Biothevapeutics Recent Developments using Chemical and Molecular Biology

Edited by

Lyn H. Jones R&D WorldWide Medicinal Chemistry Cambridge, MA USA Email: [email protected]

and

Andrew J. McKnight AnaptysBio, Inc. San Diego, CA USA Email: [email protected]

RSC Publishing Contents

Chapter 1 Synthetic Immunology 1 Thihan R. Padukkavidana, Patrick J. McEnaney and David A. Spiegel

1.1 Introduction and Scope 1 1.2 Synthetic Ligands for Pattern Recognition Receptors (PRRs) 2 1.2.1 Synthetic Mimics of TLR Ligands 2 1.2.2 Synthetic NOD Agonists 6 1.2.3 Peroxisome Proliferator-activated Receptor Gamma 8 1.2.4 C-Type Lectin Receptors (CLRs) 9 1.3 Synthetic Systems for Controlling Cell-Cell Communication: Cytokine Mimetics 10 1.4 Synthetic Molecules that Modulate the Complement System 13 1.5 Mimicking and Hijacking Function 15 1.6 Synthetic Molecules that Modulate T- and B-cell Responses 17 1.7 Concluding Remarks 22 References 22

Chapter 2 Immunopharmacotherapy for Nicotine Addiction 36 Jonathan W. Lockner and Kim D. Janda

2.1 Introduction 36 2.2 Nicotine and the Body 37 2.3 Nicotine and the Brain 38 2.4 Nicotine and the Immune System 40

RSC Drug Discovery Series No. 36 Biotherapeutics: Recent Developments using Chemical and Molecular Biology Edited by Lyn H. Jones and Andrew J. McKnight © The Royal Society of Chemistry 2013 Published by the Royal Society of Chemistry, www.rsc.org

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2.5 Nicotine and the Antibody 41 2.6 Preclinical Studies of Nicotine Immunopharmacotherapy 43 2.6.1 Janda 44 2.6.2 Pentel 45 2.6.3 Sanderson 46 2.6.4 Bunce/Bond/Aker 47 2.6.5 Maurer/Bachmann 47 2.6.6 Cerny 48 2.6.7 Svensson/de Villiers 48 2.6.8 Crystal 49 2.7 Clinical Trials of Nicotine 49 2.7.1 Cytos Biotechnology AG 50 2.7.2 ImmuLogic/Cantab/Xenova/Celtic Pharma 50 2.7.3 Nabi Biopharmaceuticals/Biota Pharmaceuticals 51 2.7.4 Chilka Limited 51 2.7.5 Independent Pharmaceutica AB 52 2.7.6 Selecta Biosciences 52 2.7.7 Pfizer Vaccines Ltd 52 2.8 New Directions to Improve Immunogenicity 53 2.8.1 Carrier 54 2.8.2 Delivery Method 54 2.8.3 Adjuvants 55 2.8.4 Hapten Design 56 2.8.5 Caveat: No Nicotine Should Be Viewed as a "Magic Bullet" Cure 57 2.8.6 Miscellany 58 2.9 Conclusions and Future Prospects 59 Acknowledgements 60 References 60

Chapter 3 Carbohydrate Vaccines 68 Benjamin Schumann, Chakkumkal Anish, Claney L. Pereira and Peter H. Seeberger

3.1 Introduction 68 3.1.1 Cell Surface Glycans as Vaccine Candidates 69 3.1.2 Immunology of Carbohydrate-based Vaccines 72 3.1.3 Production and Manufacture of Glycoconjugate Vaccines 74 3.1.4 Opportunities and Challenges of Vaccines Based on Synthetic Oligosaccharides 77 3.2 Chemical Immunology of Glycoconjugate Vaccines 77 3.2.1 Effect of the Glycan on the Immune Response 78 3.2.1.1 Saccharide Length 78 3.2.1.2 Epitope Charge 79 Contents ix

3.2.1.3 Exposed Epitopes 80 3.2.1.4 Monosaccharide Modifications 82

3.2.2 Effects of Conjugation on the Immune Response 83 3.2.2.1 Conjugation Chemistry 83 3.2.2.2 Immunogenicity of Linker/Spacer Constructs 83 3.2.2.3 Density 83 3.2.2.4 Epitopes 84 3.2.2.5 Carrier Priming and Carrier-Induced Epitope Suppression 85 3.3 Novel Developments in Vaccine Design 86 3.3.1 Multicomponent Vaccines 86 3.3.2 Novel Carrier Platforms 87 3.3.3 Rational Vaccine Design 88 3.4 Conclusions 89 Acknowledgements 90 References 90

Chapter 4 Generation and Maturation of Therapeutic via In Vitro Somatic Hypermutation 105 David J. King

4.1 Antibodies as Therapeutic Agents 105 4.2 Approaches to the Generation of Therapeutic Antibodies 106 4.2.1 Recovery of Antibodies from In Vivo Sources 108 4.2.2 Recovery of Antibodies from In Vitro Sources 110 4.3 Mammalian Cell Display 113 4.4 Somatic Hypermutation (SHM) 117 4.5 Combining Mammalian Cell Display with In Vitro SHM 119 References 123

Chapter 5 Synthetic Protein Biologies 130 Benjamin G. Davis

5.1 Introduction and Strategy 130 5.1.1 as a Parallel to Synthetic Organic Chemistry 130 5.1.2 Synthetic Biology as the Driver for Chemical Medicine 132 5.1.3 Current Strategic Small-Mindedness and Opportunities Beyond It 133 5.1.4 Current Limitations Provide Further Opportunities 134 X Contents

5.2 Synthetic Vaccines 135 5.3 Pegylation Technology and the Pegylation of Proteins 137 5.4 Synthetic Biologies with Payloads 139 5.5 Future Prospects and Conclusions 140 References 142

Chapter 6 Recent Advances in Antibody-Drug Conjugates 145 Edmund I. Graziani and L. Nathan Tumey

6.1 Introduction 145 6.2 Recent Developments in ADC Linker Technology 146 6.2.1 Cleavable Linkers 146 6.2.2 Noncleavable Linkers 153 6.2.3 Impact of Linkers on Biophysical Properties 155 6.3 Recent Developments in Site Specific Conjugation of ADCs 156 6.3.1 Engineered Cysteines 157

6.3.2 Unnatural Amino Acids as Reactive Handles for Antibody Conjugation 160 6.3.3 Chemical and Enzymatic Methods for Site-Specific Protein Conjugation 162 6.4 Recent Developments in ADC Metabolism and PK 164 6.4.1 Payload Release In Targeted Tissues 165 6.4.2 Premature Payload Release In Circulation And Non-Targeted Tissues 166 6.4.3 Pharmacokinetics/Pharmacodynamics 167 6.4.4 ADC Clearance and Elimination 168 6.5 Summary and Future Prospects 169 References 170

Chapter 7 MicroRNA Biotherapeutics: Key Challenges from a Drug Development Perspective 176 Sterghios Athanasios Moschos

7.1 Introduction 176 7.2 MicroRNA Biogenesis 180 7.2.1 Genomic Organization of Endogenous miRNAs 181 7.2.2 Virus-Encoded miRNAs 182 7.2.2.1 Herpesviruses 182 7.2.2.2 Adenovirus 183 7.2.2.3 RNA Viruses: an Unlikely, yet Flexible Group of Vectors 183 7.2.3 Canonical miRNA Maturation 184 7.2.3.1 Excision of miRNA Precursors from Primary Transcripts 185 7.2.3.2 Nuclear Export of Pre-miRNAs 186 Contents xj

7.2.3.3 Removal of the Hairpin Loop 186 7.2.3.4 Selection and Loading to Effector Protein Complexes 187 7.2.3.5 Guide Strand Selection 188 7.2.4 EXP5 and Safety of Ectopic RNAi Mediator Expression 189 7.2.5 Alternative Maturation Pathways 190 7.2.5.1 Mirtrons and Simtrons 190 7.2.5.2 miRNAs Encoded in Small Nucleolar RNAs 192 7.2.5.3 miR-451 193 7.2.5.4 miRNA Offset RNAs 193

7.2.5.5 Viral Lessons on RNAi Precursor Maturation 194 7.2.6 Post-Transcriptional Processing of miRNAs 195 7.3 miRNA Function 198 7.3.1 Molecular Actions of miRNAs: Therapeutic Challenges and Opportunities 198 7.3.1.1 Ago2-Mediated Translational Repression 198 7.3.1.2 Slicer-Independent Translational Repression 200 7.3.1.3 Other Mechanisms of miRNA Action 204 7.4 Concluding Remarks: the Impact of the Developing miRNA Knowledge Base on Biotherapeutics Development Efforts 207 References 209

Chapter 8 Novel Therapeutic Agents from Bacterial Toxins 224 John A. Chaddock

8.1 Introduction 224 8.2 Therapeutic Opportunities from Cytotoxic Bacterial Toxins 226 8.2.1 Pre-Clinical Studies with Cytotoxic Bacterial Toxin Domains 226 8.2.1.1 Diphtheria Toxin-Based Opportunities 226 8.2.1.2 Pseudomonas Exotoxin-Based Opportunities 227 8.2.2 Clinical Application of Cytotoxic Bacterial Toxin Domains 228 8.3 Therapeutic Opportunities from Non-Cytotoxic Bacterial Toxins 229 8.3.1 Clinical Application of Natural Toxin Products 229 xii Contents

8.3.2 The Domain Structure of BoNT as a Template for New Product Engineering 231 8.3.3 Utilising Toxin Domains to Deliver Therapeutic Cargo 232 8.3.4 Harnessing the Properties of Clostridial Neurotoxin Domains in Engineered Hybrids 233 8.3.4.1 Creation of Improved Product Opportunities 233 8.3.5 Targeted Secretion Inhibitors 234 8.3.5.1 Concept and Potential 234 8.3.5.2 Implementing the Concept at the Research Phase 234 8.3.5.3 Development of Recombinant TSI 235 8.3.5.4 Pre-Clinical Evidence of TSI Development 236 8.3.5.5 Targeted Secretion Inhibitors in the Clinic 237 8.4 Conclusions 238 References 239

Chapter 9 Phage Selection of Mono- and Bicyclic Peptide Ligands 241 Shiyu Chen and Christian Heinis

9.1 Introduction 241 9.2 Phage Selection of Monocyclic Peptides 242 9.2.1 Phage Display Systems 242 9.2.2 Disulfide-Cyclized Peptide Phage Libraries 245 9.2.3 Phage Panning of Disulfide-Cyclized Peptide Libraries 246 9.3 Examples of Phage-Selected Monocyclic Peptides 247 9.3.1 Ligands of Vascular Endothelial Growth Factor 247 9.3.2 Cyclic Peptide Erythropoietin Receptor Agonists 248 9.4 Phage Selection of Bicyclic Peptides 250 9.4.1 Bicyclic Peptides 250 9.4.2 Bicyclization of Peptides on Phage 252 9.4.3 Bicyclic Peptide Phage Libraries 252 9.4.4 Phage Panning of Bicyclic Peptide Phage Libraries 254 9.5 Examples of Phage-Selected Bicyclic Peptides 254 9.5.1 Inhibitors of Plasma Kallikrein 255 9.5.2 Inhibitors of Urokinase-Type Plasminogen Activator 256 9.6 Conclusions 257 Acknowledgments 258 References 258 Contents xiii

Chapter 10 Chemical Approaches for Localization, Characterization and Mimicry of Peptide Epitopes 263 Paul R. Werkhoven and Rob M.J. Liskamp

10.1 Introduction, Definitions and Different Types of Epitopes 263 10.2 Shape and Structure of Epitopes 266 10.3 Determination of the Primary, Secondary or Tertiary Structure of Epitopes 268 10.4 Applications of Peptide Epitope Mimics 270 10.4.1 Epitope Mimics as Synthetic Vaccines 270 10.4.2 Epitope Mimics as Inhibitors (Antagonists) of Protein-Protein Interactions 271 10.4.3 Paratope Mimics as Synthetic Antibodies 271 10.5 Mimicry of Epitopes by Peptides 272 10.5.1 Single Linear Peptides as Continuous Epitope Mimics 272 10.5.2 Constraining Peptides for Optimization of Structural Mimicry 272 10.5.3 Assembly of Peptides for Mimicry of Discontinuous Epitopes 274 10.5.3.1 Assembly of Peptides by the Preparation of Dimers or Multimers 275 10.5.3.2 Assembly of Peptides by Scaffolding 275 10.5.3.3 Scaffolds 276 10.5.4 Requirements for Discontinuous Epitope Containing Protein Mimics 277 10.6 Conclusions 278 References 278

Chapter 11 Cystine-Knot Micro-Proteins 285 Bill Eldridge, Simon Robins and Duncan McGregor

11.1 Cystine-Knot Micro-Proteins 285 11.1.1 Conotoxins and Venom-Derived CK Micro-Proteins 286 11.1.2 Human CK Micro-Proteins 287 11.1.3 Plant-Derived CK Micro-Proteins 287

11.2 Use as Scaffolds 288 11.2.1 Loop Grafting 288 11.2.2 Display Libraries Using Loop Replacement 288 xjv Contents

11.2.3 MCOTI-II as a Loop Replacement Scaffold 289 11.3 Stability of Selected CK Micro-Proteins 290 11.4 Potential Therapeutic Applications of CK Micro-Proteins 293 References 294

Subject Index 298