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Supplementary Figure 1: Serum levels of AMA-MMAE in mice. Supplementary Figure 2: Correlation plot of tumor growth inhibition versus specific tumor uptake. Supplementary Table 1: Overview results. Supplementary Table 2: Tumor growth inhibition study of mice bearing OVCAR3-X2.1 cells comparing the efficacy of 5mg/kg and 20mg/kg doses

Purpose: AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 (89Zr) or fluorescent dye to evaluate the tumor-targeting properties.Experimental Design: 89Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μg 89Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, 89Zr-Mec14, served as a negative control. 89Zr-AMG211 integrity was determined in tumor lysates ex vivo. Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, 89Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199.Results: 89Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μg 89Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of 89Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. 89Zr-Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured 89Zr-AMG211 fulfilled release specifications.Conclusions: 89Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior. Clin Cancer Res; 24(20); 4988–96. ©2018 AACR.

Purpose: To provide proof of principle of safety, breast tumor–specific uptake, and positive tumor margin assessment of the systemically administered near-infrared fluorescent tracer bevacizumab–IRDye800CW targeting VEGF-A in patients with breast cancer.Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab–IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and coregistration of tumor tissue and healthy tissue.Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared with those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level.Conclusions: Our study shows that systemic administration of the bevacizumab–IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor margin uptake as evaluated by a systematic validation methodology. The findings are a step toward a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in the tumor tissue. Clin Cancer Res; 23(11); 2730–41. ©2016 AACR.

Supplemental Materials and Methods; Suppl. Fig. 1. Comparison of tumor vs. skin fluorescence obtained from human breast tissue specimen; Suppl. Fig 2: Magnified comparison between fluorescence images obtained from 3mm thick paraffin slices and 4microm paraffin slices.

Purpose: The mTOR pathway is frequently activated in ovarian cancers. mTOR inhibitors, such as everolimus, can reduce VEGF-A production by cancer cells. We investigated whether early everolimus treatment effects could be monitored by positron emission tomography (PET) with 89Zr-bevacizumab.Experimental Design: The effect of everolimus on VEGF-A secretion was determined in a panel of human ovarian cancer cell lines and in A2780luc+ ovarian cancer cells xenografted subcutaneously in BALB/c mice. Mice received daily 10 mg/kg everolimus intraperitoneally (i.p.) for 14 days. PET scans with the tracer 89Zr-labeled bevacizumab were conducted before and after treatment. Ex vivo89Zr-bevacizumab biodistribution and correlative tissue analyses were conducted. Tumor VEGF-A levels were measured with ELISA and mean vascular density (MVD) was determined with immunohistochemistry.Results: Everolimus treatment reduced VEGF-A levels in the supernatant of all cell lines. Everolimus lowered 89Zr-bevacizumab tumor uptake by 21.7% ± 4.0% [mean standardized uptake value (SUVmean) 2.3 ± 0.2 vs. 2.9 ± 0.2, P < 0.01]. Ex vivo biodistribution also showed lower tracer uptake in the tumors of treated as compared with control animals (7.8 ± 0.8%ID/g vs. 14.0 ± 1.7%ID/g, P < 0.01), whereas no differences were observed for other tissues. This coincided with lower VEGF-A protein levels in tumor lysates in treated versus untreated tumors (P = 0.04) and reduced MVD (P < 0.01).Conclusion: Tumor VEGF-A levels are decreased by everolimus. 89Zr-bevacizumab PET could be used to monitor tumor VEGF-A levels as an early biomarker of the antiangiogenic effect of mTOR inhibitor therapy. Clin Cancer Res; 18(22); 6306–14. ©2012 AACR.

Near-Infrared Optical Imaging of Positive Tumor Margin.

PDF file - 20K, Schematic representation of the study design

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and nonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623–31. ©2016 AACR.

Co-localization of Bevacizumab-IRDye800CW, hematoxylin / eosin (H/E), VEGF-A, Collagen and CD34 in Ductal Carcinoma In Situ (DCIS).

Co-localization of Bevacizumab-IRDye800CW, hematoxylin/eosin (H/E), VEGF-A, Collagen and CD34 in Breast Cancer and a Satellite Lesion.

Supplementary Figures S1-S4 and Supplementary Table S1 including Supplementary Figure legend and Supplementary Table caption Figure S1. Flow chart of the drug substance (N-sucDf-AMG211) manufacturing process and drug product (89Zr-AMG211) formulation and filling process. Figure S2. Quality control of 89Zr AMG211. Representative size exclusion high performance liquid chromatography chromatogram of 89Zr AMG211 with 280 nm signal (top panel), radiochemical signal (middle panel) and the overlay (lower panel). Figure S3. Immunoreactivity of 89Zr AMG211. A) Representative competition assay using an effective N sucDf:AMG 211 ratio of 2:1. Curve fit with 95% confidence interval is visualized. B) Immunoreactivity towards CEA of different ratios N sucDf:AMG 211. Data are mean {plus minus} SD. Figure S4. Membrane binding and internalization of 89Zr AMG211 after CEA binding on LS174T cells (n = 3). Membrane bound and internalized 89Zr-AMG211 are expressed as percentage of initial cell associated activity. Data are mean {plus minus} SD. Figure S5. Expression of CEA on LS174T, BT474 and HL 60 cell lines (n = 3). Membrane expression is expressed as percentage of LS174T signal. Data are mean {plus minus} SD Table S1. GMP manufacturing of N sucDf AMG211 and 89Zr AMG211. Release criteria are fulfilled for batch 1, 2, and 3. In addition stability are shown for N sucDf AMG211 stored at 80{degree sign}C for 6 months, all quality criteria are still met.

Supplementary Material and Methods / Legends of Supplemental Figures

Supplementary Data

Multiplex pathology imaging of white-light microscopy, bevacizumab-IRDye800CW fluorescence imaging (green pseudocolor), VEGF-A (red pseudocolor), collagen (red pseudocolor) and CD34 (blue pseudocolor) in breast cancer, including overlay images is shown in a stepwise manner for reasons of clarity.

Intraoperative and Ex Vivo Optical Imaging of Axillary Lymph Nodes.

Purpose: We evaluated biodistribution and tumor targeting of 89Zr-lumretuzumab before and during treatment with lumretuzumab, a human epidermal growth factor receptor 3 (HER3)–targeting monoclonal antibody.Experimental Design: Twenty patients with histologically confirmed HER3-expressing tumors received 89Zr-lumretuzumab and underwent positron emission tomography (PET). In part A, 89Zr-lumretuzumab was given with additional, escalating doses of unlabeled lumretuzumab, and scans were performed 2, 4, and 7 days after injection to determine optimal imaging conditions. In part B, patients were scanned following tracer injection before (baseline) and after a pharmacodynamic (PD)-active lumretuzumab dose for saturation analysis. HER3 expression was determined immunohistochemically in skin biopsies. Tracer uptake was calculated as standardized uptake value (SUV).Results: Optimal PET conditions were found to be 4 and 7 days after administration of 89Zr-lumretuzumab with 100-mg unlabeled lumretuzumab. At baseline using 100-mg unlabeled lumretuzumab, the tumor SUVmax was 3.4 (±1.9) at 4 days after injection. SUVmean values for normal blood, liver, lung, and brain tissues were 4.9, 6.4, 0.9 and 0.2, respectively. Saturation analysis (n = 7) showed that 4 days after lumretuzumab administration, tumor uptake decreased by 11.9% (±8.2), 10.0% (±16.5), and 24.6% (±20.9) at PD-active doses of 400, 800, and 1,600 mg, respectively, when compared with baseline. Membranous HER3 was completely downregulated in paired skin biopsies already at and above 400-mg lumretuzumab.Conclusions: PET imaging showed biodistribution and tumor-specific 89Zr-lumretuzumab uptake. Although, PD-active lumretuzumab doses decreased 89Zr-lumretuzumab uptake, there was no clear evidence of tumor saturation by PET imaging as the tumor SUV did not plateau with increasing doses. Clin Cancer Res; 23(20); 6128–37. ©2017 AACR.

Microscopic Biodistribution of Bevacizumab-IRDye800CW

Co-localization of Hematoxylin / Eosin (H/E), VEGF-A, and Bevacizumab-IRDye800CW in Tumor-negative Lymph Nodes.

Supplemental Figures and Tables

Purpose:Incomplete oncologic resections and damage to vital structures during colorectal cancer surgery increases morbidity and mortality. Moreover, neoadjuvant chemoradiotherapy has become the standard treatment modality for locally advanced rectal cancer, where subsequent downstaging can make identification of the primary tumor more challenging during surgery. Near-infrared (NIR) fluorescence imaging can aid surgeons by providing real-time visualization of tumors and vital structures during surgery.Experimental Design:We present the first-in-human clinical experience of a novel NIR fluorescent peptide, cRGD-ZW800-1, for the detection of colon cancer. cRGD-ZW800-1 was engineered to have an overall zwitterionic chemical structure and neutral charge to lower nonspecific uptake and thus background fluorescent signal. We performed a phase I study in 11 healthy volunteer as well as a phase II feasibility study in 12 patients undergoing an elective colon resection, assessing 0.005, 0.015, and 0.05 mg/kg cRGD-ZW800-1 for the intraoperative visualization of colon cancer.Results:cRGD-ZW800-1 appears safe, and exhibited rapid elimination into urine after a single low intravenous dose. Minimal invasive intraoperative visualization of colon cancer through full-thickness bowel wall was possible after an intravenous bolus injection of 0.05 mg/kg at least 2 hours prior to surgery. Longer intervals between injection and imaging improved the tumor-to-background ratio.Conclusions:cRGD-ZW800-1 enabled fluorescence imaging of colon cancer in both open and minimal invasive surgeries. Further development of cRGD-ZW800-1 for widespread use in cancer surgery may be warranted given the ubiquitous overexpression of various integrins on different types of tumors and their vasculature.

Representative Fluorescence Flatbed Scans of Paraffin Blocks of all Patients (N=20) and Macroscopic Segmentation of Fluorescence Tissue Localization and Target-to-Background Ratios (TBR).

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of 89Zr-trastuzumab (SKOV-3 and OE19), 89Zr-bevacizumab (SKOV-3), or 89Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment. Cancer Res; 73(11); 3347–55. ©2013 AACR.

PDF file - 53K, Biodistribution of 89Zr-Trastuzumab in the OE19 model and correlation of the in vivo PET tumor uptake with the ex vivo biodistriution tumor uptake of 89Zr-trastuzumab in the OE19 model.

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Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have improved the survival of patients with non-small cell lung cancer (NSCLC). Still, many patients do not respond to these inhibitors. PD-L1 (CD274) expression, one of the factors that influences the efficacy of immune checkpoint inhibitors, is dynamic. Here, we studied the regulation of PD-L1 expression in NSCLC without targetable genetic alterations in EGFR, ALK, BRAF, ROS1, MET, ERBB2 and RET. Analysis of RNA sequencing data from these NSCLCs revealed that inferred IFN gamma, EGFR and MAPK signaling correlated with CD274 gene expression in lung adenocarcinoma. In a representative lung adenocarcinoma cell line panel, stimulation with EGF or IFN gamma increased CD274 mRNA and PD-L1 protein and membrane levels, which were further enhanced by combining EGF and IFN gamma. Similarly, tumor cell PD-L1 membrane levels increased after coculture with activated peripheral blood mononuclear cells. Inhibition of the MAPK pathway, using EGFR inhibitors cetuximab and erlotinib or the MEK 1 and 2 inhibitor selumetinib, prevented EGF- and IFN gamma-induced CD274 mRNA and PD-L1 protein and membrane upregulation, but had no effect on IFN gamma-induced MHC-I upregulation. Interestingly, although IFN gamma increases transcriptional activity of CD274, MAPK signaling also increased stabilization of CD274 mRNA. In conclusion, MAPK pathway activity plays a key role in EGF- and IFN gamma-induced PD-L1 expression in lung adenocarcinoma without targetable genetic alterations and may present a target to improve the efficacy of immunotherapy. (c) 2019 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

// Arjan Kol 1 , Anton Terwisscha van Scheltinga 2 , Martin Pool 1 , Christian Gerdes 3 , Elisabeth de Vries 1 , Steven de Jong 1 1 Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Roche Pharma Research & Early Development, Roche Innovation Center Zurich, Schlieren, Switzerland Correspondence to: Steven de Jong, email: s.de.jong@umcg.nl Keywords: non-small cell lung cancer, EGFR, imgatuzumab, cetuximab, antibodies Received: October 19, 2016 Accepted: March 30, 2017 Published: April 17, 2017 ABSTRACT Imgatuzumab is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and EGFR signal transduction inhibition. We investigated anti-EGFR monoclonal antibodies imgatuzumab and cetuximab–induced internalization and membranous turnover of EGFR, and whether this affected imgatuzumab–mediated ADCC responses and growth inhibition of non-small cell lung cancer (NSCLC) cells. In a panel of wild-type EGFR expressing human NSCLC cell lines, membranous and total EGFR levels were downregulated more effectively by imgatuzumab when compared with cetuximab. Imgatuzumab plus cetuximab enhanced EGFR internalization and reduced membranous turnover of EGFR, resulting in an even stronger downregulation of EGFR. Immunofluorescent analysis showed that combined treatment increased clustering of receptor-antibody complexes and directed internalized EGFR to lysosomes. The antibody combination potently inhibited intracellular signaling and epidermal growth factor (EGF)-dependent cell proliferation. More importantly, robust EGFR downregulation after 72 hours with the antibody combination did not impair ADCC responses. In conclusion, imgatuzumab plus cetuximab leads to a strong downregulation of EGFR and superior cell growth inhibition in vitro without affecting antibody-induced ADCC responses. These findings support further clinical exploration of the antibody combination in EGFR wild-type NSCLC.

There is a dire need for better visualization of cancer and analysis of specific targets in vivo. Molecular imaging with fluorescence is gaining more and more attention, as it allows detection of these targets and has advantages over radioactivity, such as no radiation dose, and lower costs. A key challenge in optical imaging however, is translation of the newly developed tracers from pre-clinical phase to clinical application. We describe the development and safety testing of clinical grade bevacizumab-800CW, an antibody-based targeted agent for non-invasive imaging of vascular endothelial growth factor A (VEGF-A).Development included implementing the manufacturing process and analytical methods according to current Good Manufacturing Practice (cGMP), formulation studies, extended characterization and stability testing. For safety pharmacology an extended single dose toxicity study in mice was performed.Bevacizumab-800CW was formulated in isotonic phosphate buffered sodium chloride solution at pH 7. The production was robust and showed a reproducible labeling efficiency, and no impurities. The binding affinity to VEGF-A remained intact. The optimized product meets all release specifications, is stable up to at least 3 months and its characteristics did not significantly differ from the unlabeled bevacizumab. Toxicity testing in mice showed no remarkable findings.In conclusion, sterile bevacizumab-800CW (6 mg = 6 ml) can be produced in stock according to current Good Manufacturing Practice. It is ready for first-in-human use. (C) 2016 Elsevier B.V. All rights reserved.

// Simon-Peter Williams 1 , Annie Ogasawara 1 , Jeff N. Tinianow 1 , Judith E. Flores 1 , David Kan 1 , Jeffrey Lau 1 , MaryAnn Go 1 , Alexander N. Vanderbilt 1 , Herman S. Gill 1 , Li Miao 1 , Joshua Goldsmith 1 , Bonnee Rubinfeld 1 , Weiguang Mao 1 , Ron Firestein 1 , Shang-Fan Yu 1 , Jan Marik 1 , Anton G.T. Terwisscha van Scheltinga 1, 2 1 Genentech Research and Early Development, Genentech, Inc., South San Francisco, CA, 94080, USA 2 Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, University of Groningen, Groningen, 9700RB, The Netherlands Correspondence to : Simon-Peter Williams, e-mail: williams.simon@gene.com Keywords: antibody-drug conjugates, immunoPET, TENB2, STEAP1, zirconium-89 Received: December 10, 2015 Accepted: March 04, 2016 Published: March 26, 2016 ABSTRACT The efficacy of antibody-drug conjugates (ADCs) targeted to solid tumors depends on biological processes that are hard to monitor in vivo . 89 Zr-immunoPET of the ADC antibodies could help understand the performance of ADCs in the clinic by confirming the necessary penetration, binding, and internalization. This work studied monomethyl auristatin E (MMAE) ADCs against two targets in metastatic castration-resistant prostate cancer, TENB2 and STEAP1, in four patient-derived tumor models (LuCaP35V, LuCaP70, LuCaP77, LuCaP96.1). Three aspects of ADC biology were measured and compared: efficacy was measured in tumor growth inhibition studies; target expression was measured by immunohistochemistry and flow cytometry; and tumor antibody uptake was measured with 111 In-mAbs and gamma counting or with 89 Zr-immunoPET. Within each model, the mAb with the highest tumor uptake showed the greatest potency as an ADC. Sensitivity between models varied, with the LuCaP77 model showing weak efficacy despite high target expression and high antibody uptake. Ex vivo analysis confirmed the in vivo results, showing a correlation between expression, uptake and ADC efficacy. We conclude that 89 Zr-immunoPET data can demonstrate which ADC candidates achieve the penetration, binding, and internalization necessary for efficacy in tumors sensitive to the toxic payload.

AMG 110, a bispecific T cell engager (BITE) antibody construct, induces T cell-mediated cancer cell death by cross-linking epithelial cell adhesion molecule (EpCAM) on tumor cells with a cluster of differentiation 3 epsilon (CD3 epsilon) on T cells. We labeled AMG 110 with Zr-89 or near infrared fluorescent dye (IRDye) 800CW to study its tumor targeting and tissue distribution. Methods: Biodistribution and tumor uptake of Zr-89-AMG 110 was studied up to 6 d after intravenous administration to nude BALB/c mice bearing high EpCAM-expressing HT-29 colorectal cancer xenografts. Tumor uptake of Zr-89-AMG 110 was compared with uptake in head and neck squamous cell cancer FaDu (intermediate EpCAM) and promyelocytic leukemia HL60 (EpCAM-negative) xenografts. Intratumoral distribution in HT-29 tumors was studied using 800CW-AMG 110. Results: Tumor uptake of Zr-89-AMG 110 can be clearly visualized using small-animal PET imaging up to 72 h after injection. The highest tumor uptake of Zr-89-AMG 110 at the 40-mu g dose level was observed at 6 and 24 h (respectively, 5.35 +/- 0.22 and 5.30 +/- 0.20 percentage injected dose per gram; n = 3 and 4). Tumor uptake of Zr-89-AMG 110 was EpCAM-specific and correlated with EpCAM expression. 800CW-AMG 110 accumulated at the tumor cell surface in viable EpCAM-expressing tumor tissue. Conclusion: PET and fluorescent imaging provided real-time information about AMG 110 distribution and tumor uptake in vivo. Our data support using Zr-89 and IRDye 800CW to evaluate tumor and tissue uptake kinetics of bispecific T cell engager antibody constructs in preclinical and clinical settings.

Purpose: AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 (89Zr) or fluorescent dye to evaluate the tumor-targeting properties. Experimental Design: 89Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μg 89Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, 89Zr-Mec14, served as a negative control. 89Zr-AMG211 integrity was determined in tumor lysates ex vivo. Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, 89Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199. Results: 89Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μg 89Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of 89Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. 89Zr-Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured 89Zr-AMG211 fulfilled release specifications. Conclusions: 89Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior. Clin Cancer Res; 24(20); 4988–96. ©2018 AACR.

Contains fulltext : 170986pub.pdf (Publisher’s version ) (Closed access) Small and flat adenomas are known to carry a high miss-rate during standard white-light endoscopy. Increased detection rate may be achieved by molecular fluorescence endoscopy with targeted near-infrared (NIR) fluorescent tracers. The aim of this study was to validate vascular endothelial growth factor A (VEGF-A) and epidermal growth factor receptor (EGFR)-targeted fluorescent tracers during ex vivo colonoscopy with an NIR endoscopy platform. METHODS: VEGF-A and EGFR expression was determined by immunohistochemistry on a large subset of human colorectal tissue samples-48 sessile serrated adenomas/polyps, 70 sporadic high-grade dysplastic adenomas, and 19 hyperplastic polyps-and tissue derived from patients with Lynch syndrome-78 low-grade dysplastic adenomas, 57 high-grade dysplastic adenomas, and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116(luc) tumors received intravenous bevacizumab-800CW (anti-VEGF-A), cetuximab-800CW (anti-EGFR), control tracer IgG-800CW, or sodium chloride. Three days later, 8 resected HCT116(luc) tumors (2-5 mm) were stitched into 1 freshly resected human colon specimen and followed by an ex vivo molecular fluorescence colonoscopy procedure. RESULTS: Immunohistochemistry showed high VEGF-A expression in 79%-96% and high EGFR expression in 51%-69% of the colorectal lesions. Both targets were significantly overexpressed in the colorectal lesions, compared with the adjacent normal colon crypts. During ex vivo molecular fluorescence endoscopy, all tumors could clearly be delineated for both bevacizumab-800CW and cetuximab-800CW tracers. Specific tumor uptake was confirmed with fluorescent microscopy showing, respectively, stromal and cell membrane fluorescence. CONCLUSION: VEGF-A is a promising target for molecular fluorescence endoscopy because it showed a high protein expression, especially in sessile serrated adenomas/polyps and Lynch syndrome. We demonstrated the feasibility to visualize small tumors in real time during colonoscopy using a NIR fluorescence endoscopy platform, providing the endoscopist a wide-field red-flag technique for adenoma detection. Clinical studies are currently being performed in order to provide in-human evaluation of our approach.

Mesothelin is a tumor differentiation antigen expressed by epithelial tumors, including pancreatic cancer. Currently, mesothelin is being targeted with an antibodydrug conjugate (ADC) consisting of a mesothelin-specific antibody coupled to a highly potent chemotherapeutic drug. Considering the toxicity of the ADC and reduced accessibility of pancreatic tumors, non-invasive imaging could provide necessary information. We therefore developed a zirconium-89 (Zr-89) labeled anti-mesothelin antibody (Zr-89-AMA) to study its biodistribution in human pancreatic tumor bearing mice. Biodistribution and dose-finding of Zr-89-AMA were studied 144 h after tracer injection in mice with subcutaneously xenografted HPAC. MicroPET imaging was performed 24, 72 and 144 h after tracer injection in mice bearing HPAC or Capan-2. Tumor uptake and organ distribution of Zr-89-AMA were compared with nonspecific 111In-IgG. Biodistribution analyses revealed a dose-dependent Zr-89-AMA tumor uptake. Tumor uptake of Zr-89-AMA was higher than 111In-IgG using the lowest tracer dose. MicroPET showed increased tumor uptake over 6 days, whereas activity in blood pool and other tissues decreased. Immunohistochemistry showed that mesothelin was expressed by the HPAC and CAPAN-2 tumors and fluorescence microscopy revealed that AMA-800CW was present in tumor cell cytoplasm. Zr-89-AMA tumor uptake is antigen-specific in mesothelin-expressing tumors. Zr-89-AMA PET provides non-invasive, real-time information about AMA distribution and tumor targeting.

More than 50 monoclonal antibodies (mAbs), including several antibody-drug conjugates, are in advanced clinical development, forming an important part of the many molecularly targeted anticancer therapeutics currently in development. Drug development is a relatively slow and expensive process, limiting the number of drugs that can be brought into late-stage trials. Development decisions could benefit from quantitative biomarkers, enabling visualization of the tissue distribution of (potentially modified) therapeutic mAbs to confirm effective whole-body target expression, engagement, and modulation and to evaluate heterogeneity across lesions and patients. Such biomarkers may be realized with positron emission tomography imaging of radioactively labeled antibodies, a process called immunoPET. This approach could potentially increase the power and value of early trials by improving patient selection, optimizing dose and schedule, and rationalizing observed drug responses. In this review, we summarize the available literature and the status of clinical trials regarding the potential of immunoPET during early anticancer drug development. (C) 2015 by American Society of Clinical Oncology

Purpose c-MET and its ligand hepatocyte growth factor are often dysregulated in human cancers. Dynamic changes in c-MET expression occur and might predict drug efficacy or emergence of resistance. Noninvasive visualization of c-MET dynamics could therefore potentially guide c-MET-directed therapies. We investigated the feasibility of 89Zr-labelled one-armed c-MET antibody onartuzumab PET for detecting relevant changes in c-MET levels induced by c-MET-mediated epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib resistance or heat shock protein-90 (HSP90) inhibitor NVP-AUY-922 treatment in human non-small-cell lung cancer (NSCLC) xenografts. Methods In vitro membrane c-MET levels were determined by flow cytometry. HCC827ErlRes, an erlotinib-resistant clone with c-MET upregulation, was generated from the exon-19 EGFR-mutant human NSCLC cell line HCC827. Mice bearing HCC827 and HCC827ErlRes tumours in opposite flanks underwent 89Zr-onartuzumab PET scans. The HCC827-xenografted mice underwent 89Zr-onartuzumab PET scans before treatment and while receiving biweekly intraperitoneal injections of 100 mg/kg NVP-AUY-922 or vehicle. Ex vivo, tumour c-MET immunohistochemistry was correlated with the imaging results. Results In vitro, membrane c-MET was upregulated in HCC827ErlRes tumours by 213 ± 44% in relation to the level in HCC827 tumours, while c-MET was downregulated by 69 ± 9% in HCC827 tumours following treatment with NVP-AUY-922. In vivo, 89Zr-onartuzumab uptake was 26% higher (P

The human epidermal growth factor receptor 3 (HER3) is an interesting target for antitumor therapy. For optimal HER3 signaling inhibition, a biparatopic Nanobody construct (MSB0010853) was developed that binds 2 different HER3 epitopes. In addition, MSB0010853 contains a third HER3 epitope that binds albumin to extend its circulation time. MSB0010853 is cross-reactive with HER3 and albumin of mouse origin. We aimed to gain insight into MSB0010853 biodistribution and tumor uptake by radiolabeling the Nanobody construct with Zr-89. Methods: MSB0010853 was radiolabeled with Zr-89. Dose-and time-dependent tumor uptake was studied in nude BALB/c mice bearing a subcutaneous HER3 overexpressing H441 non-small cell lung cancer xenograft. Dose-dependent biodistribution of Zr-89-MSB0010853 was assessed ex vivo at 24 h after intravenous injection. Protein doses of 5, 10, 25, 100, and 1,000 mgwere used. Time-dependent biodistribution of MSB0010853 was analyzed ex vivo at 3, 6, 24, and 96 h after intravenous administration of 25 mg of Zr-89-MSB0010853. PET imaging and biodistribution were performed 24 h after administration of 25 mu g of Zr-89-MSB0010853 to mice bearing human H441, FaDu (high HER3 expression), or Calu-1 (no HER3 expression) tumor xenografts. Results: Radiolabeling of MSB0010853 with Zr-89 was performed with a radiochemical purity of greater than 95%. Ex vivo biodistribution showed protein dose-and time-dependent distribution of Zr-89-MSB0010853 in all organs. Uptake of Zr-89-MSB0010853 in H441 tumors was only time-dependent. Tumor could be visualized up to at least 96 h after injection. The highest mean SUV of 0.6 +/- 0.2 was observed at 24 h after injection of 25 mu g of Zr-89-MSB0010853. Zr-89-MSB0010853 tumor uptake correlated with HER3 expression and was highest in H441 (6.2 +/- 1.1 percentage injected dose per gram [% ID/g]) and lowest in Calu-1 (2.3 +/- 0.3 % ID/g) xenografts. Conclusion: Zr-89-MSB0010853 organ distribution and tumor uptake in mice are time-dependent, and tumor uptake correlates with HER3 expression. In contrast to tumor uptake except for kidney uptake, organ distribution of Zr-89-MSB0010853 is protein dose-dependent for the tested doses. Zr-89-MSB0010853 PET imaging gives insight into the in vivo behavior of MSB0010853.

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

The human epidermal growth factor receptor (HER) family members are targeted by a growing numbers of small molecules and monoclonal antibodies. Resistance against the epidermal growth factor receptor (EGFR) and HER2-targeting agents is a clinically relevant problem forcing research on optimizing targeting of the HER family. In view of its overexpression in tumors, and compensatory role in HER signaling, HER3 has gained much interest as a potential additional target within the HER family. It is the only member of the HER family lacking intrinsic tyrosine kinase activity and therefore its role in cancer has long been underestimated. Drugs that block HER3 or interfere with HER3 dimer signaling, including fully human anti-HER3 antibodies, bispecific antibodies and tyrosine kinase inhibitors (TKIs), are currently becoming available. Several compounds have already entered clinical trial. In the meantime potential biomarkers are tested such as tumor analysis of HER3 expression, functional assays for downstream effector molecules and molecular imaging techniques. This review describes the biology and relevance of HER3 in cancer, agents targeting HER3 and potential biomarkers for effect of HER3-targeting.

Anticalins are a novel class of biopharmaceuticals, displaying highly desirable attributes as imaging agents. The anticalin PRS-110 was rationally engineered to target the oncogene MET with high affinity and specificity. The aim of this study was to visualize MET expression and analyze biodistribution of Zr-89-labeled PRS-110 in human tumor-bearing mice. Methods: Zr-89-PRS-110 was generated. For biodistribution studies (96 h after injection of tracer) 10 mu g of Zr-89-PRS-110 (with 0-490 mu g of unlabeled PRS-110) were injected into BALB/c mice bearing high MET-expressing H441 non-small cell lung cancer xenografts. Further characterization with PET imaging was performed at 6, 24, 48, and 96 h after injection of 50 mu g of Zr-89-PRS-110 into mice bearing H441, primary glioblastoma U87-MG (intermediate MET), or ovarian cancer A2780 (low MET) xenografts. Drug distribution was also analyzed ex vivo using fluorescently labeled PRS-110. Results: Biodistribution analyses showed a dose-dependent tumor uptake of Zr-89-PRS-110, with the highest fractional tumor uptake at 10 mu g of Zr-89-PRS-110, with no unlabeled PRS-110. Small-animal PET imaging supported by biodistribution data revealed specific tumor uptake of Zr-89-PRS-110 in the METexpressing H441 and U87-MG tumors whereas the MET-negative A2780 tumor model showed a lower uptake similar to a non-MET binder anticalin control. Tumor uptake increased up to 24 h after tracer injection and remained high, whereas uptake in other organs decreased over time. Ex vivo fluorescence revealed intracellular presence of PRS-110. Conclusion: Zr-89-PRS-110 specifically accumulates in MET-expressing tumors in a receptor density-dependent manner. PET imaging provides real-time noninvasive information about PRS-110 distribution and tumor accumulation in preclinical models.

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (Zr-89) we aimed to visualize in vivo HER 3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of 89Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER 3-positive). Dose-dependency of Zr-89-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg Zr-89-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER 3 expression. The excised tumors were analyzed for HER 3 expression. Biodistribution analyses showed a dose-and time-dependent Zr-89-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of Zr-89-RG7116 was observed in the 0.05 mg/kg dose group with 27.5% ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of Zr-89-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER 3 expression levels. In conclusion, Zr-89-RG7116 specifically accumulates in HER 3 expressing tumors. PET imaging with this tracer provides realtime non-invasive information about RG7116 distribution, tumor targeting and tumor HER 3 expression levels.

White-light surveillance colonoscopy is the standard of care for the detection and removal of premalignant lesions to prevent colorectal cancer, and the main screening recommendation following treatment for recurrence detection. However, it lacks sufficient diagnostic yield, exhibits unacceptable adenoma miss-rates and is not capable of revealing functional and morphological information of the detected lesions. Fluorescence molecular guidance in the near-infrared (NIR) is expected to have outstanding relevance regarding early lesion detection and heterogeneity characterization within and among lesions in these interventional procedures. Thereby, superficial and sub-surface tissue biomarkers can be optimally visualized due to a minimization of tissue attenuation and autofluorescence by comparison with the visible, which simultaneously enhance tissue penetration and assure minimal background. At present, this potential is challenged by the difficulty associated with the clinical propagation of disease-specific contrast agents and the absence of a commercially available endoscope that is capable of acquiring wide-field, NIR fluorescence at video-rates. We propose two alternative flexible endoscopic fluorescence imaging methods, each based on a CE certified commercial, clinical grade endoscope, and the employment of an approved monoclonal antibody labeled with a clinically applicable NIR fluorophore. Pre-clinical validation of these two strategies that aim at bridging NIR fluorescence molecular guidance to clinical translation is demonstrated in this study. (C) 2013 Optical Society of America

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of 89Zr-trastuzumab (SKOV-3 and OE19), 89Zr-bevacizumab (SKOV-3), or 89Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment. Cancer Res; 73(11); 3347–55. ©2013 AACR.

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 ((89)Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on(89)Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to (89)Zr-mAb3481 administration. Subsequently, ex vivo (89)Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). (89)Zr-mAb3481 BT474 tumor uptake was remarkably high at SUVmean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg (89)Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect (89)Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. (89)Zr-mAb3481 PET accurately reflected HER3 tumor status. (89)Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.

// Martin Pool 1 , Arjan Kol 1 , Marjolijn N. Lub-de Hooge 2, 3 , Christian A. Gerdes 4 , Steven de Jong 1 , Elisabeth G.E. de Vries 1 , Anton G.T. Terwisscha van Scheltinga 2 1 Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 4 Department of Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Schlieren, Switzerland Correspondence to: Anton G.T. Terwisscha van Scheltinga, email: a.g.t.terwisscha.van.scheltinga@umcg.nl Keywords: imgatuzumab, EGFR, 89 Zr, immunoPET, shedding Received: January 06, 2016 Accepted: August 27, 2016 Published: September 02, 2016 ABSTRACT Preclinical positron emission tomography (PET) imaging revealed a mismatch between in vivo epidermal growth factor receptor (EGFR) expression and EGFR antibody tracer tumor uptake. Shed EGFR ectodomain (sEGFR), which is present in cancer patient sera, can potentially bind tracer and therefore influence tracer kinetics. To optimize EGFR-PET, we examined the influence of sEGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody 89 Zr-imgatuzumab in varying xenograft models. Human cancer cell lines A431 (EGFR overexpressing, epidermoid), A549 and H441 (both EGFR medium expressing, non-small cell lung cancer) were xenografted in mice. Xenografted mice received 10, 25 or 160 μg 89 Zr-imgatuzumab, co-injected with equal doses 111 In-IgG control. MicroPET scans were made 24, 72 and 144 h post injection, followed by biodistribution analysis. sEGFR levels in liver and plasma samples were determined by ELISA. 89 Zr-imgatuzumab uptake in A431 tumors was highest (29.8 ± 5.4 %ID/g) in the 160 μg dose group. Contrary, highest uptake in A549 and H441 tumors was found at the lowest (10 μg) 89 Zr-imgatuzumab dose. High 89 Zr-imgatuzumab liver accumulation was found in A431 xenografted mice, which decreased with antibody dose increments. 89 Zr-imgatuzumab liver uptake in A549 and H441 xenografted mice was low at all doses. sEGFR levels in liver and plasma of A431 bearing mice were up to 1000-fold higher than levels found in A549, H441 and non-tumor xenografted mice. 89 Zr-imgatuzumab effectively visualizes EGFR-expressing tumors. High sEGFR levels can redirect 89 Zr-imgatuzumab to the liver, in which case tumor visualization can be improved by increasing tracer antibody dose.

Antibody–drug conjugates (ADC) use monoclonal antibodies (mAb) as vehicles to deliver potent cytotoxic drugs selectively to tumor cells expressing the target. Molecular imaging with zirconium-89 (89Zr)-labeled mAbs recapitulates similar targeting biology and might help predict the efficacy of these ADCs. An anti-mesothelin antibody (AMA, MMOT0530A) was used to make comparisons between its efficacy as an ADC and its tumor uptake as measured by 89Zr immunoPET imaging. Mesothelin-targeted tumor growth inhibition by monomethyl auristatin E (MMAE), ADC AMA-MMAE (DMOT4039A), was measured in mice bearing xenografts of ovarian cancer OVCAR-3×2.1, pancreatic cancers Capan-2, HPAC, AsPC-1, and HPAF-II, or mesothelioma MSTO-211H. Ex vivo analysis of mesothelin expression was performed using immunohistochemistry. AMA-MMAE showed the greatest growth inhibition in OVCAR-3×2.1, Capan-2, and HPAC tumors, which showed target-specific tumor uptake of 89Zr-AMA. The less responsive xenografts (AsPC-1, HPAF-II, and MSTO-211H) did not show 89Zr-AMA uptake despite confirmed mesothelin expression. ImmunoPET can demonstrate the necessary delivery, binding, and internalization of an ADC antibody in vivo and this correlates with the efficacy of mesothelin-targeted ADC in tumors vulnerable to the cytotoxic drug delivered. Mol Cancer Ther; 16(1); 134–42. ©2016 AACR.

Purpose: The mTOR pathway is frequently activated in ovarian cancers. mTOR inhibitors, such as everolimus, can reduce VEGF-A production by cancer cells. We investigated whether early everolimus treatment effects could be monitored by positron emission tomography (PET) with 89Zr-bevacizumab. Experimental Design: The effect of everolimus on VEGF-A secretion was determined in a panel of human ovarian cancer cell lines and in A2780luc+ ovarian cancer cells xenografted subcutaneously in BALB/c mice. Mice received daily 10 mg/kg everolimus intraperitoneally (i.p.) for 14 days. PET scans with the tracer 89Zr-labeled bevacizumab were conducted before and after treatment. Ex vivo89Zr-bevacizumab biodistribution and correlative tissue analyses were conducted. Tumor VEGF-A levels were measured with ELISA and mean vascular density (MVD) was determined with immunohistochemistry. Results: Everolimus treatment reduced VEGF-A levels in the supernatant of all cell lines. Everolimus lowered 89Zr-bevacizumab tumor uptake by 21.7% ± 4.0% [mean standardized uptake value (SUVmean) 2.3 ± 0.2 vs. 2.9 ± 0.2, P < 0.01]. Ex vivo biodistribution also showed lower tracer uptake in the tumors of treated as compared with control animals (7.8 ± 0.8%ID/g vs. 14.0 ± 1.7%ID/g, P < 0.01), whereas no differences were observed for other tissues. This coincided with lower VEGF-A protein levels in tumor lysates in treated versus untreated tumors (P = 0.04) and reduced MVD (P < 0.01). Conclusion: Tumor VEGF-A levels are decreased by everolimus. 89Zr-bevacizumab PET could be used to monitor tumor VEGF-A levels as an early biomarker of the antiangiogenic effect of mTOR inhibitor therapy. Clin Cancer Res; 18(22); 6306–14. ©2012 AACR.

Fluorescence imaging is currently attracting much interest as a method for intraoperative tumor detection, but most current tracers lack tumor specificity. Therefore, this technique can be further improved by tumor-specific detection. With tumor-targeted antibodies bound to a radioactive label, tumor-specific SPECT or PET is feasible in the clinical setting. The aim of the present study was to apply antibody-based tumor detection to intraoperative optical imaging, using preclinical in vivo mouse models. METHODS: Anti-vascular endothelial growth factor (VEGF) antibody bevacizumab and anti-human epidermal growth factor receptor (HER) 2 antibody trastuzumab were labeled with the near-infrared (NIR) fluorescence dye IRDye 800CW. Tumor uptake of the fluorescent tracers and their (89)Zr-labeled radioactive counterparts for PET was determined in human xenograft-bearing athymic mice during 1 wk after tracer injection, followed by ex vivo biodistribution and pathologic examination. Intraoperative imaging of fluorescent VEGF- or HER2-positive tumor lesions was performed in subcutaneous tumors and in intraperitoneal dissemination tumor models. RESULTS: Tumor-to-background ratios, with fluorescent imaging, were 1.93 ± 0.40 for bevacizumab and 2.92 ± 0.29 for trastuzumab on day 6 after tracer injection. Real-time intraoperative imaging detected tumor lesions at even the submillimeter level in intraperitoneal dissemination tumor models. These results were supported by standard histology, immunohistochemistry, and fluorescence microscopy analyses. CONCLUSION: NIR fluorescence-labeled antibodies targeting VEGF or HER2 can be used for highly specific and sensitive detection of tumor lesions in vivo. These preclinical findings encourage future clinical studies with NIR fluorescence-labeled tumor-specific antibodies for intraoperative-guided surgery in cancer patients.

Purpose: Triple negative breast cancer (TNBC) is biologically characterised by heterogeneous presence of molecular pathways underlying it. Insulin-like growth factor receptor-1 (IGF-1R) expression and vascular endothelial growth factor-A (VEGF-A) have been identified as key factors in these pathways in TNBC. In this study, we aimed at in vivo PET imaging the effect of heat shock protein (Hsp) 90 inhibition by means of NVP-AUY922 on these pathways, with zirconium-89 (Zr-89) labelled antibodies targeting IGF-1R and VEGF-A.Materials and methods: In vitro NVP-AUY922 effects on cellular IGF-1R expression and VEGF-A secretion were determined in MCF-7 and MDA-MB-231 cell lines. Moreover human TNBC bearing MDA-MB-231 mice received 50 mg/kg NVP-AUY922 or vehicle q3d intraperitoneally for 21 days. PET scans with Zr-89-MAB391 and Zr-89-bevacizumab for visualisation of IGF-1R and VEGF-A were performed before and during treatment. Ex vivo biodistribution and correlative tissue analyses were performed.Results: NVP-AUY922 treatment reduced IGF-1R expression and VEGF-A excretion in both cell lines. Hsp90 inhibition lowered tumour uptake on Zr-89-MAB391-PET by 37.3% (P Conclusion: Zr-89-MAB391 and Zr-89-bevacizumab PET reflect effect of Hsp90 inhibitors and can therefore potentially be used to monitor therapeutic effects of Hsp90 inhibitor therapy in TNBC. (C) 2014 Elsevier Ltd. All rights reserved.

Background: Platinum-based chemotherapy in combination with surgery is the cornerstone of treatment for advanced high-grade serous ovarian cancer. Recurrence is common and additional treatments have been developed such as angiogenesis inhibition. Small subgroups of ovarian cancer patients may benefit from upfront selection based on tumor target expression. Here, we focus on near-infrared fluorescence (NIRF) dual imaging of the tumor secreted human vascular endothelial growth factor (VEGF-A) and the membrane-bound insulin-like growth factor 1 receptor (IGF-1R), simultaneously. The aim of our study is to determine tracer uptake and kinetics in relation to target expression in ovarian cancer patient-derived xenograft (PDX) models and the effect of cisplatin treatment on these parameters. Methods: Monoclonal antibody bevacizumab (anti-human VEGF-A) and MAB391 (anti-human IGF-1R) were coupled to NIRF dyes IRDye-800CW and IRDye-680RD, respectively. Specific tumor uptake was determined in a panel of subcutaneous ovarian cancer PDX models (established from 10 patients) in NOD SCID gamma mice during 1 week after intravenous co-injection of these tracers. In addition, two PDX models were treated weekly with cisplatin (4 mg/kg dose intraperitoneal administration) followed by NIRF dual imaging. Imaging results were compared with ELISA and immunostaining of VEGF-A and IGF1R. Results: We found large differences in average radiance for bevacizumab-800CW (range 2.53 - 23.3 x 106) and for MAB391-680RD (range 1.5 - 15.5 x 107) between PDX models at 24 hrs. In vivo kinetics of bevacizumab-800CW displayed a rapid tracer decline in PDX tumors 24 hrs after co-injection. MAB391-680RD, however, resided for over 6 days in PDX tumors depending on the IGF-1R positivity of tumors, indicating receptor-mediated tracer trapping. Moreover, IgG labeled with IRDye-800CW or IRDye-680RD showed similar kinetics as the bevacizumab-800CW. Elevated levels of both tracers were found in cisplatin-treated PDX tumors 24 hrs after co-injection. The higher levels of bevacizumab-800CW corresponded with higher intratumoral human VEGF-A levels in PDX tumors after cisplatin treatment. However, tracer decline kinetics in tumors did not change. Enhanced MAB391-680RD accumulation was related to higher IGF-1R tumor levels in the less responsive PDX model. A rapid decline of MAB391-680RD, similar to bevacizumab-800CW, was observed in the cisplatin-sensitive PDX model, which was related to reduced tumor proliferation and viability. Conclusions: These results indicate that levels and kinetics of targeted NIRF tracers can be used to dissect growth factor receptor-positive from -negative tumors. This encourages further exploration of multi-wavelength NIRF imaging to select targets for personalized medicine in small subgroups of ovarian cancer patients using PDX models. This work was supported by the Dutch Cancer Society (KWF) grants RUG 2010-4833 and RUG 2011-5231. Citation Format: Tushar Tomar, Nicolette G. Alkema, Jolanda A.L. Visser, Anton G. Terwisscha van Scheltinga, Gert Jan Meersma, Harry G. Klip, Evelien W. Duiker, Elisabeth G.E. De Vries, Ate G.J. Van der Zee, Steven De Jong. Dual wavelength near-infrared fluorescence imaging of VEGF-A and IGF-1R in ovarian cancer patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A37.

Introduction: Activation of T-cells is accompanied by a strong up-regulation of interleukin-2 (IL2) receptor (CD25). Therefore PET imaging of IL2 receptors might be a suitable imaging biomarker for T-cell activation. 18F-IL2 PET could detect CD25-positive T-cells and the migration of these T-cells to distant sites of inflammation in SCID mice subcutaneously injected with human peripheral blood mononuclear cells1 and NOD mice with insulitis. Also a strong correlation was found between the accumulation of 18F-IL2 and the number of injected activated T-cells in immune-competent rats.2 In tumor bearing mice, 18F-IL2 PET could detect treatment-induced accumulation of activated T-cells in the tumor following local radiotherapy and/or vaccination. Cancer immunotherapy is increasingly obtaining a place in clinical practice. However not all patients benefit. A biomarker for upfront or early response prediction for these immunotherapies might support patient selection before and during therapy. Potentially 18F-IL2 PET might serve this purpose. Therefore we aimed to accommodate the production of 18F-IL2 for use in clinical imaging studies. Material and methods: In order to produce a GMP-compliant tracer the production is being implemented on the Eckert & Ziegler PharmTracer synthesis module. In this synthesis module, disposable cassettes, reactors and vials are used to avoid cross-contamination between productions. First the prosthetic group N-succinimidyl 4-fluorobenzoate (18F-SFB) is produced in 3 steps from cyclotron-produced 18F-fluoride. Subsequently, 18F-SFB is conjugated to human recombinant IL2 (Proleukin®). Various methods for synthesis and purification of 18F-SFB have been evaluated. Also purification of 18F-IL2 has been optimized. Quality control has been performed using ultra performance liquid chromatography (UPLC) and Thin Layer Chromatography (TLC). Results: 18F-SFB was successfully synthesized with the Eckert & Ziegler PharmTracer synthesis module with decay-corrected radiochemical yields comparable to literature (range 28-64%). Major challenges have been encountered, most importantly regarding the purification of the 18F-SFB and 18F-IL2, stability of the IL2 and specific activity. The activated ester 18F-SFB was purified by high performance liquid chromatography (HPLC) to remove any impurities that could interfere with the conjugation. 18F-IL2 has been purified using PD-10 columns with PBS containing 0.05% SDS as mobile phase. Conclusions: Several challenges for the GMP-compliant production of 18F-IL2 have been overcome. In the near future this tracer will be used in preclinical and clinical studies to non-invasively image activated T-cells before and during cancer immunotherapy. This can provide insight in the effects of cancer immunotherapy on the immune response. References: 1. Di Gialleonardo V, et al. J Nucl Med.2012;53(5):679-86. 2. Di Gialleonardo V, et al. Eur J Nucl Med Mol Imaging.2012;39(10):1551-60. Citation Format: Elly L. van der Veen, Petra Maarsingh, Anton G.T. Terwisscha van Scheltinga, Marjolijn N. Lub-de Hooge, Geke A.P. Hospers, Erik F.J. de Vries, Elisabeth G.E. de Vries. Development of 18F-IL2: a PET radiotracer for imaging activated T-cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4208.