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MULTIVALENT MONOCLONAL ANTIBODIES AS IMPROVED CANCER THERAPIES: PROOF‐OF‐CONCEPT AND MECHANISTIC STUDIES USING RITUXIMAB‐LIPID NANOPARTICLES by Jesse Popov B.Sc., B.Mus., The University of Western Ontario, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Interdisciplinary Oncology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2012 © Jesse Popov, 2012 Abstract This body of work describes a novel methodology for discovering and developing new cancer drugs based on therapeutic monoclonal antibodies. Such antibodies generally contain two sites where they bind to their target, but interesting improvements are often observed when the valence (number of target‐binding sites) is increased above two. The methodology outlined in this dissertation involves using liposomes to prepare multivalent antibody‐lipid nanoparticle formulations of different valence that can be utilized for preclinical drug development. As a proof‐of‐concept, we applied the methodology to rituximab, a therapeutic antibody used to treat lymphomas and leukemias. For the same dose of rituximab, multivalent rituximab‐ lipid nanoparticles with valences up to ~250 showed significantly elevated anticancer activity from enhanced complement‐dependent cytotoxicity, antibody‐dependent cell‐mediated cytotoxicity, and direct induction of apoptosis. A valence‐dependent improvement in apoptosis in lymphoma cells was observed up to levels that were 21‐fold higher than those observed after treatment with bivalent rituximab. We subsequently employed the different valences of multivalent rituximab to investigate its poorly defined direct mechanism of action. We uncovered a novel mechanism consisting of upregulation and activation of CD120a which led to ensuing apoptosis. Effector cells of the immune system were capable of hypercrosslinking rituximab on lymphoma cells and reproducing this mechanism, suggesting that it contributes to the in vivo cytotoxicity of regular bivalent rituximab therapy. The methodology described in this dissertation can therefore serve to identify antibodies that are more active as multivalent rather than bivalent molecules, define the optimal valence of such antibodies, and elucidate the mechanism of action of the new multivalent drugs. Furthermore, we illustrate that this information applies to other types of constructs with similar valences, enabling use of the methodology for advancing both liposomal and non‐liposomal multivalent antibody formulations to preclinical maturity. Finally, this work suggests that every therapeutic antibody may have a different valence where it shows optimal therapeutic activity. For example, antibodies directed against targets that exert therapeutic effects upon clustering may show maximum activity at valences above two. This methodology can easily be applied to other antibodies in an effort to develop superior therapies against nearly any type of cancer. • ii Preface A version of Chapter 2 has been published [Popov J, Kapanen AI, Turner C, Ng R, Tucker C, Chiu G, Klasa R, Bally MB, Chikh G. Multivalent rituximab lipid nanoparticles as improved lymphoma therapies: Indirect mechanisms of action and in vivo activity. Nanomedicine 2011 Nov;6(9):1575‐91]. I collaborated primarily with Dr. Ghania Chikh for collection of data in this article, and I wrote the manuscript based on a preliminary draft by Dr. Chikh. I obtained the content in Chapters 3–6 independently; I planned and carried out all experiments, performed data analysis, prepared figures, and wrote manuscripts for future publication. A separate article resulting from collaboration with the laboratory of Dr. Ruth Signorell (Department of Chemistry, University of British Columbia) was also published and referenced in this dissertation [Weiss A, Preston TC, Popov J, Li Q, Wu S, Chou KC, Burt HM, Bally MB, Signorell R. Selective recognition of rituximab‐functionalized gold nanoparticles by lymphoma cells studied with 3D imaging. J Phys Chem C 2009 Oct 21;113(47):20252‐8]. All experiments involving the use of animals were completed in accordance with the current guidelines of the Canadian Council of Animal Care. The University of British Columbia Animal Care Committee reviewed and approved certificate numbers A05‐1582 and A06‐1537 for the animal studies carried out in Chapter 2 (in vivo efficacy of rituximab and rituximab‐lipid nanoparticles, and pharmacokinetics of rituximab‐lipid nanoparticles). This document is written using United States English spelling. • iii Table of Contents Abstract ......................................................................................................................................... ii Preface .......................................................................................................................................... iii Table of Contents .......................................................................................................................... iv List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................. xiii List of Abbreviations ..................................................................................................................... xv Acknowledgements .................................................................................................................... xvii Dedication ................................................................................................................................. xviii 1 Introduction: Developing novel therapies for treating cancer ................................................. 1 1.1 Overview ................................................................................................................................. 1 1.2 Liposomal formulations .......................................................................................................... 2 1.2.1 Liposomes in cancer treatment .................................................................................... 2 1.2.2 What are Ab‐LNPs? ....................................................................................................... 3 1.3 Therapeutic monoclonal Abs .................................................................................................. 6 1.3.1 Rituximab and CD20 ..................................................................................................... 8 1.4 The mechanisms of action of therapeutic monoclonal Abs ................................................... 9 1.4.1 Complement‐dependent cytotoxicity ........................................................................ 10 1.4.2 Ab‐dependent cell‐mediated cytotoxicity .................................................................. 10 1.4.3 Direct mechanisms of action of Rtx and other therapeutic Abs ................................ 12 1.5 Apoptosis as a target in cancer therapy ............................................................................... 14 1.6 Multivalent Abs as improved therapies ................................................................................ 15 1.6.1 Characteristics of multivalent Ab constructs and a definition of valence .................. 15 1.6.2 Examples of multivalent interactions in biology and their physical basis .................. 17 • iv 1.6.3 Descriptions of multivalent Abs that exhibit superior efficacies ............................... 19 1.7 Multivalent Ab‐LNPs and the content of this dissertation ................................................... 20 2 Indirect mechanisms and in vivo activity of multivalent rituximab‐lipid nanoparticles ...........22 2.1 Synopsis ................................................................................................................................ 22 2.2 Background ........................................................................................................................... 22 2.2.1 Complement‐dependent cytotoxicity of Rtx .............................................................. 22 2.2.2 Ab‐dependent cell‐mediated cytotoxicity of Rtx ....................................................... 23 2.2.3 Does the creation of Rtx‐LNPs affect the indirect mechanisms and in vivo properties of Rtx? ....................................................................................................... 24 2.3 Materials and methods ......................................................................................................... 24 2.3.1 Reagents and cell lines ............................................................................................... 24 2.3.2 Preparation of multivalent Ab‐LNPs ........................................................................... 26 2.3.3 Measurement of cell‐surface protein expression levels ............................................ 28 2.3.4 In vivo xenograft models to assess Rtx efficacy ......................................................... 28 2.3.5 Quantification of Ab‐LNPs bound to cells .................................................................. 28 2.3.6 Annexin‐V/PI flow cytometry‐based apoptosis assay ................................................ 29 2.3.7 Assay for measuring levels of complement‐dependent cytotoxicity ......................... 30 2.3.8 Assay for the quantification

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