Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges -- Contents -- Preface -- Contributors -- PART I: INTRODUCTION -- 1 Fusion Proteins: Applications and Challenges -- 1.1 History -- 1.2 Definitions and Categories -- 1.3 Patenting -- 1.4 Design and Engineering -- 1.4.1 Orientation of Fusion Proteins -- 1.4.2 Linker Engineering -- 1.4.3 Oligomerization of Fusion Proteins -- 1.4.4 Immunogenicity -- 1.4.5 Mutagenesis for Molecule Optimization -- 1.5 Manufacturing -- 1.5.1 Upstream Process -- 1.5.2 Downstream Process -- 1.5.3 Formulation -- 1.5.4 Process Economies -- 1.5.5 Glycosylation -- 1.6 Regulatory Challenges -- 1.7 Competition and Market -- 1.8 Conclusion and Future Perspective -- References -- 2 Analyzing and Forecasting the Fusion Protein Market and Pipeline -- 2.1 Introduction -- 2.2 Market Sales Dynamics of the FP Market -- 2.2.1 Enbrel Founded the FP Market Following Its Launch in 1998 -- 2.2.2 We Forecast the FP Market Will Grow to 10.1 Billion by 2016 -- 2.2.3 Growth Drivers and Restrictors that Impact the FP Market -- 2.3 Individual Drug Sales Analysis -- 2.3.1 Enbrel -- 2.3.2 Orencia -- 2.3.3 Nplate -- 2.3.4 Dulaglutide and Albiglutide -- 2.3.5 VEGFtrap-Eye -- 2.4 Pipeline Database Analysis -- 2.4.1 Breakdown by Stage of Development: Half of the FP Pipeline is in Phase II -- 2.4.2 Therapeutic Focus of FPs: Oncology and Autoimmune Dominate -- 2.4.3 FPs Developers and Partners: Many Big Pharma/Big Biotech Companies Have at Least One FP in Phase II Development or Above -- 2.4.4 FP Development: Less Attractive for Earlier-Stage Drug Developers, but More Attractive for Big Pharma Licensees -- 2.4.5 FPs Deal Analysis: We Believe FPs are an Attractive Target for Multinational Drug Companies -- Disclaimer -- Acknowledgment -- References.

3 Structural Aspects of Fusion Proteins Determining the Level of Commercial Success -- 3.1 Classification of FPs -- 3.1.1 Pharmacokinetic, Targeting, and Activity FP Components -- 3.1.2 FPs Classified by the Function of Each Component -- 3.1.3 Half-Life Extension Represents a Dominant Theme (1c-7c) -- 3.1.3.1 Fc Fusion is the Half-Life Extension Standard -- 3.1.3.2 Albumin Fusion is the Most Advanced non-FcFP Half-Life Approach -- 3.1.3.3 Nonfusion Alternatives -- 3.1.4 Decoy Receptors are the Largest FP Group and Well Validated (1c) -- 3.1.5 Extended Half-Life Ligands (4c) -- 3.1.6 FPs Offer a Wide Range of Cell Death Mechanisms (3a-7b) -- 3.1.6.1 Targeted Toxins (3a and 3b) -- 3.1.6.2 Targeted Ligands (4a and 4b) -- 3.1.6.3 Antigen-Mediated Cell Death (5a and 5b) -- 3.1.7 Antibody Fragment Fusions (6b and 7b) -- 3.1.7.1 Bispecific Abs: Ab-Binding Domain Fused to Ab-Binding Domain (6b) -- 3.1.7.2 Miniaturized Abs: Ab-Binding Domain Fused to Fc Effector (7b) -- 3.1.7.3 Other Ab Fragment Structures -- 3.1.8 Ab- and non-Ab-Targeting Decisions -- 3.1.9 Targeted Enzymes (2a) -- 3.2 Factors for Commercial Success -- 3.2.1 Administration -- 3.2.2 Efficacy Profile -- 3.2.3 Safety Profile -- 3.2.4 Cost Factors -- 3.2.4.1 Manufacturing and Cost of Goods Sold -- 3.2.4.2 Intellectual Property: Freedom to Operate and Protection from Copies -- References -- 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 -- 4.2.1 Length of Linker Sequence -- 4.2.2 Preference of Amino Acid Residues in Linker Sequence -- 4.2.3 Hydrophobicity of Linkers -- 4.2.4 Secondary Structures of Linkers -- 4.3 Empirical Linkers in Recombinant Fusion Proteins -- 4.3.1 Flexible Linkers -- 4.3.1.1 Flexible Glycine and Serine Linkers.

4.3.1.2 Other Flexible Linkers -- 4.3.2 Rigid Linkers -- 4.3.2.1 Alpha Helix-Forming Linker (EAAAK)n -- 4.3.2.2 Rigid (XP)n Linkers -- 4.3.3 In Vivo Cleavable Linkers -- 4.3.3.1 In Vivo Cleavable Disulfide Linker -- 4.3.3.2 In Vivo Cleavable Protease-Sensitive Linkers -- 4.4 Functionality of Linkers in Fusion Proteins -- 4.4.1 Linkers Can Improve Biological Activity of Fusion Proteins -- 4.4.2 Linkers Can Increase Expression Yield of Fusion Proteins -- 4.4.3 Linkers Can Affect the PK of Fusion Proteins -- 4.5 Conclusions and Future Perspective -- References -- 5 Immunogenicity of Therapeutic Fusion Proteins: Contributory Factors and Clinical Experience -- 5.1 Introduction -- 5.2 Basis of Therapeutic Protein Immunogenicity -- 5.2.1 Relevance to Fusion Proteins -- 5.3 Tools for Immunogenicity Screening -- 5.3.1 In Silico Analysis Tools -- 5.3.2 In Vitro Analysis Tools -- 5.4 Approaches for Risk Assessment and Minimization -- 5.4.1 Importance to Product Development -- 5.4.2 De-immunization by Epitope Modification -- 5.4.3 Tolerance Induction -- 5.4.4 Regulatory T-Cell Epitopes in IgG Framework Regions: Tregitopes -- 5.5 Case Study and Clinical Experience -- 5.5.1 Observed Versus Predicted Immunogenicity of 21 Antibodies in Clinical Use -- 5.5.2 Correlation of Clinical Immunogenicity of Fusion Proteins with In Silico Risk Estimates -- 5.6 Preclinical and Clinical Immunogenicity Assessment Strategy -- 5.6.1 Strategy and Recommendation -- 5.7 Conclusions -- Acknowledgment -- References -- PART II THE TRIPLE T PARADIGM: TIME, TOXIN, TARGETING -- IIA TIME: FUSION PROTEIN STRATEGIES FOR HALF-LIFE EXTENSION -- 6 Fusion Proteins for Half-Life Extension -- 6.1 Introduction -- 6.2 Half-Life Extension Through Size and Recycling -- 6.2.1 Direct Genetic Fusions -- 6.2.1.1 Albumin Fusions -- 6.2.1.2 Fc Fusions -- 6.2.1.3 Transferrin Fusions.

6.2.2 Half-Life Extension Through Attachment to Large Proteins -- 6.3 Half-Life Extension Through Increase of Hydrodynamic Radius -- 6.3.1 Repetitive Peptide Fusions -- 6.3.2 Glycosylated Peptides -- 6.4 Aggregate Forming Peptide Fusions -- 6.5 Other Concepts -- 6.6 Conclusions and Future Perspective -- References -- 7 Monomeric Fc-Fusion Proteins -- 7.1 Introduction -- 7.2 FcRn and Monomeric Fc-Fusion Proteins -- 7.2.1 FcRn Expression and Function -- 7.2.2 FcRn for the Delivery of Fc-Fusion Proteins -- 7.3 Typical Applications -- 7.3.1 Interferon Beta-Fc Monomer -- 7.3.2 Interferon α-Fc Monomer -- 7.3.3 Factor VIII-Fc Monomer -- 7.3.4 Factor IX-Fc Monomer -- 7.4 Alternative Applications -- 7.4.1 Follicle Stimulating Hormone-Fc-Fusion Proteins -- 7.4.2 Cytokine-Fc-Fusion Proteins -- 7.5 Expression and Purification of Monomeric Fc-Fusion Proteins -- 7.6 Conclusions and Future Perspectives -- References -- 8 Peptide-Fc Fusion Therapeutics: Applications and Challenges -- 8.1 Introduction -- 8.2 Peptide Drugs -- 8.2.1 The Limitations of Native Endogenous Peptides in Drug Development -- 8.2.2 Chemical Modification of Peptides to Reduce Proteolytic Degradation -- 8.2.3 Constraining Peptides to be More Resistant to Protease Cleavage -- 8.2.4 Peptide Therapeutics Play an Important Role in Drug Development -- 8.2.5 Most Peptide Drugs Act as Receptor Agonists -- 8.3 Technologies Used for Reducing In Vivo Clearance of Therapeutic Peptides -- 8.3.1 PEGylation -- 8.3.2 Human Serum Albumin Fusions -- 8.3.3 Other Approaches -- 8.4 Fc-Fusion Proteins in Drug Development -- 8.4.1 The Structure of Fc-Fusion Proteins -- 8.4.2 Fc-Fusion Therapeutics -- 8.4.2.1 Enbrel® -- 8.4.2.2 Amevive® -- 8.4.2.3 Orencia® -- 8.4.2.4 Arcalyst® (Also Known as IL-1 Trap or Rilonacept) -- 8.4.2.5 Other Fc-Fusion Protein Therapeutics -- 8.5 Peptide-Fc-Fusion Therapeutics -- 8.5.1 Peptibody.

8.5.1.1 NPlate® (Romiplostim, AMG531) -- 8.5.1.2 AMG 386 (2xCon4(C)) -- 8.5.1.3 A-623 (AMG 623) -- 8.5.1.4 Dulaglutide (LY2189265) -- 8.5.2 MimetibodyTM Technology -- 8.5.2.1 CNTO 528/CNTO 530 -- 8.5.2.2 CNTO736 -- 8.6 Considerations and Challenges for Engineering Peptide-Fc-Fusion Therapeutics -- 8.6.1 Improving Activity -- 8.6.2 Improving Stability -- 8.6.3 Improving Pharmacokinetic Profile -- 8.6.4 Improving Solubility and Reducing Aggregation -- 8.6.5 Reducing Heterogeneity -- 8.6.6 Isotype Selection -- 8.6.7 Tailoring Fc-Effector Functions to Fit Individual Needs -- 8.6.8 Reducing Immunogenicity -- 8.7 Conclusions -- Acknowledgment -- References -- 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 -- 9.3.1 Proof-of-Concept for High-Affinity Traps for Ligands Utilizing Multicomponent Receptor Systems -- 9.3.2 Turning Heterodimeric Soluble Receptor-Based Ligand Traps into Therapeutics -- 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 -- 9.6.1 Multiple Ligand Binding -- 9.6.2 Signaling of Receptor-Fc Fusions -- 9.6.3 Immunogenicity -- 9.7 Conclusion -- Acknowledgment -- References -- 10 Recombinant Albumin Fusion Proteins -- 10.1 Concept -- 10.2 Technological Aspects -- 10.3 Typical Applications and Indications -- 10.3.1 Albumin Fusion Proteins in Late-Stage Development -- 10.3.1.1 Interferon-α -- 10.3.1.2 Glucagon-Like Peptide-1 -- 10.3.1.3 Granulocyte Colony-Stimulating Factor.

10.3.2 Albumin Fusion Proteins in Early-Stage Development.