REDIRECTED T CELL ACTIVITY BY HIGH AFFINITY TCR-ANTI-CD3 BISPECIFIC CANDIDATE THERAPEUTICS A Thesis submitted in requirement of Cardiff University for the Degree of Doctor of Philosophy ***** Katherine Jane Adams 2013 School of Medicine 1 ACKNOWLEDGEMENTS I owe my deepest gratitude to my supervisor, Professor Andrew Sewell, and to Dr. Bent Jakobsen of Immunocore Ltd, who have given me the opportunity to undertake this long held ambition. I would like to thank my co-workers at Immunocore Ltd who have been so supportive and helpful throughout this endeavour. Special thanks goes to Dr. Giovanna Bossi who has provided excellent scientific advice and opportunities for many fruitful discussions. Huge thanks also goes to Dr. Andrew Knox for his Microsoft Word troubleshooting skills. This journey would not have been possible without the love and support of my family so I would like to thank my father, and my brother, Paul, for being there for me. Finally I would like to acknowledge my late mother who sadly died of cancer in 2009. She has been a true inspiration to me. 2 DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ………………………………………… (candidate) Date ………………… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of …………………………(insert MCh, MD, MPhil, PhD etc, as appropriate) Signed ………………………………………… (candidate) Date ………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ………………………………………… (candidate) Date ………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………… (candidate) Date ………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ………………………………………… (candidate) Date ………………… 3 ABSTRACT T cell antigen receptors (TCRs) on CD8+ T cells recognise endogenously processed peptides bound to major histocompatibility complex (pMHC) antigens presented on the cell surface on almost all types of cells in the body, including tumour cells. The majority of tumour-associated peptide antigens (TAPAs) are derived from non-mutated self-proteins and are therefore subject to immunological tolerance, mainly through negative selection of high avidity T cells in the thymus. In addition, there is low presentation of pMHC on the surface of cancer cells. As a result, T cell responses tend to be weak and ineffective at killing tumour cells. ImmTACs (Immune mobilising monoclonal T cell receptors Against Cancer) are bispecific soluble biologics comprising a soluble TCR with an enhanced affinity for tumour-associated pMHCI fused to a humanised anti-CD3 single-chain antibody fragment (scFv) which redirect and activate T cells to lyse tumour cells. In this study, the potency, sensitivity, and specificity of ImmTACs was investigated for pMHCI epitopes derived from four tumour associated antigens (TAAs): (1) gp100, (2) MAGE-A3, (3) Melan-A/MART-1, and (4) NY-ESO-1/LAGE-1. A comprehensive range of assays and methodologies have been established to characterise the ImmTAC reagents. Cytokine release assays such as IFN-γ and Granzyme B ELISpot were used to evaluate the specificity and biological activity of ImmTACs. In concentration-response experiments, all four ImmTACs produced EC50 values in the range of 100 picomolar or lower demonstrating a high degree of sensitivity despite low epitope numbers. Killing assays, including LDH-release for assessing short-term lysis and IncuCyte technology to visualise long- term killing kinetics in real time, show that redirected T cells potently kill their targets. Furthermore, in vitro screening against a panel of antigen negative, primary human cell lines have shown that ImmTACs are highly specific and only activate T cells against target cells presenting their cognate pMHC. The potency of ImmTACs was also investigated using tumour infiltrating lymphocytes (TILs) extracted from tumour specimens and with tumour-derived cancer cells as targets. An HLA-A2, gp100 specific ImmTAC has received phase I clinical trial regulatory approval in the UK and in the US on the basis of this in vitro data, which has been used to determine minimal anticipated biological effect level (MABEL). The clinical trial is currently in progress. 4 TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................... 2 DECLARATION ............................................................................................................ 3 ABSTRACT ..................................................................................................................... 4 TABLE OF CONTENTS ............................................................................................... 5 LIST OF FIGURES ...................................................................................................... 11 LIST OF TABLES ........................................................................................................ 14 ABBREVIATION LIST ............................................................................................... 15 CHAPTER 1 INTRODUCTION ................................................................................. 19 1.1 The immune system and cancer therapy: General introduction ........................ 19 1.2 Cancer ...................................................................................................................... 24 1.2.1 Cancer and cancer cells .......................................................................................... 24 1.2.2 Types of cancer ...................................................................................................... 25 1.2.2.1 Melanoma ............................................................................................... 25 1.2.2.2 Multiple myeloma ................................................................................... 28 1.3 T cell antigens in cancer ......................................................................................... 30 1.3.1 Classification of human tumour-associated T cell antigens .................................. 32 1.3.1.1 Unique Tumour Antigens ....................................................................... 32 1.3.1.2 Tumour-specific Antigens (also known as Cancer/Testis (CT) Antigens) ............................................................................................................................ 32 1.3.1.3 Differentiation Antigens ......................................................................... 33 1.3.1.4 Overexpressed Antigens ......................................................................... 33 1.3.2 Investigated tumour-associated antigens ............................................................... 36 1.3.2.1 NY-ESO-1 (and LAGE-1) ...................................................................... 36 1.3.2.2 Gp100 and Melan-A/MART-1 ............................................................... 37 1.3.2.3 MAGE-A3 .............................................................................................. 38 1.3.3 Cancer immune escape .......................................................................................... 39 1.4 Cell surface antigen presentation .......................................................................... 42 1.4.1 Type of cell surface antigen recognised by antibodies and T cell receptor (TCR) 42 1.4.2 MHC class I antigen processing ............................................................................ 42 1.4.3 MHC class I antigen presentation .......................................................................... 44 1.4.3.1 Structure and peptide binding of MHC class I molecules ...................... 45 1.5 T cells and the T cell receptor (TCR) .................................................................... 47 5 1.5.1 Structure of the T cell receptor .............................................................................. 47 1.5.2 Hypervariability ..................................................................................................... 48 1.5.3 TCR antigen recognition ........................................................................................ 51 1.5.4 TCR degeneracy .................................................................................................... 52 1.5.5 TCR thymic selection ............................................................................................ 53 1.5.6 T cell activation in the periphery ........................................................................... 55 1.5.6.1 The immunological synapse ................................................................... 55 1.5.6.2 TCR triggering and considerations for designing bispecific activator molecules ............................................................................................................ 58 1.5.7 T cell effector functions ........................................................................................