Characterisation of anti-glycan monoclonal antibodies

263 Pages·2017·5.09 MB·English

by  Christine Middleton

Characterisation of anti-glycan

monoclonal antibodies
Philip .W. Noble B.Sc (Hons)

Thesis submitted to the University of Nottingham

for the degree of Doctor of Philosophy, April 2011
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I dedicate thesis in memory of my Nan, Janet Ellis
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Table of Contents
Table of Contents ..................................................................................................................................... i
Abstract .................................................................................................................................................. vi
Abbreviations ....................................................................................................................................... viii
Acknowledgements .............................................................................................................................. xiii
Chapter 1 Introduction ........................................................................................................................... 1
1.1 Cancer ........................................................................................................................................... 1
1.1.1 Treatment of colorectal and ovarian cancer .......................................................................... 1
1.2 Immunity ....................................................................................................................................... 2
1.2.1 The B cell ............................................................................................................................... 2
1.2.2 B cell Maturation .................................................................................................................. 4
1.2.3 Humoral immune response ................................................................................................. 8
1.2.4 Antibody Structure ............................................................................................................. 11
1.2.5 Antibody Glycosylation ...................................................................................................... 13
1.2.6 Effector mechanisms of antibodies ................................................................................... 15
1.2.7 Complement ........................................................................................................................ 16
1.2.8 Complement cascade ......................................................................................................... 16
1.2.9 Antibody dependent cellular cytotoxicity ........................................................................ 20
1.2.10 Antibody dependent cellular phagocytosis .................................................................... 22
1.2.11 Monoclonal antibodies .................................................................................................... 22
1.2.12 Monoclonal antibody production ................................................................................... 22
1.2.13 Monoclonal antibodies for therapeutic use ................................................................... 23
1.2.14 Approved monoclonal antibodies ................................................................................... 28
1.3 Glycolipids ................................................................................................................................... 37
1.3.1 Synthesis and structure of GSL.......................................................................................... 39
1.3.2 Function of GSLs ................................................................................................................. 41
1.3.3 Mechanisms of altered expression of glycans .................................................................. 45
1.3.4 Aberrant expression of glycosyl epitopes in cancer ........................................................ 46
1.3.5 Glycolipids as targets for monoclonal antibodies ............................................................ 50
1.4 Cell death .................................................................................................................................... 53
1.4.4 Apoptotic Pathways ............................................................................................................. 55
1.4.2 Intrinsic Pathway .................................................................................................................. 56
1.4.3 Extrinsic pathway ................................................................................................................. 58
1.4.1 Oncosis ................................................................................................................................. 61
1.4.2Autophagy ............................................................................................................................. 61
1.4.3 Pryoptosis ............................................................................................................................. 62
Aim of thesis ......................................................................................................................................... 63
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Chapter 2 Materials and Methods ........................................................................................................ 65
2.1 mAbs and Human cancer cell line culture .................................................................................. 65
2.1.1 Commercial mAbs ................................................................................................................ 65
2.1.2 In house production of mAbs ............................................................................................... 65
2.1.3 Biotin and FITC labelling of mAbs ........................................................................................ 66
2.2 Cell culture .................................................................................................................................. 67
2.2.1 Cell lines and hybridomas .................................................................................................... 67
2.2.2 Maintenance of cell lines and hybridomas .......................................................................... 68
2.3 Immunohistochemical analysis ................................................................................................... 69
2.3.1 Tissue preparation ............................................................................................................... 69
2.3.2 Antigen retrieval .................................................................................................................. 71
2.3.3 Prevention of non-specific binding ...................................................................................... 71
2.3.4 Application of primary antibody .......................................................................................... 72
2.3.5 Washing................................................................................................................................ 72
2.3.6 ABC method ......................................................................................................................... 73
2.3.7 Visualisation of the primary antibody .................................................................................. 74
2.3.8 IHC protocol ......................................................................................................................... 75
2.3.10 Evaluation of staining ......................................................................................................... 76
2.3.11 Data processing .................................................................................................................. 76
2.4 Immunohistochemical staining of colorectal TMA for cleaved capsase-3 and MHC-II expression
.......................................................................................................................................................... 77
2.4.1 Patient study and design ...................................................................................................... 77
2.4.2 Clinicopathological variables for the patient cohort (n = 462) ............................................ 78
2.4.3 Monoclonal antibody binding to ovarian cancer and normal tissue TMA ........................... 80
2.5 Screening of mAbs on The Consortium for Functional Glycomics array..................................... 82
2.5.1 Screening on glycan array .................................................................................................... 82
2.6 In vitro functional studies of mAbs, murine serum and hybridoma supernatants ..................... 82
2.6.1 Analysis of antibody binding to cancer cells ........................................................................ 83
2.6.3 Analysis of binding to cancer cell glycolipid extraction ....................................................... 86
2.6.4 Analysis of antibody mediated direct cell death .................................................................. 89
2.6.5 Antibody dependent cellular cytotoxicity and complement dependent cytotoxicity assays
...................................................................................................................................................... 92
2.7 Immunisations............................................................................................................................. 93
2.7.1 Preparation of whole cancer cells ........................................................................................ 93
2.7.2 Preparation of glycolipid ...................................................................................................... 93
2.7.3 Preparation of liposomes ..................................................................................................... 94
2.7.4 Adjuvants ............................................................................................................................. 94
2.7.5 Immunisation protocol......................................................................................................... 95
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2.7.6 Analysis of antibody response to immunisations ................................................................ 95
2.7.7 Isolation of splenocytes ....................................................................................................... 95
2.7.8 Collection of large quantities of sera from immunised mice ............................................... 96
2.7.9 Fusion of Mice splenocytes with NS0 myeloma cells .......................................................... 96
2.8 Statistical analysis of data ........................................................................................................... 97
2.8.1 Immunohistochemical analysis ............................................................................................ 97
2.8.2 Other analysis ...................................................................................................................... 97
Chapter 3 Cleaved caspase-3 expression in colorectal cancer ............................................................. 98
3.1 Background ................................................................................................................................. 98
3.2 Results ....................................................................................................................................... 101
3.2.1 Clinical and pathological data ............................................................................................ 101
3.2.2 CC3 expression ................................................................................................................... 103
3.2.3 Markers of apoptosis and inflammation: Bcl2, p53, CD3, CD68, CD16 and MHC-II
expression ................................................................................................................................... 105
3.2.4 Relationship between CC3 expression and standard clinicopathological variables .......... 105
3.2.5 Relationship between MHC-II expression and standard clinicopathological variables ..... 106
3.2.6 Relationship between CC3 expression and markers of immune activation and apoptosis
.................................................................................................................................................... 107
3.2.7 Relationship between MHC-II expression and markers of immune activation and apoptosis
.................................................................................................................................................... 107
3.2.8 Relationship between CC3 expression and survival .......................................................... 108
3.2.9 Relationship between MHC-II expression and survival ..................................................... 110
3.2.10 Multivariate analysis of CC3 expression and standard clinicopathological variables ...... 110
3.3 Discussion .................................................................................................................................. 112
Chapter 4 Characterisation of Lewis y/b mAb specificity and in vitro functionality ........................... 116
4.1 Introduction .............................................................................................................................. 116
4.2 Results ....................................................................................................................................... 118
4.2.1 Defining the epitopes recognised by the Lewis y/b antibodies using the Consortium for
Functional Glycomics glycan array .............................................................................................. 118
4.2.2 Binding characteristics of anti-Lewis y/b mAbs on a range of antigen positive cell lines . 124
4.2.3 Determining self-binding (homophilic binding) of the Lewis antibodies. .......................... 126
4.2.4 Induction of ADCC and CDC in cancer cells by 692/29, BR96 and 2-25 LE......................... 131
4.2.5 Induction of cell death in colorectal cancer cells by 692/29, BR96 and not 2-25 LE ......... 133
4.2.6 Cross-linking the IgG mAb 2-25 LE does not induce direct cell death .............................. 136
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4.2.7 692/29 and BR96 induce cell death via an alternative mechanism to apoptosis death ... 137
4.3 Discussion .................................................................................................................................. 141
Chapter 5 Lewis a mAbs ...................................................................................................................... 146
5.1 Introduction .............................................................................................................................. 146
5.2 Results ....................................................................................................................................... 147
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5.2.1 Analysis of 505/4 therapeutic value by normal and colorectal cancer distribution .......... 147
5.2.2 Characterisation of 505/4 antigen ..................................................................................... 152
5.2.3 Analysis of 505/4, CA19.9 and 7LE binding to the Consortium for Functional Glycomics
array ............................................................................................................................................ 153
5.2.4 Analysis of 505/4, CA19.9 and 7LE colorectal cell line binding .......................................... 157
5.2.5 Direct killing ability of 505/4, 7LE and CA19.9 mAbs ......................................................... 159
5.2.6 Mechanism of killing of colorectal cancer cells by 505/4, 7LE and CA19.9mAbs .............. 162
5.3 Discussion .................................................................................................................................. 165
Chapter 6 The production of monoclonal antibodies targeted against ovarian cancer glycolipid ..... 170
6.1 Background ............................................................................................................................... 170
6.2 Results ....................................................................................................................................... 171
6.2.1 Glycolipid extracted from C170 cells binds both 692/29 and 505/4 mAbs ....................... 171
6.2.2 Immunisation of BALB/c mice with ovarian cancer cells ................................................... 172
6.2.2 Fusion (F012) of OVCA 433 immunised mouse splenocytes yields anti-ovarian cancer
glycolipid hybridomas ................................................................................................................. 173
6.2.3 Immunisation with the mAb TIB 207 depletes mouse T cells in vivo ................................ 175
6.2.4 Depletion of CD4 cells is ineffective at increasing the anti-ovarian cancer glycolipid
response ...................................................................................................................................... 176
6.2.5 Fusion (F013) of TIB-207/OVCA 433 immunised mouse splenocytes yields anti-ovarian
cancer cell but not anti-glycolipid hybridomas ........................................................................... 177
6.2.7 Immunisation of BALB/c mice with an NK cell adjuvant .................................................... 178
6.2.9 Immunisation of colorectal cancer glycolipid extract does not give an anti-glycolipid
response ...................................................................................................................................... 179
6.2.10 Ovarian cancer cell glycolipid immunisation ................................................................... 181
6.2.11 Preparation of ovarian cancer glycolipid-containing liposomes for immunisation ......... 183
6.2.12 Immunisation with OVCA glycolipid-containing glycolipid with αGalCer ........................ 184
6.2.13 Fusion of splenocytes from mice 2-1R and 2-1L .............................................................. 185
6.2.14 Immunisation with 2.5% OVCA glycolipid and αGalCer containing liposomes boosted with
OVCAR-4 cells .............................................................................................................................. 187
6.2.15 Fusion of splenocytes from 2-1L and initial screening ..................................................... 189
6.2.16 Further characterisation and cloning of F019/1A7 and F019/1D3 .................................. 190
6.2.17 Screening of F019/1A7 against ovarian cancer tissue array ............................................ 192
6.2.18 Staining of normal tissue array with F019/1A7 ............................................................... 195
6.2.19 Evaluation of direct killing with F019/1A7 ....................................................................... 200
6.2.20 F019/1A7 cytotoxicity at different temperatures and with cross-linking........................ 202
6.2.21 Ability of F019/1A7 to induce ADCC in target cells with cross-linking ............................. 203
6.3 Discussion .................................................................................................................................. 204
Chapter 7 Final Discussion .................................................................................................................. 210
What is the therapeutic potential of mAbs? .................................................................................. 210
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Why target glycolipids? ................................................................................................................... 210
What are the ideal antigens? .......................................................................................................... 211
Functionality of the anti-tumour mAb ............................................................................................ 212
Human mAbs ................................................................................................................................... 215
Serum half-life ................................................................................................................................. 216
Other potential targets ................................................................................................................... 217
Bibliography ........................................................................................................................................ 223

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Abstract
The aims of this thesis are to establish the therapeutic value of two anti-glycan mAbs produced in-
house, to develop an immunisation protocol with the aim of improving the immunogenicity tumour-
associated glycolipids with the intention of producing therapeutically valuable mAbs and to
determine the implication of a mAb with the ability to induce apoptosis in colorectal cancer.
The anti-glycan mAbs 692/29 and 505/4 have previously been produced in-house and this study
aimed to determine their fine specificity using a glycan array. 692/29 displayed binding
predominantly to Lewis b as well as Lewis y-containing glycans. 505/4 was discovered to bind to
sialyl Lewis a as well as sialyl di-Lewis a, with no cross-reactivity with other blood group antigens.
This was compared to other anti-Lewis mAbs, with differences in specificity being observed.
Characterisation of 505/4 mAb distribution showed binding to 80% of colorectal tumours and low
levels of binding to normal tissues by IHC, suggesting it may be therapeutically useful.
This thesis aimed to assess the ability of 505/4 and 692/29 to meditate immune mediated and non-
immune mediated cell death as well as to determine whether non-immune-mediated cell death
would be a desirable therapeutic property. Resistance to apoptosis is one of the hallmarks of cancer
cells and mAbs stimulating apoptosis may not be very effective. To investigate the significance of
apoptosis in cancer a large tissue microarray of colorectal tumours was assessed for apoptosis and
its relationship to patient prognosis. Cleaved caspase-3 is a good marker of apoptosis as it is the
executioner caspase for both the extrinsic and intrinsic pathways. Immunohistochemical analysis of
colorectal tumour samples revealed that a high expression of cleaved caspase-3 in tumour was
associated with good prognosis in colorectal cancer. This suggested that some tumours were still
susceptible to apoptotic death but some are resistant and an alternative mechanism of cell death
may be an advantage in these tumours. High expression of cleaved caspase-3 in the tumour-
associated stroma was also an independent marker of good prognosis in colorectal cancer. This may
be because apoptosis of the tumour-associated stroma reduces the level of pro-tumour signals
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originating from tumour-associated immune cells and stromal cells. As the tumour
microenvironment can act in an immunosuppressive and pro-tumour manner, the ability of a mAb to
induce direct cell death without the need for effector cells or complement would be an advantage.
Lewis y and Lewis b are blood group antigens commonly overexpressed on the surface of a range of
cancers. Characterisation of effector functions of 505/4 and 692/29 demonstrated that both mAbs
have the ability to mediate apoptosis by antibody dependent cellular cytotoxicity, complement
dependent cytotoxicity and cause direct cell death in an oncosis-like manner. Comparison with other
anti-Lewis mAbs demonstrated that a number of anti-Lewis mAbs can induce direct cell death
independently of apoptosis. Thus, they could effectively target apoptotic sensitive and resistant
colorectal cancers.
Tumours aberrantly express glycolipids and these molecules may be involved in a number of cellular
pathways. In addition a large proportion of anti-glycan mAbs, including 505/4 and 692/29 in this
thesis, have displayed the ability to induce direct cell death. Therefore this thesis aimed to develop
an immunisation protocol capable of increasing the immunogenicity of tumour-associated glycolipid
for the production of anti-tumour glycolipid mAbs directed against ovarian cancer. This study
suggests that the incorporation of tumour glycolipid into liposomes and their immunisation along
with the iNKT cell adjuvant α-galactosylceramide, elicits an anti-tumour glycolipid immune response,
which can yield IgG mAbs capable of binding a high proportion of ovarian cancers.
In summary, this thesis confirmed specificity of 692/29 to Lewis y and Lewis b and 505/4 to sialyl
Lewis a and sialyl di-Lewis a. Furthermore, this thesis demonstrated a promising tissue distribution
of 505/4 in vitro. Characterisation of mAb effector functions suggest that both Lewis y and sialyl
Lewis a directed mAbs have the ability to cause direct cell death, independently of apoptosis in
antigen positive cells, as well as the ability to cause immune-mediated cell death. This may be an
important factor in the immune-suppressive tumour microenvironment. Furthermore, this thesis
provides the basis for the production of new anti-glycolipid antibodies that may also be able to
induce direct cell death.
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