Methods of Hybridoma Formation Contemporary Biomedicine Methods of Hybridoma Formation Edited by Arie H

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Methods of Hybridoma Formation Contemporary Biomedicine Methods of Hybridoma Formation Edited by Arie H. Bartal and Yashar Hirshaut, 1987 Monoclonal Antibodies in Cancer Edited by Stewart Sell and Ralph A. Reisfeld, 1985 Calcium and Contractility: Smooth Muscle Edited by A. K. Grover and E. E. Daniel, 1984 Carcinogenesis and Mutagenesis Testing Edited by J. F. Douglas, 1984 The Human Teratomas: Experimental and Clinical Biology Edited by Ivan Damjanov, Barbara B. Knowles, and Davor Salter, 1983 Human Cancer Markers Edited by Stewart Sell and Britta Wahren, 1982 Cancer Markers: Diagnostic and Developmental Significance Edited by Stewart Sell, 1980 Methods of Hybridoma Formation

Edited by Arie H. Bartal and Yashar Hirshaut

Humana Press . Clifton, New Jersey Library of Congress Cataloging in Publication Data

Main entry under title:

Hybridoma Formation: Methods and Mechanisms

(Contemporary biomedicine) Includes bibliographies and index. 1. Hybridomas. 2. Antibodies, Monoclonal. 3. Immunology-Technique. I. Bartal, Arie H. II. Hirshaut, Yashar. III. Series. QR185.8.H93M48 1987 616.07'93 87-3588 ISBN-13: 978-1-4612-9179-4 e-ISBN-13: 978-1-4612-4826-2 DOl: 10.1007/978-1-4612-4826-2

Crescent Manor PO Box 2148 Clifton, NJ 07015

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Preface

Laymen often consider modern laboratory research to be based on an endless array of sophisticated technologies whose complex capabilities are as important to the outcome of any project as the inventiveness and creativity of the scientists who employ them. Scientists at times may share this point of view until they are con• fronted by unexpected findings that demand new approaches, and they discover that yesterday's "sophisticated tools" are today's "blunt instruments." This experience provides a more sobering view of the current state of our scientific methods. It also serves as an impetus for the further development of technology that prepares us for the next stage of advance. Immunologists were confronted by such a technological crises in the late 1970s when they finally were forced to admit that poly• clonal antibodies, although quite sensitive reagents, were not spe• cific enough to answer many of the questions then confronting virologists and tumor biologists. The answer to the need for specific• ity came with the development of monoclonal antibody technology. In the last ten years there have been considerable advances in monoclonal antibody techniques. Today these reagents are much more versatile than they were initially and can be applied to a broad range of problems. Still, most workers who are using these anti• bodies are convinced that their potential is far from exhausted, and that at least in some fields we are currently in the early stages of learning how to use them properly. This book is a practical volume that can introduce the novice to a technology that will serve him or her well for many years, and at the same time provide the established investigator with helpful insights into novel present and future applications of monoclonal antibody techniques. The range of topics covered is comprehen• sive. Consideration is given to the entire sequence of monoclonal antibody preparation, from the selection of antigen to the purifica• tion of the end-product for clinical use. Each chapter includes both

v vi Preface theoretical background material and practical details for the bench worker. Many of the authors have pioneered work in the fields that they review. One of the pleasures in reading this book is to discover the new perspectives it provides into old problems. For example, how does one achieve more effective fusions? Presented are chemical, hor• monal, and even electrical solutions. What growth factors are critical to generate and maintain functioning hybridomas? Sometimes adding materials is important, sometimes it is equally valuable to subtract materials, for example, by developing serum-free media. How do you ensure monoclonality? Several methods are described. How do you recover hybridomas that are beginning to fade away or freeze early hybridomas without losing them when you thaw? Again several approaches are considered. For the future, the most important problem to solve is probably how to develop useful human-human hybridomas. Steps on the way include learning how to fuse cells of different species or non• human and human cells. Also needed are more efficient immor• talized human fusion partners and methods for selectively im• munizing human cells, perhaps in vitro. Several chapters deal in depth with these issues. All that has been mentioned is only a small sampling of the useful information that can be found in this practical guide to mono• clonal antibody generation. We hope you will find it to be a welcome companion in the laboratory. Arie H. Barta! Yashar Hirshaut DEDICATION

To all the patients who have already benefited from the hybridoma technology and to those that will. Contents

Preface v Contributors ..... xxiii

Current Methodologies in Hybridoma Formation ...... 1 Arie H. Bartal and Yashar Hirshaut 1. Introduction ...... 1 2. Homokaryons, Heterokaryons, Synkaryons ...... 2 3. Selection of Hybridomas ...... 4 4. The Art of Making Hybridomas ...... 5 4.1. Hybridoma Planning ...... 6 5. Phases in Hybridoma Formation ...... 7 5.1. Preparatory Phase ...... 8 5.2. Prefusion Phase ...... 14 5.3. Cell Fusion Phase ...... 16 5.4. Early Postfusion Phase ...... 20 5.5. Late Postfusion Phase ...... 23 5.6. Long-Term Hybridoma Cultivation and Preservation .. 28 5.7. Utilization of Hybridoma-Derived Antibodies ...... 30 6. Difficulties in Hybridoma Formation ...... 31 6.1. Contamination ...... 31 6.2. Equipment Failure ...... 31 6.3. Methodological Difficulties ...... 31 6.4. Basic Biological Problems ...... 31 6.5. Technical Skill ...... 32 6.6. Miscellaneous Factors ...... 32 7. Conclusions ...... 32 References ...... 33 ix x Contents

Retroviruses and Hybridoma Formation: Bystanders or Active Participants? 41 Arie H. Bartal and Yashar Hirshaut 1. Introduction ...... 41 2. Retroviruses, Definition, and Characterization ...... 42 3. Morphology of Retroviruses ...... 43 ...... 3.1. A-Type Particles ...... 43 3.2. B-Type Particles ...... 44 3.3. C-Type Particles ...... 44 3.4. D-Type Particles ...... 44 4. Retroviruses in Animals and Cell Lines ...... 45 5. Retroviruses in Hybridomas ...... 45 6. Hybridoma Retroviruses: Bystanders or Active Participants . 51 7. Hazards Associated With Hybridoma Retroviruses ...... 53 8. Toward Obtaining Purified Virus-Free Monoclonal Antibodies ...... 55 9. Recommendations for Monoclonal Antibody Preparations . 57 References ...... 57

Mechanisms of Cell Fusion and Selection in the Generation of Hybridomas ...... 63 Jerry W. Shay 1. Introduction ...... 63 2. Historical Review ...... 63 3. Fusion Techniques ...... 65 ...... 3.1. PEG ...... 65 3.2. Electrofusion ...... 66 4. Hybrid Selection Techniques ...... , 67 4.1. HAT ...... 67 4.2. Taggart Hybridoma Technology (AAT Selection) .. .. 67 4.3. Irreversible Biochemical Inhibitors ...... 69 4.4. Toxin-Antitoxin Selection ...... 69 4.5. Cell Sorting ...... 70 5. Future Directions and Conclusions ...... 71 References ...... 72 Contents xi Chemically Mediated Cell Fusion 77 Robert J. Klebe and Kevin L. Bentley 1. Introduction ...... 77 2. Mechanism of Polyethylene Glycol-Mediated Cell Fusion .. 78 2.1. Fusogens and Hybridogens ...... 78 2.2. Hydrophilic Character of Hybridogens ...... 79 3. Relationship of Hybridogens to Cryoprotective Agents, Genetic Transformation Agents, and Inducers of Erythroleukemia Cell Differentiation ...... 82 4. Mechanism of PEG-Mediated Cell Fusion ...... 85 5. Hybridoma Formation by PED-Mediated Cell Fusion ..... 86 5.1. Preparation of PEG ...... 88 5.2. Cell Fusion Procedure ...... 90 5.3. Biochemical Selection for Hybridomas ...... 90 References ...... 92

Electrofusion of Cells 97 U. Zimmermann 1. Introduction ...... 97 2. Electric Field Conditions ...... 101 3. Electrode Material and Fusion Media ...... 117 4. Enzyme Pretreatment ...... 120 5. Breakdown and Postbreakdown Conditions ...... 123 6. Viability of Electrofused Hybrids ...... 126 7. Selection of Antibody-Secreting Lymphocytes ...... 128 8. DNA Transfection ...... 130 9. Conclusion ...... 137 Appendix: Murine Hybridoma Electrofusion Protocol for the GCA-Electrofusion System ...... 141 References ...... 143

Enhancement of Hybridoma Formation 151 Arie H. Bartal and Yashar Hirshaut 1. Historical Introduction ...... 151 xii Contents

2. Modes of Hybridoma Enhancement ...... 152 2.1. Enhancement at the Prefusion Phase ...... 153 2.2. Enhancement at the Cell Fusion Phase ...... 154 2.3. Enhancement at the Early Postfusion Phase ...... 155 2.4. Enhancement at the Late Postfusion Phase and in the Long-Term Cultivation of Hybridoma ...... 156 References ...... 158

Modification of HAT Medium and Hybridoma Formation ..... 163 Arie H. Bartal, Carl Feit, and Yashar Hirshaut 1. Introduction ...... 163 2. Experimental Design ...... 164 3. Addition of Dexamethasone to HAT Medium (HAT-DEX) 164 4. Dexamethasone Dose Is Critical ...... 165 5. Timing of Dexamethasone Addition Is Critical ...... 166 6. Stability of Dexamethasone-Treated Hybridoma Clones .. 166 7. Dexamethasone and Hybridoma Formation ...... 168 8. Addition of Insulin to HAT Medium (HIAT) ...... 169 9 . HIAT and Mouse-Mouse Hybridoma Formation ...... 169 10. HIAT in Human-Mouse Hybridoma Formation ...... 174 11. HIAT in Human-Human Hybridoma Formation ...... 174 12. Interferon and Hybridoma Formation (HAT-INF) ...... 175 13. Summary ...... 176 ...... References ...... 177

Culture Methods for the Selection and Isolation of Stable Antibody-Producing Murine Hybridomas ...... 181 R. Thomas Taggart 1. Introduction ...... 181 2. The Nature and Origins of Hybrid Cell Chromosome Instability ...... 182 3. Chromosome Instability Reduces the Yield of Antibody- Producing Hybridomas ...... 184 4. Methods for Isolation of Stable Antibody-Producing Hybridomas ...... 185 Contents xiii

4.1. Syntenic Culture Selection of the Mouse Heavy Chain Immunoglobulin Locus ...... 185 4.2 Limiting Dilution Cloning of Hybridomas ...... 191 5. Conclusions ...... 192 References ...... 193

Selection of Growth Factors and Myelomas To Enhance Monoclonal Antibody-Producing Hybridoma Formation ...... 195 Janet H. Ransom and Martin V. Haspel 1. Introduction ...... 195 2. Selection of Murine Myelomas for Fusion Partners ...... 196 2.1. MOPC 21 (P3-X67 Ag8) ...... 197 2.2. Sp2/0 (Sp2/0-Ag14) ...... 198 2.3. NS-l (P3/NS1/1Ag4-1) ...... 198 2.4.653 (P3/X63-Ag8.653) ...... 198 2.5. FO ...... 198 2.6. S194/5.XXO.BU-1 ...... 199 2.7. FOX-NY ...... 199 3. Growth Factors ...... 199 4. Feeder Cells ...... 200 4.1. Peritoneal Macrophages ...... 200 4.2. Thymocytes and Splenocytes ...... 200 4.3. Cell Lines ...... 201 5. Soluble Growth Supplements ...... 201 5.1. Human Endothelial Cell Growth Supplement (HECS) 201 5.2. Bovine Endothelial Cell Growth Supplement (ECGS) 202 6. Myeloma-Conditioned Medium ...... 202 7. Medium Nutrient Supplements ...... 203 7.1. Growth in Low Molecular Weight Serum Protein Media ...... 203 8. B-Cell Growth Factors ...... 204 9. Technology Notes ...... 204 References ...... 205

Proliferation and Immune Secretion of B-Cell Hybridomas ...... 209 Regine J. J. M. Westerwoudt xiv Contents

1. Introduction ...... 209 2. Stimulation of B-Cell Lymphocytes in Different Phases of the Cell Cycle ...... 210 3. Hybridization and Limiting Dilution ...... 210 4. Cell Cycle of Hybridomas ...... 211 5. Growth Promoters ...... 211 5.1. Dextran Sulfate - A Stimulator of Resting B-Cells .. 211 5.2. Cell-Cell Interaction ...... 212 5.3. Lipopolysaccharide - A Stimulator of Activated B-Cells ...... 213 5.4. Macrophage Supernatant ...... 214 5.5. Human Endothelial Culture Supernatant and Human Umbilical Cord Serum ...... 218 6. Comparison of Normal B-Cells to Tumor B-Cells ...... 219 7. Proliferation and Differentiation of Hybridomas ...... 220 8. Conclusion ...... 221 References ...... 224

Statistical Assessment of Hybridoma Monoclonality After Subcloning by the Limiting Dilution Technique ...... 231 Hilary A. Coller and Barry S. Coller 1. Introduction ...... 231 2. Statistical Assessment ...... 232 References ...... 235

In Vitro Immunization for the Generation of Hybridomas Using Serum-Free Medium ...... 237 Frederick J. Darfler 1. In~oduction ...... 237 2. Growth of Lymphoid Cells and Hybridomas in Serum- Free Media ...... 238 3. Theoretical and Practical Aspects of In Vi~o Immunization 239 4. Methods ...... 239 4.1. Preparation of Serum-Free Medium ...... 239 Contents xv

4.2. In Vitro Immunization Protocol Using Serum-Free Medium ...... 240 4.3. Fusion Protocol ...... 240 5. Comparison of Serum-Containing vs Serum-Free Media for Hybridoma Formation ...... 241 6. Rate of Growth of Nascent Hybridomas in Serum- Containing vs Serum-Free Medium ...... 241 6.1. Cloning in Serum-Free Medium ...... 242 6.2. Antibody Subtype Identification ...... 242 6.3. Immunosorbent Assay for Levels of Monoclonal Antibody ...... 243 6.4. Purification of Monoclonal Antibodies From Serum- Free Medium ...... 243 6.5. Cryopreservation in Serum-Free Medium ...... 245 7. Conclusions ...... 245 7.1.Advantages ...... 245 7.2. Disadvantages ...... 246 7.3. Future Prospects ...... 247 References ...... 247

Intrasplenic Immunization for the Production of Monoclonal Antibodies ...... 249 M. Spitz and Lidia Spitz 1. Introduction ...... 249 2. Materials and Equipment ...... 250 2.1. Preparation of Alum-Precipitated Immunogen ...... 250 3. Method ...... 251 3.1. Surgical Intervention ...... 251 4. Technical Comments ...... 252 5. Discussion ...... 254 References ...... 254

Use of Heteromyelomas in the Enhancement of Human Monoclonal Antibody Production ...... 257 Nelson N. H. Teng and Marcia Bieber 1. In~oduction ...... 257 xvi Contents

2. Principles and Perspectives of Heteromyeloma ...... 259 3. Antigen-Primed Human Fusion Partners ...... 262 4 . Technical Aspects ...... 262 4.1. Preparation of Human Fusion Partners ...... 262 4.2. Fusion Procedure ...... 265 4.3. Cloning Procedure ...... 266 4.4. Mainteinance of Heteromyeloma and Hybridoma Cell Lines ...... 267 References ...... 267

Human Hybridomas: Fusion Partners 273 Danuta Kozbor and Carlo M. Croce 1. Introduction ...... 273 2. Selection of a Fusion Partner ...... 274 3. Morphology of Plasmacytoma and Lymphoblastoid Cells . 276 4. Hybridomas Generated With Human Plasmacytomas .... 277 5. Hybridomas Generated With Lymphoblastoid Cell Lines . 278 6. Lymphoblastoid vs Plasmacytoma Cells as Fusion Partners ...... 280 7. Development of a Better Fusion Partner ...... 281 7.1. Double Drug-Resistant Cell Lines ...... 281 7.2. Non-Ig-Secreting Partners ...... 281 7.3. Hybrid Fusion Partners ...... 284 8. Large-Scale Production of Human Mabs ...... 286 8.1. Ascites Production ...... 286 8.2. Serum-Free Culture ...... 288 9. Advantages of Human Monoclonal Antibodies ...... 289 10. Limitations and Future Developments ...... 290 References ...... 292

Antibody-Secreting Human-Human Hybridomas: Technical Progress, Generation Obstacles, and Applications ...... 299 Lennart Olsson 1. Introduction ...... 299 2. Malignant Fusion Partners ., ...... 300 3. Immunization Procedures ...... 302 Contents xvii

4. Production of Human Mabs by DNA Recombinant Technclogy ...... 303 5. Some Areas of Application of Human Mabs ...... 304 5.1. In Vivo Application in Nonneoplastic Diseases ..... 305 6. HMabs in Cancer Biology ...... 306 6.1. HMabs and Oncogenes ...... 308 6.2. The Antiidiotype Approach ...... 309 6.3. Antigenic Modulation ...... 310 6.4. Intratumoral Phenotypic Diversity and Metastatic Activity ...... 311 7. Conclusions ...... 311 References ...... 312

Interspecific Hybridomas 317 John A. Sogn 1. Introduction ...... 317 2. Reasons for Preparing Interspecific Hybridomas ...... 318 3. Applications of Interspecific Hybridomas ...... 319 3.1. Interrodent Hybridomas ...... 320 3.2. Human-Mouse Hybridomas ...... 321 3.3. Rabbit-Mouse Hybridomas ...... 321 3.4. Other Interspecific Hybridomas ...... 323 4. Species-Specific Chromosome Loss in Interspecific Hybridomas ...... 323 5. Approaches to Minimizing the Problem of Chromosome Loss in Interspecific Hybridomas ...... 325 6. Summary and Opportunities for Future Studies ...... 329 References ...... 332

Rat-Rat Hybridoma Formation and Rat Monoclonal Antibodies ...... 337 Harve Bazin 1. Introduction ...... 337 2. Rat Immunoglobulins ...... 338 2.1. Physicochemical Properties ...... 339 2.2. Biological Properties ...... 341 2.3. Allotypes of Rat Immunoglobulins ...... 341 xviii Contents

3. LOUIC Ileocecal Immunocytomas ...... 341 3.1. Origin of the Louvain Rat Strain ...... 344 3.2. LOUIC Ileocecal Malignant Immunocytomas ...... 344 4. Genetic Studies on the Susceptibility of Rats to Immunocytomas ...... 347 5. Transplantation of LOUIC Rat Immunocytomas ...... 348 6. Storage of LOUIC Immunocytoma Cells ...... 348 7. In Vitro Culture ...... 349 8. Biosynthesis of Myeloma Immunoglobulins ...... 349 9. Production of Monoclonal Immunoglobulins ...... 349 10. Rat Hybridomas ...... 351 11. Fusion Cell Lines ...... 351 12. Immune Lymphocytes-Rat Strains ...... 352 13. Immunization Protocol ...... 352 14. Preparation of the Fusion Line Cells ., ...... 353 15. Media ...... 353 16. Feeder Layer ...... 355 17. Fusion ...... 355 18. Screening Assays ...... 356 19. Cloning ...... 356 20. Freezing and Thawing ...... 356 21. Production of Rat Monoclonal Antibodies ...... 357 22. Determination of Antibody Isotype ...... 358 23. Purification of Rat Monoclonal Antibodies ...... 359 24. Euglobulin Property ...... 359 25. Stability ...... 360 26. Major Strategies ...... 360 27. Purification of Rat Monoclonal Antibodies From In Vitro Culture Supernatant ...... 361 28. Purification of Rat Monoclonal Antibodies From Ascitic Fluid or Serum by Kappa Allotype Immunoaffinity Chromatography ...... 363 29. Purification of Rat Monoclonal Antibodies by Using Anti• Isotype Immunoaffinity Chromatography ...... 364 . . . . 30. Purification of Rat Monoclonal Antibodies by Protein A Affinity Chromatography ...... 364 31. Conventional Techniques of Rat Monoclonal Antibody Purification ...... 365 32.lgM ...... 365 33.IgD ...... 365 34.lgE ...... 365 35.IgA ...... 366 Contents xix

36.IgG1 ...... 366 37. IgG2a ...... 366 38.IgG2b ...... 367 39. IgG2c ...... 367 40. Fragmentation of Rat Monoclonal Antibodies ...... 367 41. Sensitivity to Reduction ...... 367 42. Enzymatic Proteolysis ...... 367 43. Conclusions ...... 368 References ...... 369 ......

Generation of Mutant Monoclonal Antibodies ...... 379 Gad Spira, Hector L. Aguila, Ellyn Ffschberg, and Matthew D. Scharff 1. Introduction ...... 379 2. Modifications in Hybridoma Technology ...... 381 3. Mutant Monoclonal Antibodies ...... 384 4. Class and Subclass Switch Variants ...... 385 5 . Structural Mutants With Changes in Effector Functions and Antigen Binding ...... 391 6. Conclusion ...... 394 References ...... 394

Ranking the Affinities of Monoclonal Antibodies ...... 399 Veronica van Heyningen and Simon van Heyningen 1. Introduction ...... 399 2. Theoretical Aspects ...... 400 2.1. Definition of Affinity ...... 400 2.2. Methods of Measuring Affinity Constants ...... 401 2.3. Calculating K from Binding Curves ...... 402 3 . Practical Applications ...... 404 . . . . . 3.1. SUitability of Antigen ...... 404 3.2. The Need for Measuring the Concentration of Monoclonal Immunoglobulin ...... 406 3.3. Final Choice of Monoclonal Antibody ...... 410 References ...... 411 xx Contents

Intrasplenic Inoculation: A Method for Rescuing Hybridoma Clones ...... 413 Rosalie Ber and Pamela L. Witte 1. Introduction ...... 413 2. Technical Procedure and Remarks ...... 414 2.1. Materials ...... 414 2.2. Intrasplenic Inoculation Procedures ...... 415 3. Hybridoma Cell Inoculum ...... 415 4. Harvesting Ascitic Fluid ...... 416 5. Discussion ...... 416 References ...... 417

Cryopreservation in Hybridoma Production 419 L. de Lefj, E. Schwander, and T. H. The 1. Introduction ...... 419 2. Materials and Methods ...... 420 2.1. Growth of Cells ...... 420 2.2. Fusion Protocol ...... 421 3. Results ...... 421 3.1. Strategy for the Production of Hybridomas ...... 421 3.2. Cryopreservation and Thawing Procedures ...... 422 4. Discussion ...... 425 References ...... 427

Early and Late Freezing of Hybridoma Clones ...... 429 David E. Wells 1. Introduction ...... 429 2. Freezing Cells ...... 431 3. Methods for Freezing Hybridoma Clones ...... 432 4. Conclusions ...... 434 References ...... 435 Contents xxi

Flow Cytometry in Hybridoma Technology 437 Michael Andreeff and Edith Espiritu 1. Introduction ...... 437 References ...... 444

Regulatory Issues Surrounding Therapeutic Use of Monoclonal Antibodies: Points To Consider in the Manufacture of Injectable Products Intended for Human Use ... 447 Thomas Hoffman 1. Introduction ...... 447 2. Development and Characterization of Hybridoma Cell Lines ...... 448 3. Quality Control Tests ...... 449 3.1. Special Considerations Regarding Viral Contamination ...... 450 3.2. Special Considerations Regarding Nucleic Acid Contamination ...... 451 4. Preclinical Animal Testing ...... 452 5. Preclinical Laboratory Testing ...... 453 6. Special Considerations for Monoclonal Antibodies of Human Origin ...... 454 7. Summary and Conclusion ...... 455 References ...... 455

Human-Mouse Hybridoma Formation .... 457 William Cieplinski 1. Introduction ...... 457 2. Somatic Cell Hybridization of Immunoglobulin-Producing Lines ...... 458 3. Production and Characterization of Human-Mouse Hybrids ...... 461 3.1. Production of Hybrids ...... 461 3.2. Stability of Human-Mouse Hybrids ...... 461 xxii Contents

4. Expression of Human Enzymes ...... 462 5. Karyotype of Human-Mouse Hybrids ...... 463 6. Stability of Immunoglobulin Secretion ...... 464 7. Membrane Transport by Human-Mouse Hybrids ...... 465 8. Applications of Human-Mouse Hybridoma Technology ... 466 8.1. Human Genetic Analysis and Gene Mapping ...... 466 8.2. Production of Specific Human Monoclonal Antibodies ...... 467 8.3. Other Biological Research Applications of Human- Mouse Hybrids ...... 468 8.4. Human-Mouse Monoclonals in Cancer ...... 468 9. Conclusions ...... 470 References ...... 470 Index ...... 477 CONTRIBUTORS

HECTOR L. AGUILA . Department of Cell Biology and the Irvington House Institute, Albert Einstein College of Medicine, Bronx, New York MICHAEL ANDREEFF . Leukemia Cell Biology Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York ARIE H. BARTAL . Biotherapeutics, Inc., Franklin, Tennessee and Hybridoma Laboratory, Northern Israel Oncology Center, Rambam Medical Center, Haifa, Israel HERvE BAZIN . Experimental Immunology Unit, Faculty of Medi• ine, University of Louvain, Brussels, Belgium KEVIN L. BENTLEY . Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, Texas ROSALIE BER . Technion-Isreal Institute of Technology, Faculty of Medicine, Rappaport Family Institute of Research in the Medical Sciences, Haifa, Israel MARCIA BIEBER . Cancer Biology Research Laboratory, Departments of Radiology and Gynecology and Obstetrics, Stanford University, Stan• ford, California WILLIAM CIEPLINSKI . Veterans Administration Medical Center, Newington, Connecticut BARRY S. COLLER . Division of Hematology, State University of New York at Stony Brook, Stony Brook, New York HILARY A. COLLER . Half Hollow Hills High School West, Dix Hills, New York and Division of Hematology, State University of New York at Stony Brook, Stony Brook, New York CARLO M. CROCE . The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania FREDERICK J. DARFLER . Laboratory of Cellular and Developmental Biology, National Institutes of Health, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland L. de LEIJ . Department of Clinical Immunology, University Hospital, Groningen, The Netherlands EDITH ESPIRITU . Leukemia Cell Biology Laboratory, Memorial Sloan-Kettering, New York, New York

xxiii xxiv Contributors

CARL FElT Faculty of Biology, Yeshiva University, New York, New York ELLYN FISCHBERG . Department of Cell Biology and the Irvington House Institute, Albert Einstein College of Medicine, Bronx, New York MARTIN V. HASPEL . Litton Institute of Applied Biotechnology, Litton Bionetics, Inc., Rockville, Maryland YASHAR HIRSHAUT . Sloan-Kettering Cancer Center, New York, New York THOMAS HOFFMAN . Laboratory of Cell Biology, Food and Drug Administration, Bethesda, Maryland ROBERT J. KLEBE . Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, Texas DANUTA KOZBOR . The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania LENNART OLSSON . Cancer Biology Laboratory, State Universi• ty Hospital, Copenhagen, Denmark JANET H. RANSOM . Litton Institute of Applied Biotechnology, Lit• ton Bionetics, Inc., Rockville, Maryland MATTHEW D. SCHARFF . Department of Cell Biology and the Ir• vington House Institute, Albert Einstein College of Medicine, Bronx, New York E. SCHWANDER . Department of Clinical Immunology, Universi• ty Hospital, Groningen, The Netherlands JERRY W. SHAY . Department of Cell Biology, University of Texas Health Science Center at Dallas, Dallas, Texas JOHN A. SOGN . Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland GAD SPIRA . The Faculty of Medicine and The Rappaport Family Institute for Research in the Medical Sciences, Technion, Israel Institute of Technology, Haifa, Israel LIDIA SPITZ . St. Thomas' Hospital, Medical School, London, Great Britain M. SPITZ . National Institute for Biological Standards and Control, London, Great Britain R. THOMAS TAGGART . Human Genetics Research, Veterans Ad• ministration Medical Center, Sepulveda, California and Department of Medicine, University of California Los Angeles, Los Angeles, California Contributors xxv

NELSON N. H. TENG . Cancer Biology Research Laboratory, Departments of Radiology and Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California T. H. THE . Department of Clinical Immunology, University Hospital, Groningen, The Netherlands SIMON van HEYNINGEN . Department of Biochemistry, Univer• sity of Edinburgh, Edinburgh, Great Britain VERONICA van HEYNINGEN . MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh, Great Britain DAVID E. WELLS . Division of Bacterial Diseases, Center for Infec• tious Diseases, Centers for Disease Control, US Department of Health and Human Services, Public Health Service, Atlanta, Georgia REGINE J. J. M. WESTERWOUDT . Laboratory for Electron Microscopy, University of Leiden, Leiden, The Netherlands PAMELA L. WITTE . Cancer Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma U. ZIMMERMANN . Lehrstuhl fur Biotechnologie der Universitat Wurzburg, Wurzburg, Republic of Germany