 and COLO 25 Tumor Models Six to eight week-old female nu/nu mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained in a pathogen-free animal facility with water and low-fluorescence mouse chow (Harlan Tekland, Madison, WI). Handling of mice and experimental procedures were in accordance with PerkinElmer IACUC guidelines and approved veterinarian requirements for animal care and use. To induce tumor growth, mice were injected in the dorsal flank with 5x1 5 SKOV-3 (ovarian cancer) or COLO 25 (colorectal cancer) tumor cells, yielding tumor masses within a few weeks (Figure 1). In Vivo Imaging and Analysis Tumor-bearing mice were anesthetized using inhaled isoflurane, injected intravenously with HER2Sense, and positioned for imaging in either the IVIS Spectrum or the FMT 4 in vivo imaging system (both systems from PerkinElmer, Waltham, MA). Fluorescence images were captured at a variety of time points post-her2sense injection, and for bioluminescence imaging mice were injected intraperitoneally with 15 mg/kg Xenolight Rediject D-Luciferin (PerkinElmer), and 2D bioluminescence images were acquired minutes after injection. Imaging Data Analysis The collected FMT fluorescence images were reconstructed by FMT system software (TrueQuant v3., PerkinElmer, Waltham, MA) for the quantification of three-dimensional fluorescence signal within the tumors. Three-dimensional regions of interest (ROI) were drawn encompassing the relevant flank region. IVIS Spectrum data were analyzed by Living Image software (PerkinElmer) and analyzed for 2D bioluminescence and epifluorescence. SKOV-3 or COLO 25 Cells Nu/nu mice A few weeks 24 h IV Imaging Agent Injection Tumor Mice Figure 1. Flank tumor xenograft model. 2

3 Introduction and Results A red fluorescent dye (VivoTag 645; abs/em max 643/ 66 nm) was used to label Trastuzumab, which is currently used to treat breast and stomach cancer. The red-labeled Trastuzumab (HER2Sense 645) preferentially bound to HER2+ SKOV-3 human ovarian adenocarcinoma cells over HER2- human colorectal adenocarcinoma COLO 25 cells (1 fold), and the specificity of binding was confirmed by control experiments using free dye, labeled non-specific IgG, and competitive blockade with unlabeled excess trastuzumab. Fluorescence microscopy confirmed the expected membrane localization of fluorescence. In vivo and ex vivo imaging by fluorescence molecular tomography (FMT), showed significantly higher signal within the tumors, peaking at 6-72 hours following intravenous injection of 1.6 mg/kg HER2Sense, decreasing thereafter with a tissue half-life of 3 days. In summary, HER2Sense selectively targets HER2, allowing both imaging in vitro and the non-invasive real-time tomographic imaging and quantification in vivo of HER-2 expression. Dorsal view, FL Axial Slice, FL Dorsal view, BLI 6. x Microscopy and Flow Cytometry Figure 2 shows that HER2-positive SKOV-3 cells are effectively labeled with HER2Sense, and the fluorescent signal is readily quantified by flow cytometry as ~7- fold increase in fluorescence (Figure 1A). In contrast, HER2-negative COLO 25 cells showed little labeling with HER2Sense. Results were confirmed by fluorescence microscopy (Figure 1B). A. Mean Fluorescence (RFU) B. SKOV-3 COLO Colo-25 SKOV-3 Cells only Control IgG HER2Sense Figure 2. SKOV-3 (HER2 + ) or COLO-25 (HER2 - ) were incubated with HER2Sense 645 (.25 μm), VivoTag 645-labeled IgG or VivoTag 645 (not shown) for 1 h. Cells were analyzed by A. flow cytometry and B. fluorescence microscopy using appropriate lasers and filters appropriate for detection of 645 nm wavelength. HER2Sense only labels SKOV-3 cells (shown in red). Cell nuclei are stained with DAPI (blue). Figure 3. Scid/beige mice were implanted with human ovarian SKOV-3 luc tumors and injected with 4 µg (1.6 mg/kg) HER2Sense when tumors reached desired volume. A representative mouse is shown that was injected with HER2Sense and imaged by IVIS FLIT (3D fluorescence [FL] imaging) at 24 h post-injection. Bioluminescence (BLI) imaging of the same mouse confirmed tumor specific localization of HER2Sense [see inset]. MicroCT co-registration provides anatomical reference. In Vivo Imaging: Fluorescence Localization Figure 3 highlights the in vivo ability of HER2Sense to detect HER2 positive SKOV-3 tumor xenografts. These results confirmed appropriate localization of signal, with good tumor definition and >3 fold target to background ratios (Figure 4) and 4-fold selectivity between HER2-positive and negative tumors. The optimal imaging time was 24 h post-injection, with a tissue residence half-life of approximately 72 h (not shown). Tumor Fluorescence (Avg. Radiant Efficiency) 3.5E+9 3.E+9 2.5E+9 2.E+9 1.5E+9 1.E+9 5.E+8 SKOV-3 IVIS Quantification Background pmol source intensity.e Time Post Injection Figure 4. SKOV-3-bearing scid/beige mice from Figure 3 were imaged by IVIS Spectrum at 3, 5, 24 and 48 h relative to agent injection. Tumor signal was quantified and represented over time ± standard error of the mean. 3

4 In Vivo Selectivity Figure 5 shows that HER2-positive SKOV-3 tumors are effectively labeled in vivo with HER2Sense, and the fluorescent signal (Figure 5A) is readily quantified by 3D FMT imaging (Figure 5B). In contrast, HER2-negative COLO 25 tumors showed ~6-fold lower HER2Sense fluorescence as quantified in absolute pmoles of fluorescence per tumor (Figure 5AB). Excellent tumor definition was achieved with 4 µg of the agent, and the optimal imaging time point was 24 h post injection. Near complete clearance of HER2Sense from tissue was observed by 7 days (not shown). In Vivo Biodistribution and Pharmacokinetics Tissue biodistribution was determined by 2D imaging of excised tissues from SKOV-3-bearing mice that received HER2Sense 24 h prior to sacrifice (Figure 6A). Tissues were imaged on the FMT 4 using the 2D epifluorescence acquisition mode and quantified using TrueQuant software. Blood pharmacokinetics was assessed in normal CD-1 mice following blood sampling at several timepoints from to 72 h. Fluorescence was assessed by microplate fluorescence assay. Results confirmed that optimal imaging could be performed at 24 h, with biodistribution assessment showing predominant fluorescence targeting to the tumor as compared to tissue/blood levels in other tissues (Figure 6A). Ex vivo tissue epifluorescence images serve to illustrate the predominant tumor biodistribution (Figure 6B). In Vivo Competition with Unlabeled Trastuzumab To further confirm the specificity of HER2Sense localization to tumors, we used 5X excess unlabeled Trastuzumab, injected 5 minutes prior to HER2Sense, to block HER2 receptors in vivo in SKOV-3 tumor-bearing nu/nu mice. Mice were imaged 24 h after agent injection as optimized by previous experiments. Although tumor size was unaffected by this very short-term HER2 blockade, the targeting of HER2Sense to the tumors was decreased by approximately 7%. The residual, unblocked signal was consistent with the circulating agent still present at 24 h, and this study confirms the selectivity of the agent for HER2 + tumor cells. A. Imaging SKOV-3 and COLO 25 HER2 - COLO-25 HER2 + SKOV A. Mean Fluorescence (Counts/Energy) Biodistribution and PK Percent of Injected Dose Blood Levels Distribution t 1/2 = 12 h Terminal t 1/2 = 2 h Time (hrs) 4 nm tumor spleen liver calvaria intestines heart lung brain salivary gland fat muscle kidney ovaries/uterus skin lymph nodes bladder pancreas B. Tumor Fluorescence (pmoles) Quantification SKOV-3 COLO Imaging Time (hours) B. Tumor Liver Tissue Epifluorescence Images Pancreas Skin Spleen Lung Fat Kidney Epifluorescence (counts/energy) Figure 5. Nu/Nu mice were implanted with human ovarian SKOV-3 or colorectal COLO 25 tumors. When tumors reached desired volume, mice were injected intravenously with 4 µg HER2Sense and imaged tomographically (FMT 4) at 6, 24 and 48 h. A. Shown are 3 representative mice per group at 24 h. B. Tomographic imaging datasets were used to quantify tumor region fluorescence associated with HER2 expression. Figure 6. Control and SKOV-3-bearing Nu/Nu mice were injected intravenously with 2 mg/kg HER2Sense and blood/tissues were removed at described times to assess blood and tissue biodistribution. A. PK and quantified tissue biodistribution. B. Epifluorescence tissue images. 4

5 Conclusions Approximately 1 in 8 U.S. women (just under 12%) will develop invasive breast cancer over the course of a lifetime. This makes breast cancer the second most common cancer among women in the United States and one of the leading causes of cancer death among women of all races. Twenty to thirty percent of all breast cancers express the HER2 protein due to a gene mutation which promotes the growth of cancer cells. This gene mutation and the elevated levels of HER2 that it causes can also occur in other types of cancer, especially ovarian, stomach, and uterine cancer. There is mounting evidence of the role of HER2 overexpression in cancer, and it has been solidly correlated to poor outcomes and more aggressive disease. The ability to detect expression of HER2 non-invasively in animal models of cancer will provide an important tool for biomedical research, so we developed a red fluorescent imaging agent specific for tumor HER2 by conjugating a red fluorescent dye (VivoTag 645; ex/em = 645/66 nm) to the HER2-specific antibody, Trastuzumab, used clinically to treat breast cancer. This imaging agent was characterized in vitro and in vivo, revealing the ability to selectively detect HER2-expressing human tumor xenografts (and not HER2-negative tumors) implanted in nu/nu mice. Optimal imaging was achieved at 24 h post injection with a favorable biodistribution profile including little or no accumulation in a variety of other tissues, including liver and kidneys. Selectivity was further confirmed by blockade with prior injection of 5X excess of unlabeled Trastuzumab. The pairing of HER2Sense with PerkinElmer imaging systems, and other novel red and near infrared (NIR) imaging agents, allows the detection and quantification of multiple important biological processes in the context of tumor HER2 expression. A. FMT Images B. FMT Quantification Tumor size (mm 3 ) In Vivo Competition HER2Sense p <.2 Competition Figure 7. Tomographic imaging datasets were used to quantify tumor region fluorescence in SKOV-3 tumor-bearing mice injected with HER2Sense (4 µg/ mouse) in the absence (HER2Sense) or presence (competition) of excess unlabeled Trastuzumab (2 µg) injected 5 min before the agent. Tumor volumes were determined using caliper measurements and the formula volume = length x width 2 / [nm] HER2Sense Fluorescence (pmol) 5

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