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 Targeted Toxins, 324
Disclosure, 325
References, 325
21 Immunokinases 329
Stefan Barth, Stefan GattenlEURohner, and Mehmet Kemal Tur
21.1 Introduction, 329
21.2 Protein Kinases, Apoptosis, and Cancer, 330
21.3 Therapeutic Strategies to Restore Missing Kinase Expression, 331
21.4 Analysis of Immunokinase Efficacy, 333
21.5 Outlook, 334
References, 334
22 ImmunoRNase Fusions 337
Wojciech Ardelt
22.1 Introduction, 337
22.2 Development of ImmunoRNase Fusion Proteins as Biopharmaceuticals, 339
22.3 Aspects of ImmunoRNase Design and Production, 344
22.4 Alternatives, 346
22.5 Conclusions and Future Perspectives, 347
References, 347
23 Antibody-Directed Enzyme Prodrug Therapy (ADEPT) 355
Surinder K. Sharma
23.1 Introduction, 355
23.2 The Components, 355
23.3 ADEPT Systems with Carboxypeptidase G2 (CPG2), 357
23.4 Fusion Proteins, 359
23.5 Immunogenicity, 360
23.6 Conclusions and Future Outlook, 361
Acknowledgments, 361
References, 361
24 Tumor-Targeted Superantigens 365
Gunnar Hedlund, GEURoran Forsberg, Thore Nederman, Anette Sundstedt, Leif Dahlberg, Mikael Tiensuu, and Mats Nilsson
24.1 Introduction: Tumor-Targeted Superantigens-AUnique Concept of Cancer Treatment, 365
24.2 Structure and Production of Tumor-Targeted Superantigens, 366
24.3 Tumor-Targeted Superantigens are Powerful Targeted Immune Activators and Useful for all Types of Malignancies, 367
24.4 Increasing the Therapeutic Window and Exposure by the Creation of a Novel TTS Fusion Protein with Minimal MHC Class II Affinity; Naptumomab Estafenatox, 370
24.5 Clinical Experience with TTS Therapeutic Fusion Proteins, 371
24.6 Combining TTS with Cytostatic and Immunomodulating Anticancer Drugs, 377
24.7 Conclusions, 379
References, 379
IIC TARGETING: FUSION PROTEINS ADDRESSING SPECIFIC CELLS, ORGANS, AND TISSUES 383
25 Fusion Proteins with a Targeting Function 383
Stefan R. Schmidt
25.1 Introduction, 383
25.2 Targeting Organs, 383
25.3 Intracellular Delivery, 388
25.4 Oral Delivery, 391
25.5 Conclusions and Future Perspectives, 392
References, 393
26 Cell-Penetrating Peptide Fusion Proteins 397
Andres Mu~noz-Alarcon, Henrik Helmfors, Kristin Karlsson, and EURU lo Langel
26.1 Introduction, 397
26.2 Typical Applications and Indications, 397
26.3 Technological Aspects, 399
26.4 Successes and Failures in Preclinical and Clinical Research, 402
26.5 Alternatives/Variants of This Approach, 405
26.6 Conclusions and Future Perspectives, 405
Acknowledgments, 406
References, 406
27 Cell-Specific Targeting of Fusion Proteins through Heparin Binding 413
Jiajing Wang, Zhenzhong Ma, and Jeffrey A. Loeb
27.1 Why Target Heparan-Sulfate Proteoglycans with Fusion Proteins?, 413
27.2 Heparan Sulfate Structure and Biosynthesis Create Diversity and a Template for Targeting Specificity, 415
27.3 Tissue-Specific Expression of HSPGs and the Enzymes That Modify Them, 416
27.4 Heparin-Binding Proteins and Growth Factors, 416
27.5 Viruses Target Cells Through Heparin Binding, 417
27.6 Dissecting Heparin-Binding Protein Domains for Tissue-Specific Targeting, 418
27.7 Fusion Proteins Incorporating HBDs, 418
27.8 The Neuregulin 1 Growth Factor Has a Unique and Highly Specific HBD, 419
27.9 Using Neuregulin's HBD to Generate a Targeted Neuregulin Antagonist, 419
27.10 Tissue Targeting and Therapeutic Efficacy of a Heparin-Targeted NRG1 Antagonist Fusion Protein, 420
27.11 Conclusions and Future Perspectives, 423
References, 424
28 Bone-Targeted Alkaline Phosphatase 429
Jose Luis Millan
28.1 Detailed Description of the Concept, 429
28.2 Technical Aspects, 430
28.3 Applications and Indications, 432
28.4 Preclinical and Clinical Research, 433
28.5 Alternatives/Variants of This Approach, 434
28.6 Challenges in Production and Development, 436
28.7 Conclusions and Future Perspectives, 436
Acknowledgments, 437
References, 437
29 Targeting Interferon-a to the Liver: Apolipoprotein A-I as a Scaffold for Protein Delivery 441
Jessica Fioravanti, Jesus Prieto, and Pedro Berraondo
29.1 Detailed Description of the Concept, 441
29.2 Technological Aspects, 447
29.3 Typical Applications and Indications, 447
29.4 Alternatives and Variants of This Approach, 448
29.5 Conclusions and Future Perspectives, 448
References, 448
PART III BEYOND THE TRIPLE T-PARADIGM 453
IIIA NOVEL CONCEPTS, NOVEL SCAFFOLDS 455
30 Signal Converter Proteins 455
Mark L. Tykocinski
30.1 Introduction, 455
30.2 Historical Roots of Signal Conversion: Artificial Veto Cell Engineering and Protein Painting, 455
30.3 Trans Signal Converter Proteins, 458
30.4 Expanding Trans Signal Conversion Options: Redirecting Signals, 459
30.5 From Trans to Cis Signal Conversion: Driving Auto-Signaling, 460
30.6 Mechanistic Dividends of Chimerization, 461
30.7 Targeting Multiple Diseases with Individual Signal Converters, 462
30.8 Structural Constraints in SCP Design, 463
30.9 Coding SCP Functional Repertoires, 463
30.10 Expanding the Catalog of Inhibitory SCP, 464
30.11 Immune Activating SCP, 466
30.12 Experimental Tools for Screening SCP Candidates, 467
30.13 SCP Frontiers: Mining the Surface Protein Interactome, Rewiring Cellular Networks, 467
References, 468
31 Soluble T-Cell Antigen Receptors 475
Peter R. Rhode
31.1 Soluble T-cell Antigen Receptor (STAR) Fusion Technology and Utilities, 475
31.2 Expression and Purification of Recombinant Star Fusion Proteins, 477
31.3 Clinical and Research Product Applications, 478
31.4 Preclinical Testing Using Star Fusion Proteins, 481
31.5 Clinical Development of ALT-801, 487
31.6 Alternatives/Variants of This Approach, 488
31.7 Challenges, 489
31.8 Conclusions and Future Perspectives, 490
Acknowledgments, 490
References, 490
32 High-Affinity Monoclonal T-Cell Receptor (mTCR) Fusions 495
Nikolai M. Lissin, Namir J. Hassan, and Bent K. Jakobsen
32.1 Introduction: The T Cell Receptor (TCR) as a Targeting Molecule, 495
32.2 Engineered High-Affinity Monoclonal TCRs (mTCR), 497
32.3 mTCR-Based Fusion Proteins for Therapeutic Applications, 500
32.4 Immune-Mobilizing Monoclonal TCRs Against Cancer (ImmTAC), 500
32.5 Conclusions and Future Perspectives, 503
Acknowledgments, 504
References, 504
33 Amediplase 507
Stefano Evangelista and Stefano Manzini
33.1 Introduction, 507
33.2 Source, Physico-Chemical Properties and Formulation, 508
33.3 Preclinical Studies, 510
33.4 Human Studies, 512
33.5 Historical Comparison with Other Thrombolytics, 517
33.6 Conclusions and Future Perspectives, 517
Acknowledgment, 517
References, 517
34 Breaking New Therapeutic Grounds: Fusion Proteins of Darpins and Other Nonantibody Binding Proteins 519
Hans Kaspar Binz
34.1 Introduction, 519
34.2 Novel Scaffolds-Alternatives to Antibodies, 519
34.3 New Therapeutic Concepts with Nonantibody Binding Proteins, 523
34.4 Scaffold-Fusion Proteins Beyond Antibody Possibilities, 525
Acknowledgments, 526
References, 526
IIIB MULTIFUNCTIONAL ANTIBODIES 529
35 Resurgence of Bispecific Antibodies 529
Patrick A. Baeuerle and Tobias Raum
35.1 A Brief History of Bispecific Antibodies, 529
35.2 Asymmetric IgG-Like Bispecific Antibodies, 530
35.3 Symmetric IgG-Like Bispecific Antibodies, 531
35.4 IgG-Like Bispecific Antibodies with Fused Antibody Fragments, 533
35.5 Bispecific Constructs Based on the Fcg Fragment, 534
35.6 Bispecific Constructs Based on Fab Fragments, 535
35.7 Bispecific Constructs Based on Diabodies or Single-Chain Antibodies, 536
35.8 Bifunctional Fusions of Antibodies or Fragments with Other Proteins, 538
35.9 Bispecific Antibodies for Various Functions: How to Select the Right Format?, 539
References, 541
36 Novel Applications of Bispecific DART1 Proteins 545
Syd Johnson, Bhaswati Barat, Hua W. Li, Ralph F. Alderson, Paul A. Moore, and Ezio Bonvini
36.1 Introduction, 545
36.2 DART1 Proteins, 546
36.3 Application of DART1 to Cross-Link Inhibitory and Activating Receptors, 546
36.4 Application of Bispecific Antibodies in Oncology, 547
36.5 U-DART Concept for Screening DART1 Candidate Targets and mAbs, 549
36.6 U-DART Concept for Applications in Autoimmune and Inflammatory Disease, 549
36.7 Conclusions and Future Perspectives, 554
References, 554
37 Strand Exchange Engineered Domain (Seed): A Novel Platform Designed to Generate Mono and Multispecific Protein Therapeutics 557
Alec W. Gross, Jessica P. Dawson, Marco Muda, Christie Kelton, Sean D. McKenna, and Björn Hock
37.1 Introduction, 557
37.2 Technical Aspects, 558
37.3 Potential Therapeutic Applications, 562
37.4 Future Perspectives, 566
37.5 Conclusions, 567
Acknowledgments, 567
References, 567
38 CovX-Bodies 571
Abhijit Bhat, Olivier Laurent, and Rodney Lappe
38.1 The CovX-Body Concept, 571
38.2 Technological Aspects, 571
38.3 Applications of the CovX-Body Technology, 578
References, 581
39 Modular Antibody Engineering: Antigen Binding Immunoglobulin Fc CH3 Domains as Building Blocks for Bispecific Antibodies (mAb2) 583
Maximilian WoisetschlEURager, Florian REURuker, Geert C. Mudde, Gordana Wozniak-Knopp, Anton Bauer, and Gottfried Himmler
39.1 Introduction, 583
39.2 Immunoglobulin Fc as a Scaffold, 583
39.3 Design of Libraries Based on the Human IgG1 CH3 Domain, 584
39.4 TNF-a-Binding Fcab: Selection and Characterization of Fcab TNF353-2, 585
39.5 Conclusions and Future Perspectives, 588
Acknowledgments, 588
References, 589
40 Designer Fusion Modules for Building Multifunctional, Multivalent Antibodies, and Immunoconjugates: The Dock-and-Lock Method 591
Edmund A. Rossi, David M. Goldenberg, and Chien-Hsing Chang
40.1 Introduction, 591
40.2 DDD/AD Modules Based on PKA and AKAP, 592
40.3 Advantages and Disadvantages of the DNL Method, 592
40.4 Fab-Based Modules, 593
40.5 IgG-AD2-Modules, 594
40.6 Hexavalent Antibodies, 595
40.7 More Antibody-Based-Modules and Multivalent Antibodies, 596
40.8 Nonantibody-Based DNL Modules, 597
40.9 IFN-a2b-DDD2 Module and Immunocytokines, 597
40.10 Variations on the DNLTheme, 598
40.11 Conclusions and Future Perspective, 599
References, 599
INDEX 603
