Immune presentation and recognition of class I MHC phosphopeptide antigens by Daniel Henry Stones A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Supervisor: Professor Benjamin E. Willcox University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Alterations to metabolic pathways, in particular post-translational modification, are a recognised hallmark of diseases such as autoimmunity, inflammation and cancer, and potentially provide a source of altered self antigens that can stimulate immune responses. Most notably, phosphorylated peptides have emerged as a group of tumour associated antigens which can be presented by MHC molecules and recognised by T-cells, and therefore represent promising candidates for future cancer immunotherapy strategies. However, how antigen phosphorylation impacts upon antigen presentation and recognition remains unclear. During this study I demonstrated that the phosphate moiety of phosphopeptides bearing the canonical P4 phosphorylation is more structurally diverse in its binding mode than previously thought. Strikingly, two epitopes exhibited a major conformational change upon addition of the phosphate moiety, effectively creating “conformational neoantigens”. This occurred through a similar mechanism for each epitope, whereby the presence of the phosphate moiety raised the position of the P4 Serine, allowing phosphate-mediated contacts with MHC residues and distorting the conformation of the central epitope region most critical for T- cell receptor recognition. Finally, I found that recognition of phosphopeptides can be both phosphate-dependent and epitope-specific at the level of the T-cell receptor. Therefore, this study shows that phosphorylation can have a profound and diverse effect on antigen binding, epitope identity and T-cell receptor recognition. In summary, my studies suggest that phosphopeptides are not only tumour associated but also highly antigenically distinct, establishing them as attractive candidates for cancer immunotherapy strategies. For Leanne and Ella Acknowledgements I would firstly like to thank my supervisor Ben Willcox for giving me the opportunity to undertake a PhD as part of his group and for all his support both in and out of the Lab. Also, thanks to Fiyaz Mohammed for mentoring in all things crystallography related and for all the collaborative efforts and late nights/early mornings spent shooting X-rays at crystals. A big thanks also to the past and present members of the Willcox lab; especially Carrie Willcox, Mahboob Salim and Sarah Nicholls, for all their help and advice in the lab and for mutual support during those much needed coffee breaks. Thanks to Mark Cobbold for discussions regarding HLA-B7 phosphoantigens and the cellular aspects of phosphoantigen presentation and recognition. I would like to thank Angela Zarling for providing the TCR gene constructs used for cloning into expression vectors and also Victor Engelhard and Kara Cummings for providing peptide affinity data on all of the phospho/nonphospho-peptides. Finally I would like to thank all my friends and family who have supported me over the course of this thesis, even when I was at my most stressed and irritable, this applies especially to Leanne, Ella and my parents, without your constant love and support this thesis would not have been possible. Contents 1 Introduction 1 1.1 Cancer and the immune system 1 1.1.1 Tumour biology 1 1.1.2 Evidence for tumour suppression by the immune system 3 1.2 Vertebrate Immunity 6 1.3 Antigen Presentation 13 1.3.1 Proteasomal degradation of cytosolic antigens 13 1.3.2 TAP transport 17 1.3.3 Antigen processing in the endoplasmic reticulum 18 1.3.4 Class I MHC structure 21 1.3.5 MHC-peptide binding and complex stability 24 1.3.6 Cross Presentation 25 1.4 Immunotherapy 26 1.4.1 Immunotherapy strategies 26 1.4.2 Challenges facing immunotherapy 28 1.5 Targeting post-translationally modified antigens as novel tumour antigens for immunotherapy 31 1.5.1 Types of post-translational modification 31 1.5.2 Phosphorylation 31 1.5.3 Presentation of phosphopeptide antigens 32 1.5.4 T-cell recognition of post-translational modified antigens 35 1.5.5 Phosphopeptides as novel tumour antigens for immunotherapy 36 1.6 Aims 37 2 Methods 39 2.1 Molecular Cloning Techniques 39 2.1.1 Plasmids 39 2.1.2 Bacterial cell culture 40 2.1.3 Preparation of chemically competent bacteria 40 2.1.4 Bacterial transformation with plasmid DNA 41 2.1.5 Insect S2 cell culture and transfection with plasmid DNA 41 2.1.6 Isolation of plasmid DNA 42 2.1.7 Polymerase chain reaction 42 2.1.8 Agarose gel electrophoresis 43 2.1.9 Gel purification of DNA 43 2.1.10 Restriction enzyme DNA digests and DNA ligation 44 2.1.11 DNA sequencing 44 2.2 General Protein Techniques 44 2.2.1 S2 insect cell protein expression 44 2.2.2 Ni-NTA column purification of His tagged proteins 45 2.2.3 E.coli protein expression 45 2.2.4 Inclusion body purification 46 2.2.5 SDS-PAGE 47 2.2.6 Quantification of protein 48 2.2.7 Protein refolding 49 2.2.8 Size exclusion chromatography 51 2.2.9 Anion and Cation exchange chromatography 51 2.2.10 Western blotting 51 2.2.11 3C cleavage of T-cell receptors 52 2.2.12 Biotinylation of protein complexes 52 2.3 Surface Plasmon Resonance 53 2.4 Overview of X-ray crystallography 53 2.4.1 Crystal generation 53 2.4.2 X-ray diffraction 58 2.4.3 Data processing, model building and refinement 58 2.4.4 Structural Verification, Analysis and Figure creation 59 3 Structural plasticity of phosphopeptide binding to HLA-A2 60 3.1 Background 60 3.2 HLA-A2-phosphopeptide complex production 61 3.3 Crystallisation of HLA-A2-phosphopeptide complexes 63 3.4 Data collection and processing 68 3.5 HLA-A2-phosphopeptide complex structure solution 68 3.6 HLA-A2-phosphopeptide structure refinement 71 3.7 Overall structure of HLA-A2-phosphopeptide complexes 74 3.8 Analysis of canonical phosphopeptide binding mode 77 3.9 Phosphate moiety effects on epitope binding affinity 86 3.10 Conclusions 88 4 Phosphate induced effects on HLA-A2 antigen identity 91 4.1 Background 91 4.2 HLA-A2-nonphosphopeptide complex production 92 4.3 Crystallisation 93 4.3.1 Crystallisation of HLA-A2-non-phosphopeptide complexes using commercial screens 93 4.3.2 Crystallisation of HLA-A2-non-phosphopeptide complexes with LILRB1 98 4.4 X-ray Data collection and Processing 102 4.5 HLA-A2-non-phosphopeptide complex structure solution 104 4.6 HLA-A2-non-phosphopeptide structure refinement 106 4.7 Overall Structure of HLA-A2-non-phosphopeptide complexes 108 4.8 Phosphate induced effects on epitope identity 112 4.9 Conclusions 130 5 Phosphopeptide antigen recognition by phosphopeptide specific T-cell receptors 134 5.1 Background 134 5.2 Production of soluble T-cell receptors 134 5.2.1 Expression vector cloning 134 5.2.2 Soluble T-cell receptor expression 135 5.3 IRS2 TCR binding studies 142 5.4 IRS2BK T-cell binding studies 144 5.5 CDC25B T-cell clone binding studies 148 5.6 Conclusions 162 6 Discussion 167 Appendices 177 References 199 List of Figures 1. Introduction Figure 1.1 Hallmarks of cancer 2 Figure 1.2 Cancer Immunoediting 4 Figure 1.3 Clonal Selection Theory 9 Figure 1.4 The proteasome 14 Figure 1.5 TAP transport of peptides 19 Figure 1.6 Pockets of the MHC-class I binding groove 23 2. Methods Figure 2.1 Two dimensional crystallisation phase diagram 55 Figure 2.2 Diagram of the hanging drop vapour diffusion method 57 3. Structural plasticity of phosphopeptide binding to HLA-A2 Figure 3.1 SDS-PAGE analysis of purified HLA-A2 and β2M inclusion bodies 62 Figure 3.2 Size exclusion chromatography purification of a typical HLA-A2- Phosphoeptide complex 64 Figure 3.3 SDS-PAGE analysis of refolded HLA-A2-phosphopeptide complex following purification by size exclusion chromatography 65 Figure 3.4 HLA-A2-phosphopeptide complex crystals used for X-ray diffraction 67 Figure 3.5 Typical example of HLA-A2-phosphopeptide X-ray diffraction pattern 69 Figure 3.6 Unbiased features of electron density maps 72 Figure 3.7 Ramachandran plot for HLA-A2-RLQpS complex 75 Figure 3.8 Overall structures of HLA-A2-phosphopeptide complexes 76 Figure 3.9 Individual phosphopeptide epitope structures 78 Figure 3.10 Orthogonal view of peptide binding groove 79 Figure 3.11 Plasticity of the canonical phosphopeptide binding mode 81 Figure 3.12 Phosphate mediated contacts 82 Figure 3.13 The accommodation of anchor residues in HLA-A2 phosphopeptides 84 4. Phosphate induced effects on HLA-A2 antigen identity Figure 4.1 Size exclusion chromatography purification of a typical HLA-A2- nonphosphopeptide complex 94 Figure 4.2 SDS-PAGE analysis of purified HLA-A2-nonphosphopeptide complex used for crystallisation 95 Figure 4.3 Crystals of HLA-A2-nonphosphopeptide complexes 97 Figure 4.4 Comparison of HLA-A2 structure with and without LILRB1 99 Figure 4.5 X-ray diffraction patterns of HLA-A2-RQISnp and LILRB1-HLA-A2-RTFSnp complexes 103 Figure 4.6 Overall structure of HLA-A2-nonphosphopeptide and LILRB1-HLA-A2-