CEA-Targeted Monoclonal Antibody Therapy in Colorectal Cancer

286 Pages·2010·2.98 MB·English

CEA-Targeted Monoclonal Antibody
Therapy in Colorectal Cancer

Philip J Conaghan
Green College

A thesis in submission for the degree of
Doctor of Medicine, University of Oxford

Michelmas 2009

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To Elizabeth, Joshua, Emma & Edward

and to the glory of the Almighty God

who gave them to me.
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Abstract

Introduction
The adjuvant treatment of colorectal cancer (CRC) has seen little improvement in terms
of mortality of the disease in the last 40 years. There has been a resurgence in research
into the use of monoclonal antibodies in the treatment of CRC. Carcinoembryonic antigen
(CEA) is a useful target in cancer immunotherapy. The distribution of CEA in CRC
differs from that in normal colorectal tissue. In normal colorectal tissue CEA is found
only on the luminal surface of the cell which is inaccessible to intravenous antibody,
whereas in CRC, CEA is found on all borders of the cell membrane and so becomes
accessible to intravenous antibody. However, anti-CEA antibodies are prone to
sequestration by circulating CEA. The anti-CEA antibody, PR1A3, binds only
membrane-bound CEA and thus is able to overcome this problem. The aim of my
research was to assess whether PR1A3 is suitable to be considered as a therapeutic agent
in the treatment of CRC and what its mechanism of action might be.

Methods
The level of expression of CEA on a panel of cell lines was determined under different
conditions using a solid-phase ELISA and FACS analysis. Humanized PR1A3 (hPR1A3)
was assessed in a variety of in vitro cytotoxicity assays with colorectal cell lines
expressing varying levels of CEA, using peripheral blood mononuclear cells and purified
natural killer cells as sources of effector cells. The mechanism of action of PR1A3 was
investigated by modifying the Fc fragment of the antibody and using antibodies to block
the FcIIIA receptor on the effector cells.
PR1A3 was also investigated in combination with a humanised A33 antibody.

Results
A panel of colorectal cell lines was found to have a range of CEA expression which could
be upregulated in certain cell lines by growing the cell line beyond confluence and by
treatment with the chemotherapeutic agent, 5-fluorouracil. The in vitro assays
demonstrated hPR1A3 antibody-dependent and CEA-specific killing of tumour cell lines
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by human PBMC. The effect increased with increasing concentration of antibody and was
lost by using the parent murine IgG1 PR1A3. Using 50g/ml hPR1A3, tumour cell lysis
was increased by more than 3-fold above spontaneous killing (p<0.001) in a high CEA-
expressing cell line. Both antibody-dependent and antibody-independent (spontaneous)
killing was blocked by using whole antibody to the Fc-IIIA receptor, although the
spontaneous killing was restored when a F(ab‟) was used instead of whole antibody.
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hPR1A3 and the A33 antibody showed potential synergy when used in combination
against a high-CEA and a moderate-A33 expressing cell line.

Conclusion
The monoclonal antibody hPR1A3 causes CEA-specific lysis of human colorectal cancer-
derived cell lines in the presence of human PBMCs. This lysis is dependent on the dose of
the antibody, requires a compatible Fc-receptor and is inhibited by blockade of the
FcγIIIA receptor. These findings show that hPR1A3 can kill tumour cells by antibody-
mediated cellular cytotoxicity (ADCC) and implicate NK cells as a major contributor to
this effect.
The results support the development of hPR1A3 for therapy of colorectal cancer.

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Acknowledgements

I should like to thank the numerous people who provided me with support, help and
advice during the execution of this thesis but in particular the following people and
institutions:

Members of the Cancer and Immunogenetics Laboratory, Weatherall Institute of
Molecular Medicine, Oxford for their experience and advice at various stages of the
project

Dr Jenny Wilding, Cancer and Immunogenetics Laboratory, for her invaluable teaching,
guidance, supervision and advice throughout the entire project and during the writing of
this thesis

Dr Elma Tchilian, the Jenner Institute, Oxford University, for encouragement and helping
me with the FACS analysis

Messrs Matthew Tytherleigh and Shazad Ashraf, my predecessor and successor
respectively

The staff and supporters of the CORE charity who awarded me a Research Fellowship
and funded me during my period of research

The staff and supporters of Cancer Research UK who funded the work in the Cancer and
Immunogenetics Laboratory

The Jacqueline Seroussi Foundation and the Oxfordshire Health Services Research
Committee for financial support of this work

Prof Neil Mortensen, Professor of Colorectal Surgery, John Radcliffe Hospital, Oxford
for his help, support and encouragement throughout not only my research but also my
surgical career and for his supervision during this period of research

Prof Sir Walter Bodmer, Cancer and Immunogenetics Laboratory, for supervising this
thesis, for his unerring judgement, for guidance and encouragement and for being a
constant source of ideas and enthusiasm
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CONTENTS

Chapter 1: Introduction

Chapter 2: Materials and Methods

Chapter 3: Assessment of CEA expression on colorectal cell lines and
factors influencing this expression

Chapter 4: ADCC of cancer cell lines using PR1A3

Chapter 5: The importance of the FcA receptor and the antibody Fc-
fragment in the action of PR1A3

Chapter 6: Combination therapy: PR1A3 with other antibodies

Chapter 7: Future directions

Chapter 8: Conclusions
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CHAPTER 1 Introduction

1.1 Colorectal cancer and its treatment
1.1.1 Incidence
1.1.2 Aetiology
1.1.3 Natural history of CRC
1.1.4 Presentation of CRC
1.1.5 Investigation
1.1.6 Pre-operative staging
1.1.7 Pathology and post-operative staging
1.1.8 Screening for bowel cancer
1.1.9 Current treatment of colorectal cancer
1.1.9.1 Early rectal cancer
1.1.9.2 Surgery with curative intent
1.1.9.3 Neoadjuvant treatment for rectal cancer
1.1.9.4 Adjuvant treatment for colorectal cancer
1.1.9.5 Metastatic disease
1.1.9.6 Palliative treatment
1.1.10 Novel treatments
1.1.10.1 Tailoring treatment based on genetic profiles
1.1.10.2 Targeting the cyclo-oxygenase (COX) enzymes
1.1.10.3 Small molecule inhibitors

1.2 Antibody based cancer therapy
1.2.1 Introduction
1.2.2 Antibody structure and function
1.2.3 Monoclonal antibodies
1.2.4 Suggested mechanisms of naked antibody-based cancer therapy
1.2.4.1 Antibody-dependent cellular cytotoxicity
1.2.4.2 Complement-dependent cytotoxicity
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1.2.4.3 Anti-idiotypic networks
1.2.4.4 Receptor modulation
1.2.4.5 Activation of the adaptive immune response
1.2.4.6 Effect on angiogenesis
1.2.5 Naked antibodies as anti-cancer drugs in clinical practice
1.2.5.1 Rituximab
1.2.5.2 Trastuzumab
1.2.5.3 Cetuximab
1.2.5.4 Bevacizumab
1.2.5.5 Panitumumab
1.2.6 Radioimmunotherapy
1.2.6.1 The antibody
1.2.6.2 The radionuclide
1.2.6.3 Radioimmunotherapy and colorectal cancer
1.2.7 Antibodies conjugated with other ligands
1.2.7.1 Pretargeting strategies
1.2.8 Immunotherapy with antibody fragments
1.2.9 Models of colorectal cancer for antibody-based cancer therapy
1.2.9.1 Cell-lines
1.2.9.2 Animal models
1.2.9.2.1 Xenografts
1.2.9.2.2 Syngeneic models
1.2.9.2.3 Transgenic models
1.2.9.2.4 Ideal animal models

1.3 The Fc receptor family
1.3.1 The FcIIIA receptor in ADCC in humans
1.3.2 Role of NK cells in FcIIIA-mediated cytotoxicity in humans

1.4 Carcinoembryonic Antigen
1.4.1 CEA family of cell surface glycoproteins
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1.4.2 Expression of CEA in normal and neoplastic tissues
1.4.3 Proposed functions of CEA
1.4.4 Advantages of CEA as a target for ADCC

1.5 The monoclonal antibody PR1A3
1.5.1 The biology of PR1A3
1.5.2 Binding of PR1A3 to colorectal tissue
1.5.3 Animal studies using mPR1A3 in the Apcmin+/-CEA.tg+/- mouse model of
colorectal cancer
1.5.4 Radioimmunoscintigraphy using PR1A3
1.5.5 Radioimmunoguided surgery
1.5.6 PR1A3 and human trials
1.5.7 PR1A3 and antibody-based cancer treatments

1.6 Factors influencing ADCC
1.6.1 Antibody
1.6.1.1 Isotype
1.6.1.2 Binding affinity with target and binding site barrier
1.6.1.3 Glycosylation
1.6.1.4 Fc sequence modification
1.6.1.5 Prolonging antibody half-life
1.6.1.6 Internalisation of Ab-target complex
1.6.2 Effector cell
1.6.2.1 Enhancing Fca expression
1.6.2.2 High-binding Fca polymorphism
1.6.2.3 Inhibiting FcRIIB
1.6.3 Local tumour environment
1.6.3.1 Other inhibitors of ADCC
1.6.3.2 Cytokines
1.6.3.3 Tumour vascularisation
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1.7 Project aims

CHAPTER 2 Materials and Methods

2.1 Cancer cell lines
2.1.1 Biography of cell lines used in study
2.1.2 Cell culture conditions

2.2 Human effector cells
2.2.1 Peripheral blood mononuclear cells (PBMC)
2.2.1.1 Isolation of PBMCs from fresh whole blood
2.2.1.2 Isolation of PBMCs from leucodepletion filters
2.2.2 Isolation of CD56+ CD3- cells (Natural Killer subset)

2.3 Antibodies
2.3.1 ADCC assays
2.3.2 ELISA
2.3.3 Fluorescence-activated cell sorting (FACS) analysis

2.4 Antibody-dependent cellular cytotoxicity (ADCC) assays
2.4.1 51Chromium release assay
2.4.1.1 Target cell preparation
2.4.1.2 Optimisation of labelling
2.4.1.3 Effector cell and antibody preparation
2.4.1.4 Assay conditions and measurement of cytotoxicity
2.4.2 Fluorescence-based assay using Europium-TDA
2.4.2.1 Target cell preparation
2.4.2.2 Optimisation of labelling
2.4.2.3 Effector cell and antibody preparation
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