Table of Contents

Cover

Title Page

Preface

References

A Personal Foreword

Acknowledgments

Part I: Introduction to Protein Therapeutics: Past and Present

Chapter 1: Early Recombinant Protein Therapeutics
1. 1.1 Introduction
2. 1.2 The Birth of Genetic Engineering
3. 1.3 Recombinant Human Insulin
4. 1.4 Recombinant Human Growth Hormone
5. 1.5 Recombinant Human Interferons
6. 1.6 Recombinant Human Erythropoietin
7. 1.7 Recombinant Tissue – Type Plasminogen Activator
8. 1.8 Recombinant Hepatitis B Virus (HBV) Vaccine
9. 1.9 Postscript
10. Acknowledgments
11. References
Chapter 2: Evolution of Antibody Therapeutics
1. 2.1 Overview of Antibody Therapeutics Development
2. 2.2 Polyclonal Antibodies – Laying the Foundation for mAb Therapy
3. 2.3 Evolution of Monoclonal Antibody Therapeutics
4. 2.4 Fc Fusion Proteins
5. 2.5 Evolution of Concepts in Antibody Pharmacology
6. 2.6 Future Directions for Antibody Therapeutics Development
7. Acknowledgments
8. References

Part II: Antibodies: The Ultimate Scaffold for Protein Therapeutics

Chapter 3: Human Antibody Structure and Function
1. 3.1 Introduction
2. 3.2 General Sequence and Structural Features of Antibodies
3. 3.3 Antibody Diversity
4. 3.4 Canonical Structures of CDR Loops
5. 3.5 Crystal Structures of Antibody–Antigen Interactions
6. 3.6 Glycosylation
7. 3.7 Role of the Fc and Fc Receptors
8. 3.8 Conclusions and Outlook
9. Acknowledgments
10. References
Chapter 4: Antibodies from Other Species
1. 4.1 Introduction
2. 4.2 Mammals
3. 4.3 Reptiles
4. 4.4 Amphibians
5. 4.5 Fish
6. 4.6 Conclusions
7. References

Part III: Discovery and Engineering of Protein Therapeutics

Chapter 5: Human Antibody Discovery Platforms
1. 5.1 Introduction to Therapeutic Human Antibody Platforms
2. 5.2 Properties of Human Antibody Genes
3. 5.3 New Technologies Driving Changes and Improvements in Human Antibody Discovery
4. 5.4 Antigen-Specific Human mAbs from Human B Cells
5. 5.5 Human Antibody Libraries
6. 5.6 Human Antibodies from Transgenic Animals
7. 5.7 Summary and Future Directions
8. References
Chapter 6: Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development
1. 6.1 Introduction
2. 6.2 Motivation for Developing Antibody Alternatives
3. 6.3 Non-antibody Scaffolds with Homogenous Secondary Structure
4. 6.4 Non-antibody Scaffolds with Mixed Secondary Structure
5. 6.5 Conclusions and Considerations
6. Acknowledgments
7. References
8. Further Reading
Chapter 7: Protein Engineering: Methods and Applications
1. 7.1 Introduction
2. 7.2 General Approaches for Protein Optimization
3. 7.3 Engineering for Affinity and Specificity
4. 7.4 Optimizing IgG Serum Half-Life
5. 7.5 Engineering IgG Effector Function
6. 7.6 Conclusion
7. Acknowledgments
8. References
Chapter 8: Bispecifics
1. 8.1 Introduction: Continuing Evolution of Antibody-based Therapeutics
2. 8.2 Enhancing Antibody Therapeutics by Addition of Functional Moieties
3. 8.3 Format Selection – Pairing Function and Target Biology
4. 8.4 Engineering Considerations
5. 8.5 Conclusion
6. Disclosure of Potential Conflict of Interest
7. References
Chapter 9: Antibody–drug Conjugates (ADCs)
1. 9.1 Introduction
2. 9.2 General Mode of Action
3. 9.3 The Components of an Antibody–Drug Conjugate
4. 9.4 Assembling the ADC
5. 9.5 Approved ADCs: Adcetris and Kadcyla
6. 9.6 Developing the ADC Platform
7. 9.7 New Warheads and Payloads with a Novel Mechanism of Action
8. 9.8 Conclusion
9. References
Chapter 10: Pharmacokinetics of Therapeutic Proteins
1. 10.1 Absorption
2. 10.2 Distribution
3. 10.3 Metabolism and Elimination
4. 10.4 Pharmacokinetic Modeling of Therapeutic Proteins
5. References

Part IV: Physiological and Manufacturing Considerations for Biologics

Chapter 11: Safety Considerations for Biologics
1. 11.1 Introduction
2. 11.2 Small Molecules versus Large Molecules – A Comparison
3. 11.3 Toxicity Related to Exaggerated Pharmacology – Importance of Species Selection
4. 11.4 Toxicity Unrelated to Exaggerated Pharmacology
5. 11.5 Regulatory Guidance
6. 11.6 Development Considerations Due to Biological Characteristics
7. 11.7 First in Human (FIH) to Registration
8. 11.8 Selection of a Safe Starting Dose for First Time in Human Clinical Study
9. 11.9 Summary
10. References
Chapter 12: Immunogenicity of Biologics
1. 12.1 Introduction
2. 12.2 Mechanistic View of Immunogenicity: Innate and Adaptive Immunity
3. 12.3 Immunogenicity of Protein Therapeutics in Autologous Cell Therapies
4. 12.4 Regulatory Context
5. 12.5 Application of the “Risk-Based Approach” for Undesirable Immunogenicity
6. 12.6 Clinical Proof of Concept and Beyond
7. 12.7 Future Perspectives
8. References
Chapter 13: Expression Systems for Recombinant Biopharmaceutical Production by Mammalian Cells in Culture
1. 13.1 Introduction
2. 13.2 Host Cell Systems
3. 13.3 Mammalian Cell Transfection
4. 13.4 Controlling Recombinant Gene Expression
5. 13.5 Selection and Amplification Systems
6. 13.6 Transient Production Systems
7. 13.7 Protein Purification
8. 13.8 CHO Cell Engineering for Enhanced Bioprocessing Properties
9. Abbreviations
10. References
Chapter 14: Stability, Formulation, and Delivery of Biopharmaceuticals
1. 14.1 Introduction
2. 14.2 Stability
3. 14.3 Drug Product Development
4. 14.4 Handling and Administration Considerations
5. 14.5 Summary and Conclusion
6. References
Chapter 15: Protein Therapeutics in Autoimmune and Inflammatory Diseases
1. 15.1 Introduction
2. 15.2 Rheumatoid Arthritis
3. 15.3 Psoriasis
4. 15.4 TNF-α Antagonist Therapy
5. 15.5 Anti-IL-12/IL-23 Therapies
6. 15.6 Anti-IL-17 Therapies
7. 15.7 Atopic Dermatitis
8. 15.8 Inflammatory Bowel Disease (IBD)
9. 15.9 Systemic Lupus Erythematosus
10. 15.10 Conclusions
11. References

Part V: Clinical Applications

Chapter 16: Antibody-Based Therapeutics in Oncology
1. 16.1 Introduction
2. 16.2 Targeting Cell-Surface Signaling Pathways in Solid Tumors
3. 16.3 Targeting of Immune Modulators
4. 16.4 Bispecific Antibodies
5. 16.5 Conclusions and Future Directions
6. References
Chapter 17: Protein Therapeutics in Respiratory Medicine
1. 17.1 Introduction
2. 17.2 Asthma
3. 17.3 Th2-Targeted Therapies
4. 17.4 Mepolizumab
5. 17.5 Other Anti-IL-5-Targeted Treatments
6. 17.6 Other Respiratory Uses of Monoclonal Antibodies
7. 17.7 Summary
8. References
Chapter 18: Antibodies for the Prevention, Treatment, and Preemption of Infectious Diseases
1. 18.1 Prophylaxis and Precision Medicine
2. 18.2 Antibacterial Immune Therapy – A Nineteenth Century Breakthrough
3. 18.3 Other Potential Anti-infective mAbs
4. References
Chapter 19: Rescue Therapies
1. 19.1 Introduction
2. 19.2 Antidotes/Reversal Agents
3. 19.3 Antivenoms and Antitoxins
4. 19.4 Conclusion
5. References
Chapter 20: Biosimilars
1. 20.1 Introduction
2. 20.2 Concept and Definition of Biosimilars
3. 20.3 Rationale and Significance of Biosimilars
4. 20.4 Current Approvals and Trends
5. 20.5 Challenges and Future Trends
6. References

Part VI: Future Horizons

Chapter 21: Future Horizons and New Target Class Opportunities
1. 21.1 Introduction
2. 21.2 Targeting the Central Nervous System
3. 21.3 Intracellular Biologics
4. 21.4 Building on the Success of Traditional Monoclonal Antibodies
5. References
Index

End User License Agreement

List of Illustrations

Chapter 1: Early Recombinant Protein Therapeutics

Figure 1.1 Paul Berg's construction of hybrid genome.
Figure 1.2 From left to right: Keiichi Itakura, Art Riggs, David Goeddel, and Roberto Crea.
Figure 1.3 Production of insulin from separate genes encoding the A and B chains of human insulin.
Figure 1.4 Zinc crystals of recombinant human insulin produced from the separate chains recombination process.
Figure 1.5 Yeast expression plasmid for dibasic insulin precursors.
Figure 1.6 Assembly of the plasmid containing the human growth hormone gene. For detailed explanation, see Ref. [61].
Figure 1.7 The fibrinolytic system. The proenzyme plasminogen is activated to the active enzyme plasmin by tissue-type or urokinase-type plasminogen activator. Plasmin degrades fibrin into soluble fibrin degradation products. Inhibition of the fibrinolytic system may occur through plasminogen activators, by plasminogen activator inhibitors, or more directly via plasmin, mainly by α2-antiplasmin.

Chapter 2: Evolution of Antibody Therapeutics

Figure 2.1 Chronological, technological, and conceptual evolution of antibody therapeutics from 1891 to 2016. Rather than presenting the evolution linearly and chronologically only (Y-axis, from top to bottom), a second dimension (X-axis) has been added to better highlight the technological evolutions (gray boxes), which led to new concepts and therapeutic principles (pink boxes) and a large panel of therapeutic uses, which are categorized into neutralizing Abs (blue boxes), cytolytic Abs (red-brown boxes), antagonist Abs (green boxes), and bispecific Abs (dark gray box). The Y-axis starts in 1891, year of the first clinical use of serotherapy. The trajectory of the thin arrows indicates how today's applications originate from older therapeutic concepts, having gradually benefited from new technological inputs. The thick arrows indicate the importance of the deployment of the applications.

Chapter 3: Human Antibody Structure and Function

Figure 3.1 Schematic representation of a prototypic antibody IgG, which contains four polypeptide chains, two copies of each with two different chains, heavy (H) and light (L). The heavy chain has four domains: one variable (V_H) followed by three constant domains (C_H1, C_H2, and C_H3). The heavy chain also contains the hinge region. The light chain has one variable (V_L) and one constant domain (C_L). The N-terminal variable domains of both chains have hypervariable regions, also known as CDRs, interspersed with framework regions. Disulfide bonds connect the four chains. N-linked glycosylation site is found at the C_H2 domain.
Figure 3.2 Five major isotypes of antibodies. All subtypes contain light and heavy chains and each with one variable domain (V_H) and different number of constant domains (C_H1–C_H4). Generally, two heavy (H) and two light (L) chains form a monomer Ig module as in IgG, IgD, and IgE. IgM is a pentamer with four constant domains, as in IgE, and an additional polypeptide, the J chain, connecting two monomer Ig units. The J chain is also found in IgA linking two units to form the IgA dimer. Carbohydrate moieties attached to N-glycosylation site of the penultimate constant domains are not shown in this figure.
Figure 3.3 (a) Cartoon ribbon diagram of an intact human antibody IgG1. The variable domains of heavy (V_H) and light (V_L) chains at the N-terminal are shown blue and purple, respectively. The constant domains of light (C_L1) and heavy (C_H1, C_H2, and C_H3) chains at the C-terminal are shown in orange. IgG contains the two Fab units that are linked to Fc. The carbohydrates (in green sticks) are found between the C_H2 domains of the IgG. The flexibility of IgG is achieved through the hinge regions that connect the Fab to the Fc and the elbow regions along the peptides linking V_H to C_H1 and V_L to C_L1, as shown. (b) Structural comparison of variable and constant domains of IgG. Both domains share similar topology comprising of a pair of β sheets in which the β strands are labeled with A–G. The variable domain has CDR loops, whereas those loops in the constant domains are shorter and invariable. The CDR1 connects the β strands B and C, while CDR3 connects the β strands F and G. The variable domain has two additional β strands, C′ and C′′, that supports CDR2. The conserved intradomain disulfide bond is shown in green.
Figure 3.4 (a) Crystal structure of a Fab depicted in ribbons. The variable domains of heavy (V_H) and light (V_L) chains at the N-terminal are shown blue and purple, respectively, while the constant domains are in orange. (b) Close-up view showing the antigen-binding site as formed by the six CDR loops. The CDR H3 is located centrally and forms the primary antigen-binding loop of all CDRs. Note that CDR H3 makes significant interactions with other CDRs.
Figure 3.5 Ribbon diagrams of different IgG Fc fragments. X-ray crystal structures of human IgG Fc fragment (PDB 1H3X), monomeric Fc (PDB 4J12), monomeric and unglycosylated CH2 domain (PDB 3DJ9), monomeric CH3 (PDB 4B53), and CH3 dimer (PDB 5HSF).
Figure 3.6 Antibody diversity generated by genetic recombination, junctional mechanisms, and heavy/light chain pairing. Theoretical diversity could be estimated as of up to 10¹² different antibodies based on the number of germline gene segments contributing to functional antibodies and junctional diversity.
Figure 3.7 Five of the six CDR loops (L1, L2, L3, H1, and H2) with standard conformations, called canonical structures. The CDR H3 is highly variable in length, sequence, and structure, and therefore does not have any canonical structures. (a–e) The most abundant canonical structures for the five CDRs are shown. (f) For CDR H3, it is generally divided into three conformations – base, torso, and head (tip or crown region). The base region has always a kinked conformation or sometimes an extended conformation, and the torso region is either budged or non-bulged. The head region generally forms a classical β-turn motif or different conformations based on the torso region as well as its interactions with antigen and/or other CDRs.
Figure 3.8 Co-crystal structures of fully human antibody fragments (Fabs) with viral envelope glycoproteins: (a) HIV-1 gp120-Fab X5 (PDB 2B4C), (b) SARS CoV RBD-Fab 396 (PDB 2DD8), (c) HeVG-Fab m102.3 (PDB XXX), and (d) MERS CoV RBD-Fab m336 (PDB 4XAK). The antibodies against different infectious diseases were generated by the selection of human antibody fragments from in vitro libraries. Structural analysis revealed neutralizing epitopes on the viral envelope glycoproteins or its receptor binding domains (RBDs) recognized by CDRs of those antibodies (paratopes) as indicated by rectangular boxes. (e) Close-up views of amino acid residues that make up the interacting paratope and epitope surfaces.
Figure 3.9 (a) View of human IgG with a representative oligosaccharide structure attached to the N-glycosylation site at Asn297. (b) Commonly observed oligosaccharides on recombinant IgGs produced by standard cell lines.
Figure 3.10 Comparison of the complex crystal structures of (a) IgG Fc–FcγRI (PDB 4ZNE), (b) IgG Fc–FcγRIII (PDB 1T83), (c) IgE Fc–FcϵRI (PDB 1F6A), and (d) IgG Fc–FcαR1 (PDB 1OW0). The extracellular domains of the Fc receptors are shown in green, whereas one heavy chain of each Fc region is shown in yellow and the other in red. Carbohydrates are shown as gray sticks.

Chapter 4: Antibodies from Other Species

Figure 4.1 Phylogenetic tree of species of immunological interest. Appearance of unique features, isotypes, and binding structures are indicated in blue. Appearance of lymphoid organs and molecular mechanisms involved in antibody diversity are indicated in the bars on the right. AID stands for the activation-induced cytidine deaminase and GALT for gut-associated lymphoid tissue.
Figure 4.2 Comparison of different species' antibody structures. Common IgG antibody structure found in most species. The heavy chain only antibodies, or HCAbs, are found in camels, which only possess C_H2 and C_H3. The heavy chain only antibodies, IgNAR, are found in sharks, have 3–5 C_H (C_H1–5) regions. The stalk and knob structure found in ultralong cow antibodies. Cartoon (a) of the antibody and ribbon (b) diagrams of the variable region of each antibody type are shown. The CDR H3 regions are highlighted in each ribbon diagram. The PDB codes of the structures are indicated below each graphic.

Chapter 5: Human Antibody Discovery Platforms

Figure 5.1 Total and human monoclonal antibodies approved by the United States Food and Drug Administration (US FDA) by year. Clear bar: total number of antibodies approved in a given year; Solid bar: number of human antibodies approved in a given year; Line with solid circles: accumulated total of human antibodies approved over time.

Chapter 6: Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development

Figure 6.1 Non-antibody scaffolds with β-sheet secondary structure. Engineered variable domains are highlighted in red. (a) Monobody/Adnectin/FN3 domain (PDB: 1FNF); (b) Fynomer/SH3 Domain (PDB ID: 1M27); (c) Anticalin/Lipocalin (PDB: 2HZQ); (d) Nanobody/VHH Domain (PDB: 4KRL).
Figure 6.2 Non-antibody scaffolds with α-helix secondary structure. Engineered variable domains are highlighted in red. (a) DARPin/Ankyrin repeats (PDB ID: 3HG0), (b) Affibody (PDB: 1LP1).
Figure 6.3 Non-antibody scaffolds with mixed secondary structure. Engineered variable domains are highlighted in red. Disulfide linkages are highlighted in yellow and represented as sticks. The GP2 protein in F is isolated because it does not contain disulfide linkages. (a) Avimer/A-domain binders (PDB ID: 1AJJ), (b) EETI-II knottin (PDB ID: 2IT7), (c) MCoTI-II cyclotide (PDB ID: 4GUX), (d) Kringle domain (PDB ID: 1I5K), (e) Kunitz domain (PDB ID: 1AAP), (f) GP2 (PDB ID: 2WNM).
Figure 6.4 Drugs derived from non-antibody scaffolds in clinical development. Each slice corresponds to a clinical target. Individual colors delineate phase of development. White circles represent a single drug in a certain phase of clinical development. AMD: Age-related macular degeneration, HAE: Hereditary angioedema. “Other” includes cachexia, pain, anemia, IBD, plaque psoriasis, and acute respiratory distress syndrome.

Chapter 7: Protein Engineering: Methods and Applications

Figure 7.1 Cross section of an FcRn-expressing endothelial cell showing (a) IgG recycling and (b) IgG transcytosis. After pinocytosis, IgG molecules enter acidic endosomes, bind to FcRn, and are rescued from a lysosomal degradation pathway. IgG-containing vesicles then cycle back or migrate to the cell surface where the IgG molecules are released.
Figure 7.2 Model of an Fc fragment derived from a human IgG1, based upon the X-ray structure corresponding to protein database ID number 1HZH [214]. Colored residues correspond to the various positions where single or multiple substitutions were shown to improve the affinity of the (a) human IgG/FcRn, (b) human IgG/CD16A, (c) human IgG/C1q, and (d) human IgG/CD32A interaction. Carbohydrates are shown as sticks.
Figure 7.3 Cross section of a cell showing the fate of (a) a conventional antibody binding to a membrane-bound antigen and (b) binding to a soluble antigen. In both situations, the antibody–antigen complex is degraded via proteolysis in the lysosome. A pH-dependent antigen binding antibody will dissociate from its antigen, either membrane-bound (c) or soluble (d), in the acidic endosome and is recycled back to the plasma via FcRn, enabling it to bind to another antigen.

Chapter 9: Antibody–drug Conjugates (ADCs)

Figure 9.1 ADC mode of action (for DNA-targeting warhead).
Figure 9.2 ADC components.
Figure 9.3 Auristatin-based warheads and payloads.
Figure 9.4 Kadcyla (where the mAb is trastuzumab).
Figure 9.5 Mylotarg.
Figure 9.6 Mafodotin (MMAF payload).
Figure 9.7 Maytansine-based payloads.
Figure 9.8 Govitecan payload.
Figure 9.9 Pyrrolobenzodiazepine-based warheads.
Figure 9.10 SYD985.
Figure 9.11 Release of duocarmycin payload from SYD985.
Figure 9.12 Tubulysin-based warheads and payloads.

Chapter 10: Pharmacokinetics of Therapeutic Proteins

Figure 10.1 Schematics for two separate case examples showing the effect of ADA on exposure by between-subject comparison. (a) Between-subject comparison of exposure time course. (b) Within-subject comparison of exposure at two different time points.
Figure 10.2 Binding of TGN1412 with the co-stimulatory receptor CD28.
Figure 10.3 Full TMDD model comprising drug distribution and drug–receptor interaction.

Chapter 11: Safety Considerations for Biologics

Figure 11.1 Key activities during early discovery of biologics.

Chapter 12: Immunogenicity of Biologics

Figure 12.1 Human monocyte-derived dendritic cells (white arrows) produced by in vitro culture of monocytes with IL-4, GM-CSF, and TNFα.
Figure 12.2 Peptide binding to HLA class II molecules. (a) Top view of peptide (stick model) sitting in the binding groove of MHC class II (surface model). The surface is colored to indicate the depth of cavities, with dark blue indicating the deepest pockets. (b) Lateral view of peptide orientation in the binding groove. Residues with side chains oriented downward make contact with MHC class II, whereas side chains oriented upward potentially contact the TCR [63]. Figure prepared using Swiss-PdbViewer (Guex, N. and Peitsch, M.C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18, 2714–2723.) using PDB structure file 1FV1.
Figure 12.3 Potential interacting CMC variables that may influence immunogenicity.

Chapter 17: Protein Therapeutics in Respiratory Medicine

Figure 17.1 Stepwise approach to increasing asthma medications. Modified from GINA guidelines. SABA, short acting β-2 agonist; ICS, inhaled corticosteroid; LABA, long-acting β-2 agonist; LTRA, leukotriene receptor antagonist.
Figure 17.2 Pathways leading to Th1 (neutrophilic) and Th2 (eosinophilic) inflammation. Naïve Th0 cells differentiate following interaction with antigen -APCs and cytokine influence. The Th1 and Th2 pathways inhibit each other via IFN-γ and IL-4, respectively. APC, antigen presenting cell; Th, T helper; IL, interleukin; IFN-γ, interferon gamma.
Figure 17.3 Proposed parallel mechanisms of eosinophilic inflammation due to interactions of antigens and irritants at the air/epithelial layer interface. Antigens and irritants can induce eosinophilic inflammation via the IgE-mediated hypersensitivity pathway and also by IL-25- and IL-33-mediated activation of ILC-2 cells. IgE, immunoglobulin E; PGD2, prostaglandin D2; IL, interleukin; Th2, T-helper type 2; ILC2, innate lymphoid cell type 2; LTE4, leukotriene E4; CRTH2, chemokine receptor homologous molecule expressed on Th2 lymphocytes.
Figure 17.4 Cumulative number of exacerbations in each treatment group – DREAM study, 2012 [25]. Significantly reduced exacerbation rate at all doses of mepolizumab.
Figure 17.5 (a) IL-4Rα Type 1 and Type 2 receptors. Type 1 comprises the IL-4Rα and common cytokine receptor γ-chain (γc). Type 2 receptor is made from IL-4Rα subunit and IL-13Rα1. IL-4 binds to Type 1 and Type 2 receptors, while IL-13 binds to only the Type 2 receptor. (b) Dupilumab binds to the IL-4Rα subunit of both Type 1 and Type 2 receptors, stopping the binding of IL-4 and IL-13. IL, interleukin.

Chapter 20: Biosimilars

Figure 20.1 Number of mAbs clinical studies in the United States.
Figure 20.2 Major patents expiry for top-selling mAbs.
Figure 20.3 Herceptin price change in India.
Figure 20.4 Dossier required for biosimilar approval.
Figure 20.5 How similar is “highly similar”?

List of Tables

Chapter 2: Evolution of Antibody Therapeutics

Table 2.1 Monoclonal antibody therapeutics approved or in review in the European Union or the United States
Table 2.2 Fc fusion protein therapeutics approved in the European Union or the United States

Chapter 3: Human Antibody Structure and Function

Table 3.1 Different numbering schemes for antibody variable domain sequence annotation
Table 3.2 Properties and function of five different antibody isotypes
Table 3.3 Definitions of different antibody fragments and domains.
Table 3.4 Germline V_H–V_L pairing occurrences as observed in 766 human antibodies as derived from the Protein Data Bank
Table 3.5 Canonical structures for CDRs H1, H2, L1, L2, and L3 are classified into different CDR clusters as implemented in PyIgClassify.
Table 3.6 Databases and tools for analyzing antibody sequence and structural data

Chapter 4: Antibodies from Other Species

Table 4.1 Different immunoglobulin isotypes found in each species
Table 4.2 Presence and use of light chains in various species

Chapter 5: Human Antibody Discovery Platforms

Table 5.1 Human antibodies approved for marketing in major markets as of January 2016.^a
Table 5.2 Source of variable sequences for approved and Phase III clinical candidate mAbs.^a
Table 5.3 Use of genes in formation of serum Igs
Table 5.4 Examples of antibodies in preclinical and clinical trials that have been sourced from human B cells
Table 5.5 Examples of human antibody libraries and associated display technologies
Table 5.6 Companies with transgenic animal platforms producing human antibodies upon immunization

Chapter 6: Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development

Table 6.1 Characteristic qualities of prominent non-antibody scaffolds

Chapter 8: Bispecifics

Table 8.1 Description of DT and PE variants used for immunotoxin generation
Table 8.3 Ongoing immunotoxin clinical trials
Table 8.4 Recently completed immunotoxin clinical trials
Table 8.2 Examples of Bispecifics
Table 8.5 Immunocytokines in the clinic

Chapter 9: Antibody–drug Conjugates (ADCs)

Table 9.1 Tabulated summary of vedotin ADCs in Phase II clinical trials
Table 9.2 Summary of PBD ADC clinical trial

Chapter 11: Safety Considerations for Biologics

Table 11.1 Comparison between small molecules and biologics
Table 11.2 Key considerations for an early safety prediction evaluation
Table 11.3 Examples demonstrating variations in species specificity
Table 11.4 Benefits and considerations of alternate approaches to nonclinical safety assessment.

Chapter 12: Immunogenicity of Biologics

Table 12.1 TLR ligands identified from natural, synthetic, and protein therapeutic sources
Table 12.2 Values provided are the total number of peptide contacts (either side chain or main chain) from six crystal structures including both human and murine peptide/MHC II/TCR complexes (1u3h, 1j8h, 1fyt, 1ymn, 1zgl, 1d9k) [74]
Table 12.3 Identified immunogenicity risks for approved therapeutic proteins and antibodies

Chapter 14: Stability, Formulation, and Delivery of Biopharmaceuticals

Table 14.1 Example of a stability program for sterile drug products intended to be stored in a refrigerator (5 °C)
Table 14.2 Overview of analytical methods possibly to study drug product stability targeting different instabilities

Chapter 16: Antibody-Based Therapeutics in Oncology

Table 16.1 Solid Tumor Cell-Surface Targeting Antibodies in Clinical Development
Table 16.2 Check-point blockade antibodies in clinical development
Table 16.3 Costimulatory antibodies in clinical development
Table 16.4 Bispecific antibodies in clinical development or approved for the treatment of cancer

Chapter 17: Protein Therapeutics in Respiratory Medicine

Table 17.1 Biomarkers of eosinophilic inflammation used in RCTs with monoclonal antibodies to preselect patients in adult asthma
Table 17.2 Anti-IL-5 programs
Table 17.3 Anti-IL-5 efficacy and safety
Table 17.4 Effects of different cytokine blockers on clinical measures and biomarkers

Chapter 18: Antibodies for the Prevention, Treatment, and Preemption of Infectious Diseases

Table 18.1 mAbs in development

Chapter 19: Rescue Therapies

Table 19.1 Antidotes/reversal agents acting through high-affinity binding
Table 19.2 Antidotes/reversal agents acting as degrading enzymes
Table 19.3 Molecular weights and estimated pharmacokinetic properties of antibodies and fragments
Table 19.4 Examples for antitoxins/antisera

Chapter 20: Biosimilars

Table 20.1 Characteristics of small molecule drugs compared to biologics
Table 20.2 Definitions of biosimilar products worldwide
Table 20.3 Top 10 best-selling drugs of 2012
Table 20.4 EMA approved biosimilar products.
Table 20.5 Guidance comparison between EMA, FDA, and WHO

Chapter 21: Future Horizons and New Target Class Opportunities

Table 21.1 Key parameters for functional intracellular delivery of therapeutic proteins

Methods and Principles in Medicinal Chemistry

Edited by R. Mannhold, G. Folkers, H. Buschmann

Editorial Board

J. Holenz, H. Kubinyi, H. Timmerman, H. van de Waterbeemd, John Bondo Hansen

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Preface

The number of marketed protein therapeutics [1–3] has increased enormously since the introduction of the first recombinant protein, human insulin, into the clinic several decades ago. Protein therapeutics play a very significant role in many various fields of medicine, and their use continues to steadily broaden. There are several key advantages of protein therapeutics over small-molecule drugs that contribute to this [1]:

Proteins often exhibit highly specific and complex functions that cannot be mimicked by simple chemical compounds.
The larger binding interface of a protein therapeutic enables them to be engineered with high affinity for their target.
Due to their high level of specificity, there is often less potential for protein therapeutics to interfere with normal biological processes and cause off-target effects.
Recombinant technology allows the production of proteins that provide a novel function or activity.
Because the body naturally produces many of the proteins that are used as therapeutics, these agents are often well tolerated and are less likely to elicit immune responses.
For diseases in which the product of a gene is absent or defective, protein therapeutics can provide an effective replacement treatment.
The clinical development and approval time for protein therapeutics may be faster than for small-molecule drugs [4].
Recombinant proteins can also be used in combination with both other large molecule, or indeed small-molecule, drugs to provide additive or synergistic benefit.

Many successful uses of protein therapeutics are documented in this volume. However some challenges still remain for the discovery and development of protein therapeutics: (i) The current route of administration is typically parenteral. Development of oral biologics remains largely an aspiration at this time. (ii) Although the production of recombinant proteins is becoming increasingly efficient and cost–effective, it remains relatively expensive compared to that of small molecules. (iii) The body may mount an immune response against the therapeutic protein. In some cases, this immune response can neutralize the protein and reduce the efficacy of the potential drug.

Taken together, the early success of recombinant insulin production in the 1970s created an atmosphere of enthusiasm and hope, which was followed by an era of disappointment when the vaccine attempts, nonhumanized monoclonal antibodies, and cancer trials in the 1980s were largely unsuccessful. Despite these setbacks, significant progress has been made. With the large number of protein therapeutics both in current clinical use and in clinical trials for a range of disorders, one can confidently predict that protein therapeutics will have a further expanding role in future medicine and may – together with cell and gene therapy – dominate over classical therapeutic approaches based on small-molecule drugs.

Accordingly, this is an appropriate time to review our current knowledge and future perspectives of protein therapeutics as realized in this volume by experts in the field both from industry and academia. It is organized in six sections, first of which introduces the past and present development of protein therapeutics in the chapters on “Early Recombinant Protein Therapeutics” and “Evolution of Antibody Therapeutics.” The second section is dedicated to antibodies as the ultimate scaffold for protein therapeutics and is covered in two chapters on “Human Antibody Structure and Function” as well as “Antibodies from Other Species.” Discovery and engineering of protein therapeutics are described in the next section comprising detailed chapters on “Human Antibody Discovery Platforms,” “Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development,” “Protein Engineering: Methods and Applications,” “Bispecifics,” and on “Antibody–Drug conjugates.” Physiological and manufacturing considerations are given in the follow-up section including overviews on “Pharmacokinetics,” “Safety Considerations,” “Immunogenicity,” “Expression Systems for Manufacture,” and a chapter on “Stability, Formulation, and Delivery.” The section on Clinical Applications discusses in detail “Protein Therapeutics in Autoimmune and Inflammatory Diseases, Oncology, Respiratory, and Infectious Diseases.” Chapters on “Rescue Therapies” and “Biosimilars” supplement this section. Future horizons and new target class opportunities are the topics of the final section.

The series editors thank Tristan Vaughan, Jane Osbourn, and Bahija Jallal for organizing this volume and for identifying and working with such excellent authors. Last but not least we thank Frank Weinreich and Waltraud Wüst from Wiley-VCH for their valuable contributions to this project and to the entire book series.

May 2017

Raimund Mannhold, Düsseldorf
Gerd Folkers, Zürich
Helmut Buschmann, Aachen

References

1 Leader, B., Baca, Q.J., and Golan, D.E. (2008) Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug Discov., 7, 21–39.
2 Tsomaia, N. (2015) Peptide therapeutics: targeting the undruggable space. Eur. J. Med. Chem., 94, 459–470.
3 Carter, P.J. (2011) Introduction to current and future protein therapeutics: a protein engineering perspective. Exp. Cell Res., 317, 1261–1269.
4 Reichert, J.M. (2003) Trends in development and approval times for new therapeutics in the United States. Nat. Rev. Drug Discov., 2, 695–702.

A Personal Foreword

To a diabetic patient, it is hard to imagine a world without biosynthetic insulin. For a new parent of a premature baby, Synagis provides potentially life-saving prevention of respiratory syncytial virus. And for someone suffering the devastating effects of rheumatoid arthritis, Humira, the world's first fully human antibody, introduced in 2002, is so effective that it has become the world's best-selling medicine. Today, biologics such as these account for nearly half of all new drug approvals across the globe and nearly 25% of overall sales. In fact, in 2015, 6 biologics were among the top 10 best-selling drugs worldwide. With more than 500 biopharmaceuticals on the market, biologics represent the fastest growing sector of this industry, targeting illnesses such as cancer, asthma, cardiovascular disease, infectious diseases, multiple sclerosis, hepatitis, inflammatory disease, and so many others.

As a student of physiology and biochemistry in Paris, I was struck by the possibility of modifying proteins to increase their potential to treat illness. My postdoctoral work in molecular biology and oncology at the Max-Planck Institute for Biochemistry in Munich allowed me to focus on the analysis of epidermal growth factor receptor (EGF-R and HER2) signaling and to investigate the antitumor properties of the novel secreted tumor-associated antigen 90K.

My later work building the translational sciences function at Sugen further heightened my interest. But the day I met a patient with renal cell carcinoma who was successfully being treated with Sutent, a multitargeted receptor tyrosine kinase (RTK) inhibitor, made me realize that my true calling was to work in the biopharmaceutical industry to discover new drugs to help patients. Today, as the head of MedImmune, the global biologics early research and development unit of AstraZeneca, I am even more excited by the possibilities of using protein therapeutics to not just treat or prevent disease but to provide a durable cure for so many illnesses affecting patients across the globe.

This book dissects the field of protein therapeutics, from its early struggles to its promising future, and provides a thorough look into this dynamic industry. It touches on exciting developments around immuno-therapies for oncology – advancements I never thought were possible in my lifetime. It delves into the considerable energy going into the development of antibody drug conjugates and their potential for new therapies. Other topics include human and nonhuman antibodies; technological advances in protein therapeutics, including human antibody discovery platforms, nonantibody scaffolds and antibody mimetics; protein engineering and physiological and manufacturing considerations around pharmacokinetics, immunogenicity, safety, and manufacturing; and clinical applications for protein therapeutics.

Protein Therapeutics concludes with a view into innovation of the future, including the potential to target protein therapeutics across the blood–brain barrier for the treatment of diseases ranging from brain tumors to Alzheimer's disease and how protein therapeutics could be delivered intracellularly to gain a better understanding of protein interactions and, for example, modify the RAS/MAPK pathway that could be potentially transformative for the treatment of leukemia and other cancers.

With contributions from recognized academic and industry experts, including Professor Pierre De Meyts, the leading academician in the field of insulin research and one of the fathers of protein therapeutics, and Herren Wu, MedImmune's chief technology officer who offers just a glimpse at potential future innovations, Protein Therapeutics will be a valuable addition to a field that is profoundly changing the way we treat disease.

Gaithersburg, MD

December 2016

Bahija Jallal