Fusion Protein Technologies for Biopharmaceuticals

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 xxiiiCONTRIBUTORS xxvPART I INTRODUCTION 11 Fusion Proteins: Applications and Challenges 3Stefan R. Schmidt1.1 History, 31.2 Definitions and Categories, 41.3 Patenting, 51.4 Design and Engineering, 61.5 Manufacturing, 101.6 Regulatory Challenges, 151.7 Competition and Market, 161.8 Conclusion and Future Perspective, 17References, 182 Analyzing and Forecasting the Fusion Protein Market and Pipeline 25Mark Belsey and Giles Somers2.1 Introduction, 252.2 Market Sales Dynamics of the FP Market, 252.3 Individual Drug Sales Analysis, 272.4 Pipeline Database Analysis, 32Disclaimer, 36Acknowledgment, 36References, 363 Structural Aspects of Fusion Proteins Determining the Level of Commercial Success 39Giles Somers3.1 Classification of FPs, 393.2 Factors for Commercial Success, 49References, 544 Fusion Protein Linkers: Effects on Production, Bioactivity, and Pharmacokinetics 57Xiaoying Chen, Jennica Zaro, and Wei-Chiang Shen4.1 Introduction, 574.2 Overview of General Properties of Linkers Derived From Naturally Occurring Multidomain Proteins, 584.3 Empirical Linkers in Recombinant Fusion Proteins, 594.4 Functionality of Linkers in Fusion Proteins, 664.5 Conclusions and Future Perspective, 70References, 715 Immunogenicity of Therapeutic Fusion Proteins: Contributory Factors and Clinical Experience 75Vibha Jawa, Leslie Cousens, and Anne S. De Groot5.1 Introduction, 755.2 Basis of Therapeutic Protein Immunogenicity, 755.3 Tools for Immunogenicity Screening, 775.4 Approaches for Risk Assessment and Minimization, 815.5 Case Study and Clinical Experience, 835.6 Preclinical and Clinical Immunogenicity Assessment Strategy, 855.7 Conclusions, 87Acknowledgment, 87References, 87PART II THE TRIPLE T PARADIGM: TIME, TOXIN, TARGETING 91IIA TIME: FUSION PROTEIN STRATEGIES FOR HALF-LIFE EXTENSION 936 Fusion Proteins for Half-Life Extension 93Stefan R. Schmidt6.1 Introduction, 936.2 Half-Life Extension Through Size and Recycling, 946.3 Half-Life Extension Through Increase of Hydrodynamic Radius, 1006.4 Aggregate Forming Peptide Fusions, 1026.5 Other Concepts, 1036.6 Conclusions and Future Perspective, 103References, 1047 Monomeric Fc-Fusion Proteins 107Baisong Mei, Susan C. Low, Snejana Krassova, Robert T. Peters, Glenn F. Pierce, and Jennifer A. Dumont7.1 Introduction, 1077.2 FcRn and Monomeric Fc-Fusion Proteins, 1087.3 Typical Applications, 1097.4 Alternative Applications, 1147.5 Expression and Purification of Monomeric Fc-Fusion Proteins, 1167.6 Conclusions and Future Perspectives, 118References, 1188 Peptide-Fc Fusion Therapeutics: Applications and Challenges 123Chichi Huang and Ronald V. Swanson8.1 Introduction, 1238.2 Peptide Drugs, 1248.3 Technologies Used for Reducing In Vivo Clearance of Therapeutic Peptides, 1268.4 Fc-Fusion Proteins in Drug Development, 1278.5 Peptide-Fc-Fusion Therapeutics, 1318.6 Considerations and Challenges for Engineering Peptide-Fc-Fusion Therapeutics, 1338.7 Conclusions, 138Acknowledgment, 138References, 1389 Receptor-Fc and Ligand Traps as High-Affinity Biological Blockers: Development and Clinical Applications 143Aris N. Economides and Neil Stahl9.1 Introduction, 1439.2 Etanercept as a Prototypical Receptor-Fc-Based Cytokine Blocker, 1449.3 Heteromeric Traps for Ligands Utilizing Multicomponent Receptor Systems with Shared Subunits, 1449.4 Development and Clinical Application of an Interleukin 1 Trap: Rilonacept, 1519.5 Development and Clinical Application of a VEGF Trap, 1519.6 “To Trap Or Not To Trap?” Advantages and Disadvantages of Receptor-Fc Fusions and Traps Versus Antibodies, 1529.7 Conclusion, 155Acknowledgment, 155References, 15510 Recombinant Albumin Fusion Proteins 163Thomas Weimer, Hubert J. Metzner, and Stefan Schulte10.1 Concept, 16310.2 Technological Aspects, 16410.3 Typical Applications and Indications, 16410.4 Successes and Failures in Preclinical and Clinical Research, 17210.5 Challenges, 17310.6 Future Perspectives, 17410.7 Conclusion, 174Acknowledgment, 174References, 17411 Albumin-Binding Fusion Proteins in the Development of Novel Long-Acting Therapeutics 179Adam Walker, Grainne Dunlevy, and Peter Topley11.1 Introduction, 17911.2 Clinically Validated Half-Life Extension Technologies—An Overview, 18011.3 Interferon-a Fused to Human Serum Albumin or AlbudAb—A Direct Comparison of HSA and AlbudAb Fusion Technologies, 18211.4 Nanobodies in the Development of Alternative Half-Life Extension Technologies Based on Single Immunoglobulin Variable Domains, 18611.5 Novel Half-Life Extension Technologies—Alternative Approaches to Single Immunoglobulin Variable Domains, 18711.6 Conclusions, 188References, 18912 Transferrin Fusion Protein Therapies: Acetylcholine Receptor-Transferrin Fusion Protein as a Model 191Dennis Keefe, Michael Heartlein, and Serene Josiah12.1 Disease Overview, 19112.2 Fusion Protein SHG2210 Design, 19212.3 Characterization of SHG2210, 19312.4 Applications and Indications, 19612.5 Future Perspectives, 19712.6 Conclusion, 198References, 19813 Half-Life Extension Through O-Glycosylation 201Fuad Fares13.1 Introduction, 20113.2 The Role of O-Linked Oligosaccharide Chains in Glycoprotein Function, 20213.3 Designing Long-Acting Agonists of Glycoprotein Hormones, 20313.4 Conclusions, 207References, 20714 ELP-Fusion Technology for Biopharmaceuticals 211Doreen M. Floss, Udo Conrad, Stefan Rose-John, and J€urgen Scheller14.1 Introduction, 21114.2 ELP-based Protein Purification, 21214.3 ELPylated Proteins in Medicine and Nanobiotechnology, 21514.4 Molecular Pharming: a New Application for ELPylation, 21714.5 Challenges and Future Perspectives, 22114.6 Conclusion, 222References, 22215 Ligand-Receptor Fusion Dimers 227Sarbendra L. Pradhananga, Ian R. Wilkinson, Eric Ferrandis, Peter J. Artymiuk, Jon R. Sayers, and Richard J. Ross15.1 Introduction, 22715.2 The GHLR-Fusions, 22815.3 Expression and Purification, 22915.4 Analysis of the LR-Fusions, 22915.5 LR-Fusions: The Next Generation in Hormone Treatment, 23415.6 Conclusion, 234References, 23416 Development of Latent Cytokine Fusion Proteins 237Lisa Mullen, Gill Adams, Rewas Fatah, David Gould, Anne Rigby, Michelle Sclanders, Apostolos Koutsokeras, Gayatri Mittal, Sandrine Vessillier, and Yuti Chernajovsky16.1 Introduction, 23716.2 Description of Concept, 23816.3 Limitations of the Latent Cytokine Technology, 24016.4 Generation of Latent Cytokines, 24216.5 Applications and Potential Clinical Indications, 24416.6 Alternatives/Variants of Approach, 24616.7 Challenges (Production and Development), 24716.8 Conclusions and Future Perspectives, 248Acknowledgments, 249References, 249IIB TOXIN: CYTOTOXIC FUSION PROTEINS 25317 Fusion Proteins with Toxic Activity 253Stefan R. Schmidt17.1 Introduction, 25317.2 Toxins, 25417.3 Immunocytokines, 25817.4 Human Enzymes, 25917.5 Apoptosis Induction, 26117.6 Fc-Based Toxicity, 26317.7 Peptide-Based Toxicity, 26417.8 Conclusions and Future Perspectives, 265References, 26518 Classic Immunotoxins with Plant or Microbial Toxins 271Jung Hee Woo and Arthur Frankel18.1 Introduction, 27118.2 Toxins Used in Immunotoxin Preparation, 27218.3 Immunotoxin Design and Synthesis, 27418.4 Clinical Update of Immunotoxin Trials, 27818.5 Challenges and Perspective of Classic Immunotoxins, 28418.6 Conclusions, 286References, 28619 Targeted and Untargeted Fusion Proteins: Current Approaches to Cancer Immunotherapy 295Leslie A. Khawli, Peisheng Hu, and Alan L. Epstein19.1 Introduction, 29519.2 Immunotherapeutic Strategy for Cancer: Fusion Proteins, 29619.3 Immunotherapeutic Applications of Antibody-Targeted and Untargeted Fc Fusion Proteins, 29719.4 Combination Fusion Proteins Therapy, 30519.5 Mechanism of Action: Immunoregulatory T-Cell (Treg) Depletion and Fusion Protein Combination Therapy, 30619.6 Future Directions, 30919.7 Conclusion, 309Acknowledgments, 310References, 31020 Development of Experimental Targeted Toxin Therapies for Malignant Glioma 315Nikolai G. Rainov and Volkmar Heidecke20.1 Introduction, 31520.2 Targeted Toxins—General Considerations, 31620.3 Delivery Mode and Pharmacokinetics of Targeted Toxins in the Brain, 31620.4 Preclinical and Clinical Studies with Targeted Toxins, 31820.5 Conclusions and Future Developments of Targeted Toxins, 324Disclosure, 325References, 32521 Immunokinases 329Stefan Barth, Stefan Gattenl€ohner, and Mehmet Kemal Tur21.1 Introduction, 32921.2 Protein Kinases, Apoptosis, and Cancer, 33021.3 Therapeutic Strategies to Restore Missing Kinase Expression, 33121.4 Analysis of Immunokinase Efficacy, 33321.5 Outlook, 334References, 33422 ImmunoRNase Fusions 337Wojciech Ardelt22.1 Introduction, 33722.2 Development of ImmunoRNase Fusion Proteins as Biopharmaceuticals, 33922.3 Aspects of ImmunoRNase Design and Production, 34422.4 Alternatives, 34622.5 Conclusions and Future Perspectives, 347References, 34723 Antibody-Directed Enzyme Prodrug Therapy (ADEPT) 355Surinder K. Sharma23.1 Introduction, 35523.2 The Components, 35523.3 ADEPT Systems with Carboxypeptidase G2 (CPG2), 35723.4 Fusion Proteins, 35923.5 Immunogenicity, 36023.6 Conclusions and Future Outlook, 361Acknowledgments, 361References, 36124 Tumor-Targeted Superantigens 365Gunnar Hedlund, G€oran Forsberg, Thore Nederman, Anette Sundstedt, Leif Dahlberg, Mikael Tiensuu, and Mats Nilsson24.1 Introduction: Tumor-Targeted Superantigens—AUnique Concept of Cancer Treatment, 36524.2 Structure and Production of Tumor-Targeted Superantigens, 36624.3 Tumor-Targeted Superantigens are Powerful Targeted Immune Activators and Useful for all Types of Malignancies, 36724.4 Increasing the Therapeutic Window and Exposure by the Creation of a Novel TTS Fusion Protein with Minimal MHC Class II Affinity; Naptumomab Estafenatox, 37024.5 Clinical Experience with TTS Therapeutic Fusion Proteins, 37124.6 Combining TTS with Cytostatic and Immunomodulating Anticancer Drugs, 37724.7 Conclusions, 379References, 379IIC TARGETING: FUSION PROTEINS ADDRESSING SPECIFIC CELLS, ORGANS, AND TISSUES 38325 Fusion Proteins with a Targeting Function 383Stefan R. Schmidt25.1 Introduction, 38325.2 Targeting Organs, 38325.3 Intracellular Delivery, 38825.4 Oral Delivery, 39125.5 Conclusions and Future Perspectives, 392References, 39326 Cell-Penetrating Peptide Fusion Proteins 397Andres Mu~noz-Alarcon, Henrik Helmfors, Kristin Karlsson, and €U lo Langel26.1 Introduction, 39726.2 Typical Applications and Indications, 39726.3 Technological Aspects, 39926.4 Successes and Failures in Preclinical and Clinical Research, 40226.5 Alternatives/Variants of This Approach, 40526.6 Conclusions and Future Perspectives, 405Acknowledgments, 406References, 40627 Cell-Specific Targeting of Fusion Proteins through Heparin Binding 413Jiajing Wang, Zhenzhong Ma, and Jeffrey A. Loeb27.1 Why Target Heparan-Sulfate Proteoglycans with Fusion Proteins?, 41327.2 Heparan Sulfate Structure and Biosynthesis Create Diversity and a Template for Targeting Specificity, 41527.3 Tissue-Specific Expression of HSPGs and the Enzymes That Modify Them, 41627.4 Heparin-Binding Proteins and Growth Factors, 41627.5 Viruses Target Cells Through Heparin Binding, 41727.6 Dissecting Heparin-Binding Protein Domains for Tissue-Specific Targeting, 41827.7 Fusion Proteins Incorporating HBDs, 41827.8 The Neuregulin 1 Growth Factor Has a Unique and Highly Specific HBD, 41927.9 Using Neuregulin’s HBD to Generate a Targeted Neuregulin Antagonist, 41927.10 Tissue Targeting and Therapeutic Efficacy of a Heparin-Targeted NRG1 Antagonist Fusion Protein, 42027.11 Conclusions and Future Perspectives, 423References, 42428 Bone-Targeted Alkaline Phosphatase 429Jose Luis Millan28.1 Detailed Description of the Concept, 42928.2 Technical Aspects, 43028.3 Applications and Indications, 43228.4 Preclinical and Clinical Research, 43328.5 Alternatives/Variants of This Approach, 43428.6 Challenges in Production and Development, 43628.7 Conclusions and Future Perspectives, 436Acknowledgments, 437References, 43729 Targeting Interferon-a to the Liver: Apolipoprotein A-I as a Scaffold for Protein Delivery 441Jessica Fioravanti, Jesus Prieto, and Pedro Berraondo29.1 Detailed Description of the Concept, 44129.2 Technological Aspects, 44729.3 Typical Applications and Indications, 44729.4 Alternatives and Variants of This Approach, 44829.5 Conclusions and Future Perspectives, 448References, 448PART III BEYOND THE TRIPLE T-PARADIGM 453IIIA NOVEL CONCEPTS, NOVEL SCAFFOLDS 45530 Signal Converter Proteins 455Mark L. Tykocinski30.1 Introduction, 45530.2 Historical Roots of Signal Conversion: Artificial Veto Cell Engineering and Protein Painting, 45530.3 Trans Signal Converter Proteins, 45830.4 Expanding Trans Signal Conversion Options: Redirecting Signals, 45930.5 From Trans to Cis Signal Conversion: Driving Auto-Signaling, 46030.6 Mechanistic Dividends of Chimerization, 46130.7 Targeting Multiple Diseases with Individual Signal Converters, 46230.8 Structural Constraints in SCP Design, 46330.9 Coding SCP Functional Repertoires, 46330.10 Expanding the Catalog of Inhibitory SCP, 46430.11 Immune Activating SCP, 46630.12 Experimental Tools for Screening SCP Candidates, 46730.13 SCP Frontiers: Mining the Surface Protein Interactome, Rewiring Cellular Networks, 467References, 46831 Soluble T-Cell Antigen Receptors 475Peter R. Rhode31.1 Soluble T-cell Antigen Receptor (STAR) Fusion Technology and Utilities, 47531.2 Expression and Purification of Recombinant Star Fusion Proteins, 47731.3 Clinical and Research Product Applications, 47831.4 Preclinical Testing Using Star Fusion Proteins, 48131.5 Clinical Development of ALT-801, 48731.6 Alternatives/Variants of This Approach, 48831.7 Challenges, 48931.8 Conclusions and Future Perspectives, 490Acknowledgments, 490References, 49032 High-Affinity Monoclonal T-Cell Receptor (mTCR) Fusions 495Nikolai M. Lissin, Namir J. Hassan, and Bent K. Jakobsen32.1 Introduction: The T Cell Receptor (TCR) as a Targeting Molecule, 49532.2 Engineered High-Affinity Monoclonal TCRs (mTCR), 49732.3 mTCR-Based Fusion Proteins for Therapeutic Applications, 50032.4 Immune-Mobilizing Monoclonal TCRs Against Cancer (ImmTAC), 50032.5 Conclusions and Future Perspectives, 503Acknowledgments, 504References, 50433 Amediplase 507Stefano Evangelista and Stefano Manzini33.1 Introduction, 50733.2 Source, Physico-Chemical Properties and Formulation, 50833.3 Preclinical Studies, 51033.4 Human Studies, 51233.5 Historical Comparison with Other Thrombolytics, 51733.6 Conclusions and Future Perspectives, 517Acknowledgment, 517References, 51734 Breaking New Therapeutic Grounds: Fusion Proteins of Darpins and Other Nonantibody Binding Proteins 519Hans Kaspar Binz34.1 Introduction, 51934.2 Novel Scaffolds—Alternatives to Antibodies, 51934.3 New Therapeutic Concepts with Nonantibody Binding Proteins, 52334.4 Scaffold-Fusion Proteins Beyond Antibody Possibilities, 525Acknowledgments, 526References, 526IIIB MULTIFUNCTIONAL ANTIBODIES 52935 Resurgence of Bispecific Antibodies 529Patrick A. Baeuerle and Tobias Raum35.1 A Brief History of Bispecific Antibodies, 52935.2 Asymmetric IgG-Like Bispecific Antibodies, 53035.3 Symmetric IgG-Like Bispecific Antibodies, 53135.4 IgG-Like Bispecific Antibodies with Fused Antibody Fragments, 53335.5 Bispecific Constructs Based on the Fcg Fragment, 53435.6 Bispecific Constructs Based on Fab Fragments, 53535.7 Bispecific Constructs Based on Diabodies or Single-Chain Antibodies, 53635.8 Bifunctional Fusions of Antibodies or Fragments with Other Proteins, 53835.9 Bispecific Antibodies for Various Functions: How to Select the Right Format?, 539References, 54136 Novel Applications of Bispecific DART1 Proteins 545Syd Johnson, Bhaswati Barat, Hua W. Li, Ralph F. Alderson, Paul A. Moore, and Ezio Bonvini36.1 Introduction, 54536.2 DART1 Proteins, 54636.3 Application of DART1 to Cross-Link Inhibitory and Activating Receptors, 54636.4 Application of Bispecific Antibodies in Oncology, 54736.5 U-DART Concept for Screening DART1 Candidate Targets and mAbs, 54936.6 U-DART Concept for Applications in Autoimmune and Inflammatory Disease, 54936.7 Conclusions and Future Perspectives, 554References, 55437 Strand Exchange Engineered Domain (Seed): A Novel Platform Designed to Generate Mono and Multispecific Protein Therapeutics 557Alec W. Gross, Jessica P. Dawson, Marco Muda, Christie Kelton, Sean D. McKenna, and Bjo¨rn Hock37.1 Introduction, 55737.2 Technical Aspects, 55837.3 Potential Therapeutic Applications, 56237.4 Future Perspectives, 56637.5 Conclusions, 567Acknowledgments, 567References, 56738 CovX-Bodies 571Abhijit Bhat, Olivier Laurent, and Rodney Lappe38.1 The CovX-Body Concept, 57138.2 Technological Aspects, 57138.3 Applications of the CovX-Body Technology, 578References, 58139 Modular Antibody Engineering: Antigen Binding Immunoglobulin Fc CH3 Domains as Building Blocks for Bispecific Antibodies (mAb2) 583Maximilian Woisetschl€ager, Florian R€uker, Geert C. Mudde, Gordana Wozniak-Knopp, Anton Bauer, and Gottfried Himmler39.1 Introduction, 58339.2 Immunoglobulin Fc as a Scaffold, 58339.3 Design of Libraries Based on the Human IgG1 CH3 Domain, 58439.4 TNF-a-Binding Fcab: Selection and Characterization of Fcab TNF353-2, 58539.5 Conclusions and Future Perspectives, 588Acknowledgments, 588References, 58940 Designer Fusion Modules for Building Multifunctional, Multivalent Antibodies, and Immunoconjugates: The Dock-and-Lock Method 591Edmund A. Rossi, David M. Goldenberg, and Chien-Hsing Chang40.1 Introduction, 59140.2 DDD/AD Modules Based on PKA and AKAP, 59240.3 Advantages and Disadvantages of the DNL Method, 59240.4 Fab-Based Modules, 59340.5 IgG-AD2-Modules, 59440.6 Hexavalent Antibodies, 59540.7 More Antibody-Based-Modules and Multivalent Antibodies, 59640.8 Nonantibody-Based DNL Modules, 59740.9 IFN-a2b-DDD2 Module and Immunocytokines, 59740.10 Variations on the DNLTheme, 59840.11 Conclusions and Future Perspective, 599References, 599INDEX 603

“Overall, this book is a “bona fide” companion for newcomers, as well as for experts in the pharmaceutical industry, in biotechnology or universities with affiliations to industry and medicine.”  (mAbs, 15 April 2015)