Glycomimetic Langerin Ligands for Langerhans Cell Targeting Inauguraldissertation to obtain the academic degree Dr. rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Eike-Christian Wamhoff from Bassum, Germany Berlin, June 2017 The work presented in this dissertation was conducted between April 2013 and May 2017 at the Max Planck Institute of Colloids and Interfaces under the supervision of Dr. Christoph Rademacher. 1st Reviewer: Dr. Christoph Rademacher 2nd Reviewer: Prof. Dr. Christian Freund Date of Defense: Acknowledgements Acknowledgements Foremost, I would like to express my sincere gratitude to Dr. Christoph Rademacher for his mentorship, his trust and his patience. His passion for science has been inspirational and his integrity exemplary. It was an honor to work on my dissertation under Dr. Christoph Rademacher’s supervision and I am certain that he will be an outstanding mentor for future generations of Ph. D. students. I would like to thank Prof. Peter H. Seeberger for providing an excellent interdisciplinary and international scientific environment at the Max Planck Institute of Colloids and Interfaces. Moreover, I am grateful for his career advice and support during application processes. I deeply acknowledge the friendship, the professionalism and the lasting memories I have shared with my fellow Ph. D. students and co-workers in the Structural Glycobiology Group. Thank you, Dr. Jonas Aretz, Hannes Baukmann, Dr. Jonas Hanske, Jessica Schulze and Dr. Robert Wawrzinek! The last years have been an exciting time and would not have been the same without you. My gratitude furthermore extends to the ever-motivated and talented students I had the opportunity to supervise. Felix Fuchsberger, David Hartmann, Lena Kilian, Julia Mastouri, Mareike Rentzsch, Lennart Schnirch and Christian Sommereisen have conducted important experiments and data analyses. Moreover, the help provided by the administrative and technical staff has been invaluable and I would like to specifically thank Dorothee Böhme, René Genz, Eva Settels, Felix Hentschel and Olaf Niemeyer. Many collaborators have contributed substantially to the work presented in this dissertation. Dr. Oliver Schwardt and Prof. Beat Ernst have generously hosted me in their laboratory at the Universität Basel and their advice has been instrumental for the design and synthesis of several promising glycomimetics. Additionally, Prof. Beat Ernst and Dr. Alexander Titz have provided a focused glycomimetic library for screening against Langerin. Dr. Andrea Volkamer provided valuable insight into in silico druggability analysis. Gunnar Bachem and Prof. Oliver Seitz as well as Kira Neuhaus and Prof. Laura Hartmann have designed and synthesized multivalent glycomimetics. Here, I particularly acknowledge the productive discussions with Gunnar Bachem and his excellent synthetic work to enable the utilization of glycomimetics in cell-based in vitro and ex vivo experiments. These experiments were devised and conducted by Jessica Schulze, Lydia Bellmann, Dr. Martin Hermann and Assoc. Prof. Patrizia Stoitzner and lend this dissertation much of its relevance in the context of cancer immunotherapy. Finally, Maurice Grube and Dr. Daniel Varón Silva have patiently introduced me to the field of carbohydrate chemistry during my first year at the Max Planck Institute of Colloids and Interfaces. 1 Acknowledgements Most importantly, I would like to thank my parents and my brother for their everlasting support and love. I greatly appreciate your continued encouragement and the sacrifices you endured to enable my education. Last but certainly not least, I would like express my deepest gratitude to Anna Behrens, who motivates me to be the best version of myself and gives me the opportunity to be happy. Thank you for your patience over the last months - I am excited to begin a new chapter in our lives. 2 Summary Summary Immune evasion represents an important hallmark of cancer progression. In this context, the induction of tumor-specific cytotoxic T cell immunity to overcome immunological tolerance has become a focal point of cancer immunotherapy. Langerhans cells (LCs) constitute a dendritic cell (DC) subset residing in the epidermis of the human skin. They have been recognized for their capacity to endocytose and cross-present exogenous antigens via MHC-I to efficiently prime naïve CD8+ T cells. Langerin, an endocytic C-type lectin receptor (CLR) involved in the Ca2+-dependent recognition of both pathogen- and self-associated glycans, displays an expression profile highly restricted to LCs. Hence, the targeted delivery of tumor-associated antigens (TAAs) to Langerin represents an intriguing approach to develop novel vaccination strategies. Over the last decades, liposomes have emerged as versatile delivery platforms that can be targeted to LCs via the conjugation to Langerin ligands. As glycan interactions with CLRs are typically weak and highly promiscuous, liposomal targeting required the discovery of potent and specific glycomimetic ligands. However, the onerous synthesis of carbohydrate analogs as well as the hydrophilicity and high solvent exposure of carbohydrate binding sites render glycomimetic ligand design challenging. The structure-based in silico analysis of 21 X-ray structures presented in this dissertation corroborated the classification of CLRs as undruggable or challenging targets. Druggable secondary binding pockets adjacent to the carbohydrate binding site were exclusively identified for CLRs of limited therapeutic relevance. Several strategies were employed to address the challenges outlined above and to discover potent carbohydrate analogs for Langerin. The structure-based in silico screening of substituents in C2 of the mannose (Man) scaffold served to design an initial focused glycomimetic library. The structure- activity relationship (SAR) of this scaffold was further elucidated via determination of affinities for an existing library of Man analogs derivatized in C1 and C6. Alternatively, a structure-based design strategy guided the exploration of substituents in C2 of glucsoamine-2-sulfate (GlcNS). These investigations ultimately led to the discovery of potent Man (KI = 0.25±0.07 mM) and GlcNS (KI = 0.24±0.03 mM) analogs displaying a 40- to 42-fold affinity increase over naturally occurring carbohydrate ligands. The multivalent organization of carbohydrates or their synthetic analogs to match the geometry of the Langerin trimer represents an attractive strategy to optimize their specificity and potency. The GlcNS analog was conjugated to nucleic acid scaffolds to design divalent glycomimetics (IC50 = 23±2 µM) resulting in an additional distance-dependent 12-fold avidity increase. In an alternative approach, trivalent Man-bearing glycoclusters (IC50 = 0.20±0.08 mM) displayed an 80-fold avidity increase over naturally occurring carbohydrate ligands. 3 Summary 19 The development of a sensitive F R2-filtered nuclear magnetic resonance (NMR) assay enabled the determination of KI and IC50 values for Langerin. Importantly, the optimization of the assay setup with respect to throughput and material consumption proved instrumental for the discovery of potent carbohydrate analogs and multivalent glycomimetics. Moreover, the implementation of an explorative 19 F R2-filtered NMR fragment screening led to the identification of the first non-carbohydrate inhibitor reported for Langerin. The assay was successfully transferred to the CLR DC-SIGN to evaluate the specificity of designed carbohydrate analogs. 19 While F R2-filtered NMR experiments served as the primary screening and characterization assay, affinities were validated via saturation transfer difference and 15N heteronuclear single quantum coherence NMR. Furthermore, an integrated strategy combining these NMR experiments with molecular docking studies was implemented to analyze the Ca2+-dependent binding mode of the designed Man and GlcNS analogs. These investigations enabled the development of suitable conjugation strategies for liposomal formulations. 19 The GlcNS analog displayed remarkable specificity against DC-SIGN in F R2-filtered NMR experiments and was thus utilized for the preparation of targeted liposomes. Flow cytometry studies were employed to optimize liposomal formulations and to validate the binding of these liposomes to Langerin+ model cells in vitro. Finally, ex vivo experiments demonstrated their capacity to specifically target LCs in the human skin. The liposomes were efficiently endocytosed and thus represent a promising TAA delivery platform. In conclusion, the integration of carbohydrate chemistry, structure-based in silico methods and NMR experiments enabled the discovery of carbohydrate analogs and multivalent glycomimetics as potent and specific ligands of the endocytic CLR Langerin. These ligands were demonstrated to specifically target liposomes to LCs in the human skin and to promote endocytosis. Consequently, the findings presented in this dissertation constitute an important advancement for the research field of DC immunology and the development of novel cancer immunotherapies. 4 Publications Publications Parts of this dissertation have been published or will be included in journal articles: Wamhoff, E.-C., Schulze, J., Bellmann, L., Bachem, G., Seitz, O., Stoitzner, P., and Rademacher, C. (2017) A glycomimetic Langerin ligand for Langerhans cell targeting. (in preparation). Hanske, J., Wawrzinek, R., Geissner, A., Wamhoff,