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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.

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.

GA201 is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) that is optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and inhibition of EGFR signal transduction. Recently, combinations of EGFR mAbs were shown to be more potent in inducing EGFR downregulation and superior in controlling growth in several tumor types, including breast and lung cancer. Currently, it is unknown how internalization and membrane recycling of EGFR after treatment with GA201, and in combination with the anti-EGFR mAb cetuximab, will affect GA201-induced ADCC response and tumor growth inhibition. In the present study we have investigated the effects of the GA201 and cetuximab on EGFR expression and endosomal trafficking in non-small cell lung cancer cell lines (NSCLC). Effects of GA201, cetuximab and the combination of these mAbs on EGFR were studied in a panel of NSCLC cell lines (A549, H441, H322 and H292). The influence of mAb binding on EGFR membrane expression, internalization and endocytic trafficking was determined using flow cytometry and immunofluorescence. For the internalization experiments, cells were pre-incubated with the mAbs for one hour at 4 °C. The effects on total EGFR protein levels and downstream signaling were studied using Western blotting. Treatment of cells with GA201 or cetuximab at 37 °C triggered EGFR internalization as demonstrated with flow cytometry. During treatment with either antibody alone for 1, 2 and 4 hours, EGFR reappeared at the cell surface. Immunofluorescence demonstrated the presence of GA201 and cetuximab in early endosomes and recycling endosomes, which can explain the EGFR reappearance on the cell surface. Interestingly, after 24 and 72 hours incubation with GA201 membranous EGFR levels were diminished to 50%, whereas cetuximab had no effect on membranous EGFR expression levels. Treating cells with the combination of the two mAbs resulted in EGFR internalization, downregulation of membranous EGFR levels with 80% and a 2-fold reduction in cellular EGFR protein levels as shown by Western blotting. When combined, most of the internalized GA201 and cetuximab colocalized with the lysosomes, and were almost absent in early endosomes and recycling endosomes. Moreover, the combination efficiently inhibited EGF-induced phosphorylation of downstream signaling molecules. In conclusion, GA201 downregulated membranous EGFR levels, whereas cetuximab had no effect. GA201 in combination with cetuximab leads to stronger downregulation of membranous and cellular EGFR levels by inhibiting endosomal recycling and increasing EGFR degradation in the endosmal/lysosomal compartment compared to both antibodies separately. We are currently investigating the effects of the single agents and the combination on EGFR signaling, ADCC response and tumor growth. Funded by Hoffmann-La Roche AG and ERC grant OnQview Citation Format: Arjan Kol, Steven de Jong, Martin Pool, Elisabeth G.E. de Vries, Christian A. Gerdes, Anton G.T. Terwisscha van Scheltinga. Differential effects of GA201 and cetuximab on EGFR expression and endosomal recycling in non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2014-4511

Background: Mesothelin (MSLN) is a tumor differentiation antigen that is highly expressed by cells of many epithelial tumors, with limited expression in normal human tissues. Our understanding of therapeutic antibodies targeting MSLN might benefit from immunoPET imaging of antibody uptake. We developed and preclinically validated an 89Zr labeled anti-MSLN antibody (“89Zr-AMA”) for this noninvasive imaging of tumor and normal organ uptake. Methods: 89Zr was attached to an anti-MSLN humanized IgG1 monoclonal antibody derivatized with the bifunctional chelator reagent N-succinyldesferrioxamine-B-tetrafluorphenol. The 89Zr-AMA was characterized in terms of conjugation ratio, aggregation, radiochemical purity, stability, and immunoreactivity. Two human MSLN-expressing pancreatic tumor cell lines, HPAC and CAPAN-2, were used for xenograft studies in mice. Tumor uptake and organ distribution of 89Zr-AMA were studied in the HPAC line at three protein doses (10, 25 and 100 μg) labeled with 1 MBq 89Zr and results were compared with nonspecific 111In-IgG. After dose-finding, CAPAN-2 and HPAC tumor xenograft-bearing mice were scanned with μPET at 1, 3, and 6 days after tracer injection of the optimal AMA dose labeled with 5 MBq 89Zr, followed by ex vivo biodistribution at day 6. Tracer uptake was quantified and expressed as mean standardized uptake values (SUVmean). Results: 89Zr-AMA formed with high specific activity (> 500 MBq/mg), high yield (> 90% without further purification), and high purity (> 95% determined by SE-HPLC analysis). In vitro validation of 89Zr-AMA showed a fully preserved immunoreactivity with a long (> 1 week) stability in 0.9% NaCl. Biodistribution analyses of the dose-finding groups revealed a dose-dependent 89Zr-AMA tumor uptake, with the highest fractional tumor uptake in the 10 μg dose group, 14.2 %ID/g on day 6. Tumor uptake of the non-specific control antibody, 111In-IgG, was lower than that of the 89Zr-AMA (P < 0.05, paired t test). Day 6 89Zr-AMA biodistribution data from the animals that underwent μPET showed ex vivo tumor uptake of 12.0 %ID/g in HPAC and 11.8 %ID/g in CAPAN-2 tumors and 4.6 and 4.4 %ID/g in blood. Uptake of the nonspecific control 111In-IgG was 5.7 %ID/g for HPAC and 3.6 %ID/g for CAPAN-2 tumors, and 10.0 and 7.5 %ID/g for their respective blood pools. MicroPET imaging was consistent with the biodistribution data. 89Zr-AMA showed a progressive increase in tumor uptake over time, whereas the activity in the blood pool decreased; in liver, spleen and kidney it remained stable. Conclusion: 89Zr-AMA tumor uptake is antigen-specific in MSLN-expressing tumors. This tracer can be translated to the clinic for serial non-invasive PET imaging. Citation Format: Eva J. ter Weele, Marjolijn N. Lub-de Hooge, Daniel Maslyar, Anton G.T. Terwisscha van Scheltinga, Jos G.W. Kosterink, Elisabeth G.E. de Vries, Simon P. Williams. Imaging human pancreatic tumor xenografts with 89Zr-labeled anti-mesothelin antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2659. doi:10.1158/1538-7445.AM2013-2659

The oncogene c-Met is a clinically validated target having been shown to be involved in tumorigenesis. Imaging agents capable of quantifying c-Met expression in human tumors would be a valuable tool for aiding diagnosis, drug scheduling and monitoring the response to targeted therapies. Anticalins are a novel class of biopharmaceuticals based on the human lipocalin scaffold with many properties which make them desirable imaging agents including their smaller size and ease of manufacturing when compared to antibodies. PRS-110 is a c-Met specific anticalin which is being developed as a therapeutic and imaging modality. The aim of this study was to evaluate the utility of the c-Met anticalin as a positron emission tomography (PET) imaging agent and analyze its biodistribution in human tumor bearing mice. Methods: A zirconium-89 (89Zr) labeled version of PRS-110 was generated and biodistribution studies were performed 96 h after single dose injection of the tracer (10 to 500 μg) into Balb/c mice bearing subcutaneous c-Met expressing human H441 non-small cell lung cancer tumors. PET imaging was executed at 6, 24, 48 and 96 h after injection of 50 μg 89Zr-PRS-110 to mice bearing H441 (c-Met ++), primary glioblastoma U87-MG (c-Met +) or ovarian cancer A2780 (c-Met -) xenografts. After the final scan, biodistribution analysis was performed. The excised tumors were analyzed for c-Met expression by immunohistochemistry and fluorescent PRS-110-800CW distribution by fluorescence microscopy. Results: Biodistribution analyses showed a PRS-110 dose-dependent 89Zr-PRS-110 H441-tumor uptake, with the highest tumor uptake at 10 μg PRS-110 resulting in 7.5 %ID/g at 96 h after tracer injection. MicroPET imaging revealed specific tumor uptake of 89Zr-PRS-110 in the c-Met expressing H441 and U87 tumors while imaging of the c-Met negative A2780 tumor model showed background level similar to a non-specific anticalin control. Biodistribution data supported the microPET findings, showing ex vivo tumor uptake of 89Zr-PRS-110 of 5.9, 1.8 and 1.7 %ID/g in H441, U87 and A2780 xenografts respectively, which correlated with c-Met expression levels. Tumor : blood ratios of 89Zr-PRS-110 compared to 89Zr-Tlc-PEG confirmed these finding. Ex vivo fluorescence revealed intracellular presence of PRS-110 96 h after tracer injection. Conclusion: 89Zr-PRS-110 specifically accumulates in c-Met expressing tumors. PET imaging with this tracer provides real-time non-invasive information about PRS-110 distribution and tumor accumulation. This makes 89Zr-PRS-110, which is ready for clinical evaluation, of interest for the clinical development of PRS-110. Citation Format: Anton G.T. Terwisscha van Scheltinga, Elisabeth G.E. de Vries, Marlon Hinner, Remy B. Verheijen, Andrea Allesdorfer, Laurent Audoly, Wouter B. Nagengast, Carolien P. Schröder, Martin Hülsmeyer, Jos G.W. Kosterink, Marjolijn N. Lub-de Hooge, Shane A. Olwill. Next generation PET imaging agents: The development of radiolabeled c-Met specific Anticalin 89Zr-PRS-110 with diagnostic and therapeutic drug monitoring applications. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2013-2664

The mammalian target of rapamycin (mTOR) pathway is activated in the majority of ovarian cancers and is involved in tumor angiogenesis. Inhibitors of mTOR, like everolimus, are potentially interesting drugs as they can exert antitumor activity in part through reducing downstream vascular endothelial growth factor-A (VEGF-A) production. We investigated whether early effects of everolimus treatment could be monitored with 89Zr-bevacizumab positron emission tomography (VEGF-PET). Methods: The effect of everolimus on VEGF-A secretion was determined in three human ovarian cancer cell lines and in A2780luc+ ovarian cancer cells xenografted subcutaneously in BALB/c mice. Mice received daily everolimus (10 mg/kg intraperitoneally) for 14 days. PET scans with the tracer 89Zr-labeled bevacizumab were performed to monitor tumor VEGF-A expression before (baseline) and after treatment. Images were obtained 6 days after tracer injection. Tracer uptake was quantified and expressed as mean standardized uptake values (SUVmean). For ex vivo 89Zr-bevacizumab biodistribution and correlative tissue analyses, control animals were sacrificed after the baseline scans. Tumor VEGF-A levels were measured with ELISA in tumor lysates and mean vascular density (MVD) was determined with immunohistochemistry. Results: Everolimus treatment lowered VEGF-A levels in the supernatant of all cell lines. Everolimus lowered 89Zr-bevacizumab tumor uptake by 21.7 ± 4.0% (SUVmean 2.26 ± 0.18 versus 2.89 ± 0.20, p < 0.01). Ex vivo 89Zr-bevacizumab biodistribution showed less tracer uptake in the tumors of treated compared to control animals (7.78 ± 0.84 %ID/g versus 14.02 ± 1.68 %ID/g, p < 0.01), while no differences were observed for other tissues. VEGF-A protein levels in tumor lysates were lower in treated versus untreated tumors (p = 0.04), as was the MVD (p < 0.01). Conclusion: 89Zr-bevacizumab PET showed reduced tumor VEGF-A levels in vivo in response to everolimus therapy, coinciding with inhibition of tumor angiogenesis. Currently there are 2 clinical trials ongoing to study the value of 89Zr-bevacizumab PET to monitor tumor VEGF-A levels as an early biomarker of response to mTOR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2431. doi:1538-7445.AM2012-2431

Background: Extensive angiogenesis, defective vascular architecture, impaired lymphatic drainage and increased vascular permeability are characteristics unique for solid tumors. These characteristics result in enhanced tumor permeability and retention (EPR) effect. The EPR effect has been recognized as an important factor for intratumoral delivery of macromolecular drugs, like antibodies. Rapid changes in tumor vasculature and permeability due to antiangiogenic treatment could possibly restrain the EPR effect and thereby lead to a decreased uptake of antibodies. This would have implications for combining antiangiogenic therapy with monoclonal antibodies. We therefore investigated the effect of bevacizumab treatment on the tumor uptake of antibodies by means of immunoPET imaging. Methods: The effect of bevacizumab treatment on the antibody tumor uptake was studied with in vivo PET imaging of 89Zr-trastuzumab, 89Zr-bevacizumab and 89Zr-IgG in a SKOV-3 human xenograft tumor model. PET images were made before and during bevacizumab (5mg/kg; 2qw) or placebo (normal saline) treatment. All animals also received trastuzumab, bevacizumab or IgG labeled with the near-infrared fluorescence dye IRDye 800CW as a co-injection. Immunohistochemistry was performed to determine the mean vascular density (MVD). Immunofluorescence was used to visualize trastuzumab-800CW, bevacizumab-800CW and IgG-800CW and to determine vessel normalization by vessel pericyte coverage. Results: Bevacizumab treatment decreased 89Zr-trastuzumab, 89Zr-bevacizumab and 89Zr-IgG tumor uptake with 41 ± 5% (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2438. doi:1538-7445.AM2012-2438

Zirconium-89 (89Zr) labeled bevacizumab visualizes tumor vascular endothelial growth factor (VEGF) excretion in vivo, and can therefore serve as a potential biomarker for targeted drugs affecting VEGF levels. TNBC is more dependent upon alternative routes for their maintenance, such as angiogenesis, than hormone receptor or HER2 positive breast cancer. HSP90 inhibitors are known to impact the angiogenesis pathway via degradation of HSP90 client proteins such as HIF1α and VEGFRs, and in vitro studies have shown that breast cancer cells (including TNBC) are sensitive to these compounds. The aim of this study was to image modulation of VEGF expression in response to HSP90 inhibition with NVP-AUY922, a highly potent non-geldamycin HSP90 inhibitor, in a mouse model with a TNBC xenograft. Methods: Three groups of animals were used in this study, a control (n = 6), treated (n = 7) and placebo (n = 6) group. Twenty-two days after inoculation of TNBC cell line MDA MB-231, all mice received co-injection of 89Zr-bevacizumab and Indium-111 (111In) labeled IgG as aspecific control. VEGF-PET was performed 144 h after tracer injection. Ex vivo biodistribution studies were performed in the control group after the first scan week, for the treatment and placebo group after the second scan week, which was after 3 weeks of treatment. The control group received no treatment, the treatment and placebo group received respectively 50 mg/kg NVP-AUY922 or vehicle every-third-day (q3d) administrated intraperitoneally. Treatment effect was verified with histological response. Results: VEGF-PET showed a decreased 89Zr-bevacizumab tumor uptake of 44% (P = 0.0021) after 3 weeks NVP-AUY922 treatment compared to the baseline scan. Placebo treated mice showed no significant difference in bevacizumab uptake. These in vivo imaging data were supported by ex vivo biodistribution data. IgG tumor uptake was in all groups consistent, indicating that there was no influence on tumor perfusion due to the anti-angiogenic treatment. VEGF downregulation in the tumors was shown by immunohistochemistry of VEGF-A. Conclusion: NPV-AUY922 downregulates VEGF in a TNBC mouse model. VEGF-PET using 89Zr-bevacizumab can be used in vivo to monitor downregulation of VEGF excretion in response to HSP90 inhibition, and can therefore potentially serve as biomarker for early HSP90 inhibition effect in TNBC. Supported by a grant of the Dutch Cancer Society. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5307. doi:10.1158/1538-7445.AM2011-5307