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Deciphering Binding Patterns of Therapeutic Antibodies with Immune Cells Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1648 Deciphering Binding Patterns of Therapeutic Antibodies with Immune Cells From Method Development to Application SINA BONDZA ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0902-6 UPPSALA urn:nbn:se:uu:diva-406875 2020 Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds Väg 20, Uppsala, Thursday, 7 May 2020 at 09:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Mark Cragg (Academic Unit of Cancer Sciences, University of Southampton). Abstract Bondza, S. 2020. Deciphering Binding Patterns of Therapeutic Antibodies with Immune Cells. From Method Development to Application. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1648. 68 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0902-6. Reversible binding, for example between signaling molecules and receptors on the cell surface, is one of the main means to communicate information in cellular systems. Knowledge about how molecules interact is crucial for both understanding biological function and for therapeutic intervention. The cellular environment often makes ligand-receptor interactions complex with the membrane providing structural support and containing other components that interfere with the interaction. One of the fastest growing drug classes for targeting cellular receptors are monoclonal antibodies (mAb), in particular within oncology. Therapeutic mAbs can have direct effects on target cells mediated via the Fab-domain and immune-related effects that are mediated via the Fc-domain. An example of the latter is activation of the complement system by binding of its first component C1q to Fc-domains. Furthermore, immune cells can recognize Fc-domains via Fc-receptors and cause target cell death by a process called antibody-dependent cellular cytotoxicity (ADCC). Increased understanding about structure-binding-function relationships facilitates rational drug design, as has been demonstrated with the development of next-generation mAbs that harbor a structural modification on their Fc-domain that strengthens the interaction with immune cells thereby increasing ADCC efficacy. In this thesis, assays for characterizing mAb binding and mAb mediated interactions on live cells were developed and applied to illustrate how detailed knowledge about binding processes helps to understand the relation between binding and biological function. Paper I describes a protocol for real-time interaction analysis of antibodies with live immune cells enabling binding measurements in a relevant cellular context with the data resolution needed to study complex binding processes. Paper II presents a novel real-time proximity assay that allows to study binding kinetics in connection with receptor dimerization and clustering thereby aiding in decipher complex interactions. In paper III, binding patterns of the CD20 mAbs rituximab, ofatumumab and obinituzumab were established on cells revealing that the fraction of bivalently bound mAbs differed resulting in dose-dependent affinities for rituximab and obinituzumab. In paper IV, a C1q binding assay to mAb opsonized cells was developed and it was shown that a higher degree of bivalent binding correlated with stronger C1q binding for the CD20 mAbs evaluated in paper III. In paper V, an assay to study mAb mediated cell-cell interactions was set-up and it was found that neutrophil engagement with target cells was similar for antibodies of IgG and IgA isotype. Keywords: affinity, binding kinetics, CD20, cell-based assay, immunology, receptor-ligand interaction, therapeutic antibody Sina Bondza, Department of Immunology, Genetics and Pathology, Medical Radiation Science, Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden. © Sina Bondza 2020 ISSN 1651-6206 ISBN 978-91-513-0902-6 urn:nbn:se:uu:diva-406875 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-406875) To all who supported me List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Bondza, S., Foy, E., Brooks, J., Andersson, K., Robinson, J., Richalet, P., Buijs, J. (2017) Real-time Characterization of An- tibody Binding to Receptors on Living Immune Cells. Frontiers in Immunology, 8:455 II Bondza, S., Björkelund, H., Nestor, M., Andersson, K., Buijs, J. (2017) Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells. Analytical Chemistry, 89(24):13212–13218 III Bondza S., ten Broeke T., Leusen J.H.W., Buijs J. Bivalent Binding on Cells varies between CD20 Antibodies and is Dose- dependent. submitted Manuscript IV Bondza S., Buijs J. Degree of Bivalent Binding correlates with C1q Binding Strength for CD20 Antibodies, Rituximab, Ofatu- mumab and Obinituzumab. Manuscript V Brandsma, A.M., Bondza, S., Evers, M., Koutstaal, R., Ne- derend, M., Jansen, J.H.M., Rösner, T., Valerius, T., Leusen, J.H.W., ten Broeke, T. (2019) Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG. Frontiers in Immunology, 10:704 Reprints were made with permission from the respective publishers. Papers not included in this Thesis Mansouri, L., Sutton, L.-A., Ljungström, V., Bondza, S., Arngården, L., Bhoi, S., Larsson, J., Cortese, D., Kalushkova, A., Plevova, K., Young, E., Gunnars- son, R., Falk-Sörqvist, E., Lönn, P., Muggen, A.F., Yan, X.-J., Sander, B., Enblad, G., Smedby, K.E., Juliusson, G., Belessi, C., Rung, J., Chiorazzi, N., Strefford, J.C., Langerak, A.W., Pospisilova, S., Davi, F., Hellström, M., Jern- berg-Wiklund, H., Ghia, P., Söderberg, O., Stamatopoulos, K., Nilsson, M., Rosenquist, R., (2014) Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia. Journal of Experimental Medicine, 212(6):833–843 Bondza, S., Stenberg, J., Nestor, M., Andersson, K., Björkelund, H. (2014) Conjugation effects on antibody-drug conjugates: evaluation of interaction ki- netics in real time on living cells. Molecular Pharmaceutics, 11(11): 4154– 4163 Contents Introduction ................................................................................................... 11 Cellular communication and signaling ..................................................... 11 Kinetics of pharmaceutical drugs ............................................................. 13 Real-time Interaction analysis and homogenous interactions .................. 14 Heterogenous interactions ........................................................................ 18 1:2 interaction model ........................................................................... 18 1:1, 2-state interaction model .............................................................. 20 Bivalent interaction model and avidity ................................................ 20 Interaction Map .................................................................................... 21 Techniques for biomolecular real-time interaction analysis .................... 23 Biophysical measurement techniques .................................................. 23 Cell-based techniques .......................................................................... 24 LigandTracer ....................................................................................... 25 Cancer and therapy options ...................................................................... 26 Monoclonal antibodies for cancer therapy ............................................... 28 Mechanisms of action of mAb therapy – with a focus on CD20 mAbs ... 30 Fab mediated effects ............................................................................ 30 Antibody-drug conjugates ................................................................... 31 Fc-mediated effects .............................................................................. 32 Antibody dependent complement activation ................................... 32 Antibody dependent cellular cytotoxicity and phagocytosis ........... 33 Clinical relevance of Fc-mediated effects ....................................... 35 Improving therapy with next-generation mAbs and alternative formats .. 36 IgA isotype for antibody therapy ......................................................... 36 Bispecific antibodies ............................................................................ 37 Scope of Thesis ............................................................................................. 39 Summary of Findings .................................................................................... 41 Paper I ...................................................................................................... 41 Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells ........................................................................... 41 Aim and Background ...................................................................... 41 Results ............................................................................................. 41 Discussion ....................................................................................... 42 Paper II ..................................................................................................... 43 Novel Real-Time Proximity Assay for Characterizing Multiple Receptor
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