This volume of Methods in Enzymology looks at Protein Engineering for Therapeutics. The chapters provide an invaluable resource for academics, researchers and students alike. With an international board of authors, this volume is split into sections that cover subjects such as Antibodies, Protein conjugates, Peptides, Enzymes and Scaffolds
1. 1. Introduction 2. 2. Materials 3. 3. Methods 4. Generation of Dual-Variable-Domain Immunoglobulin Molecules for Dual-Specific Targeting 5. 1. Introduction 6. 2. Design of DVD-Ig™ Molecules 7. 3. Cloning and Expression of DVD-Ig™ Molecules 8. 4. Molecular Characterization of DVD-Ig™ Proteins 9. 5. Functional Characterization of DVD-Ig™ Proteins 10. 6. Concluding Remarks 11. Acknowledgments 12. Discovery of Internalizing Antibodies to Tumor Antigens from Phage Libraries 13. 1. Introduction 14. 2. Selection of Tumor Cell-Specific Internalizing scFv from Phage Antibody Libraries 15. 3. Profiling the Specificity of Phage Antibodies on Tumor Cells Using Flow Cytometry 16. 4. Identification of the Cognate Antigen Recognized by Tumor-Specific scFv by Using Mass Spectrometry 17. 5. Selection of Antigen-Specific Phage Antibodies by Sequential Selection on Tumor Cells Followed by Selection on Yeast Cells Displaying Specific Tumor Antigens 18. Optimizing Properties of Antireceptor Antibodies Using Kinetic Computational Models and Experiments 19. 1. Introduction: Valence, Affinity, and Avidity in Antibodies 20. 2. Quantifying Antibody Cross-linking Strength Using “VFC” Models of Antibody Binding to Cell-Surface Receptors 21. 3. Workflow for Quantifying Antibody Cross-linking Strength 22. 4. Model Applications and Insights 23. Acknowledgments 24. Cell-Specific siRNA Delivery by Peptides and Antibodies 25. 1. Introduction 26. 2. Cationic Cell Permeable Peptides 27. 3. Solid Biodegradable Polymer and Vesicular (Liposomal) Systems 28. Acknowledgments 29. Conjugation of Anticancer Drugs Through Endogenous Monoclonal Antibody Cysteine Residues 30. 1. Antibody Reduction 31. 2. Monitoring Antibody Reduction 32. 3. Conjugation with Maleimido Drug-Linker 33. 4. Purification Methods 34. 5. Small-Scale Conjugations for Early ADC Discovery 35. 6. Procedure for Solid-Phase Conjugation 36. 7. Determination of Drug Loading 37. 8. Determination of Unconjugated Drug Content 38. 9. Conclusions 39. Building and Characterizing Antibody-Targeted Lipidic Nanotherapeutics 40. 1. Introduction 41. 2. Preparation of Immunoliposomes 42. 3. Physicochemical Characterization of Immunoliposomes 43. 4. In Vitro and In Vivo Characterization of Immunoliposomal Therapeutics 44. Cell-Targeting Fusion Constructs Containing Recombinant Gelonin 45. 1. Introduction 46. 2. Engineered Proteins Targeting Her2/neu 47. 3. rGel/BLyS Targeting Malignant B-Cells 48. 4. VEGF121/rGel Targeting Tumor Vasculature and Skeletal Metastases 49. Fusions of Elastin-Like Polypeptides to Pharmaceutical Proteins 50. 1. Introduction 51. 2. Protein ELP Fusion Design 52. 3. Recursive Directional Ligation by Plasmid Reconstruction 53. 4. Expression and Purification of Protein ELP Fusions 54. 5. Physical Characterization of Protein ELP Fusions 55. 6. Conclusions 56. Acknowledgment 57. Oral Enzyme Therapy for Celiac Sprue 58. 1. Introduction 59. 2. Lead Identification 60. 3. Assay Development 61. 4. Gram-Scale Production and Formulation 62. 5. Lead Optimization for Next-Generation Proteases 63. 6. Summary and Outlook 64. Rational Design and Evaluation of Mammalian Ribonuclease Cytotoxins 65. 1. Introduction 66. 2. Attributes of Cytotoxic ptRNases 67. 3. Assays to Evaluate the Cytotoxicity of ptRNases 68. 4. Prospectus 69. Acknowledgments 70. Engineering Reduced-Immunogenicity Enzymes for Amino Acid Depletion Therapy in Cancer 71. 1. Introduction: Amino Acid Depletion as a Chemotherapeutic Strategy 72. 2. Method I. Combinatorial Deimmunization 73. 3. Method II. Engineering Therapeutically Relevant Substrate Specificities into a Human Enzyme 74. 4. Method III. Improving the Kinetics and Stability of a Human Enzyme 75. 5. Concluding Remarks
This volume of Methods in Enzymology looks at Protein Engineering for Therapeutics. The chapters provide an invaluable resource for academics, researchers and students alike. With an international board of authors, this volume is split into sections that cover subjects such as Antibodies, Protein conjugates, Peptides, Enzymes and Scaffolds
1. 1. Introduction 2. 2. Materials 3. 3. Methods 4. Generation of Dual-Variable-Domain Immunoglobulin Molecules for Dual-Specific Targeting 5. 1. Introduction 6. 2. Design of DVD-Ig™ Molecules 7. 3. Cloning and Expression of DVD-Ig™ Molecules 8. 4. Molecular Characterization of DVD-Ig™ Proteins 9. 5. Functional Characterization of DVD-Ig™ Proteins 10. 6. Concluding Remarks 11. Acknowledgments 12. Discovery of Internalizing Antibodies to Tumor Antigens from Phage Libraries 13. 1. Introduction 14. 2. Selection of Tumor Cell-Specific Internalizing scFv from Phage Antibody Libraries 15. 3. Profiling the Specificity of Phage Antibodies on Tumor Cells Using Flow Cytometry 16. 4. Identification of the Cognate Antigen Recognized by Tumor-Specific scFv by Using Mass Spectrometry 17. 5. Selection of Antigen-Specific Phage Antibodies by Sequential Selection on Tumor Cells Followed by Selection on Yeast Cells Displaying Specific Tumor Antigens 18. Optimizing Properties of Antireceptor Antibodies Using Kinetic Computational Models and Experiments 19. 1. Introduction: Valence, Affinity, and Avidity in Antibodies 20. 2. Quantifying Antibody Cross-linking Strength Using “VFC” Models of Antibody Binding to Cell-Surface Receptors 21. 3. Workflow for Quantifying Antibody Cross-linking Strength 22. 4. Model Applications and Insights 23. Acknowledgments 24. Cell-Specific siRNA Delivery by Peptides and Antibodies 25. 1. Introduction 26. 2. Cationic Cell Permeable Peptides 27. 3. Solid Biodegradable Polymer and Vesicular (Liposomal) Systems 28. Acknowledgments 29. Conjugation of Anticancer Drugs Through Endogenous Monoclonal Antibody Cysteine Residues 30. 1. Antibody Reduction 31. 2. Monitoring Antibody Reduction 32. 3. Conjugation with Maleimido Drug-Linker 33. 4. Purification Methods 34. 5. Small-Scale Conjugations for Early ADC Discovery 35. 6. Procedure for Solid-Phase Conjugation 36. 7. Determination of Drug Loading 37. 8. Determination of Unconjugated Drug Content 38. 9. Conclusions 39. Building and Characterizing Antibody-Targeted Lipidic Nanotherapeutics 40. 1. Introduction 41. 2. Preparation of Immunoliposomes 42. 3. Physicochemical Characterization of Immunoliposomes 43. 4. In Vitro and In Vivo Characterization of Immunoliposomal Therapeutics 44. Cell-Targeting Fusion Constructs Containing Recombinant Gelonin 45. 1. Introduction 46. 2. Engineered Proteins Targeting Her2/neu 47. 3. rGel/BLyS Targeting Malignant B-Cells 48. 4. VEGF121/rGel Targeting Tumor Vasculature and Skeletal Metastases 49. Fusions of Elastin-Like Polypeptides to Pharmaceutical Proteins 50. 1. Introduction 51. 2. Protein ELP Fusion Design 52. 3. Recursive Directional Ligation by Plasmid Reconstruction 53. 4. Expression and Purification of Protein ELP Fusions 54. 5. Physical Characterization of Protein ELP Fusions 55. 6. Conclusions 56. Acknowledgment 57. Oral Enzyme Therapy for Celiac Sprue 58. 1. Introduction 59. 2. Lead Identification 60. 3. Assay Development 61. 4. Gram-Scale Production and Formulation 62. 5. Lead Optimization for Next-Generation Proteases 63. 6. Summary and Outlook 64. Rational Design and Evaluation of Mammalian Ribonuclease Cytotoxins 65. 1. Introduction 66. 2. Attributes of Cytotoxic ptRNases 67. 3. Assays to Evaluate the Cytotoxicity of ptRNases 68. 4. Prospectus 69. Acknowledgments 70. Engineering Reduced-Immunogenicity Enzymes for Amino Acid Depletion Therapy in Cancer 71. 1. Introduction: Amino Acid Depletion as a Chemotherapeutic Strategy 72. 2. Method I. Combinatorial Deimmunization 73. 3. Method II. Engineering Therapeutically Relevant Substrate Specificities into a Human Enzyme 74. 4. Method III. Improving the Kinetics and Stability of a Human Enzyme 75. 5. Concluding Remarks
