The characterization of soluble t cell receptors specific for the parasite toxoplasma gondii

THE CHARACTERIZATION OF SOLUBLE T CELL RECEPTORS SPECIFIC FOR THE PARASITE TOXOPLASMA GONDII TAN ZHEN WEI (B.Sc.(Hons.), NTU A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Tan Zhen Wei 31 July 2014 I   Acknowledgements Being able to write this page would mean the end of a four and a half year journey, a journey which I will not be able to make alone Here, I would like to express my heartfelt thanks to these people First and foremost, I would like to thank my supervisor, Dr Gijsbert Grotenbreg This journey would not have started without him With his guidance, I could see myself grow over the years, learning the skill sets expected of a researcher He was a mentor but never appeared too far to approach and always willing to provide advice Next, I would also like to thank the members of the lab, past and present Special thanks to Ming Yan and Kai Yee, both of whom showed me the ropes when I first joined the lab To Ling yun, for offering help and support as a fellow post-grad To Jia Wei, for being in charge of peptide synthesis, taking care of the lab and making sure the lab is always in running order, not to forget being a very good gym buddy To Socks, for always willing to drive me to NTU for experiments, teaching me on SPR, as well as providing advice whenever needed To Kenneth and Lionel, for offering advice on experiments and football discussions To Joanna, Gladys, Ping Ping and all other members whom I did not list but have helped me in one way or the other in my journey Next, I would like to thank Dr Johnathan Rapley and Dr Rob Meijers (European Molecular Biology Laboratory, Hamburg Germany) for solving the crystal structure of the Rop7c1 TCR This allowed me to the supercharging part of my project I would also like to thank Dr Lee Kim Swee and Dr Hidde Ploegh (Whitehead Institute of Biomedical Research, Cambridge USA) for agreeing to share data on the effector function of the T cell clones, as well as II   the chance to collaborate on a publication currently under review I would also like to thank Dr David Thompson and Dr David Liu (Howard Hughes Medical Institute, Harvard University USA) for kindly sharing the AvNAPSA program for our supercharging experiments I would like to take this opportunity to thank Dr Markus Wenk and Dr Cynthia He as well, both of whom were ever so friendly and provided insightful advice during my TAC meetings with them I would like to thank my parents for their support during this time, for trusting in me in my decision to a Ph.D Last, I would like to thank my fiancée, Yi Zhen, for her support and her understanding that I was always late for our dates whenever I was in lab III   Table of Contents Summary……… ……………………………………………………… VII List of Tables……….………………………………… IX List of Figures………………………………………… X List of Illustrations………………………………………………… XII List of Abbreviations and symbols………………………………… XIII List of Publications…………………………………………………… XVII Chapter 1: Introduction .1 1.1 Adaptive immune system………………………………………………1 1.2 BCR recognition and diversity……………………………………… 1.3 BCR therapeutics: Monoclonal Antibodies………………………… 1.4 TCR recognition and diversity……………………………………… 14 1.5 TCR therapeutics: Adoptive T cell transfer…………………………18 1.6 TCR therapeutics: Soluble TCRs…………………………………….21 1.7 Supercharging to improve stabilities of soluble TCRs…………… 27 Chapter 2: Production and characterization of TCR clones with identical antigen specificities 2.1 Introduction…………………………………………………………… 31 2.2 Materials and Methods……………………………………………… 35 2.2.1 Cloning of TCR α and β chains…………………………………….35 2.2.2 Expression and purification of TCR α and β chains.…………….36 2.2.3 Refolding and purification of soluble TCRs……………………….36 IV   2.2.4 TCR-tetramer binding assay……………………………………….37 2.2.5 Surface Plasmon Resonance………………………………………38 2.3 Results………………………………………………………………….38 2.3.1 Cloning, expression and purification of individual TCR chains…38 2.3.2 Refolding and purification of the soluble TCR clones…………42 2.3.3 Binding specificities of refolded TCRs…………………………….48 2.3.4 Binding affinity between the three TCRs and Ld Rop7 MHC… 51 2.4 Discussion…………………………………………………………… 55 Chapter 3: Production and characterization of supercharged TCRs 3.1 Introduction………………………………………………………… 60 3.2 Materials and Methods……………………………………… 65 3.2.1 Design of supercharged Rop7c1 TCR chains………………… 65 3.2.2 Production and purification of supercharged TCR chains………66 3.2.3 Refolding of supercharged TCRs………………………………….66 3.2.4 Assessing functional avidities of supercharged TCRs………… 66 3.2.5 RAW cells staining with TCR tetramers………………………… 67 3.2.6 Stability assay of supercharged TCRs……………………………67 3.3 Results……………………………………………………………… 68 3.3.1 Crystal Structure of Rop7c1 TCR………………………………….68 3.3.2 Production of supercharged Rop7c1 TCR α and β chains…… 68 3.3.3 Production and purification of supercharged Rop7c1 TCRs……73 3.3.4 Functional avidity and specificity of supercharged TCRs……….84 3.3.5 Stabilities of supercharged Rop7c1 TCRs……………………… 88 3.4 Discussion…………………………………………………………… 98 V   Chapter 4: Comparing TCR binding affinity and effector function 4.1 Introduction……………………………………………………………105 4.2 Materials and Methods………………………………………………110 4.2.1 Surface Plasmon Resonance…………………………………….110 4.2.2 Structural modeling of Altered Peptide Ligands……………… 110 4.2.3 Stability assays for pMHCs……………………………………….110 4.3 Results……………………………………………………………… 111 4.3.1 Rop7c1, Rop7c2 and Rop7c3 recognize their cognate ligands differently…………………………………………………………….111 4.3.2 Stabilities of the Ld MHC presenting APLs…………………… 116 4.3.3 TCR binding affinity is not indicative of effector function………119 4.4 Discussion…………………………………………………………….122 Conclusion……………………………………………………………….130 Future Work………………………………………………………………131 Bibliography…………………………………………………………… 132 Annex A………………………………………………………………… 143 VI   Summary CD8+ T cells are important for resolving attacks by pathogens such as viruses and parasites Recently, transnuclear mice monoclonal for each of three TCRs recognizing the Rop7 antigenic peptide from the parasite Toxoplasma gondii were generated To better understand T cell immunity against the parasite, I characterized the binding affinities of these three T Cell Receptors (TCRs) against the Rop7 peptide MHC molecule I observed a range of binding affinities for these three TCRs Moreover, binding kinetic studies also revealed that they contact the peptide Major Histocompatibility Complex (pMHC) for different periods of time These data indicate that during an infection by the parasite, T cells expressing TCRs with a range of binding affinities are activated Thus, T cell activation is not solely dependent on the binding affinity of the TCR to its cognate ligand With the soluble TCRs generated from the binding affinity and kinetics studies, I sought to increase the stabilities of soluble TCRs so as to increase their attractiveness as a potential alternative immunoconjugate platform to monoclonal antibodies To this end, I selected the Rop7c1 TCR, which has the highest refolding yield as well as binding affinity, to try and improve its stability Increasing the surface charges of a protein have been shown to increase the thermostabilities of some proteins Thus, I increased the surface charges of the Rop7c1 TCR by selecting and mutating highly surface exposed residues to either positively or negatively charged residues However, increasing the surface charges did not improve the stability of the TCR VII   Several factors such as free energy and surface charge-charge interactions play a role in protein stability as well and thus, solely increasing the surface charges of the TCR may not be sufficient to improve its stability With our collaborator’s data, I was also able to compare the binding affinities of the three TCR clones with the effector function of the T cells expressing them I observed that the binding affinities of the TCR not correlate positively with the strength of their effector function This implies T cell effector function may not be dependent on TCR binding affinity but on other possible intrinsic factors More experiments will be required to identify these factors VIII   List of Tables Table 1: TCR gene usage .40 Table 2: AvNAPSA scores for Rop7c1 TCR 70 Table 3: Attempted refolds of supercharged TCRs………………………81 Table 4: Buffer conditions for stability assays……………………………90 IX   References 10 11 12 13 14 15 16 17 18 19 20 21 22 Cooper, M D & Alder, M N The evolution of adaptive immune systems Cell (2006) Starr, T K & Jameson, S C Positive and negative selection of T cells Annual review 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KTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNA TYPSS Predicted p.I.: 5.88 Molecular weight: 21.71 kDa Rop7c1β MEAAVTQSPRNKVTVTGGNVTLSCRQTNSHNYMYWYRQDTGHGLR LIHYSYGAGNLQIGDVPDGYKATRTTQEDFFLLLELASPSQTSLYFCAS SEAGDTEVFFGKGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATL VCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYALSSR LRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW GRALPETGGLNDIFEAQKIEWHE   Predicted p.I.: 6.14 Molecular weight: 29.03 kDa Rop7c2α MGQQVQQSPASLVLQEGENAELQCNFSTSLNSMQWFYQRPGGSLVSL FYNPSGTKQSGRLTSTTVIKERRSSLHISSSQTTDSGTYLCAMEQGNNR IFFGDGTQLVVKPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPK TMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNAT YPSS Predicted p.I.: 5.14 Molecular weight: 21.66 kDa Rop7c2β MEAAVTQSPRNKVAVTGGKVTLSCNQTNNHNNMYWYRQDTGHGLR LIHYSYGAGSTEKGDIPDGYKASRPSQENFSLILELATPSQTSVYFCASG DETKSSYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATL VCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYALSSR LRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW GRALPETGGLNDIFEAQKIEWHE   Predicted p.I.: 6.31 Molecular weight: 29.18 kDa Rop7c3α 143   MAQKVQQSPESLSVPEGGMASLNCTSSDRNFQYFWWYRQHSGEGPK ALMSIFSDGDKKEGRFTAHLNKASLHVSLHIRDSQPSDSALYFCAVSA GGSNAKLTFGKGTKLSVKSNIQNPEPAVYQLKDPRSQDSTLCLFTDFD SQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIF KETNATYPSS Predicted p.I.: 6.89 Rop7c3β Molecular weight: 22.01 kDa MGGIITQTPKFLIGQEGQKLTLKCQQNFNHDTMYWYRQDSGKGLRLI YYSITENDLQKGDLSEGYDASREKKSSFSLTVTSAQKNEMAVFLCASS RDWGYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLV CLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYALSSRLR VSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGR ALPETGGLNDIFEAQKIEWHE Predicted p.I.: 6.40 Molecular weight: 29.40 kDa Molecular Weights and p.I.s of refolded TCRs TCR Rop7c1 Rop7c2 Rop7c3 Molecular Weight (kDa) 50.72 50.82 29.40 p.I 6.08 5.97 6.40 Amino acid sequence, molecular weight and p.I of supercharged TCR chains: Rop7c1α (-25) MGQQVEQSPASLVLQEGEDAELQCNFSTSLNSMQWFYQRPGGSLVSL FYNPSGTKESGRLTSTTVIDEERSSLHISSSQTTDSGTYLCAMGDTNAY KVIFGKGTHLHVLPNIEDPEPAVYQLKDPDSDDSTLCLFTDFDSDIEVP DTMESGTFITDKCVLDMEAMDSKSNGAIAWSEETSFTCQDIFEETDAT YPSS Predicted p.I.: 3.98 Molecular weight: 21.63 kDa Rop7c1α (-16) MGQQVQQSPASLVLQEGENAELQCNFSTSLNSMQWFYQRPGGSLVSL FYNPSGTKQSGRLTSTTVIDERRSSLHISSSQTTDSGTYLCAMGDTNAY KVIFGKGTHLHVLPNIEDPEPAVYQLKDPDSDDSTLCLFTDFDSQIEVP DTMESGTFITDKCVLDMEAMDSKSNGAIAWSNQTSFTCQDIFEETNAT YPSS Predicted p.I.: 4.23 Molecular weight: 21.65 kDa 144   Rop7c1α (+6) MGQQVQQSPASLVLQEGENAELQCNFSTSLNSMQWFYQRPGGSLVSL FYNPSGTKQSGRLTSTTVIKKRRSSLHISSSQTTDSGTYLCAMGDTNAY KVIFGKGTHLHVLPNIKRPEPAVYQLKDPRSRKSTLCLFTDFDSQIKVP KTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNA TYPSS Predicted p.I.: 9.10 Molecular weight: 21.81 kDa Rop7c1α (+23) MGQQVRQSPASLVLQEGRKARLQCNFSTSLNSMQWFYQRPGGSLVSL FYNPSGTKRSGRLTSTTVIKKRRSSLHISSSQTTDSGTYLCAMGDTNAY KVIFGKGTHLHVLPNIKRPRPAVYQLKDPRSRKSTLCLFTDFDSRIKVP KTMESGTFITDKCVLDMKAMKSKSNGAIAWSRKTSFTCQDIFKKTKA TYPSS Predicted p.I.: 10.32 Molecular weight: 22.05 kDa Rop7c1β (-11) MEAAVTQSPRNKVTVTGGNVTLSCRQTNSHNYMYWYRQDTGHGLR LIHYSYGAGNLQIGDVPDGYKATRTTQEDFFLLLELASPSQTSLYFCAS SEAGDTEVFFGKGTRLTVVEDLDNVTPPKVSLFEPSKAEIANKQKATL VCLARGFFPDHVELSWWVNGEEVHSGVCTDPQAYKESEYSYALSSRL RVSATFWHNPRNHFRCQVQFHGLSEEDEWPEGSPKPVTQNISAEAWG RALPETGGLNDIFEAQKIEWHE Predicted p.I.: 5.27 Molecular weight: 29.00 kDa Rop7c1β (-25) MEAAVTQSPRNKVTVTGGEVTLSCEQTNSHNYMYWYRQDTGHGLRL IHYSYGAGNLQIGDVPDGYEATRTTQEDFFLLLELASPSQTSLYFCASS EAGDTEVFFGDGTRLTVVEDLDNVTPPEVSLFEPSDAEIANKDKATLV CLARGFFPDHVELSWWVNGEEVHSGVCTDPQAYKESEYSYALSSRLR VSATFWHNPDNHFRCQVQFHGLSEEDEWPEGSPKPVTQNISAEAWGR ALPETGGLNDIFEAQKIEWHE Predicted p.I.: 4.50 Molecular weight: 28.91 kDa Rop7c1β (+5) MEAAVTQSPRNKVTVTGGNVTLSCRQTNSHNYMYWYRQDTGHGLR LIHYSYGAGNLQIGDVPKGYKATRTTQEDFFLLLELASPSQTSLYFCAS SEAGDTEVFFGKGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATL VCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESKYSYALSSR 145   LRVSATFWHNPRNHFRCQVQFHGLSRKDKWPRGSPKPVTQNISAEAW GRALPETGGLNDIFEAQKIEWHE Predicted p.I.: 8.89 Molecular weight: 29.11 kDa Rop7c1β (+9) MEAAVTQSPRNKVTVTGGRVTLSCRQTNSHNYMYWYRQDTGHGLR LIHYSYGAGNLQIGDVPKGYKATRTTQEDFFLLLRLASPSQTSLYFCAS SEAGDTEVFFGKGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKKKATL VCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESKYSYALSSR LRVSATFWHNPRNHFRCQVQFHGLSRKDKWPRGSPKPVTQNISAEAW GRALPETGGLNDIFEAQKIEWHE Predicted p.I.: 9.33 Molecular weight: 29.18 kDa Molecular Weights and p.I.s of refolded supercharged TCRs TCR Rop7c1α 1β (-11) Rop7c1α 1β (-25) Rop7c1α (-16) 1β (-11) Rop7c1α 1β (+5) Molecular Weight (kDa) 50.64 50.55 50.63 50.74 146   p.I 5.42 4.73 4.74 8.23 ... stages of the parasite Toxoplasma gondii Toxoplasma exists as oocysts, the sexual stage of the parasite, in the guts of cats They can then infect humans or animals that come into close contact with... causing death to the patient receiving the therapy 75 In addition, it is important to choose the appropriate subsets of T cells to transfer given that there are different types of T cell subsets such... selection of cells for transfer to maximize the full therapeutic potential of this approach 20   1.6 TCR therapeutics: Soluble TCRs Another arm of T cell therapeutics would be soluble TCRs They