“‘I hope we will establish valid imaging protocols & workflows for the preclinical evaluation and validation of newly developed immunotracers. ”

Manfred Kneilling from the University of Tübingen is leading work package 4 of the Immune-Image project together with Bastian Zinnhardt from Roche. In this work package they evaluate the specificity and suitability of new immunotracers targeting different immune cells for non-invasive in vivo imaging in cancer and inflammatory diseases. We interviewed Manfred about his research, the different work packages the University of Tübingen is involved in, and the immune cell imaging techniques he is developing.

Manfred Kneiling

Can you briefly introduce yourself?

“I am a consultant physician in Dermatology, heading the Allergology Section of the Department of Dermatology at the University hospital of the Eberhard Karls University Tübingen. In addition, I am leading the group for Inflammation & Immunology at the Werner Siemens Imaging Center of the Department for Preclinical Imaging and Radiopharmacy.”

What does your research focus on?

“My research focuses on non-invasive in vivo imaging of inflammatory processes and whole body tracking of immune cells (mainly T cells) employing multimodal imaging methods (PET/MRI, OI, etc.). My team is developing new highly advanced combined immunotherapies for mice with progressed solid cancers by combining tumor-antigen specific T cells, oncolytic viruses, different combinations of immune-checkpoint inhibitors, whole body or fractionated irradiation, and further immune modulating agents. One very important aim is to identify therapy responders to immune-checkpoint inhibitors in carcinoma bearing mice as well as metastasized cancer patients by holistic non-invasive in vivo imaging focusing on the primary, secondary and tertiary lymphatic organs. In this regard, I am especially interested in translational studies in Immune checkpoint inhibitor-treated cancer patients with focus on the homing and distribution patterns of T cells and their activation state (e.g. CD69, OX40 expression) to uncover mechanisms of treatment response and resistance. In addition, I am highly interested in the non-invasive in vivo visualization of the cellular and molecular processes during immune mediated diseases such as T cell-driven autoimmune diseases like psoriasis vulgaris, psoriasis arthritis or rheumatoid arthritis and in researching the complex molecular processes involved in chronic inflammatory immune responses which are responsible for cancer development (such as NASH-HCC).”

How did you get involved in the Immune-Image consortium?

“About six years ago, we recognized the IMI immune ‘cell tracking’ call and discussed about potential consortium partners. At this time Bert Windhorst, the project coordinator of Immune-Image project, contacted Bernd Pichler, the head of the Werner Siemens Imaging Center, concerning the IMI call. Bert, Tony Lahoutte and Andreas Jacobs build a consortium covering the whole field of immune cell imaging. Next, the members of the consortium were invited by Bert to Amsterdam, to discuss and to exchange ideas before we started to work on the first draft of the twostep call. After our consortium was selected by EFPIA we were happy to prepare the final proposal. This was the beginning of Immune-Image.”

What is your role and the role of University of Tübingen?

“Besides the lead of WP4, the university of Tübingen is involved in different work packages within the Immune-Image consortium. Conjoint with WP2 we are involved in the positron emission tomography (PET) scanner validation at different sites and in the harmonization of imaging protocols, animal handling and data analysis. In WP3 we are improving the radiolabeling of immunotracers such as the CD4 and CD8 minibodies kindely provided by ImaginAb. Within WP5 we are studying longitudinal cell tracking in cancer and IMIDs (contact dermatitis, arthritis, colitis, vasculitis) including treatment approaches. Longitudinal cell tracking is a powerful approach to understand the biology of single cells. In WP 6, we are involved in a prospective CD206 Nanobody study in non small cell  lung cancer patients. This consists of precision in vivo targeting of tumor associated macrophages with a novel CD206-directed radiotracer.”

What is the main goal of WP4?

“WP4 is developing cutting-edge imaging protocols for specific tracers and is conducting proof-of-concept and immunotracer validation studies. The main goal of WP4 is to characterize and validate in a preclinical setting newly developed immunotracers from WP3, to provide proof-of-concept data in small animals before further validation, including therapeutic interventions, in advanced disease-specific animal models and in non-human primates in WP5 and ultimately translation towards clinical application in WP6. WP4 is well imbedded between WP3 and WP5 and works closely together with these and other workpackages.”

How do you conduct this research?

“First, we evaluate the in vivo distribution and pharmacokinetics of the newly developed immunotracers in wild-type, immunodeficient or, when cross-reactivity is not achievable, target-specific knock-in mice – where the extracellular part of the murine target is replaced by that of the human target protein via methods such as CRISPR/CAS. As imaging modalities, we apply PET/magnetic resonance imaging (MRI), PET/computed tomography (CT), single photon emission computed tomography (SPECT)/CT, MRI or optical imaging. Immunotracers with unsuitable pharmacokinetics and/or biodistribution will be returned to WP3 for kinetic immunotracer modelling and further optimization. In addition, we employ ex vivo immunohistochemistry, fluorescence microscopy and flow cytometric analysis for cross-validation of in vivo PET or optical imaging data. Next, chronic cutaneous delayed-type hypersensitivity reaction (associated with the homing of T-cells, B cells, macrophages and neutrophils) in the right ear will be employed, which will allow us to visualize the cells of interest non-invasively in vivo with the respective imaging modalities. This mouse model is specifically suitable for cross-validation of in vivo with ex vivo data sets. Immunotracers which were successfully tested and validated in the cutaneous DTHR model will be further evaluated in immunogenic cancer models and immune mediated inflammatory disease (IMID) models, where a convolution of all immune cells of interest are involved. This will enable us to optimize imaging protocols & workflows for WP5, which will focus on longitudinal cancer and IMIDs therapy monitoring studies, as well as the identification of immune therapy responders.”

What kind of immune cell imaging techniques are you developing?

“Within WP4 we are developing cutting-edge imaging protocols & workflows for already available and newly developed specific immunotracers: e.g. dosing, imaging time point, appropriate quantitative image reconstruction, correction and analysis.
This immune cell imaging techniques enable us to evaluate  the utility of already available and newly developed immunotracers in a simplified experimental mouse model of chronic cutaneous DTHR for initial validation at the time point with strongest immune cell recruitment and in other IMID and tumor models.”

What do you hope Immune-Image will accomplish after 5 years?

“I hope we will have established valid imaging protocols & workflows for the preclinical evaluation and validation of newly developed immunotracers, of which one will be brought from the scratch into clinical application within 5 years of development. In general, I hope we will have been able to foster the public visibility concerning the amenities and value of non-invasive in vivo immune cell imaging including immune cell tracking for stratification of tumor or IMID patients, therapy monitoring and identification for treatment success and failure.”

What have you achieved so far in the project?

“So far, we have determined the ideal set of multi-modality molecular imaging for immune cell tracking. We also established a wide spectrum of cancer and IMID mouse models for immunotracer validation as well as imaging protocol for already available immunotracers. Thus, we are ready to characterize and validate new immunotracers from WP3. Currently, we are characterizing and validating the available immunotracers in cancer and IMID rodent models and have recently started with new characterization and validation of newly developed immunotracers such as CD8 and CD163.”

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