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Non-Radioactive Molecular Imaging-Driven Drug Development in Oncology

Final Report Summary - ONQVIEW (Non-Radioactive Molecular Imaging-Driven Drug Development in Oncology)

Cancer remains a leading cause of death worldwide. Fortunately an abundance of cancer drugs, often molecularly targeted and rationally designed, are becoming available, making selection of the most promising compounds for effective cancer treatment one of the major challenges. Integrated cancer research and biomarker-driven adaptive and hypothesis-testing clinical trials are therefore imperative. Molecular imaging can advance this drug development by characterizing drug behavior and the targeting and functional effects. However, this approach had hardly been explored, which is at least partly due to the radiation burden to the patient caused by the conventional radioactive tracers that are currently used in clinical research. We took a radically different molecular imaging-based approach in which drug efficacy measurements are already incorporated in early preclinical and clinical development stages using non-radioactive strategies, including optical and photoacoustic imaging modalities. Our group is uniquely suited to develop this approach. We have extensive (pre)clinical experience with molecular imaging and drug development as well as advanced infrastructure that includes lasers, microscopes and state-of-the art detection systems for non-ionizing electromagnetic waves, such as light and radio waves, that pose no known health hazard. With these techniques, a wide variety of tumor characteristics can now be visualized simultaneously, for example with a novel clinical approach using monoclonal antibodies as tracers.

The central aim of OnQview was to create an integrative drug development strategy, based on a novel preclinical approach that primarily uses non-radioactive molecular imaging. This strategy will guide patient-tailored selection of drugs, measure in vivo drug and tumor behavior, and monitor effect sensors to enable dynamic treatment tuning.

OnQview involved two interconnected work packages. The first work package was the backbone of the proposed research. This largely biology-driven work package was focused on novel non-radioactive tracers based on new targeted drugs in breast and colon cancer.

Several antibodies were labeled with both 89-Zirconium (89Zr) for PET imaging and IRDye800CW for near-infrared fluorescence imaging. Radiolabeling with 89Zr and in vivo microPET validation was performed to benchmark uptake of fluorescently labeled tracers. Macroscopic analysis showed a differential distribution of pertuzumab-800CW-labeled specific tracer versus aspecific-labeled tracers in tumor tissues.

Moreover, we searched for effect sensors for visualizing HER2- or EGFR-directed treatment effects. We characterized a panel of breast cancer cell lines for EGFR inhibitor sensitivity, and used SILAC-based quantitative mass-spectrometry to identify ‘effect sensors’ of effective treatment. The glycoprotein MUC1 was identified and validated. In vivo xenograft studies revealed that MUC1 expression is elevated in erlotinib-sensitive tumors upon treatment, and that MUC1 upregulation can be measured in blood upon erlotinib treatment in tumor-bearing animals. Ongoing studies focus on developing non-invasive MUC1 imaging tools to visualize effective EGFR-targeted treatment.

In parallel, quantitative mass-spec analysis to identify effect sensors for EGFR-targeted treatment yielded novel potential biomarkers to be used as imaging agents. Additionally, mass-spec-based effect sensor identification for genotoxic agents doxorubicin and cisplatin in breast cancer models, generated multiple leads to follow-up investigation.
Tracers that were produced for clinical use and originated from this research include HER family-directed tracers cetuximab, trastuzumab as well as a HER3-directed antibody. These results indicate that we were already able to bring relevant tracers to the clinic.

The second work package focused on advancing the technical aspects of optical and photoacoustic imaging preclinically and clinically.

We showed the possibility to image and detect 800CW fluorophore dye with a photoacoustic imaging system developed for animal imaging with a detection sensitivity around 200 nM at 5 mm depth in highly scattering medium and the possibility to extract fluorophore spatial distribution inside the scattering medium. Besides, we developed a fully integrated handheld probe, integrating a laser diode and ultrasound transducer array for a portable system combining ultrasound and photoacoustic imaging modality. This new compact imaging system can be easily transported to the clinic for in vivo studies.

A tri-modality (i.e. photoacoustic, acousto-optic and ultrasound) tomographic small animal imaging system was developed and visualization and quantification of HER2 expression in human breast cancer xenografted nude mice was evaluated. It showed the multispectral imaging ability of the tri-modality tomographic imaging system, and its ability to perform functional and anatomical imaging. However, the sensitivity of the system needs to be further improved in order to detect physiologically relevant concentrations of fluorescently labeled (800CW) antibody pertuzumab. We anticipate that this can be reached with the use of more photoacoustic detectors.

We finalized a study in breast cancer patients with the near-infrared fluorescent tracer bevacizumab-IRDye800CW targeting VEGF-A, that showed that systemic administration of this tracer is safe for breast cancer guidance and confirms tumor and tumor margin uptake. In addition, the study illustrated that a quantitative approach is possible which relates fluorescence signals to malignant tissues and improves the theranostic application of fluorescence molecular imaging. Moreover, we developed a molecular-guided endoscopy approach using targeted near infrared fluorescent tracers preclinically and clinically.

In addition to fluorescence imaging, we investigated applications of label-free four-wave mixing (FWM) microscopy for molecular imaging of tumor cells and tissues showed that FWM is well-suited for cellular imaging. A comparison between vibrationally non-resonant FWM imaging with vibrational resonant coherent anti-Stokes Raman scattering (CARS) signals revealed nearly identical qualitative information in cellular imaging. This multi-modal microscopy approach enabled to correlate molecule specific imaging with dynamic biological processes related to uptake, presentation and degradation. For label-free 3-dimensional NR-FWM imaging of single cells and tissue, a theoretical overview was made and applied to a computer simulation to calculate microscopy images in three dimensions.

OnQview allowed us to create an integrative drug development strategy based on a novel approach that next to molecular imaging with radionuclides also included non-radioactive molecular imaging. This strategy will guide patient-tailored selection of drugs, measure in vivo drug and tumor behavior, and monitor effect sensors to enable dynamic treatment tuning in future studies.