Fusion Protein Technologies for Biopharmaceuticals E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1971

Veröffentlichungsjahr: 2013

Beschreibung

The state of the art in biopharmaceutical FUSION PROTEIN DESIGN Fusion proteins belong to the most lucrative biotech drugs--with Enbrel® being one of the best-selling biologics worldwide. Enbrel® represents a milestone of modern therapies just as Humulin®, the first therapeutic recombinant protein for human use, approved by the FDA in 1982 and Orthoclone® the first monoclonal antibody reaching the market in 1986. These first generation molecules were soon followed by a plethora of recombinant copies of natural human proteins, and in 1998, the first de novo designed fusion protein was launched. Fusion Protein Technologies for Biopharmaceuticals examines the state of the art in developing fusion proteins for biopharmaceuticals, shedding light on the immense potential inherent in fusion protein design and functionality. A wide pantheon of international scientists and researchers deliver a comprehensive and complete overview of therapeutic fusion proteins, combining the success stories of marketed drugs with the dynamic preclinical and clinical research into novel drugs designed for as yet unmet medical needs. The book covers the major types of fusion proteins--receptor-traps, immunotoxins, Fc-fusions and peptibodies--while also detailing the approaches for developing, delivering, and improving the stability of fusion proteins. The main body of the book contains three large sections that address issues key to this specialty: strategies for extending the plasma half life, the design of toxic proteins, and utilizing fusion proteins for ultra specific targeting. The book concludes with novel concepts in this field, including examples of highly relevant multifunctional antibodies. Detailing the innovative science, commercial realities, and brilliant potential of fusion protein therapeutics, Fusion Protein Technologies for Biopharmaceuticals is a must for pharmaceutical scientists, biochemists, medicinal chemists, molecular biologists, pharmacologists, and genetic engineers interested in determining the shape of innovation in the world of biopharmaceuticals.

Leseprobe

Contents

Cover

Title Page

Copyright

Preface

Contributors

Part I: Introduction

Chapter 1: Fusion Proteins: Applications and Challenges

1.1 History

1.2 Definitions and Categories

1.3 Patenting

1.4 Design and Engineering

1.5 Manufacturing

1.6 Regulatory Challenges

1.7 Competition and Market

1.8 Conclusion and Future Perspective

References

Chapter 2: Analyzing and Forecasting the Fusion ProteinMarket and Pipeline

2.1 Introduction

2.2 Market Sales Dynamics of the FP Market

2.3 Individual Drug Sales Analysis

2.4 Pipeline Database Analysis

Disclaimer

Acknowledgment

References

Chapter 3: Structural Aspects of Fusion Proteins Determining the Level of Commercial Success

3.1 Classification of FPs

3.2 Factors for Commercial Success

References

Chapter 4: Fusion Protein Linkers: Effects on Production, Bioactivity, and Pharmacokinetics

4.1 Introduction

4.2 Overview of General Properties of Linkers Derived from Naturally Occurring Multidomain Proteins

4.3 Empirical Linkers in Recombinant Fusion Proteins

4.4 Functionality of Linkers in Fusion Proteins

4.5 Conclusions and Future Perspective

References

Chapter 5: Immunogenicity of Therapeutic Fusion Proteins: Contributory Factors and Clinical Experience

5.1 Introduction

5.2 Basis of Therapeutic Protein Immunogenicity

5.3 Tools for Immunogenicity Screening

5.4 Approaches for Risk Assessment and Minimization

5.5 Case Study and Clinical Experience

5.6 Preclinical and Clinical Immunogenicity Assessment Strategy

5.7 Conclusions

Acknowledgment

References

Part II: The Triple T Paradigm: Time, Toxin, Targeting

IIA: Time: Fusion Protein Strategies for Half-Life Extension

Chapter 6: Fusion Proteins for Half-Life Extension

6.1 Introduction

6.2 Half-Life Extension Through Size and Recycling

6.3 Half-Life Extension Through Increase of Hydrodynamic Radius

6.4 Aggregate Forming Peptide Fusions

6.5 Other Concepts

6.6 Conclusions and Future Perspective

References

Chapter 7: Monomeric Fc-Fusion Proteins

7.1 Introduction

7.2 FcRn and Monomeric Fc-Fusion Proteins

7.3 Typical Applications

7.4 Alternative Applications

7.5 Expression and Purification of Monomeric Fc-Fusion Proteins

7.6 Conclusions and Future Perspectives

References

Chapter 8: Peptide-Fc Fusion Therapeutics: Applications and Challenges

8.1 Introduction

8.2 Peptide Drugs

8.3 Technologies Used for Reducing in Vivo Clearance of Therapeutic Peptides

8.4 Fc-Fusion Proteins in Drug Development

8.5 Peptide-Fc-Fusion Therapeutics

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

8.7 Conclusions

Acknowledgment

References

Chapter 9: Receptor-Fc and Ligand Traps as High-Affinity Biological Blockers: Development and Clinical Applications

9.1 Introduction

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

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

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

9.5 Development and Clinical Application of a Vegf Trap

9.6 “To Trap or not to Trap?” Advantages and Disadvantages of Receptor-Fc Fusions and Traps Versus Antibodies

9.7 Conclusion

Acknowledgment

References

Chapter 10: Recombinant Albumin Fusion Proteins

10.1 Concept

10.2 Technological Aspects

10.3 Typical Applications and Indications

10.4 Successes and Failures in Preclinical and Clinical Research

10.5 Challenges

10.6 Future Perspectives

10.7 Conclusion

Acknowledgment

References

Chapter 11: Albumin-Binding Fusion Proteins in the Development of Novel Long-Acting Therapeutics

11.1 Introduction

11.2 Clinically Validated Half-Life Extension Technologies—An Overview

11.3 Interferon-α Fused to Human Serum Albumin or Albudab—A Direct Comparison of HSA and AlbudAb Fusion Technologies

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

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

11.6 Conclusions

References

Chapter 12: Transferrin Fusion Protein Therapies: Acetylcholine Receptor-Transferrin Fusion Protein as a Model

12.1 Disease Overview

12.2 Fusion Protein SHG2210 Design

12.3 Characterization of SHG2210

12.4 Applications and Indications

12.5 Future Perspectives

12.6 Conclusion

References

Chapter 13: Half-Life Extension Through O-Glycosylation

13.1 Introduction

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

13.3 Designing Long-Acting Agonists of Glycoprotein Hormones

13.4 Conclusions

References

Chapter 14: ELP-Fusion Technology for Biopharmaceuticals

14.1 Introduction

14.2 ELP-Based Protein Purification

14.3 ELPylated Proteins in Medicine and Nanobiotechnology

14.4 Molecular Pharming: A New Application for Elpylation

14.5 Challenges and Future Perspectives

14.6 Conclusion

References

Chapter 15: Ligand-Receptor Fusion Dimers

15.1 Introduction

15.2 The GHLR-Fusions

15.3 Expression and Purification

15.4 Analysis of the LR-Fusions

15.5 LR-Fusions: The Next Generation in Hormone Treatment

15.6 Conclusion

References

Chapter 16: Development of Latent Cytokine Fusion Proteins

16.1 Introduction

16.2 Description of Concept

16.3 Limitations of the Latent Cytokine Technology

16.4 Generation of Latent Cytokines

16.5 Applications and Potential Clinical Indications

16.6 Alternatives/Variants of Approach

16.7 Challenges (Production and Development)

16.8 Conclusions and Future Perspectives

Acknowledgments

References

IIB: Toxin: Cytotoxic Fusion Proteins

Chapter 17: Fusion Proteins with Toxic Activity

17.1 Introduction

17.2 Toxins

17.3 Immunocytokines

17.4 Human Enzymes

17.5 Apoptosis Induction

17.6 Fc-Based Toxicity

17.7 Peptide-Based Toxicity

17.8 Conclusions and Future Perspectives

References

Chapter 18: Classic Immunotoxins with Plant or Microbial Toxins

18.1 Introduction

18.2 Toxins Used in Immunotoxin Preparation

18.3 Immunotoxin Design and Synthesis

18.4 Clinical Update of Immunotoxin Trials

18.5 Challenges and Perspective of Classic Immunotoxins

18.6 Conclusions

References

Chapter 19: Targeted and Untargeted Fusion Proteins: Current Approaches to Cancer Immunotherapy

19.1 Introduction

19.2 Immunotherapeutic Strategy for Cancer: Fusion Proteins

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

19.4 Combination Fusion Proteins Therapy

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

19.6 Future Directions

19.7 Conclusion

Acknowledgments

References

Chapter 20: Development of Experimental Targeted Toxin Therapies for Malignant Glioma

20.1 Introduction

20.2 Targeted Toxins—General Considerations

20.3 Delivery Mode and Pharmacokinetics of Targeted Toxins in the Brain

20.4 Preclinical and Clinical Studies with Targeted Toxins

20.5 Conclusions and Future Developments of Targeted Toxins

Disclosure

References

Chapter 21: Immunokinases

21.1 Introduction

21.2 Protein Kinases, Apoptosis, and Cancer

21.3 Therapeutic Strategies to Restore Missing Kinase Expression

21.4 Analysis of Immunokinase Efficacy

21.5 Outlook

References

Chapter 22: Immunornase Fusions

22.1 Introduction

22.2 Development of ImmunoRNase Fusion Proteins as Biopharmaceuticals

22.3 Aspects of ImmunoRNase Design and Production

22.4 Alternatives

22.5 Conclusions and Future Perspectives

References

Chapter 23: Antibody-Directed Enzyme Prodrug Therapy (ADEPT)

23.1 Introduction

23.2 The Components

23.3 ADEPT Systems with Carboxypeptidase G2 (CPG2)

23.4 Fusion Proteins

23.5 Immunogenicity

23.6 Conclusions and Future Outlook

Acknowledgments

References

Chapter 24: Tumor-Targeted Superantigens

24.1 Introduction: Tumor-Targeted Superantigens—A Unique Concept of Cancer Treatment

24.2 Structure and Production of Tumor-Targeted Superantigens

24.3 Tumor-Targeted Superantigens are Powerful Targeted Immune Activators and Useful for All Types of Malignancies

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

24.5 Clinical Experience with TTS Therapeutic Fusion Proteins

24.6 Combining TTS with Cytostatic and Immunomodulating Anticancer Drugs

24.7 Conclusions

References

IIC: Targeting: Fusion Proteins Addressing Specific Cells, Organs, and Tissues

Chapter 25: Fusion Proteins with a Targeting Function

25.1 Introduction

25.2 Targeting Organs

25.3 Intracellular Delivery

25.4 Oral Delivery

25.5 Conclusions and Future Perspectives

References

Chapter 26: Cell-Penetrating Peptide Fusion Proteins

26.1 Introduction

26.2 Typical Applications and Indications

26.3 Technological Aspects

26.4 Successes and Failures in Preclinical and Clinical Research

26.5 Alternatives/Variants of This Approach

26.6 Conclusions and Future Perspectives

Acknowledgments

References

Chapter 27: Cell-Specific Targeting of Fusion Proteins Through Heparin Binding

27.1 Why Target Heparan-Sulfate Proteoglycans with Fusion Proteins?

27.2 Heparan Sulfate Structure and Biosynthesis Create Diversity and a Template for Targeting Specificity

27.3 Tissue-Specific Expression of HSPGs and the Enzymes That Modify Them

27.4 Heparin-Binding Proteins and Growth Factors

27.5 Viruses Target Cells Through Heparin Binding

27.6 Dissecting Heparin-Binding Protein Domains for Tissue-Specific Targeting

27.7 Fusion Proteins Incorporating HBDs

27.8 The Neuregulin 1 Growth Factor has a Unique and Highly Specific HBD

27.9 Using Neuregulin's HBD to Generate a Targeted Neuregulin Antagonist

27.10 Tissue Targeting and Therapeutic Efficacy of a Heparin-Targeted NRG1 Antagonist Fusion Protein

27.11 Conclusions and Future Perspectives

References

Chapter 28: Bone-Targeted Alkaline Phosphatase

28.1 Detailed Description of the Concept

28.2 Technical Aspects

28.3 Applications and Indications

28.4 Preclinical and Clinical Research

28.5 Alternatives/Variants of This Approach

28.6 Challenges in Production and Development

28.7 Conclusions and Future Perspectives

Acknowledgments

References

Chapter 29: Targeting Interferon-α to the Liver: Apolipoprotein A-I as a Scaffold for Protein Delivery

29.1 Detailed Description of the Concept

29.2 Technological Aspects

29.3 Typical Applications and Indications

29.4 Alternatives and Variants of This Approach

29.5 Conclusions and Future Perspectives

References

Part III: Beyond the Triple T-Paradigm

IIIA: Novel Concepts, Novel Scaffolds

Chapter 30: Signal Converter Proteins

30.1 Introduction

30.2 Historical Roots of Signal Conversion: Artificial Veto Cell Engineering and Protein Painting

30.3 Trans Signal Converter Proteins

30.4 Expanding Trans Signal Conversion Options: Redirecting Signals

30.5 From Trans to Cis Signal Conversion: Driving Auto-Signaling

30.6 Mechanistic Dividends of Chimerization

30.7 Targeting Multiple Diseases with Individual Signal Converters

30.8 Structural Constraints in SCP Design

30.9 Coding SCP Functional Repertoires

30.10 Expanding the Catalog of Inhibitory SCP

30.11 Immune Activating SCP

30.12 Experimental Tools for Screening SCP Candidates

30.13 SCP Frontiers: Mining the Surface Protein Interactome, Rewiring Cellular Networks

References

Chapter 31: Soluble T-Cell Antigen Receptors

31.1 Soluble T-Cell Antigen Receptor (STAR) Fusion Technology and Utilities

31.2 Expression and Purification of Recombinant Star Fusion Proteins

31.3 Clinical and Research Product Applications

31.4 Preclinical Testing Using Star Fusion Proteins

31.5 Clinical Development of ALT-801

31.6 Alternatives/Variants of This Approach

31.7 Challenges

31.8 Conclusions and Future Perspectives

Acknowledgments

References

Chapter 32: High-Affinity Monoclonal T-Cell Receptor (mTCR) Fusions

32.1 Introduction: The T Cell Receptor (TCR) as a Targeting Molecule

32.2 Engineered High-Affinity Monoclonal TCRs (mTCR)

32.3 mTCR-Based Fusion Proteins for Therapeutic Applications

32.4 Immune-Mobilizing Monoclonal TCRs Against Cancer (ImmTAC)

32.5 Conclusions and Future Perspectives

Acknowledgments

References

Chapter 33: Amediplase

33.1 Introduction

33.2 Source, Physico-Chemical Properties and Formulation

33.3 Preclinical Studies

33.4 Human Studies

33.5 Historical Comparison with Other Thrombolytics

33.6 Conclusions and Future Perspectives

Acknowledgment

References

Chapter 34: Breaking New Therapeutic Grounds: Fusion Proteins of Darpins and Other Nonantibody Binding Proteins

34.1 Introduction

34.2 Novel Scaffolds—Alternatives to Antibodies

34.3 New Therapeutic Concepts with Nonantibody Binding Proteins

34.4 Scaffold-Fusion Proteins Beyond Antibody Possibilities

Acknowledgments

References

IIIB: Multifunctional Antibodies

Chapter 35: Resurgence of Bispecific Antibodies

35.1 A Brief History of Bispecific Antibodies

35.2 Asymmetric IgG-Like Bispecific Antibodies

35.3 Symmetric IgG-Like Bispecific Antibodies

35.4 IgG-Like Bispecific Antibodies with Fused Antibody Fragments

35.5 Bispecific Constructs Based on the Fcγ Fragment

35.6 Bispecific Constructs Based on Fab Fragments

35.7 Bispecific Constructs Based on Diabodies or Single-Chain Antibodies

35.8 Bifunctional Fusions of Antibodies or Fragments with Other Proteins

35.9 Bispecific Antibodies for Various Functions: How to Select the Right Format?

References

Chapter 36: Novel Applications of Bispecific DART® Proteins

36.1 Introduction

36.2 DART® Proteins

36.3 Application of DART® to Cross-Link Inhibitory and Activating Receptors

36.4 Application of Bispecific Antibodies in Oncology

36.5 U-DART Concept for Screening DART® Candidate Targets and mAbs

36.6 U-DART Concept for Applications in Autoimmune and Inflammatory Disease

36.7 Conclusions and Future Perspectives

References

Chapter 37: Strand Exchange Engineered Domain (Seed): A Novel Platform Designed to Generate Mono and Multispecific Protein Therapeutics

37.1 Introduction

37.2 Technical Aspects

37.3 Potential Therapeutic Applications

37.4 Future Perspectives

37.5 Conclusions

Acknowledgments

References

Chapter 38: CovX-Bodies

38.1 The CovX-Body Concept

38.2 Technological Aspects

38.3 Applications of the CovX-Body Technology

References

Chapter 39: Modular Antibody Engineering: Antigen Binding Immunoglobulin Fc CH3 Domains as Building Blocks for Bispecific Antibodies (mAb2)

39.1 Introduction

39.2 Immunoglobulin Fc as a Scaffold

39.3 Design of Libraries Based on the Human IgG1 CH3 Domain

39.4 TNF-α-Binding Fcab: Selection and Characterization of Fcab TNF353-2

39.5 Conclusions and Future Perspectives

Acknowledgments

References

Chapter 40: Designer Fusion Modules for Building Multifunctional, Multivalent Antibodies, and Immunoconjugates: The Dock-and-Lock Method

40.1 Introduction

40.2 DDD/AD Modules Based on PKA and AKAP

40.3 Advantages and Disadvantages of the DNL Method

40.4 Fab-Based Modules

40.5 IgG-AD2-Modules

40.6 Hexavalent Antibodies

40.7 More Antibody-Based-Modules and Multivalent Antibodies

40.8 Nonantibody-Based DNL Modules

40.9 IFN-α2b-DDD2 Module and Immunocytokines

40.10 Variations on the DNL Theme

40.11 Conclusions and Future Perspective

References

Index

Copyright © 2013 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.

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Library of Congress Cataloging-in-Publication Data:

Fusion protein technologies for biopharmaceuticals : applications and challenges / edited by Stefan R. Schmidt.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-0-470-64627-4 (cloth)

I. Schmidt, Stefan R.

[DNLM: 1. Recombinant Fusion Proteins–therapeutic use. 2. Drug Carriers. 3. Immunotoxins–pharmacokinetics. 4. Immunotoxins–therapeutic use. 5. Protein Engineering. 6. Recombinant Fusion Proteins–pharmacokinetics. QU 450]

615.7–dc23

2012039440

Preface

This book covers the applications and challenges of fusion proteins; a relatively new class of therapeutic molecules. It is written both for experienced scientists wanting to acquire a deeper understanding of the underlying principles of fusion protein design and for students looking for a comprehensive introduction to this topic. This book shall serve as a source of inspiration by describing a wide range of examples. It gives an overview on the current state of the art and summarizes the huge potential of fusion protein design and functionality. The fascination of fusion proteins is derived from the vast range of possibilities that can be generated by joining individual natural molecules through human creativity. Overall, the rise of fusion proteins is driven by the revolution of genetic engineering, the improved knowledge on protein function, and the unmet medical need of many indications.

One of the many objectives is trying to fulfil the promise of Paul Ehrlich's Magic Bullet. This book also picks up the current trend of designing next generation biopharmaceuticals to enable combination therapy by addressing two targets simultaneously. Fusion proteins offer an elegant solution to the challenge of delivering equivalent concentrations of two active molecules to a specific location while keeping manufacturing costs low.

Inspiring the design and therapeutic applications of fusion proteins is the goal of this book. In the first few chapters, the readers are introduced to the concept of fusion proteins while some challenges such as immunogenicity are addressed. The main body contains three large sections on typical paradigms such as strategies to extend the plasma half-life, the design of toxic proteins, and finally utilizing fusion proteins for highly specific targeting. Each of these sections is preceded by an introduction summarizing the respective topic. The last part of the book covers novel concepts including examples of highly relevant multifunctional antibodies. I have tried to maintain a balance between well-established molecules with clinical data and more experimental approaches that nevertheless appear very promising. All chapters are authored by experts in the field who happily share their deep understanding with the readers.

Throughout my professional life I have been studying proteins, starting with proteins whose malfunction causes disease and moving to monoclonal antibodies; always dealing with their isolation and characterization. My particular personal interest in fusion proteins was stimulated years ago when trying to optimize the half-life of an enzyme prototype. By digging deeper into the matter, I came across the many ways how to influence the plasma half-life. However, at this time no textbook about that topic was available, therefore, evaluating the different approaches was quite tedious. In the meantime, some books have been published addressing some isolated aspects that could be of interest to fusion proteins, but none really dedicated to fusion proteins in general. Identifying that gap, I started to collect material to prepare a focussed book and got into contact with several well-known colleagues working on various fusion proteins. Many stimulating discussions shaped the framework of the book. The result of these interactions is this book that covers all aspects of modern therapeutic fusion proteins.

I hope that my readers will enjoy reading this book as much as I enjoyed creating it together with so many talented people.

Finally I would like to thank my wife Gabriele and my son Felix for their encouragement and loving support.

Stefan R. Schmidt

Contributors

Gill Adams, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Ralph F. Alderson, MacroGenics, Inc., Rockville, MD, USA

Wojciech Ardelt, Tamir Biotechnology, Inc., Monmouth Junction, NJ, USA

Peter J. Artymiuk, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK

Patrick A. Baeuerle, Amgen Research (Munich) GmbH, Munich, Germany

Bhaswati Barat, MacroGenics, Inc., Rockville, MD, USA

Stefan Barth, Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, Helmholtz-Institute for Biomedical Engineering, University Hospital RWTH Aachen, Germany; Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany

Anton Bauer, f-star GmbH, Vienna, Austria

Mark Belsey, WestLB, London, UK

Pedro Berraondo, Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona; Liver Unit, University Clinic, CIBER-EHD, Pamplona, Navarra, Spain

Abhijit Bhat, CovX Research, Pfizer Worldwide R & D, San Diego, CA, USA

Hans Kaspar Binz, Molecular Partners AG, Schlieren, Switzerland

Ezio Bonvini, MacroGenics, Inc., Rockville, MD, USA

Chien-Hsing Chang, Immunomedics, Inc., Morris Plains, NJ, USA

Xiaoying Chen, Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer, San Diego, CA, USA

Yuti Chernajovsky, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Udo Conrad, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany

Leslie Cousens, EpiVax Inc., Providence, RI, USA

Leif Dahlberg, Active Biotech AB, Lund, Sweden

Jessica P. Dawson, EMD Serono Research Institute, Billerica, MA, USA

Anne S. De Groot, EpiVax Inc., Providence, RI, USA; Institute for Immunology and Informatics, University of Rhode Island, Providence, RI, USA

Jennifer A. Dumont, Biogen Idec Hemophilia, Weston, MA, USA

Gráinne Dunlevy, Biopharm R & D, Domantis Ltd., Cambridge, UK

Aris N. Economides, Regeneron Pharmaceuticals, Tarrytown, NY, USA

Alan L. Epstein, Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

Stefano Evangelista, Menarini Ricerche SpA, Preclinical Development, Florence, Italy

Fuad Fares, Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Mount Carmel, Haifa, Israel

Rewas Fatah, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Eric Ferrandis, IPSEN, Institut Henri Beaufour, Les Ulis, France

Jessica Fioravanti, Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona; Liver Unit, University Clinic, CIBER-EHD, Pamplona, Navarra, Spain

Doreen M. Floss, Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany

Göran Forsberg, Active Biotech AB, Lund, Sweden

Arthur Frankel, Cancer Research Institute of Scott and White Memorial Hospital, Texas A&M Health Science Center College of Medicine, Temple, TX, USA

Stefan Gattenlöhner, Institute of Pathology, University Hospital Giessen and Marburg GmbH (UKGM), Giessen, Germany

David M. Goldenberg, Garden State Cancer Center, Center for Molecular Medicine and Immunology, Morris Plains, NJ, USA

David Gould, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Alec W. Gross, EMD Serono Research Institute, Billerica, MA, USA

Namir J. Hassan, Immunocore Ltd, Abingdon, UK

Michael Heartlein, Shire Human Genetic Therapies, Lexington, MA, USA

Gunnar Hedlund, Active Biotech AB, Lund, Sweden

Volkmar Heidecke, Department of Neurosurgery, Klinikum Augsburg, Augsburg, Germany

Henrik Helmfors, Department of Neurochemistry, Stockholm University, Stockholm, Sweden

Gottfried Himmler, f-star GmbH, Vienna, Austria

Björn Hock, Merck Serono, Protein Engineering and Antibody Technologies, Darmstadt, Germany

Peisheng Hu, Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

Chichi Huang, Biologics Research, Centocor R&D, A Division of Johnson & Johnson Pharmaceutical Research & Development, Radnor, PA, USA

Bent K. Jakobsen, Immunocore Ltd, Abingdon, UK

Vibha Jawa, Medical Sciences, Amgen, Inc., Thousand Oaks, CA, USA

Syd Johnson, MacroGenics, Inc., Rockville, MD, USA

Serene Josiah, Shire Human Genetic Therapies, Lexington, MA, USA

Kristin Karlsson, Department of Neurochemistry, Stockholm University, Stockholm, Sweden

Dennis Keefe, Shire Human Genetic Therapies, Lexington, MA, USA

Christie Kelton, EMD Serono Research Institute, Billerica, MA, USA

Leslie A. Khawli, Department of Pharmacokinetic and Pharmacodynamic Sciences, Genentech Research and Early Development, Genentech, Inc, San Francisco, CA, USA

Apostolos Koutsokeras, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Snejana Krassova, Biogen Idec Hemophilia, Weston, MA, USA

Ülo Langel, Department of Neurochemistry, Stockholm University, Stockholm, Sweden

Rodney Lappe, CovX Research, Pfizer Worldwide R & D, San Diego, CA, USA

Olivier Laurent, CovX Research, Pfizer Worldwide R & D, San Diego, CA, USA

Hua W. Li, MacroGenics, Inc., Rockville, MD, USA

Nikolai M. Lissin, Immunocore Ltd, Abingdon, UK

Jeffrey A. Loeb, Department of Neurology, Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA

Susan C. Low, Biogen Idec Hemophilia, Weston, MA, USA

Zhenzhong Ma, Department of Neurology, Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA

Stefano Manzini, Menarini Ricerche SpA, Preclinical Development, Florence, Italy

Sean D. McKenna, EMD Serono Research Institute, Billerica, MA, USA

Baisong Mei, Biogen Idec Hemophilia, Weston, MA, USA

Hubert J. Metzner, Preclinical Research and Development, CSL Behring GmbH, Marburg, Germany

José Luis Millán, Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA

Gayatri Mittal, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Paul A. Moore, MacroGenics, Inc., Rockville, MD, USA

Marco Muda, EMD Serono Research Institute, Billerica, MA, USA; Merrimack Pharmaceuticals, Cambridge, MA, USA

Geert C. Mudde, f-star GmbH, Vienna, Austria

Lisa Mullen, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Andrés Muñoz-Alarcón, Department of Neurochemistry, Stockholm University, Stockholm, Sweden

Thore Nederman, Active Biotech AB, Lund, Sweden

Mats Nilsson, Active Biotech AB, Lund, Sweden

Robert T. Peters, Biogen Idec Hemophilia, Weston, MA, USA

Glenn F. Pierce, Biogen Idec Hemophilia, Weston, MA, USA

Sarbendra L. Pradhananga, Department of Human Metabolism, University of Sheffield, Medical School, Sheffield, UK

Jesús Prieto, Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona; Liver Unit, University Clinic, CIBER-EHD, Pamplona, Navarra, Spain

Nikolai G. Rainov, Department of Neurosurgery, Klinikum Augsburg, Augsburg, Germany

Tobias Raum, Amgen Research (Munich) GmbH, Munich, Germany

Anne Rigby, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Peter R. Rhode, Altor BioScience Corporation, Miramar, FL, USA

Stefan Rose-John, Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany

Richard J. Ross, Department of Human Metabolism, University of Sheffield, Medical School, Sheffield, UK

Edmund A. Rossi, IBC Pharmaceuticals, Inc., Morris Plains, NJ, USA

Florian Rüker, Department of Biotechnology, Christian Doppler Laboratory for Antibody Engineering, University of Natural Resources and Life Sciences, Vienna, Austria

Jon R. Sayers, Department of Infection and Immunity, University of Sheffield, Medical School, Sheffield, UK

Jürgen Scheller, Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany

Stefan R. Schmidt, Rentschler Biotechnologie GmbH, Laupheim, Germany

Stefan Schulte, Preclinical Research and Development, CSL Behring GmbH, Marburg, Germany

Michelle Sclanders, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Surinder K. Sharma, Research Department of Oncology, University College London, London, UK

Wei-Chiang Shen, Department of Pharmacology and Pharmaceutical Sciences, University of Southern California School of Pharmacy, Los Angeles, CA, USA

Giles Somers, Datamonitor, London, UK

Neil Stahl, Regeneron Pharmaceuticals, Tarrytown, NY, USA

Anette Sundstedt, Active Biotech AB, Lund, Sweden

Ronald V. Swanson, Biologics Research, Centocore R&D, A Division of Johnson & Johnson Pharmaceutical Research & Development, San Diego, CA, USA

Mikael Tiensuu, Active Biotech AB, Lund, Sweden

Peter Topley, Biopharm R&D, GlaxoSmithKline Medicines Research Centre, Stevenage, UK

Mehmet Kemal Tur, Institute of Pathology, University Hospital Giessen and Marburg GmbH (UKGM), Giessen, Germany

Mark L. Tykocinski, Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

Sandrine Vessillier, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London Medical School, London, UK

Adam Walker, Biopharm R & D, Domantis Ltd., Cambridge, UK

Jiajing Wang, Department of Neurology, Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA

Thomas Weimer, Preclinical Research and Development, CSL Behring GmbH, Marburg, Germany

Ian R. Wilkinson, Department of Human Metabolism, University of Sheffield, Medical School, Sheffield, UK

Maximilian Woisetschläger, f-star GmbH, Vienna, Austria

Jung Hee Woo, Cancer Research Institute of Scott and White Memorial Hospital, Texas A&M Health Science Center College of Medicine, Temple, TX, USA

Gordana Wozniak-Knopp, Department of Biotechnology, Christian Doppler Laboratory for Antibody Engineering, University of Natural Resources and Life Sciences, Vienna, Austria

Jennica Zaro, Department of Pharmacology and Pharmaceutical Sciences, University of Southern California School of Pharmacy, Los Angeles, CA, USA

Part I

Introduction

1

Fusion Proteins: Applications and Challenges

Stefan R. Schmidt

Rentschler Biotechnologie GmbH, Laupheim, Germany

1.1 History

Proteins as drugs have a long history. In the beginning, natural proteins were extracted from animal, human sources, or in some rare cases even from plants. Large-scale processing of human plasma became a primary source for the isolation of many proteins [1]. For instance, blood factors became available as a therapy against the different forms of hemophilia or the lack of functional (α-1 antitrypsin. The major serum component, albumin, has now been used for more than 50 years as a treatment for shock, trauma, or burns. Immune globulins isolated from human sources are also used successfully in various immunodeficiency diseases. However, despite the great success of plasma products, contaminations with the HIV or hepatitis virus in the 1970–1980s triggered more intensified efforts to prepare virus-free recombinant therapeutic proteins.

Since its identification in the 1920s until the 1980s, insulin from animals was the only treatment for diabetes patients. Particularly, the porcine insulin was widely used since there is only a single amino acid variation from the human form. Chemical processes were developed to obtain the fully human variant from the pig isoform [2]. Finally, the first recombinant human insulin was manufactured by Eli Lilly & Co in partnership with Genentech, approved in 1982 by the FDA and marketed under the name Humulin® [3]. This was also the first therapeutic recombinant protein for human use.

Since that time, the number of recombinant products and approved biopharmaceuticals has increased considerably. Initially, recombinant copies of proteins were made that replaced the natural protein, which until then was harvested from animal or human sources. With the exception of factor VIII against hemophilia, all these proteins such as human growth hormone (hGH) or follicle stimulating hormone (FSH) belonged to the class of hormones. They were soon accompanied by a growing number of first generation therapeutics that could only be obtained recombinantly such as erythropoietin (EPO), interferon (IFN) or tissue plasminogen activator (tPA), just to name a few. After this first enthusiasm and the success with reproducing natural proteins by recombinant DNA technology, researchers started to consider the de novo design of therapeutic proteins that do not occur in nature. There is one specific class that can be seen as intermediate link between natural and designed proteins, monoclonal antibodies (mABs).

Antibodies, being a major part of the organism's immune defense, are large proteins that exist in all higher animals. In 1975, a method was developed to generate murine cell lines producing antibody molecules of a single specificity, the so-called monoclonal antibodies (mABs) [4]. The first therapeutic monoclonal antibody, orthoclone OKT3, was of murine origin and approved in 1986. From then, this concept was further refined with the help of modern recombinant DNA technology to obtain the first fully human antibody against tumor necrosis factor-α (TNF-α), marketed under the name Humira® in 2002 [5]. The milestones for recombinant therapeutic proteins can be seen in Figure 1.1.

Figure 1.1 Milestones of recombinant therapeutic proteins.

Interestingly, preventing the activity of TNF-α was also the goal for the first fusion protein, Etanercept. It consists of the TNF-α receptor attached to a sequence encoding the Fc portion and hinge region of an IgG1 heavy chain. This drug has been marketed under the name Enbrel® since 1998 and is the best selling fusion protein till date [6]. The two extraordinarily successful drugs Humira and Enbrel can serve as prototype for their molecule classes, exemplifying the different ways to address the same target and present a typical case of competition between antibodies and fusion proteins in the market.

1.2 Definitions and Categories

This book focuses on fusion proteins that are generated by joining two or more genes by genetic engineering that originally code for separate proteins. The result is a single polypeptide with functional properties of both parent proteins. These recombinant proteins are combinations of unrelated domains not occurring in nature. Excluded from the content of this book are multiepitope recombinant vaccines [7], chemical conjugates [8] naturally occurring fusion proteins resulting from chromosomal rearrangements that can be observed in many cancer cells [9] or fusion tags for affinity purification [10].

Bi or multispecific antibodies are special case that do not always represent a single polypeptide chain but usually consist of the combination of heavy and light chains. The Part IIIb of this book discusses some non-natural versions that have more than a single specificity.

The most straightforward classification of these novel proteins can be based on the functions of their incorporated domains. Typically, one part serves molecular recognition or binding, whereas the other part adds certain functionalities such as extending half-life or stability, cytotoxicity, or novel targeting or delivery routes [11].

Recently, a review classified therapeutic proteins according to their pharmacologic activity to (a) replace a deficient or abnormal protein, (b) augment an existing pathway, (c) provide a novel function or activity, (d) interfere with a molecule or organism, or (e) deliver a payload such as a radionuclide, cytotoxic drug, or protein effector [12].

However, this classification is not fully suitable for the scope of this book. Most fusion proteins serve three major purposes that can be summarized under the triple T (T3) paradigm: (a) t1/2 (half-life), (b) targeting (or binding), or (c) toxicity (cell killing). Of these three elements, at least two are simultaneously present in fusion proteins (Figure 1.2). Antibodies as natural molecules combine all three aspects in a single molecule. However, antibody derivatives, fragments, or domains have also been used extensively as building blocks for fusion proteins, hence constituting a large part of the portfolio of proteins discussed here, and thus deserving their own category. The main functionality of antibodies, the binding with high affinity and selectivity to a specific epitope, has been reproduced in a number of nonantibody scaffolds that can either be used as single module or by combining two units with different specificity [13]. These molecules together with other bi- or multifunctional therapeutics that do not fit to the T3 categories are classified into the group of novel artificial molecules that is discussed in Part IIIa of this book.

Figure 1.2 The triple T paradigm of fusion proteins. Most fusion proteins are composed of mixtures of these modules to combine two functionalities. The multifunctional natural prototype is an antibody combining the constant Fc part that can contribute to half-life extension and toxicity. The Fab part is contained in many fusion proteins for targeting and binding purposes. LRF, ligand receptor fusion; CTP, C-terminal peptide; XTEN, XTENylation; ELP, elastin-like peptide; PAS, PASylation; GILT, glycosylation independent lysosomal targeting; ApoA1, apolipoprotein A1; ADEPT, antibody-directed enzyme prodrug therapy; TNF, tumor necrosis factor family.

A very practical classification is proposed by the authors of Chapter 3 about “Structural Aspects of Fusion Proteins Determining the Level of Commercial Success” in this book. They suggest sorting fusion proteins based on one of three different functional groups: activity, targeting, or half-life, of which the latter two can be summarized under delivery agents, thus being able to define a two-dimensional landscape of fusion proteins. This is quite similar to a previous scheme using the combination of an effector fragment together with a molecular recognition part as building blocks for fusion proteins [14].

But why should we deal at all with fusion proteins? Several advantages make them very attractive: the combination of two functionalities in a single molecular entity simplifies manufacture and drug delivery. Two molecules combined into one will automatically have identical biodistribution profiles instead of two separate molecules that might have a very different distribution. Furthermore, new functionalities can be created that are lacking in natural or separate proteins. This includes the modification of half-life or targeting specificity. Even economic opportunities such as life cycle extension of products with expired patents are possible. This includes also the generation of novel intellectual property for new and non-natural combinations of proteins. Therapeutic benefits derived from reduced side effects or longer dosing intervals and improved activity are strong drivers to promote the generation of fusion proteins.

But besides all these important advantages, there are also a number of challenges. The combination of unrelated proteins might prove difficult to manufacture because in some cases, the fusion partners have noncompatible properties. This can cause aggregation or misfolding of one domain while the conditions might be perfect for the other domain. Despite the fact that some modules of fusion proteins are elements of other well-proven molecules such as antibodies, the established platform processes might not be applicable because other features shield the required property. This can go so far that formulation is not possible due to conflicting stability requirements. Furthermore, it will be difficult to control and tune the relative amounts of each component thus complicating dosing for optimal efficacy and safety. Probably most important challenge is the high potential for immunogenicity due to the formation of novel epitopes at the junction between the fusion partners even if only fully human proteins are connected.

1.3 Patenting

The first generation biologics that represented a true copy of human proteins used for therapeutic applications have already lost or are about to lose their patent protection [15]. In many cases second generation molecules, for example, with improved half-life, are taking their place. A number of them are fusion proteins that are patented as well.

To be able to file a patent for an invention, three characteristics must be achieved novelty, nonobviousness, and utility or enablement [16]. In the postgenome era, the discovery of novel proteins, at least of human origin, will be difficult. This challenges the first critical parameter on the way to a patent, the novelty. If we focus on the scope of this book, the fusion proteins, novelty still seems to be easy to reach. As described in the paragraph about definitions, “joining two or more genes by genetic engineering that originally code for separate proteins,” so the generated fusion protein will be novel if nobody did the same earlier. Therefore, novelty is given on one hand through the composition of matter (the new construct, e.g., long-acting human growth hormone [hGH], consisting of hGH fused to human serum albumin [HSA]) or on the other hand by the use that results from combined features of the new molecule (e.g., to treat dwarfism with longer administration intervals). This is in contrast to a natural polypeptide with multiple functions [17].

Taking the example of antibodies as molecules, their individual characteristics, for example, target or captured epitope, affinity, half-life, or sequence of the variable part should be sufficient to enable patenting based on novelty [18]. Surprisingly, exactly these descriptive features are missing in many antibody patents, which is an effect of patenting human genes that often include hypothetical antibodies to that target in the claims [19]. It might be easier trying to patent antibodies based on unexpected advantages, that is, having lower cross-reactivity since higher affinity could be regarded as obvious [20]. This should be taken into account for patenting antibody derived fusion proteins. Particularly, tumor-specific antibodies have had only limited success so far. This led to new intellectual property of improved molecules by coupling antibodies to toxic proteins, thus combining target specificity with nonspecific cytotoxicity [21].

However, if we continue with the second parameter, the nonobviousness, it starts to get more difficult. In the context of fusion proteins in many cases, the existence or functionality of the potential fusion partners will be well known; thus representing prior art. Interestingly, when combining several “prior arts” into a new concept, this patent will only be rejected as obvious if there existed at the time of invention a known problem for which there was an obvious solution encompassed by the patent's claims [22]. The best argument to get a combination patent is to demonstrate that prior art is not providing a motivation or suggestion to prepare this combination. Even when sufficient suggestions for combinations can be found in prior art, patenting can still be possible if enabling in form of a production method can be claimed additionally. Therefore, when defining patent claims, it can be very beneficial to include methods how to manufacture the protein of interest and what formulations are useful [23].

The third parameter to demonstrate utility is relatively straightforward since the fusion protein was designed with a specific application in mind. Here, both typical aspects of utility, recognition of a benefit and the motivation to make a change to current practice, come into play. Overall it has to be described how the invention can be put into practice, which is ideally done in the form of examples.

In the past, some patent disputes ranked around protected fusion protein technology. For instance, Zymogenetics accused Bristol-Myers Squibb (BMS) to infringe their Fc-fusion technology with Orencia®. Initially, the case was settled by a lump sum payment, but finally BMS acquired Zymogenetics in 2010 together with the rights for Fc fusions [24].

1.4 Design and Engineering

In the design of a novel fusion protein a number of parameters have to be taken into account. Following questions need to be addressed: Will the proteins be functional on either the N-terminus or the C-terminus? In which orientation will the individual proteins be connected? What linker length and sequence should be used? Is there a need for a specific oligomerization? Are mutations or truncations required to enhance or eliminate certain features? In many cases rational design will guide the generation of innovative protein therapeutics [25]. One of the aims of protein design is certainly to improve the functionality of biological drug (Figure 1.3). A key question in this context is: Will the protein reach the target in a significant quantity? Taking the example of a solid tumor it becomes clear that in order to reach the tumor the protein has to be sufficiently small to penetrate the many cell layers. But on the other hand it should not be too small to be excreted too fast. This requires a delicate balancing of the molecule size and has been demonstrated experimentally with a number of different antibody derivatives [26]. Not only size but also valency of antibodies can be modified. Further details on bi-specific and multifunctional antibodies can be found in Part IIIb of this book.

Figure 1.3 Fusion protein design. The typical variations of fusion proteins comprise orientation, linkers, and oligomerization. Sometimes proteins require a free N- or C-terminus for their activity. The linker can vary in length, flexibility, and susceptibility to protease cleavage. Dimeric forms of fusion proteins can be generated by including leucine zippers in the linker region. Two examples of higher oligomerization are depicted as well; first, a trimer built from the trimerization domain of human endostatin and second, a hexamer constructed through the combination of three Fc dimers connected via an ILZ coiled coil to two trimers (1see Morris et al. [51]).

1.4.1 Orientation of Fusion Proteins

Looking at the largest group, the Fc-fusion proteins, it is well known that naturally the Fc part is positioned at the C-terminus of an antibody. But in artificial fusions it can also form the N-terminal part. Several studies have evaluated cytokine mono- or tandem fusions to full antibodies or Fc parts. Here no dependency on the selection of the respective terminus could be observed [27]. However, comparing N- or C-terminal Fc fusions of peptides blocking angiopoietin-2 (Ang-2), it was found that the N-terminal fusion had shorter half-life and weaker binding but better selectivity [28].

Generally, when combining two proteins, there are two orientations in which the fusion partners can be arranged, either at the amino or at the carboxy terminus of the first protein. In many cases the position is without influence on the functional properties, for instance albumin (HSA) can be fused to either end [29]. But during the manufacture of Albuferon®, a combination of interferon-α2b (IFN-α2b) with albumin, it was observed that due to incomplete disulfide bridge formation, aggregates formed that drastically reduced the recovery. Interestingly, this phenomenon could be completely abolished by positioning the IFN-α2b on the N-terminus instead [30]. In another case the order of two angiostatic proteins, human angiostatin (hAS) and endostatin (hES), combined in a fusion protein had to be in a specific orientation to obtain maximum activity. The fusion hES–hAS was 28% more potent than hAS-hES or 7% better than hAS and hES when administered separately [31]. Sometimes a free N-terminus is required. The second partner is then connected at the C-terminus as in the case of receptor traps such as Etanercept. Here, the extracellular receptor domain is combined with an Fc part at the C-terminus to maintain the natural conformation [6]. Another striking example for the positioning effect of fusion partners is the case of elastin-like peptides (ELP). It was observed that C-terminal fusions of ELP resulted in a higher expression level, better yield, and bioactivity. The underlying reason could be increased misfolding, induced by ELPs at the N-terminus; thus reducing the amount of active proteins and increasing their susceptibility to proteolysis [32]. Orientation has a high impact on functionality particularly when fusion proteins contain enzymes that require either a free N- or C-terminus. This has been demonstrated with the ImmunoRNAse consisting of angiogenin (ANG) and a single-chain variable domain (scFv) against CD22. Only constructs in the scFv-ANG orientation did not aggregate and were fully functional [33].

1.4.2 Linker Engineering

Again starting with Fc fusions as example, the hinge region fulfils the function of a linker, allowing some spatial flexibility [34]. Besides this exception where a part of a fusion protein ends with a flexible peptide chain, in most cases specific linkers between protein molecules have to be artificially introduced. The multiple aspects of linker design have recently been reviewed [35]. Many researchers use a simple glycine and serine (G4S)-containing linkers as proposed by a large study of natural domain separating linkers [36]. Spacer peptides that connect both modules of a fusion protein in a spatial conformation are frequently needed to maintain functionality. For instance, the highest potency could be observed when a spacer was introduced between a single chain variable domain (scFv) and ANG [37]. Since fusion proteins ideally consist only of a single polypeptide chain, a reformatting of Fab fragments to scFv is required. This is done with the help of a linker sequence that frequently consists of repeats of glycine and serine, as for example the popular (G4S)3 linker. The basis of linker design is the rational engineering of both length and conformation. A controlled distance between domains can be achieved by defined repeats of α-helical peptides A(EAAAK)nA that maintains the separation of domains in contrast to flexible linkers [38].

When evaluating the optimal linker between IFN-α2b and HSA it could be demonstrated that five amino acids (aa) are already sufficient. However, maximum activity was obtained with a helical 12 aa linker which was 1.7 times or 2.9 times better than a short rigid or the standard five aa long G4S linker [39]. The knowledge about linkers was continuously expanded [40]. Recently, a database collecting natural linker sequences and their properties could be established1. Inter domain linkers have to be differentiated from intradomain loops based on their function, but also because of their amino acid preference. Interestingly proline is the most frequent amino acid in both cases. Proline destroys α-helix and β-sheet structures as well and cannot form hydrogen bonds [41].

But linker peptides can also be engineered to contain additional functionalities. To optimise the pharmacokinetic profile of a granulocyte-colony-stimulating factor (G-CSF) fused to transferrin, a linker with an intramolecular disulfide bridge was introduced. The peptide sequence was thrombin sensitive, which allowed an in vitro cleavage by adding this protease, leaving the two domains only connect through the disulfide bridge. This labile bond could then easily be cleaved in vivo, releasing free G-CSF that was more active than the fused molecule [42]. Previously, a similar molecule was engineered to improve oral efficacy. Here, a long rigid helical linker composed of 50 aa between G-CSF and transferrin led to a 10 times lower EC50 than the initial fusion protein with a two aa spacer [43]. This particular linker also increased the expression level more than 10-fold compared to the direct fusion with only two aa in between. However, this time the construct was in an opposite orientation having transferrin at the N- and G-CSF at the C-terminus. Interestingly an unstructured 50 aa linker inhibited expression totally [44].

The orientation of domains and the impact of spacers were also studied when a scFv against human transferrin receptor was combined with the fungal ribonucleolytic toxin restrictocin. Independent of the orientation in both cases, the introduction of a protease-sensitive linker drastically improved the EC50 2- to 30-fold [45]. The influence of linkers on fusion proteins is discussed in Chapter 4.

1.4.3 Oligomerization of Fusion Proteins

The next level of design involves the correct oligomerization of the protein. In many cases, proteins are dependent on multimerization or have a higher bioactivity as a multimer. One example is the combination of stem cell factor (SCF) and macrophage-colony-stimulating factor (M-CSF) with a 12 aa flexible peptide linker. The fusion protein forms dimers that have a 10- to 20-fold higher potency than the individual monomeric proteins, also benefitting from a synergistic effect [46]. A higher cytoxicity could also be demonstrated for divalent antibody toxin fusions. Combining a Fab fragment with two molecules of a truncated Pseudomonas exotoxin A resulted in an almost 40-fold more active fusion protein than with only one toxin molecule [47]. Oligomerization can also help to improve valency of recombinant proteins. Sometimes it is required to induce cross-linking of receptors to execute specific functions such as activation or internalization. It has been demonstrated that by the right choice of linkers between VH and VL in scFv, aggregates can spontaneously form having a higher avidity [48].

Using an Fc part automatically delivers dimerization, however there are also different approaches possible to enforce dimers. Frequently leucine zippers are used for that purpose. An early example is the generation of bivalent scFv antibodies with Fos or Jun leucine zippers. A covalent bond could be introduced by positioning cysteine in proximity to the zipper. These molecules spontaneously formed dimers when secreted to Escherichia coli periplasm [49]. The leucine zipper GCN4 of Saccharomyces cerevisiae has been used to dimerize the soluble insulin receptor. This resulted in an improved binding constant, very similar to the original membrane bound native receptor [50].

As in the case of OX40 ligand (OX40L) even a trimerization can be required for full functionality. Here an active construct in the right conformation could be obtained by fusing OX40L to a GCN4 zipper domain connected to an Fc part. The final hexameric molecule consisted of three Fc-induced disulfide linked dimers that allowed the formation of two trimers [51]. Another molecule having a trimer as active natural conformation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). As before, the GCN4 zipper-induced self-assembled trimers of TRAIL based on three-stranded coiled coils [52]. However, TRAIL can also be trimerized as linear fusion in a head to tail configuration interrupted by short flexible peptide loops. To improve targeting, even a scFv could be included at the N-terminus [53]. Trimerization through tandem repeats in a single chain molecule could also be shown with TNF [54]. CD95 ligand (CD95L) as another member of the TNF homology domain (THD) family could be forced into covalent trimerization by fusion to a tenascin-C (TNC) oligomerization domain, again resulting in improved bioactivity [55]. Higher oligomerization of multiple trimers was tested with the CD40 ligand (CD40L). First a di-trimer conformation was achieved by incorporating the N-terminal part of adiponectin (Acrp30). A tetra-trimer construct could be obtained by combining the N-terminal part of mouse surfactant protein-D (SP-D) with the extracellular part of CD40L. Additional leucine zippers were not required because both Acrp30 and SP-D spontaneously self-assemble into the desired multimers. These constructs have been successfully used as adjuvant in DNA vaccines [56]. A similar approach was also initiated with soluble Fas ligand (FasL). The bioactivity of a single FasL trimer was much lower than di-trimer (hexameric) constructs that either relied on the oligomerization effects of Acrp30 or Fc [57]. Other naturally occurring trimer inducing molecules are the carboxyl-terminal noncollagenous domains (NC1 domains) of collagens XV and XVIII coding for endostatin. This was utilized to generate a multifunctional anti-angiogenic compound consisting of a scFv and a NC1 domain. Cleavage of the NC1 domain by tumor-associated proteases released endostatin from the scFv trimers, thus multiplying the anti-angiogenic effect [58]. Collagen trimers were also used to increase the binding strength of scFv 20- to1000-fold though multimerization. It was shown that this collagen-like scaffold (Gly-Pro-Pro)10 could either be fused to the N- or C-terminus. Depending on the addition of cysteins flanking the scaffold even a hexameric configuration could be obtained [59]. The trivalent human plasma protein tetranectin builds the core of a novel scaffold, the Atrimer™. So far Atrimers have not been used as basis for fusion proteins, but their multiple loops have been engineered for diverse binding applications [60].

1.4.4 Immunogenicity

Rational design of fusion proteins must also be used in order to minimize immunogenicity of the new construct. Even if using only human proteins as starting point for a modular assembly, still the region between both molecules represent a novel epitope that could elicit an immune response. Therefore, it is recommended to use at least in silico analysis to predict T- and B-cell epitopes. A lot of work on the removal of T-cell epitopes or de-immunization of fusion proteins has been conducted on immune toxins. In one recent example, a truncated version of Pseudomonas exotoxin A (PE38) fused to an anti CD22 scFv was scanned for the presence of B-cell epitopes with a huge set of antibodies. The identified major epitopes were neutralized by specific mutations [61]. Furthermore, two lysosomal protease-sensitive areas were eliminated from PE38. The resulting mutant had a drastically reduced immunogenicity and an even improved cytotoxicity [62].

A very special case is the antibody-directed enzyme prodrug therapy (ADEPT) that utilizes nonhuman enzymes to metabolize inactive prodrugs into highly potent toxins. One of the promising enzymes, a beta-lactamase had to be mutagenized to remove CD4+T-cell epitopes. Site-directed mutagenesis replacing individual aa lowered the T-cell response fivefold [63].

In general, Fc-fusion proteins should also display lower immunogenicity. This effect can be based on the presence of inhibitory Fc receptors on B lymphocytes, the FcγRIIb. This hypothesis was proven with the injection of DNA coding for a Exendin-4 Fc-fusion protein that did not result in the generation of neutralizing antibodies, which was the case for Exendin-4 alone [64].

A number of factors determine the immunogenicity potential of proteins in general. For example, protein aggregates might cross-link B-cell receptors, or increase protein internalization of antigen-presenting cells (APC), thus initiating an immune response. However, endocytosis can also happen if the target of a fusion protein is up taken. Furthermore, nonhuman modification (e.g., glcosylation) will also induce an immune reaction. Many of these reactions can be predicted in silico or analyzed in vitro with a wide range of assays [65]. Since both B- and T-cell epitopes contribute to immunogenicity, it is very advisable to identify and remove these epitopes. Due to the high polymorphism of the major histocompatibility complex (MHC) it can be difficult to remove all T-cell epitopes. B-cell epitopes are not restricted to MHC molecules; therefore, it might be easier to eliminate B-cell epitopes [66]. More details on the immunogenicity of fusion proteins can be seen in Chapter 5 of this book.

1.4.5 Mutagenesis for Molecule Optimization

A lot of work on the optimization of fusion proteins by side-directed mutagenesis is focused toward improving parameters beyond immunogenicity. An important factor is the resistance against proteases, on one hand during manufacture and on the other hand while circulating through the patient's organism. In both cases, it must be distinguished between exo- and endopeptidases. For exopeptidases that can cleave their target from either end, usually a modification of the terminal aa abolishes the degradation. For instance, the extension of a glucagon-like peptide-1 (GLP-1) fusion protein by only a single amino acid at the N-terminal prevented cleavage by dipeptidyl peptidase-IV (DPP-IV) [2]. The circulation half-life of an immunotoxin could be doubled when replacing an endoprotease-sensitive arginine residue by serine or lysine [67]. However, proteolytic processing during the endosome/lysosome trafficking is an important step in the mechanism of action of immunotoxins. Therefore, engineering of cleavage recognition sequences must be done very carefully. Recently, it was demonstrated that a deletion of a protease-sensitive region of PE38 abolishes lysosomal degradation while maintaining efficacy and increasing tolerance of high doses [68]. A combination of increased half-life and improved activity induced by single amino acid changes could also be observed with an interleukin-2 (IL-2) immunocytokine. Here, the linker peptide between the C-terminus of the antibody heavy chain and the N-terminus of IL-2 was modified, primarily with the aim to remove protease cleavage sites [69]. The nonselective toxicity of immunotoxins can be avoided by generating in-frame fusions with ubiquitin that triggers rapid degradation. However, the insertion of a cancer protease-specific cleavage sequence can stabilize the immunotoxin by removal of the ubiquitin moiety. For instance, a saporin-based immunotoxin containing a prostate-specific antigen (PSA) recognition sequence had a 10-fold higher activity in the presence of PSA-producing prostate cancer cells [70].

Other mutagenesis approaches are directed to the presence of disulfide bridges. Cysteins can be added, removed, or repositioned. In many cases, the introduction of disulfide bonds improves stability. Heterodimers between VH and VL of antibody fragments are relatively unstable but can be stabilized by adding a cystein in each of the fragments to generate a disulfide bridge. Although there are two possible positions, only one locus retained the full binding activity [71]. Even the rearrangement of existing disulfide bridges is possible as demonstrated with erythropoietin (EPO) where a cystein was moved from position 33 to 88. This modified EPO in fusion to an Fc part exhibited superior dimerization capabilities, better glycosylation stability and improved pharmacokinetic properties [72]. Stabilization by introducing disulfide bridges can also suppress immunogenicity as shown with an immunotoxin that was mutagenized in the domain III of Pseudomonas exotoxin A [73]. However in some cases the presence of an unpaired cystein can cause aggregation problems. A fusion protein between IFN-α2b and HSA (IFN-α2b-HSA) aggregated and caused immunogenicity issues. Only replacing the free cystein by serine abolished the effect, leading to a more stable and less immunogenic protein [74]. Sometimes disulfide-induced dimerization has multiple effects. The Fab-PE38 dimer with PE38 fused to the light chain and linked through a disulfide bridge in the hinge region had a 16-fold higher refolding yield and 2.5-fold better activity than the initial monomer [75].

Not only free cysteins can cause aggregation, much more frequent is the exposure of hydrophobic residues at protein surfaces that leads to aggregation. In a systematic study, these critical positions and their respective ideal aa of scFvs were identified. Two positions on the VH (82, 85) and also on the VL (36, 60) were found, whose replacement with more ideal aa resulted in a significant improvement of stability and yield [76]. A similar approach with another scFv revealed three other heavy chain and one light chain residue that contained not conserved aa. Triple VH mutants resulted in eightfold higher yields that could be increased to 20-fold, when the orientation was reversed. Interestingly, the mutations did not have a negative effect on binding affinity, but improved plasma stability [77]. Aggregation can also occur by the lack of glycosylation when proteins are expressed in E. coli. In the case of erythropoietin (EPO) this was counteracted by replacing asparagine residues with lysine. Due to the increased isoelectrical point (pI) the protein became positively charged under physiological conditions thus eliminating aggregation [78].

Mutagenesis can also have an effect on the activity profile of proteins. For instance, the replacement of the plasminogen activator inhibitor-1 binding site in Tenecteplase® by exchanging four aa resulted in a significantly longer activity, because the inhibitor could no longer bind [79].

1.5 Manufacturing

The first recombinant protein product, insulin, was initially produced as two separate chains that were conjugated chemically. But soon thereafter the commercial process utilized expression in E. coli and subsequent enzymatic maturation to generate insulin from proinsulin [80]. Since that time more than 120 therapeutic proteins, including a number of fusion proteins, have been manufactured for human use in bacteria, yeasts, or animal cells [81]. From a manufacturing perspective in most cases it is not necessary to differentiate between fusion proteins or regular singular therapeutic proteins. The production typically covers three complex steps: upstream processing (molecular biology and fermentation), downstream processing (capture and purification), and finally formulation (transforming the protein into a storable and administrable form). One of the many advantages of fusion proteins is the uninterrupted manufacturing process of a single protein molecule with several functions.

1.5.1 Upstream Process

The choice of the expression system depends heavily on the properties of the desired protein product such as glycosylation, disulfide bridges or other post-translational modifications that can only be obtained from eukaryotic cells [82]. Also protein size plays a role; usually, proteins larger than 100 kDa are by default produced in eukaryotic cells, whereas proteins below 30 kDa are expressed in bacteria. A recent analysis revealed that 39% of recombinant proteins were expressed in E. coli, 35% by Chinese hamster ovary (CHO) cells, 15% by yeasts, and 10% by other mammalian cells, but only 1% by other systems [83]. Interestingly, 17 of the 58 approved therapeutic proteins between 2006 and 2010 were manufactured in E. coli [84]. The following paragraph focuses on the two major host organisms, E. coli and CHO cells, while Pichia pastoris has been primarily used for albumin and transferrin fusion proteins [85]. Secreted Fc-fusion proteins in P. pastoris require individual optimization of upstream conditions [86].