Understanding the role for Fc gamma receptors in response to anti-CD20 monoclonal antibody ...

271 Pages·2016·6.91 MB·English

UNIVERSITY OF SOUTHAMPTON
FACULTY OF MEDICINE
Cancer sciences
Understanding the role for Fc gamma
receptors in response to anti-CD20
monoclonal antibody therapy
by
Thomas R. W. Tipton
Thesis for the degree of Doctor of Philosophy
September 2015

UNIVERSITY OF SOUTHAMPTON
ABSTRACT
FACULTY OF MEDICINE
Cancer Sciences
Thesis for the degree of Doctor of Philosophy
Understanding the role for Fc gamma
receptors in response to anti-CD20
monoclonal antibody therapy
Research into cancer therapeutics has utilized the antigen specificity of
antibodies to provide targeted cancer treatments. One notable therapeutic is
rituximab, a chimeric human IgG1 antibody that has specificity to the CD20
protein on the surface of B cells. This antibody is currently used in the
treatment of leukaemia and lymphoma. However, due to reasons not fully
understood patients often relapse and become resistant to therapy. Therefore
an increasingly important area of research is to uncover the key mechanisms of
action of anti-CD20 antibodies.
It has been demonstrated that antibody Fc: FcγR interactions are critical for
effective anti-CD20 antibody dependent cellular cytotoxicity (ADCC). During
this project the role of Fc: FcγR interactions and their ability to mediate anti-
CD20 mediated B cell depletion was explored. We assessed two different types
of anti-CD20 antibody: type I, rituximab has been shown to have high levels of
antibody:antigen internalisation following CD20 engagement. This process has
been termed antigenic modulation. In contrast, type II, obinutuzumab do not
display a high level of antigenic modulation. We assessed the ability of
rituximab and obinutuzumab to deplete normal and malignant B cells.
Additionally, macrophages and NK cells have both been implicated as key FcγR
expressing effector cells. Therefore, we assessed rituximab and obinutuzumab
using in vitro macrophage or NK cell mediated ADCC assays and looked at the
impact antigenic modulation had on effector cell activity. In vivo analysis
revealed that obinutuzumab give more sustained B cell depletion compared to
i
rituximab. Furthermore, we demonstrate that this sustained depletion is due to
a lack of antigenic modulation preventing NK cell and macrophage mediated
ADCC. In contrast rituximab undergoes significant internalisation preventing
both NK cell and macrophage mediated ADCC.
In addition, it has been well documented that the tumour microenvironment
will affect the phenotype of the surrounding stroma, particularly macrophages.
Established tumours are typically associated with low levels of apoptosis.
Therefore, we investigated the impact dysregulated apoptosis had on the
ability of macrophages to orchestrate ADCC. Using apoptosis resistant Vav Bcl-
2 mice, as well as other apoptosis defective strains, we demonstrate that there
is a decrease in the activatory to inhibitory FcγR ratio on effector cell subsets.
This was largely due to an increase in inhibitory FcγRIIb. Subsequent analysis
revealed that an increase in FcγRIIb may be due to the autoimmune-like
phenotype of the Vav Bcl-2 mouse.
This knowledge will be beneficial to future consideration of antibody selection
and provides further avenues of research into antibody and FcγR interactions.
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Table of Contents
ABSTRACT .................................................................................................... i
FACULTY OF MEDICINE ................................................................................. i
Cancer Sciences ........................................................................................... i
Thesis for the degree of Doctor of Philosophy .............................................. i
Table of Contents ....................................................................................... iii
List of tables .............................................................................................. vii
List of figures ............................................................................................. ix
DECLARATION OF AUTHORSHIP ................................................................ xiii
Acknowledgements ................................................................................... xiv
Definitions and Abbreviations .................................................................... xv
Chapter 1: General Introduction ................................................................................. 1
1.1 Background information ...................................................................... 1
1.2 B cell lymphopoiesis............................................................................ 3
1.2.1 The discovery of B cells ........................................................... 3
1.2.2 Early B cell development ......................................................... 4
1.2.3 Late B cell development .......................................................... 8
1.2.4 B cell activation ....................................................................... 9
1.2.5 Follicular lymphoma tumorigenesis ....................................... 13
1.3 Apoptosis ......................................................................................... 15
1.3.1 Mitochondrial pathway .......................................................... 16
1.3.2 Death receptor pathway ........................................................ 19
1.4 Anti-CD20 monoclonal antibody therapy ........................................... 21
1.4.1 History of monoclonal antibodies .......................................... 21
1.4.2 The structure and function of immunoglobulins.................... 22
1.4.3 Anti-CD20 monoclonal antibodies ......................................... 24
1.4.4 Mechanisms of action by anti-CD20 mAb .............................. 25
1.4.5 Resistance to anti-CD20 therapy ........................................... 28
1.5 The role of Fcγ receptors in antibody therapy .................................... 30
1.5.1 Introduction to Fc gamma receptors ..................................... 30
1.5.2 Signalling by Fc gamma receptors ......................................... 32
1.6 Macrophages .................................................................................... 34
1.6.1 The mononuclear phagocytic system .................................... 34
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1.6.2 Phagocytosis and inflammation ............................................. 36
1.6.3 Phagocytosis of apoptotic cells ............................................. 37
1.6.4 Macrophage Phenotype and polarisation ............................... 41
1.6.5 Macrophages and anti CD20 therapy ..................................... 41
1.7 Mouse Models ................................................................................... 45
1.7.1 Vav Bcl-2 mouse .................................................................... 45
1.7.2 Eµ Myc mouse ....................................................................... 46
1.8 Hypothesis and aims ......................................................................... 46
Chapter 2: Materials and Methods .......................................................................... 48
2.1 Primary CLL samples ......................................................................... 48
2.2 Animals ............................................................................................. 48
2.2.1 Animal genotyping ................................................................ 49
2.3 Antibodies ......................................................................................... 50
2.4 Tissue culture ................................................................................... 51
2.4.1 Murine L929 cells .................................................................. 51
2.4.2 Murine bone marrow derived macrophages ........................... 51
2.4.3 Monocyte derived macrophages ............................................ 52
2.4.4 Murine natural killer cells ...................................................... 53
2.5 Antibody effector function assays ...................................................... 54
2.5.1 Murine ADCP assay ............................................................... 54
2.5.2 Murine ADCC assay ............................................................... 55
2.5.3 Human ADCP assay ............................................................... 56
2.5.4 Human ADCC ........................................................................ 57
2.6 Macrophage polarisation ................................................................... 57
2.6.1 M1 polarisation ..................................................................... 57
2.6.2 M2 polarisation ..................................................................... 57
2.6.3 Detection of NO .................................................................... 58
2.6.4 Detection of M2 macrophages ............................................... 58
2.7 Adoptive transfer assay ..................................................................... 59
2.7.1 Clodronate liposome production ........................................... 60
2.8 Antigenic modulation assay ............................................................... 60
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2.9 In vivo B cell depletion experiments .................................................. 61
2.10 Enzyme linked immunosorbant assays .............................................. 61
2.10.1 Human IgG detection ............................................................ 61
2.10.2 Murine IgG detection ............................................................ 62
2.10.3 Murine IgG subset detection ................................................. 63
2.10.4 Immune complex detection ................................................... 63
2.11 Surface plasmon resonance ............................................................... 63
2.12 Size exclusion chromatography ......................................................... 64
2.13 Antibody dialysis............................................................................... 64
2.14 Generation of immune complex ........................................................ 65
2.15 Antibody deglycosylation .................................................................. 65
2.16 Detection of murine FcγR by flow cytometry ...................................... 65
2.17 Quantification of FcγR mRNA ............................................................. 66
2.18 Murine B cell subset immunophenotyping ......................................... 67
2.19 Antibody quantification ..................................................................... 67
2.20 In vitro apoptosis assay .................................................................... 68
2.21 Statistical analysis ............................................................................. 68
Chapter 3: Antigenic modulation impacts on ADCC and ADCP effector
mechanisms ............................................................................................................. 69
3.1 Introduction ...................................................................................... 69
3.2 Investigation into the impact of antibody type and class on B cell
depletion .......................................................................................... 74
3.3 Adaptation of a novel calcein based assay for detection of murine
ADCC ................................................................................................ 84
3.4 Assessment of murine and human ADCC activity in response to anti-
CD20 mAb ........................................................................................ 92
3.5 Assessment of murine and human ADCP activity in response to anti-
CD20 antibodies ............................................................................... 96
3.6 Antigenic modulation impacts on effector cell mechanisms ............. 102
3.7 Chapter discussion ......................................................................... 110
Chapter 4: Multiple FcγR are required for effective B cell depletion . 116
4.1 Chapter Introduction ....................................................................... 116
4.2 Functional redundancy between multiple FcγR................................. 118
4.2.1 Impact of silencing multiple activatory FcγR on B cell depletion
.......................................................................................... 120
4.3 De-glycosylation of 9E9 restores B cell depletion in FcγRI-/- mice ...... 127
4.4 Binding to FcγRIV is needed for cis blocking of FcγRIII ..................... 136
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4.5 Chapter Discussion ......................................................................... 138
Chapter 5: Impact of apoptosis on anti-CD20 therapy .............................. 142
5.1 Introduction .................................................................................... 142
5.2 Apoptosis resistant mice show abrogated B cell depletion ............... 144
5.3 Apoptosis resistant BMDMs are able to be polarised and can effectively
phagocytose B cell targets ............................................................... 152
5.3.1 Polarising BMDMs with apoptotic cells ................................. 157
5.4 Development of an 8 colour flow cytometry panel to investigate FcγR
expression ...................................................................................... 160
5.4.1 Antigen and Fluorochrome selection ................................... 160
5.4.2 Experimental set up ............................................................ 165
5.4.3 Anti-mouse FcγR antibody validation ................................... 167
5.5 FcγR expression in apoptosis dysregulated mice ............................. 169
5.6 Phenotyping of B cells in Vav Bcl-2 mice .......................................... 175
5.7 The impact of blocking mouse FcγRIIb on phagocytic potential in Vav
Bcl-2 mice ....................................................................................... 181
5.8 Chapter discussion .......................................................................... 185
Chapter 6: The impact of immune complex on ADCP ................................ 189
6.1 Introduction .................................................................................... 189
6.2 Apoptosis dysregulated Vav Bcl-2 mice have increased levels of
circulating IgG ................................................................................. 191
6.2.1 Vav Bcl-2 mice do not show appreciable levels of immune
complex .............................................................................. 195
6.3 Immune complex specifically block ADCP in vitro ............................ 202
6.3.1 Does immune complex block or modulate FcγR expression . 207
6.4 Chapter discussion .......................................................................... 209
Chapter 7: General discussion ............................................................................... 213
References ................................................................................................................................ 226
Appendix .................................................................................................................................... 242
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List of tables
Table 1: KD values following SPR analysis of anti- CD20 antibodies ......... 76
Table 2 List of antigen fluorochrome combinations ................................ 160
Table 3 Predicted spectral overlap between chosen fluorochromes ....... 164
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