HANDBOOK OF

MONOCLONAL ANTIBODIES

Applications inBiology and Medicine

Edited by

Soldano FerroneDepartment of Microbiology and Immunology

New York Medical CollegeValhalla, New York

Manfred P. DierichInstitute of Hygiene

University of InnsbruckInnsbruck, Austria

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Sole distribution by:Gothard House PublicationsGothard House, Henley-on-Thames,Oxon RG91AJTel: 0491 573602

NOYES PUBLICATIONSPark Ridge, New Jersey, U.S.A.

Contents and Subject Index

Preface vContributors vii

1. MONOCLONAL ANTIBODY PRODUCTION: PRINCIPLES ANDPRACTICE 1

Stephen Fazekas de St. GrothHistorical 1

The Case Against Antisera 1Production of Monoclonal Antibodies 2

Principles 2Practice, 1975 3Practice, 1983 5

Use and Misuse of Monoclonal Antibodies 7The Consequences of Heterogeneity 7

Cross Reactivity 7Affinity 7Isotypy 7

Criteria for the Effective Use of Monoclonals 8Purification of Antigens 8Antigenic Analysis 8Diagnostics 9Therapeutics 9

References 10

2. LYMPHOCYTE SUBPOPULATIONS IN RODENTS: FUNCTIONALSUBPOPULATIONS IN RODENTS 11

Mark R. Greenwood and R. Michael ParkhouseIntroduction 11Heterogeneity of T Cells 12

xi

xii Contents and Subject Index

Functional Heterogeneity of T Cells 12T Cell Markers 13

Thy-1 13Markers on Mouse T Cells 14Markers on Rat T Cells 16

Heterogeneity of B Cells 16Functional Heterogeneity in B Cells 17

Bi and B2 Cells 17The CBA/N Defect 17

.. ***" C3H/HeJ Mice 19Pre-Progenitor B Cells 19Virgin and Memory B Cells 20Immunoglobin on B Cells 20The Fc Receptor (FcR) 22la Antigens 22The Complement Receptor 23

Antisera and Monoclonal Antibodies which Recognize BCells 23

Lyb-2 23Lyb-3 24Lyb-5 24Lyb-7 24P e l 24MBLA 24Qa-1 and Qa-2 2419B5 2414.8 24DNL 19A 25RA.3-2C2 25

Concluding Remarks 25References 27

3. LYMPHOCYTE SUBPOPULATION SEPARATION BY MONOCLONALANTIBODIES 36

Rudiger W. BraunIntroduction 36Direct and Indirect Cytotoxicity 37Rosetting Procedures " 39Panning and Procedures 40Fluorescence Activated Cell Sorting 41Separation by Microspheres 42Affinity Chromatography 43Conclusions 43References 44

4. IMMUNOFLUORESCENCE ANALYSIS OF LEUCOCYTE/LYMPHO-CYTE MEMBRANE ANTIGENS WITH MONOCLONAL ANTIBODIES . .48

Wolfgang Ax and Sabine SchottlerIntroduction 48

Contents and Subject Index xiii

Materials 48Monoclonal Antibodies 49Conjugate 49Agar Slide 49Dilution Medium/Washing Solution 49

Procedure 49Lymphocyte/Leucocyte Isolation from Blood—RoomTemperature 49

Monocyte Isolation from Lymphocyte-Containing Layer ofDensity Gradient—37°C 49

Lymphocyte Fluorescence Labeling (0 - 4°C) 50Microscopic Evaluation 50

Principles 50Filter Sets for Epi-Fluorescence 51Preparation of Samples - Reading. . . . 51Microphotography 51Television 53

Discussion 53References 54

5. PHENOTYPING OF HUMAN NK CELLS WITH MONOCLONALANTIBODIES 56

Dietrich Kraft, Helmut Rumpold and OlegEreminIntroduction 56Characterization of NK Cells by MoAB Raised Against Cells of

the T, B and Myelomonocytic Cell Series 57Characterization of NK Cells by Anti-LGL MoAB 58

HNK-1 (Anti-Leu7) 58NK-8 593G8 59VEP13 59B73.1 62NKP-15 (Anti-Leulla) 64GO22 (Anti-Leullb) 64Comparison of the Anti-NK/PMNL FcryR MoAB B73.1, 3G8,

Anti-Leulla, Anti-Leullb and VEP13 64MoAB with Various Specificities 65

H25 and H366 65Anti-N901 65HNC-1A3 66

Evidence for a Third Lymphocytic Lineage, as Ascertained byStudies with MoAB 66

Summary 66References 67

6. MONOCLONAL ANTIBODIES AND HUMAN MYELOMONOCYTICCELLS -. 71

Walter KnappIntroduction 71

xiv Contents and Subject Index

Cells of the Myelomonocytic Series 71Monoclonal Antibodies Reactive with Myelomonocy tic Cells 72

Granulocytic Antibodies 72Monocytic Antibodies 74Myelomonocytic Antibodies 76Receptors for Complement Components 76Monoclonal Antibodies to Fc-Receptors 77Nomenclature of Monoclonal Antibodies to Myelomonocytic

Cells 78Differentiation of Myelomonocytic Cells 79

Progenitor Cells 79Granulocytic Differentiation 79Monocytic Differentiation 80Macrophage Differentiation 81Dendritic Cells 82

Malignant Proliferations of Myelomonocytic Cells 82Acute Myeloid Leukemias (AML) 82Chronic Myelogenous Leukemia (CML) 84

References 84

7. MONOCLONAL ANTIBODIES AS PROBES TO DEFINE HUMANT CELL SURFACE STRUCTURES AND FUNCTIONS 91

Stefan C. Meuer, Stuart F. Schlossman and Ellis L. ReinherzIntroduction 91Monoclonal Antibodies to Human T Lymphocyte Surface

Antigens 92Differentiation and Function of Human T Lymphocytes 92The Biology of the Helper and Suppressor T Cell Subsets 95The Role of Surface Molecules for Human T Cell Recognition 97Clinical Applications 100References 103

8. MONOCLONAL ANTIBODIES AS TOOLS TO STUDY RECEPTOR-TRANSDUCER-EFFECTOR SYSTEMS 109

Manfred Schmitt, Richard G. Painter and Charles G. CochraneIntroduction 109Monoclonal Antibodies as Receptor Specific Analytic Probes I l lEffects of Monoclonal Antibodies on N-Formyl ChemotacticPeptide Mediated Stimulation of Human Neutrophils 116'

References 125

9. MONOCLONAL ANTIBODIES IN ENZYMOLOGY 131Franco Celada and Boris Rotman

Introduction 131Identification and Purification of Enzymes 132Anti-Enzyme MoAB to Detect Tumor-Related Specificities 133Functional MoAB to Study Association of Molecules on Cell

Organelles and Membranes 134

Contents and Subject Index xv

Multifunctional Enzyme Molecules 134Isozymes and Enzyme Complexes 135MoABs to Study Antibody-Mediated Effects on Protein

Conformation 137Definition of the Epitope 140Dissection of an Epitope 142New Methodology, Future Developments 143References 145

10. MONOCLONAL ANTIBODIES IN THE ANALYSIS OF STRUCTUREAND FUNCTION OF COMPLEMENT COMPONENTS 150

Reinhard BurgerIntroduction 150Production of Monoclonal Antibodies to Complement

Components: Methodological Aspects 151Immunization Protocol 151Antibody Assays 152

Monoclonal Antibodies to Complement Components of theClassical Pathway 152

Antibodies to Cl 152Ab 48.3 to Purified Clq 152Antibodies to Clq Bound to Immune Complexes and to

Neoantigenic Determinants of Clq 153Ab 146 Recognizing Membrane-Associated Clq with Fc

Receptor Function 154Antibodies to C4 and C2 154Antibodies to C3 155

Antibody WM1 to C3c 155Antibodies 3 , 4 and 9 to C3c, or C3g 155Antibodies to C3d 156Antibodies Against Functionally Relevant Sites of C3 . . . . 156

Antibodies to the Terminal Components C5, C6, C7, C8and C9 158

Monoclonal Antibodies to Components of the AlternativePathway 159

Antibodies to Factor B 159Antibodies to Factor D 160

Monoclonal Antibodies to the Serum Regulatory Proteins 160An Antibody to I (C3b INA) 160Antibodies to H '161

Naturally Occurring Monoclonal Antibodies to ComplementComponents: Nephritic Factor 161

Commercially Available Monoclonal Antibodies to ComplementComponents 162

Conclusion: Further Applications of Monoclonal Antibodiesto Complement 162

References 163

xvi Contents and Subject Index

11. MONOCLONAL ANTIBODIES AND COAGULATION 166Kenneth G. Mann, Jerry A. Katzmann, W. Barry Foster and

David N. PassIntroduction 166Methods of Monoclonal Antibody Preparation and Assay 172Quantitation of Monoclonal Antibody Binding 180Isolation of Coagulation Proteins and Preparation of Immuno-

depleted Plasma Using Monoclonal Antibodies 182#, Structural Analysis with Monoclonal Antibodies 185

von Willebrand Factor and Platelet Adhesion 189Anti-Platelet Monoclonal Antibodies 190Summary 191References 191

12. MONOCLONAL ANTIBODIES AND HUMAN CHLORIONICGONADOTROPIN: MAPPING OF EPITOPES AND RECEPTOR-INTERACTION SITES 195

Siegfried Schwarz, Peter Berger, Reinhard Kofler and Georg WickIntroduction 195Production and Characterization of Monoclonal Antibodies to

hCG and bLH 198Development of a Holo-hCG Specific Immuno Radio Metric

Assay (IRMA) 199Choice of MoAB 199IRMA-Protocol 199Validation of the 22-13*-IRMA 200

Mapping of hCG Epitopes 203Analysis of Results 205

Relationship of Receptor-Interaction Sites and ImmunologicalEpitopes of hCG 208

Measurement of Different Forms of hCG by Epitope-SelectiveIRMAs 210

Concluding Remarks and Perspectives 214References 216

Books 216Journals 216

13. MONOCLONAL ANTIBODIES AND VIRUSES: APPLICATION TOTHE EPSTEIN-BARR SYSTEM 221

Nikolaus Mueller-LantzschIntroduction 221Characterization of Late EBV Proteins by Monoclonal

Antibodies 222Procedure for Antibody Production 222Monoclonal Antibodies Against EBV Envelope Proteins 223Immunological Differences of EBV Strains Analyzed by

Monoclonal Antibodies 225

Contents and Subject Index xvii

EBV Specific Membrane Antigen (MA) Immunofluorescenceby Monoclonal Antibodies 226

Neutralization Potential of EBV Specific Monoclonal Anti-bodies 227

Monoclonal Antibody Specific for Capsid Antigen of EBV . . . 228Identification of Polypeptide Components of the EBV Early

Antigen Complex with Monoclonal Antibodies 229Monoclonal Antibodies to EBV-Induced Transformation-

Associated Cell Surface-Antigen •. 229Conclusions 230References 231

14. MONOCLONAL ANTIBODIES AND HERPES SIMPLEX VIRUSINFECTIONS 235

Joseph C. Glorioso and Myron LevineIntroduction 235Production of Anti-HSV Monoclonal Antibodies and Studies

of HSV Infection 237The Antigenic Structure of HSV Glycoproteins 238

Epitopes Defined by Synthetic Oligopeptides 238Analysis of Antigenic Sites Using Monoclonal Antibodies

and Antibody Selected Variants 239Monoclonal Antibody Selection of.Glycoprotein Deficient

Mutants and the Definition of Antigenic Domains 242Monoclonal Antibodies and Diagnosis of HSV Infections . . . . 244

Serotyping of HSV Isolates 245Detection of HSV Antigens in Herpetic Lesions 245Detection of HSV Antibodies in Body Fluids 246

Monoclonal Antibodies in Immune Protection 248Monoclonal Antibodies and the Molecular Biology of HSV

Infection 250Future Prospects and Directions 251References .251

15. MONOCLONAL ANTIBODIES AND BACTERIA 261Thomas F. Schulz and Manfred P. Dierich

Introduction 261Use of Monoclonal Antibodies in Investigating Aspects of Host-

Parasite Relationship 262Introduction 262Infections Caused by Gram-Negative Organisms 262Infections Caused by Gram-Positive Organisms 265

Use of Monoclonal Antibodies to Investigate the SurfaceStructure of Bacteria 268

Attempts to Define Molecules Responsible for the Adhesionof Bacteria to Animal Cells 268

Attempts to Define Surface Structures Determining theAntigenic Phenotype of Bacteria 269

xviii Contents and Subject Index

Attempts to Investigate the Structure-Function Relationshipof Isolated Membrane Molecules 276

Monoclonal Antibodies to Bacterial Toxin 276Use of Monoclonal Antibodies in the Diagnosis of Infectious

Diseases 278Some Methods Employed in the Production of Monoclonal

Antibody to Bacteria ; 282Immunization Schedules 282Screening for Antibody Production of Hybridomas 283

f References 283

16. MONOCLONAL ANTIBODIES IN DIAGNOSTIC PATHOLOGY 293Salim E. Kabawat, Frederic I. Preffer and Atul K. Bhan

Introduction 293Staining Methods 294Characterization of Lymphocyte Subclasses 297

Analysis of Cell Mediated Reactions 301Characterization of Malignant Lymphomas 302

Monoclonal Antibodies in Tumor Diagnosis 304Differentiation Antigens 304

Hormones as Endocrine Markers 304Blood Group Antigens 305Major Histocompatibility Complex Antigens 305Antibodies to Intermediate Filaments 306Antibodies to Other Cellular Structural Proteins 310

Oncofetal and Oncoplacental Antigens 310Carcinoembryonic Antigen 310Alpha-Fetoprotein 313Human Chorionic Gonadotropin 313Other Oncoplacental Antigens 313

Tumor Associated Antigens 313Summary •. 317References 317

17. MONOCLONAL ANTIBODIES AND DETECTION OFMALIGNANCIES 329

Duane R. SchultzIntroduction 329Oncofetal Antigens 330

Alpha-Fetoprotein 330Carcinoembryonic Antigen 332Pancreatic Tumor-Associated Antigens 334

Colorectal Carcinoma-Specific Antigens 338Tumor Imaging with Monoclonal Antibodies 339Potential Problems Associated with Monoclonal Antibodies 340References 341

Contents and Subject Index xix

18. MONOCLONAL ANTIBODIES AND MELANOMAS 347Judith P. Johnson and Gert Riethmiiller

Introduction 347Melanoma Surface Antigens Defined by Monoclonal Anti-

bodies: The Evolution from Melanoma Specific toMelanoma Associated 348

la Like Antigens . 348p97 350Melanoma Associated Proteoglycan-Glycoprotein 351GD3 Ganglioside 351

••' Melanoma vs. Nevus and Melanocyte: Asking the RealQuestion 351

Looking for Tumor-Specific Markers with MonoclonalAntibodies: Asking the Right Question 353

References 354

19. MONOCLONAL ANTIBODIES AND CHARACTERIZATION OFHUMAN LYMPHOPROLIFERATIVE MALIGNANCIES 360

Daniel M. Knowles IIIntroduction 360T Lymphocyte Markers " 362OKT Monoclonal Antibodies 363Monoclonal Antibodies in the Phenotypic Characterization ofT Cell Malignancies 365

Monoclonal Antibody Leu 1/OKT1 367B Lymphocyte Markers 368Monoclonal Antibodies BA-1 and BA-2 369Monoclonal Antibodies Bl , B2, and B4 369OKB Monoclonal Antibodies 370Monoclonal Antibodies BL1, BL2, BL3 371Monoclonal Antibodies Pro-Iml and Pro-Im2 373Monoclonal Antibodies in the Phenotypic Characterization of

B Cell Malignancies 373Conclusion 376References 376

20. MONOCLONAL ANTIBODIES AND HUMAN HISTO-COMPATIBILITY ANTIGENS 382

Piergiorgio Natali, Aldo Bigotti, Carlo Russo, Kohsaku Sakaguchi,Muneo Igarashi and Soldano Ferrone

Monoclonal Antibodies to HLA Allospecificities Defined byConventional Alloantisera 385

Use of Monoclonal Antibodies in the Lymphocytotoxic Assayfor HLA Typing 388

New Information Derived from the Application of Anti HLAMonoclonal Antibodies 389

Conclusion 398References 398

xx Contents and Subject Index

21. MONOCLONAL ANTIBODIES IN THE THERAPY OF RENALTRANSPLANT REJECTION 404

Charles B. CarpenterT Cell Differentiation Antigens 405Selection of a Monoclonal Antibody for In Vivo Use 407Testing of Anti-Human Monoclonal Antibodies in Primates 409Reversal of Acute Renal AHograft Rejection in Man 410

OKT3 410Anti-T12 411

f CBL1 413Immunogenicity of Mouse Monoclonal Antibodies 414Treatment of Acute Graft-Versus-Host Disease (GVHD) 414Conclusions 415References 416

22. MONOCLONAL ANTIBODIES AS THERAPEUTIC TOOLS INMEDICINE 419

Jean-Francois Bach and Lucienne ChatenoudIntroduction 419Specific Problems of Monoclonal Antibody Production for

Clinical Use 420Experimental and Human Therapeutic Use of Monoclonal

Antibodies 421Nonspecific Immunosuppression (Antilymphocyte

Antibodies) 421Specific Immunosuppression in Autoimmune Diseases 423Immunotherapy of Leukemia and Cancer 424Serotherapy of Infectious and Parasitic Diseases 426Cure of Intoxication (Antidrug Antibodies) 427

Mechanisms of In Vivo Action of Monoclonal Antibodies 427Escape Mechanisms from the Therapeutic Effect of Monoclonal

Antibodies and Their Prevention 428Antigenic Modulation 428Xenosensitization 429Immunotoxins 431

Conclusions and Prospectives 432References 432

23. MONOCLONAL ANTIBODIES IN THE THERAPY OF MALIGNANTDISEASES 436

Robert O. Dill man and Ivor RoystonIntroduction 436

Introductory Comments 436Advantages of Monoclonal Antibodies over Antisera 436

Monoclonal Antibodies as Passive Therapy 437Passive Cytotoxic Therapy 437Heterologous Antisera Trials 438Monoclonal Antibody Therapy in Animal Models 440

Contents and Subject Index xxi

Monoclonal Antibodies as Passive Cytotoxic Therapy 441Review of Trials 441Binding to Target Cells 443Biological and Clinical Effects 443Toxicity and Side Effects 444Bioavailability and Pharmacokinetics 446Free Antigen 446Antigenic Modulation/Immunoselection 447Endogenous Anti-Mouse Antibody 447

Monoclonal Antibodies as Regulatory Therapy 447General Considerations 447Anti-Idiotype Monoclonal Antibodies 448Anti-Transferrin Receptor Therapy 450Conclusions Regarding Passive MoAB Therapy 450

Monoclonal Antibody Conjugates as Active Therapy 451Radioisotope Conjugates 451

General Considerations 451Isotope-Antisera Conjugate Experience 452Radioimmunodetection and Radioimmunotherapy with

Monoclonal Antibodies in Animal Models 453Radioimmunoscintigraphy in Humans with Monoclonal

Antibody Conjugates 453Other Considerations 455Conclusions 456

Chemotherapy Conjugates 456General Considerations 456Therapy in Animal Models 456Therapy in Man 457Conclusions 457

Immunotoxin Conjugates 457General Considerations 457Immunotoxins In Vitro 458Immunotoxins in Animal Models 458Conclusions Regarding Immunotoxins 459

Monoclonal Antibodies in Bone Marrow Transplantation 459General Considerations 459Allogeneic Marrow Transplantation 459

In Vivo Anti-GVHD 459In Vitro Anti-GVHD 460

Autologous Marrow Transplantation 460Concluding Remarks 461References 462

APPENDIX: LIST OF COMMERCIALLY AVAILABLE MONOCLONALANTIBODIES 473