STEFAN R. SCHMIDT, PhD, is Vice President for Downstream Processing at Rentschler Biotechnology. Previously, he served as CSO at ERA Biotech and Associate Director for Protein Science at AstraZeneca. Dr. Schmidt has chaired many international conferences and written several original articles, reviews, and book chapters.

PREFACE xxiii

CONTRIBUTORS xxv

PART I INTRODUCTION 1

1 Fusion Proteins: Applications and Challenges 3
Stefan R. Schmidt

1.1 History, 3

1.2 Definitions and Categories, 4

1.3 Patenting, 5

1.4 Design and Engineering, 6

1.5 Manufacturing, 10

1.6 Regulatory Challenges, 15

1.7 Competition and Market, 16

1.8 Conclusion and Future Perspective, 17

References, 18

2 Analyzing and Forecasting the Fusion Protein Market and Pipeline 25
Mark Belsey and Giles Somers

2.1 Introduction, 25

2.2 Market Sales Dynamics of the FP Market, 25

2.3 Individual Drug Sales Analysis, 27

2.4 Pipeline Database Analysis, 32

Disclaimer, 36

Acknowledgment, 36

References, 36

3 Structural Aspects of Fusion Proteins Determining the Level of Commercial Success 39
Giles Somers

3.1 Classification of FPs, 39

3.2 Factors for Commercial Success, 49

References, 54

4 Fusion Protein Linkers: Effects on Production, Bioactivity, and Pharmacokinetics 57
Xiaoying Chen, Jennica Zaro, and Wei-Chiang Shen

4.1 Introduction, 57

4.2 Overview of General Properties of Linkers Derived From Naturally Occurring Multidomain Proteins, 58

4.3 Empirical Linkers in Recombinant Fusion Proteins, 59

4.4 Functionality of Linkers in Fusion Proteins, 66

4.5 Conclusions and Future Perspective, 70

References, 71

5 Immunogenicity of Therapeutic Fusion Proteins: Contributory Factors and Clinical Experience 75
Vibha Jawa, Leslie Cousens, and Anne S. De Groot

5.1 Introduction, 75

5.2 Basis of Therapeutic Protein Immunogenicity, 75

5.3 Tools for Immunogenicity Screening, 77

5.4 Approaches for Risk Assessment and Minimization, 81

5.5 Case Study and Clinical Experience, 83

5.6 Preclinical and Clinical Immunogenicity Assessment Strategy, 85

5.7 Conclusions, 87

Acknowledgment, 87

References, 87

PART II THE TRIPLE T PARADIGM: TIME, TOXIN, TARGETING 91

IIA TIME: FUSION PROTEIN STRATEGIES FOR HALF-LIFE EXTENSION 93

6 Fusion Proteins for Half-Life Extension 93
Stefan R. Schmidt

6.1 Introduction, 93

6.2 Half-Life Extension Through Size and Recycling, 94

6.3 Half-Life Extension Through Increase of Hydrodynamic Radius, 100

6.4 Aggregate Forming Peptide Fusions, 102

6.5 Other Concepts, 103

6.6 Conclusions and Future Perspective, 103

References, 104

7 Monomeric Fc-Fusion Proteins 107
Baisong Mei, Susan C. Low, Snejana Krassova, Robert T. Peters, Glenn F. Pierce, and Jennifer A. Dumont

7.1 Introduction, 107

7.2 FcRn and Monomeric Fc-Fusion Proteins, 108

7.3 Typical Applications, 109

7.4 Alternative Applications, 114

7.5 Expression and Purification of Monomeric Fc-Fusion Proteins, 116

7.6 Conclusions and Future Perspectives, 118

References, 118

8 Peptide-Fc Fusion Therapeutics: Applications and Challenges 123
Chichi Huang and Ronald V. Swanson

8.1 Introduction, 123

8.2 Peptide Drugs, 124

8.3 Technologies Used for Reducing In Vivo Clearance of Therapeutic Peptides, 126

8.4 Fc-Fusion Proteins in Drug Development, 127

8.5 Peptide-Fc-Fusion Therapeutics, 131

8.6 Considerations and Challenges for Engineering Peptide-Fc-Fusion Therapeutics, 133

8.7 Conclusions, 138

Acknowledgment, 138

References, 138

9 Receptor-Fc and Ligand Traps as High-Affinity Biological Blockers: Development and Clinical Applications 143
Aris N. Economides and Neil Stahl

9.1 Introduction, 143

9.2 Etanercept as a Prototypical Receptor-Fc-Based Cytokine Blocker, 144

9.3 Heteromeric Traps for Ligands Utilizing Multicomponent Receptor Systems with Shared Subunits, 144

9.4 Development and Clinical Application of an Interleukin 1 Trap: Rilonacept, 151

9.5 Development and Clinical Application of a VEGF Trap, 151

9.6 "To Trap Or Not To Trap?" Advantages and Disadvantages of Receptor-Fc Fusions and Traps Versus Antibodies, 152

9.7 Conclusion, 155

Acknowledgment, 155

References, 155

10 Recombinant Albumin Fusion Proteins 163
Thomas Weimer, Hubert J. Metzner, and Stefan Schulte

10.1 Concept, 163

10.2 Technological Aspects, 164

10.3 Typical Applications and Indications, 164

10.4 Successes and Failures in Preclinical and Clinical Research, 172

10.5 Challenges, 173

10.6 Future Perspectives, 174

10.7 Conclusion, 174

Acknowledgment, 174

References, 174

11 Albumin-Binding Fusion Proteins in the Development of Novel Long-Acting Therapeutics 179
Adam Walker, Grainne Dunlevy, and Peter Topley

11.1 Introduction, 179

11.2 Clinically Validated Half-Life Extension Technologies-An Overview, 180

11.3 Interferon-a Fused to Human Serum Albumin or AlbudAb-A Direct Comparison of HSA and AlbudAb Fusion Technologies, 182

11.4 Nanobodies in the Development of Alternative Half-Life Extension Technologies Based on Single Immunoglobulin Variable Domains, 186

11.5 Novel Half-Life Extension Technologies-Alternative Approaches to Single Immunoglobulin Variable Domains, 187

11.6 Conclusions, 188

References, 189

12 Transferrin Fusion Protein Therapies: Acetylcholine Receptor-Transferrin Fusion Protein as a Model 191
Dennis Keefe, Michael Heartlein, and Serene Josiah

12.1 Disease Overview, 191

12.2 Fusion Protein SHG2210 Design, 192

12.3 Characterization of SHG2210, 193

12.4 Applications and Indications, 196

12.5 Future Perspectives, 197

12.6 Conclusion, 198

References, 198

13 Half-Life Extension Through O-Glycosylation 201
Fuad Fares

13.1 Introduction, 201

13.2 The Role of O-Linked Oligosaccharide Chains in Glycoprotein Function, 202

13.3 Designing Long-Acting Agonists of Glycoprotein Hormones, 203

13.4 Conclusions, 207

References, 207

14 ELP-Fusion Technology for Biopharmaceuticals 211
Doreen M. Floss, Udo Conrad, Stefan Rose-John, and JEURurgen Scheller

14.1 Introduction, 211

14.2 ELP-based Protein Purification, 212

14.3 ELPylated Proteins in Medicine and Nanobiotechnology, 215

14.4 Molecular Pharming: a New Application for ELPylation, 217

14.5 Challenges and Future Perspectives, 221

14.6 Conclusion, 222

References, 222

15 Ligand-Receptor Fusion Dimers 227
Sarbendra L. Pradhananga, Ian R. Wilkinson, Eric Ferrandis, Peter J. Artymiuk, Jon R. Sayers, and Richard J. Ross

15.1 Introduction, 227

15.2 The GHLR-Fusions, 228

15.3 Expression and Purification, 229

15.4 Analysis of the LR-Fusions, 229

15.5 LR-Fusions: The Next Generation in Hormone Treatment, 234

15.6 Conclusion, 234

References, 234

16 Development of Latent Cytokine Fusion Proteins 237
Lisa Mullen, Gill Adams, Rewas Fatah, David Gould, Anne Rigby, Michelle Sclanders, Apostolos Koutsokeras, Gayatri Mittal, Sandrine Vessillier, and Yuti Chernajovsky

16.1 Introduction, 237

16.2 Description of Concept, 238

16.3 Limitations of the Latent Cytokine Technology, 240

16.4 Generation of Latent Cytokines, 242

16.5 Applications and Potential Clinical Indications, 244

16.6 Alternatives/Variants of Approach, 246

16.7 Challenges (Production and Development), 247

16.8 Conclusions and Future Perspectives, 248

Acknowledgments, 249

References, 249

IIB TOXIN: CYTOTOXIC FUSION PROTEINS 253

17 Fusion Proteins with Toxic Activity 253
Stefan R. Schmidt

17.1 Introduction, 253

17.2 Toxins, 254

17.3 Immunocytokines, 258

17.4 Human Enzymes, 259

17.5 Apoptosis Induction, 261

17.6 Fc-Based Toxicity, 263

17.7 Peptide-Based Toxicity, 264

17.8 Conclusions and Future Perspectives, 265

References, 265

18 Classic Immunotoxins with Plant or Microbial Toxins 271
Jung Hee Woo and Arthur Frankel

18.1 Introduction, 271

18.2 Toxins Used in Immunotoxin Preparation, 272

18.3 Immunotoxin Design and Synthesis, 274

18.4 Clinical Update of Immunotoxin Trials, 278

18.5 Challenges and Perspective of Classic Immunotoxins, 284

18.6 Conclusions, 286

References, 286

19 Targeted and Untargeted Fusion Proteins: Current Approaches to Cancer Immunotherapy 295
Leslie A. Khawli, Peisheng Hu, and Alan L. Epstein

19.1 Introduction, 295

19.2 Immunotherapeutic Strategy for Cancer: Fusion Proteins, 296

19.3 Immunotherapeutic Applications of Antibody-Targeted and Untargeted Fc Fusion Proteins, 297

19.4 Combination Fusion Proteins Therapy, 305

19.5 Mechanism of Action: Immunoregulatory T-Cell (Treg) Depletion and Fusion Protein Combination Therapy, 306

19.6 Future Directions, 309

19.7 Conclusion, 309

Acknowledgments, 310

References, 310

20 Development of Experimental Targeted Toxin Therapies for Malignant Glioma 315
Nikolai G. Rainov and Volkmar Heidecke

20.1 Introduction, 315

20.2 Targeted Toxins-General Considerations, 316

20.3 Delivery Mode and Pharmacokinetics of Targeted Toxins in the Brain, 316

20.4 Preclinical and Clinical Studies with Targeted Toxins, 318

20.5 Conclusions and Future Developments of...