UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE Cancer Sciences Unit Volume 1 of 1 The Role of Fc Gamma Receptors in the Activity of Therapeutic Monoclonal Antibodies by Robert James Oldham Thesis for the degree of Doctor of Philosophy September 2016 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE Biomedicine Thesis for the degree of Doctor of Philosophy THE ROLE OF FC GAMMA RECEPTORS IN THE ACTIVITY OF THERAPEUTIC MONOCLONAL ANTIBODIES Robert James Oldham Fc gamma receptors (FcγRs) are the major family of receptors responsible for interacting with immunoglobulin G (IgG). They are known to be required for the anti-tumour activity of direct targeting mAbs through expression on NK cells and macrophages. Furthermore, recent work has suggested that cross-linking via FcγRs is required for the activity of agonistic, immune modulatory mAb. This thesis sought to investigate the requirement for these receptors for different aspects of mAb activity; from T cell activation to tumour depletion, using a combination of in vitro and in vivo systems. A panel of CHO-K1 cells were generated and transfected to express the polymorphic variants of human FcγRs. These were characterised for their ability to bind IgG before being used as feeder cells in T cell proliferation assays. The assays found that cross-linking of the anti-CD28 mAb, TGN1412 by FcγRIIb (CD32b) or FcγRIIa (CD32a) but not FcγRIIIa (CD16a) transfected cells induced T cell proliferation. Furthermore, this was accompanied by the release of pro-inflammatory cytokines including TNF-α, IFN-γ and IL-2. With the importance of cross-linking via CD32b demonstrated, experiments probed the mechanism of expression using Ramos and Raji cells. These experiments investigated the gene and promoter sequences as well as the effect of epigenetic inhibitors, however further investigation is required. Next a novel mouse model was developed to investigate the efficacy of anti-human (h)CD20 mAbs. The type II mAb obinutuzumab was found to give prolonged tumour clearance compared to the type I rituximab in a fully syngeneic model with the target antigen expressed on malignant and normal B cells. The use of immune compromised mice confirmed that activatory FcγRs were required for efficient anti-CD20 mediated tumour clearance. Further experiments demonstrated that anti-CD20 mAbs could be combined with the PI3Kδ inhibitor, GS9820, to prolong tumour clearance. These experiments unexpectedly revealed that hIgG1 had an abnormally short half-life in NOD SCID mice, causing the basis for this to be examined as NOD SCID mice are widely used for immunotherapy experiments, particularly patient derived xenografts. Further investigations found that half-life could be restored through genetic deletion of mouse CD32 or by reconstitution with IgG. The polymorphic variant of CD32 found in NOD SCID mice had a higher affinity for hIgG1 which could explain the short half-life. Overall the results presented here demonstrate the multiple roles played by FcγRs in the many aspects of mAb immunotherapy, from effector cell activation to cross-linking and altering mAb half-life. This knowledge will help guide the next generation of therapeutic mAbs Table of contents List of figures ......................................................................................................................... x List of tables ........................................................................................................................ xiv Declaration of Authorship .....................................................................................................xv Acknowledgements .............................................................................................................. xvi Definitions and Abbreviations ................................................................................................ 1 Chapter 1 Literature Review ................................................................................................... 5 1.1 Cancer as a disease ................................................................................................................. 5 1.1.1 The Hallmarks of Cancer .................................................................................................. 6 1.1.2 B cell malignancies ........................................................................................................... 9 1.2 Approaches to cancer therapy ..............................................................................................10 1.3 Immunotherapy ....................................................................................................................12 1.3.1 Active immunotherapy ..................................................................................................13 1.3.2 Passive immunotherapy – immune modulation ............................................................14 1.4 B cells within the human immune system ............................................................................16 1.5 B cell origins and development in humans ...........................................................................19 1.5.1 Antibody structure and diversity ...................................................................................21 1.6 Production of monoclonal antibodies ..................................................................................24 1.7 Current therapeutic monoclonal antibodies ........................................................................26 1.8 CD20 structure and function .................................................................................................31 1.8.1 Classification of anti-CD20 mAbs ...................................................................................32 1.9 Effector mechanisms of monoclonal antibodies ..................................................................33 1.9.1 Complement dependent cytotoxicity ............................................................................34 1.9.2 Direct cell death .............................................................................................................37 Page | iii 1.9.3 FcγR mediated mechanisms .......................................................................................... 39 1.10 Anti-CD20 resistance and new developments in mAb therapy.......................................... 42 1.10.1 Overcoming rituximab resistance ................................................................................ 43 1.10.2 Antibody engineering .................................................................................................. 44 1.10.3 Combination therapies to improve anti-CD20 therapy ............................................... 44 1.11 Fcγ receptors ...................................................................................................................... 45 1.11.1 FcγR structure .............................................................................................................. 46 1.11.2 Human FcγR specificity and function........................................................................... 48 1.11.3 Mouse FcγRs ................................................................................................................ 53 1.11.4 FcγR Signalling ............................................................................................................. 54 1.11.5 Human FcγR polymorphisms ....................................................................................... 57 1.11.6 Copy number variation in human FcγRs ...................................................................... 62 1.12 Antibody half-life ................................................................................................................ 62 1.12.1 FcRn ............................................................................................................................. 63 1.13 Animal models of human disease ....................................................................................... 65 1.13.1 B cell depletion in hCD20 Tg mice ............................................................................... 66 1.13.2 Tcl-1 model of CLL like disease .................................................................................... 67 1.13.3 Immune deficient mice for patient derived xenografts .............................................. 68 1.14 Hypothesis and aims of thesis ............................................................................................ 71 Chapter 2 Materials and Methods ........................................................................................ 73 2.1 Mammalian cell culture techniques ................................................................................... 73 2.1.1 Determining cell viability ................................................................................................ 74 2.1.2 Determining cell concentration ..................................................................................... 74 2.1.3 Transient transfection of HEK293F cells for surface expression .................................... 74 Page | iv 2.1.4 Transfection of Mexi-293E cells for secreted protein.....................................................75 2.1.5 Stable transfection of CHO-K1 cells ................................................................................75 2.1.6 Selection of stably transfected CHO-K1 cells ..................................................................76 2.1.7 Fluorescence