Making the effect visible - OX40 targeting nanobodies for in vivo imaging of activated T cells.

Autor:	Frecot DI; Pharmaceutical Biotechnology, University Tübingen, Tübingen, Germany.; Cluster of Excellence iFIT (EXC2180) 'Image-Guided and Functionally Instructed Tumor Therapies', University of Tübingen, Tübingen, Germany., Blaess S; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.; Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Tübingen, Germany., Wagner TR; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany., Kaiser PD; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany., Traenkle B; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany., Fandrich M; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany., Jakobi M; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany., Scholz AM; Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University Munich, Oberschleissheim, Germany., Nueske S; Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University Munich, Oberschleissheim, Germany., Schneiderhan-Marra N; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany., Gouttefangeas C; Cluster of Excellence iFIT (EXC2180) 'Image-Guided and Functionally Instructed Tumor Therapies', University of Tübingen, Tübingen, Germany.; Institute for Immunology, University of Tübingen, Tübingen, Germany.; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) partner site Tübingen, Tübingen, Germany., Kneilling M; Cluster of Excellence iFIT (EXC2180) 'Image-Guided and Functionally Instructed Tumor Therapies', University of Tübingen, Tübingen, Germany.; Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Tübingen, Germany.; Department of Dermatology, University of Tübingen, Tübingen, Germany., Pichler BJ; Cluster of Excellence iFIT (EXC2180) 'Image-Guided and Functionally Instructed Tumor Therapies', University of Tübingen, Tübingen, Germany.; Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Tübingen, Germany.; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) partner site Tübingen, Tübingen, Germany., Sonanini D; Cluster of Excellence iFIT (EXC2180) 'Image-Guided and Functionally Instructed Tumor Therapies', University of Tübingen, Tübingen, Germany.; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.; Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Tübingen, Germany.; Department of Medical Oncology and Pneumology, University of Tübingen, Tübingen, Germany., Rothbauer U; Pharmaceutical Biotechnology, University Tübingen, Tübingen, Germany.; Cluster of Excellence iFIT (EXC2180) 'Image-Guided and Functionally Instructed Tumor Therapies', University of Tübingen, Tübingen, Germany.
Jazyk:	angličtina
Zdroj:	Frontiers in immunology [Front Immunol] 2024 Oct 15; Vol. 15, pp. 1480091. Date of Electronic Publication: 2024 Oct 15 (Print Publication: 2024).
DOI:	10.3389/fimmu.2024.1480091
Abstrakt:	Purpose: Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. Methods: Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro , including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. Results: Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. Conclusion: Considering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40 + T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs. Competing Interests: DF, DS, MK, BP, TW, BT, PK, and UR are named as inventors on a patent application claiming the use of the described nanobodies for diagnosis and therapeutics filed by the Natural and Medical Sciences Institute, the Werner Siemens Imaging Center and the University of Tuebingen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2024 Frecot, Blaess, Wagner, Kaiser, Traenkle, Fandrich, Jakobi, Scholz, Nueske, Schneiderhan-Marra, Gouttefangeas, Kneilling, Pichler, Sonanini and Rothbauer.)
Databáze:	MEDLINE
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