Visualizing dual downregulation of IGF-1R and VEGF by Hsp90 inhibition effect in triple negative breast cancer

Purpose Triple negative breast cancer (TNBC) is biologically characterized 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 (89Zr) labeled antibodies targeting IGF-1R and VEGF. Experimental design 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 89Zr-MAB391 and 89Zr-bevacizumab for visualization of IGF-1R and VEGF 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 tumor uptake on 89Zr-MAB391-PET by 37.3% (P < 0.01) and on 89Zr-bevacizumab-PET by 44.4% (P < 0.01). This was confirmed by ex vivo biodistribution with a reduction of 41.3 % injected dose (ID)/g for 89Zr-MAB391 and 37.8 %ID/g for 89Zr-bevacizumab, while no differences were observed for other tissues. This coincided with reduced IGF-1R expression and mean vessel density in the NVP-AUY922 treated tumors. Conclusion 89Zr-MAB391 and 89Zr-bevacizumab PET reflect effect of Hsp90 inhibitors and can therefore potentially be used to monitor therapeutic effects of Hsp90 inhibitor therapy in TNBC

Imaging human pancreatic tumor xenografts with 89Zr-labeled anti-mesothelin antibody

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

89Zr-bevacizumab PET as an early biomarker for response to everolimus treatment in an ovarian cancer xenograft model

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

Bevacizumab-Induced Normalization of Blood Vessels in Tumors Hampers Antibody Uptake

<p>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 Zr-89-trastuzumab (SKOV-3 and OE19), Zr-89-bevacizumab (SKOV-3), or Zr-89-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. (C) 2013 AACR.</p>