Human Hybridomas and Monoclonal Antibodies, Softcover reprint of the original 1st ed. 1985

Soon after Kohler and Milstein described the use of somatic cell hybridization for the production of murine monoclonal antibodies of desired specificity, this relatively simple technique became widely applied. Indeed, production of murine monoclonal antibodies is now considered routine by immunologists and nonimmunologists alike. However, as heterologous proteins, mouse monoclonal antibodies have one major limitation: they are immunogenic in man and, hence, their use in vivo is severely limited. An obvious solution to this problem is to produce human hybridomas with the same techniques used for the production of rodent hybrids. Unfortunately, the history of human hybridomas has been marked by substantive and often exasperating tech­ nical problems, and the first reports of hybrids secreting human immu­ noglobulin of desired specificity did not appear until 1980. These reports were met with initial enthusiasm, but it soon became apparent that while human lymphocytes might be fused, their frequency, level of Ig synthesis, and stability were such that production of human antibodies with this method was neither routine nor practical. Nonetheless, a sufficient number of investiga­ tors persevered, and during the next 5 years relatively efficient B-cell fusion partners as well as improved methods of Epstein-Barr virus transformation were developed. Generation of human T -T hybrids has also been achieved, although problems of chromosomal stability remain a substantial obstacle, more so than with B-cell lines.

I B-Cell Lines, Hybridomas, and Monoclonal Antibodies.- A. Background and General Strategies.- 1 Human Hybridomas and Monoclonal Antibodies: The Biology of Cell Fusion.- I. Introduction.- II. Historical Development of Hybridomas.- III. Problems with Conventional Immunization.- IV. Advantages of the Monoclonal Antibody Technology.- V. Difficulties and Technical Problems in the Production of Monoclonal Antibodies.- VI. Human Hybridomas and Monoclonal Antibodies.- VII. Methods for Making Human Monoclonal Antibodies.- VIII. Limitations in Making Human Monoclonal Antibodies.- IX. Enrichment for Sensitized Lymphocytes and in Vitro Stimulation of Lymphocytes.- X. Fusion Variables.- XI. Selection of Hybrids.- XII. Freezing and Thawing Hybridomas.- XIII. Chromosome Stability in Hybridomas.- References.- 2 Fusion Partners for Production of Human Monoclonal Antibodies.- I. Murine Plasmacytomas as Fusion Partners.- II. Human Plasmacytomas as Fusion Partners.- III. Human Lymphoblastoid Cell Lines as Fusion Partners.- IV. Lymphoblastoid versus Myeloma Cells as Fusion Partners.- V. Search for Better Fusion Partners.- References.- 3 Production of Human Monoclonal Antibodies Using Epstein-Barr Virus.- I. Introduction.- II. The Biological Properties of Epstein-Barr Virus.- III. Human Monoclonal Antibody Production.- IV. Future Perspectives.- References.- 4 The Epstein-Barr Virus-Hybridoma Technique.- I. Epstein-Barr Virus Technology.- II. The Epstein-Barr Virus-Hybridoma Technique.- III. Anti-Tetanus Monoclonals from in Vitro Immunized Cultures.- IV. Anti-Mycobacterium leprae Monoclonals.- V. Human Monoclonal Antibodies against Lung Carcinoma Cell Lines.- VI. Human Monoclonal Antibodies against Myelin-Associated Glycoprotein.- VII. New Fusion Partners.- VIII. Ascites Growth of Human Hybridomas.- IX. Epstein-Barr Virus-Hybridoma Protocol.- X. The Advantages of Human Monoclonal Antibodies.- XI. Possible Limitations.- XII. Conclusions.- References.- 5 Strategies for Stable Human Monoclonal Antibody Production: Construction of Heteromyelomas, in Vitro Sensitization, and Molecular Cloning of Human Immunoglobin Genes.- I. Introduction.- II. General Properties of Mouse-Human Heteromyeloma and Heteromyelolymphoma Cell Lines.- III. Optimal Antigen-Primed Human B-Lymphocyte Fusion Partners.- IV. Permanent Preservation of Immunoglobulin Production.- References.- B. Applications to Infectious Diseases.- 6 Production and Characterization of Human Monoclonal Antibodies against Gram-Negative Bacteria.- I. Introduction.- II. Human Hybridomas Secreting Antibody to Escherichia coli.- III. Human Monoclonal Antibody Production in Nude Mice.- IV. Characterization of Antibody Specificity.- V. Discussion.- VI. Summary.- References.- 7 Human Monoclonal Antibodies to Defined Antigens: Toward Clinical Applications.- I. Production of Monoclonal Antibodies for Diagnostic and Therapeutic Use.- II. Peripheral Blood as a Source of Antigen-Primed Lymphocytes.- III. A Comparison of Mouse and Human Lymphoid Fusion Partners.- IV. Human Antibodies to Specific Antigens.- V. Clinical Applications.- VI. Summary.- References.- 8 Production of Human Monoclonal Antibodies Using a Human—Mouse Fusion Partner.- I. Introduction.- II. Generation of a Human-Mouse Fusion Partner.- III. Human Anti-A Red Blood Cell Antibody.- IV. Human Monoclonal Antibodies to Varicella Zoster Virus.- V. An Anti-Epstein-Barr Virus IgM Human Monoclonal Antibody.- VI. Human Monoclonal Antibodies to Mycobacterium leprae.- VII. Concluding Comments.- References.- 9 In Vitro Expansion of Human B Cells for the Production of Human Monoclonal Antibodies.- I. Introduction.- II. Methods.- III. In Vitro B-Cell Expansion with EBV.- IV. Class Switching of Human Hybridomas.- V. In Vitro Antigen-Specific Expansion of B Cells.- VI. Conclusions.- References.- 10 Cell-Driven Viral Transformation.- I. Introduction.- II. Derivation of the Driving Cell Line.- III. Application of the Cell-Driven Viral Transformation Process to the Generation of Specific Human Monoclonal Antibodies.- IV. Discussion.- References.- C. Applications to Cancer.- 11 The Generation of Human Monoclonal Antibodies and Their Use in the Analysis of the Humoral Immune Response to Cancer.- I. Introduction.- II. Properties of Drug-Marked Fusion Partners.- III. Human Monoclonal Antibodies Directed against Cellular Antigens.- IV. Summary.- References.- 12 Design and Production of Human Monoclonal Antibodies to Human Cancers.- I. Introduction.- II. Development and Characterization of UC 729–6.- III. Enzyme Immunoassay to Qualitate and Quantitate Human Immunoglobulin.- IV. Use of Regional Draining Lymph Nodes of Cancer Patients as a Source of Antitumor Antibodies.- V. Detection of Cell-Bound Antigens.- VI. Concluding Remarks.- References.- 13 Human-Human Hybridoma Technology: Five Years of Technical Improvements, and Its Application in Cancer Biology.- I. Introduction.- II. Improvement of Malignant Fusion Partners.- III. In Vitro Antigen Priming.- IV. Fusion Procedures, Cultivation of Human-Human Hybrids, and Screening for Immunoglobulin Production.- V. Human Monoclonal Antibodies against Tumor-Associated Antigens.- VI Conclusion.- References.- D. Applications to Autoimmunity.- 14 The Production of Monoclonal Antibodies by Human-Human Hybridomas: Their Application to Studies of Autoimmune Diseases.- I. Introduction.- II. Preparation of Human-Human Hybridomas.- III. Technical Considerations.- IV. Monoclonal Human Lupus Autoantibodies.- References.- 15 Human Monoclonal Autoantibodies Reactive with Multiple Organs.- I. Introduction.- II. Establishment of Continuous Cell Lines Producing Autoantibodies.- III. Comparison of Methods for Generating Monoclonal Autoantibodies.- IV. Methods of Screening for Autoantibodies.- V. Evaluation of Tissue Reactivity of Monoclonal Autoantibodies.- VI. Purification of Human Autoantigens by Immunoaffinity Chromatography.- VII. Discussion.- References.- 16 Principles of in Vitro Immunization of Human B Lymphocytes.- I. Introduction.- II. Principles That Govern the Activation of B Lymphocytes. Helper Requirements.- III. Antigens.- IV. Antigen-Specific Induction of Antibody Synthesis in Culture of Human Lymphoid Cells.- V. The Role of Suppressor Cells in the Generation of Antibody-Forming Cells in Vitro.- VI. Measures to Amplify Antibody Production.- VII. In Vitro Activation of B Cells Applied to the Production of Human Monoclonal Antibody.- References.- E. Special Topics.- 17 Human-Human Hybridomas in the Study of Immunodeficiencies.- I. Introduction.- II. Immunodeficiency Patients Used in Hybridoma Formation.- III. Lymphocyte Activation and Hybridization.- IV. Characterization of the Hybridomas Obtained from the Peripheral Blood B Cells of Immunodeficiency Patients.- V. Immunoglobulin Gene Analysis of the Hybridomas Derived from Immunodeficiency Patients.- VI. RNA Processing of µ Heavy Chain Message in the Hybridomas Derived from Immunodeficiency Patients.- VII. Discussion.- References.- II Human T-T Hybridomas.- 18 Human T-Lymphocyte Subsets and T-T Hybridomas: An Overview.- I. Introduction.- II. Functional T-Cell Subsets in Man.- III. The Role of Lymphokines in Immunoregulation.- IV. Human T-T Hybridomas.- References.- 19 Production of Human T-Cell Hybridomas by Electrofusion.- I. Introduction.- II. Fusion Partners in Polyethylene Glycol Fusion and Electrofusion.- III. Polyethylene Glycol Fusion.- IV. Electrofusion.- V. Characterization of Fusion Products Obtained by Polyethylene Glycol and Electrofusion.- VI. Fusion Protocols.- VII. Discussion.- References.- 20 Human B-Cell Growth and Differentiation Factors Secreted by T-Cell Hybridomas.- I. Introduction.- II. Assay Systems for Human B-Cell Proliferation and Differentiation.- III. Hybridization of Mitogen-Activated Human Peripheral Blood T Lymphocytes.- IV. Development of a Human T-Cell Hybridoma Secreting B-Cell Growth Factor.- V. Development of a Human T-Cell Hybridoma Secreting Both B-Cell Growth Factor and B-Cell Differentiation Factor.- VI. Application of T-Cell Hybridoma-Derived B-Cell Growth Factor and B-Cell Differentiation Factor to the Study of Human B-Cell Immunoregulation.- VII. Summary and Conclusions.- References.- 21 Generation and Characterization of Human T-Cell Hybridomas That Constitutively Produce Immune Interferon.- I. Introduction.- II. Isolation of a Parent Cell Line Deficient in Hypoxanthine Phosphoribosyltransferase.- III. Derivation and Characterization of Human T-Cell Hybridomas.- IV. Production of Immune Interferon by Hybridomas.- V. Application of Hybridomas to Structure-Function Studies of Immune Interferon.- VI, Summary.- References.- 22 Selection of Human T-Cell Hybridomas That Produce Inflammatory Lymphokines by the Emetine-Actinomycin D Method.- I. Introduction.- II. Emetine-Actinomycin D Selection Method.- III. Migration-Inhibitory Factor and Macrophage-Activating Factor Activities in Culture Supernatants of Hybridomas.- IV. Functional Stability of Human T-Cell Hybridomas.- References.- 23 Human T-T Hybridomas Specific for Epstein-Barr Virus: Generation and Function.- I. Introduction.- II. Choice and Construction of the Malignant T Lymphoblast.- III. Selection and Preparation of Epstein-Barr Virus-Specific Normal T-Cell Parent.- IV. Hybridization.- V. Proof of Hybridization.- VI. Testing for Antigen Specificity.- VII. HLA-DR Restriction of Recognition.- VIII. General Considerations.- IX. Summary.- References.- 24 Factors Generated by Human T-Cell Hybridomas Regulate B-Cell Activation, Polyclonal Differentiation, and Isotype Expression.- I. Introduction.- II. Methodology for Human T-Cell Hybridomas.- III. Factor Secretion by Human T-Cell Hybridomas.- IV. Model of B-Cell Maturation.- References.- 1. Human T- and B-Cell Lines.- 2. Preparation of Mutant Cell Lines.- 3. Freezing Human Cell Lines.- 4. Mycoplasma Testing.- 5. Peripheral Blood Lymphocyte Separation from Whole Blood or Buffy Coats.- 6. Separation of Human T and Non-T Lymphocytes from Peripheral Blood.- 7. Panning for Human T-Lymphocyte Subpopulations.- 8. Human Hybridoma Tube Fusion Protocol.- 9. Plate Fusion Technique for Nonadherent Cells.- 10. Identification of Human Chromosomes in Mouse-Human Lymphocyte Hybrids.- 11. Epstein-Barr Virus Transformation.- 12. Growth of Human Cell Lines in Mice.- 13. Methods of Large-Scale Tissue Culture.- 16. Soft Agar Cloning Protocol.- 17. Cloning by Limiting Dilution.- 18. Reverse Plaque Assay.- 19. Assay of Interleukin 1 (IL-1).- 20. Assay of Interleukin 2 (IL-2).- 21. Assay of B-Cell Growth and Differentiation Factors.- 22. Indirect Immunoglobulin ELISA Protocol.- 23. Purification of Human Immunoglobulins.- 24. A Nitrocellulose Strip Method for Isotyping Monoclonal Antibodies.- 25. Immunoprecipitation of Antigens Using Polystyrene Balls.- 26. Immunostaining Using Monoclonal Antibodies.- 27. Immunoblotting.- Manufacturers and Distributors.