Enzyme-Prodrug Strategies for Cancer Therapy
Enzyme-Prodrug Strategies for Cancer Therapy Edited by Roger G. Melton Enzacta Ltd. Porton Down Wiltshire, United Kingdom and Richard J. Knox Imperial College School of Medicine Charing Cross Campus London, United Kingdom SPRINGER SCIENCE+BUSINESS MEDIA, LLC
Llbrary of Congress Cataloglng-ln-Publlcatlon Data Enzyme-prodrug strateg ies for cancer therapy / edited by Roger G. Melton and Rfchard J. Knox. p. cm. Includes bfbliographfcal references and index. ISBN 978-1-4613-7186-1 ISBN 978-1-4615-4823-2 (ebook) DOI 10.1007/978-1-4615-4823-2 1. Antibody-directed enzyme prodrug therapy. 2. Cancer -Chemotherapy. 3. Prodrugs. 4. Antfbody-enzy.e conjugates- -Therapeutic use. 1. Melton, Roger G. II. Knox, Richard J. [DNLM, 1. Antfneoplastfc Agents--therapeutfc use. 2. Prodrugs -therapeutfc use. 3. Neoplasms--drug therapy. 4. Antfbodies- -therapeutic use. 5. Enzyoes--therapeutfc use. 6. Antigens--drug effects. ez 267 E6l 19981 RC271.A66E56 1998 616.99'406l--dc21 DNLM/DLC for Library of Congress 98-37682 CIP ISBN 978-1-4613-7186-1 1999 Springer Science+Business Media New York Origina11y published by Kluwer Academic / Plenum Publishers, New York in 1999 Softcover reprint ofthe hardcover Ist edition 1999 10987654321 A C.I.P. record for this book is available from the Library ofcongress. Ali rights reserved No part ofthis 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
Contributors K. D. Bagshawe Charing Cross & Westminster Medical School, London W6 8RP, United Kingdom P. 1. Burke Department of Medical Oncology, Charing Cross Hospital, London W6 8RF, United Kingdom K. A. Chester Department of Clinical Oncology, Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom Tom Connors Centre for Polymer Therapeutics, The School of Pharmacy, London WCIN lax, United Kingdom R. E. Hawkins Department of Oncology, MRC Centre, Cambridge CB2 2HQ, United Kingdom Richard 1. Knox Department of Medical Oncology, Charing Cross Hospital, London W6 8RP, United Kingdom Roger G. Melton Enzacta Ltd., Salisbury SP4 OIQ, United Kingdom Mark Napier The Meyerstein Institute of Oncology, The Middlesex Hospital, London WIN 8AA, United Kingdom M. A. Sims Centre for Applied Microbiology & Research, Salisbury, SP4 OJG, United Kingdom v
Preface Since the concept was first described in 1987, the use of antibody-enzyme conjugates directed at tumor-associated antigens to achieve site-specific activation of prodrugs to potent cytotoxic species, termed antibody-directed enzyme prodrug therapy (ADEPT), has attracted considerable interest. ADEPT attempts to overcome a major problem associated with the administration of cytotoxic drugs for treating cancer-the intrinsic lack of tumor selectivity. Thus, chemotherapeutic agents have the potency to kill tumor cells in vitro but, because of host toxicity, the treatment of human patients has to be discontinued at dose levels well below those which are expected to kill all of their tumor cells. In ADEPT a cytotoxic agent is generated selectively at the site of a tumor by an antibody-targeted enzyme. The antibody delivery combined with the amplification provided by the enzymatic activation of prodrugs enables adequate selection to be made between tumor and normal tissue. A rich diversity of potential enzyme and prodrug combinations exists for use in ADEPT applications, and ADEPT offers new research ideas for the chemist, enzymologist, molecular biologist, protein engineer, and the cancer chemotherapist. It offers opportunities for the therapy of systemic cancer and may be a major advance for treating solid tumors. ADEPT has shown very encouraging results in animal models and in a preliminary clinical trial. The results of more extensive trials are awaited with interest, and ADEPT systems may soon form an important element in the clinical oncologist's armament. In compiling this book we have been fortunate to have authors participate who have been involved with ADEPT since it was conceived, and we are grateful for their expert contributions. We hope this book will strengthen the understanding of this field of research and perhaps stimulate some new ideas in the area. ROGER MELTON RICHARD KNox vii
Contents Chapter 1 Introduction Richard 1. Knox and Roger G. Melton 1. Introduction... 1 1.1. The Activity of the Activated Drug... 4 1.2. Activation of the Prodrug by Unbound, Circulating Conjugate 5 1.3. Immunogenicity of the Antibody~Enzyme Conjugate... 5 References...... 7 Chapter 2 Prodrugs in Cancer Chemotherapy Tom Connors 1. Introduction... 11 2. Uses of Prodrugs in Cancer Chemotherapy... 11 2.1. Optimizing Pharmacokinetics and Tissue Distribution......... 11 2.2. Prodrugs Activated by Tumor Enzymes.................... 19 2.3. Bioreductive Prodrugs... 27 3. Clinical Use of Prodrugs... 33 References... 33 ix
x Contents Chapter 3 Factors Influencing Thmor-Selective Localization of Antibody Conjugates M. A. Sims and Roger G. Melton 1. Introduction... 39 2. Tumor Physiology... 40 2.1. Relevance of Murine Tumor Models... 41 2.2. Route of Administration... 42 2.3. Vascularization of Tumors............................... 43 2.4. Vascular Permeability of Tumors... 45 2.5. Movement of Macromolecules within Tumors............... 46 2.6. Effect of Tumor Size and Necrosis........................ 46 3. Properties of the Monoclonal Antibody Vector... 47 3.1. Affinity for Antigen.................................... 47 3.2. Antibody Class and Isotype.............................. 48 3.3. Physical Properties of Immunoconjugates... 48 3.4. Metabolism of Tumor-Bound Immunoconjugates... 49 3.5. Clinical Findings with ADEPT Systems.................... 51 3.6. Manipulation of Tumor Physiology... 52 3.7. Vascular Targeting... 55 4. Tumor Antigens... 56 4.1. Heterogeneity of Antigenic Expression... 56 4.2. Antigenic Modulation... 58 4.3. Membrane Expression and Circulating Antigen.............. 58 4.4. Antigens Potentially Useful for ADEPT Approaches... 60 5. Manipulation of Tumor Antigenic Expression... 76 6. Evaluation of Antigenic Targets for ADEPT... 78 7. In Vitro Cell Culture Problems... 79 8. Unforeseen Problems of Specificity... 79 References... 80 Chapter 4 Enzymes and Prodrugs Used for ADEPT Richard 1. Knox 1. Introduction... 97 2. Enzymes and Prodrugs Used for ADEPT....................... 98 2.1. Glucose Oxidase and Xanthine Oxidase.................... 99 2.2. Carboxypeptidase G2................................... 100
Contents xi 2.3. Carboxypeptidase A and B... 103 2.4. Aminopeptidase... 105 2.5. Alkaline Phosphatase... 106 2.6. I3-Glucuronidase... 107 2.7. I3-Glucosidase... 110 2.8. I3-Lactamase... 111 2.9. Penicillin Amidase... 113 2.10. Cytosine Dearninase... 115 2.11. Nitroreductases... 116 3. Conclusions... 122 References... 126 Chapter 5 The Design and Synthesis of Prodrugs for Antibody-Directed Enzyme Prodrug Therapy (ADEPT) p. 1. Burke 1. Introduction... 133 2. Prodrugs Used in ADEPT... 134 2.1. Prodrugs for Carboxypeptidases........................... 134 2.2. Prodrugs for Alkaline Phosphatase... 138 2.3. Prodrugs for Glycosidases... 139 2.4. Prodrugs for I3-Lactamases... 143 2.5. Prodrugs for Penicillin V/G Amidase... 147 2.6. Prodrugs for Nitroreductase... 148 2.7. Prodrugs for Cytosine Deaminase... 150 3. Surnrnary... 151 References................................................. 151 Chapter 6 Preparation and Purification of Antibody-Enzyme Conjugates for Therapeutic Applications Roger G. Melton 1. Introduction................................................ 155 2. Production of Antibody Fragments... 156 3. Chemical Coupling of Antibodies to Enzymes... 159 3.1. Choice of Linkage... 159 3.2. Insertion of Maleimide Groups into Proteins... 160
xii Contents 3.3. Thiolation of Proteins... 162 3.4. Coupling Conditions... 164 3.5. Site-Specific Coupling of Proteins... 165 4. Alternatives to Chemical Coupling... 167 4.1. Bispecific Antibodies... 168 4.2. Fusion Proteins... 168 5. Purification of Conjugates... 169 6. Postpurification Modifications... 172 7. Future Developments... 172 References............................................. 173 Chapter 7 Phage Technology for Producing Antibody-Enzyme Fusion Proteins K. A. Chester; Roger G. Melton. and R. E. Hawkins 1. Introduction................................................ 179 2. Antibody-Targeted Cancer Therapy... 180 3. Potential of Engineered Antibodies for Therapy... 180 4. Single-chain Fv Antibodies... 182 4.1. Tumor Penetration and Biodistribution... 182 4.2. Avidity of Binding... 183 5. The Phage System... 185 5.1. Immunogenicity... 186 5.2. Affinity... 188 5.3. Diversity... 188 6. Target Antigens... 189 7. Engineered Antibody-Enzyme Fusion Proteins in ADEPT... 190 8. Practical Considerations... 192 9. Conclusions................................................ 193 References............................................... 193 Chapter 8 Early Clinical Studies with ADEPT K. D. Bagshawe and M. Napier 1. Introduction................................................ 199 2. Initial Clinical Studies... 200 2.1. Study Design........................................... 200 2.2. Results... 201
Contents xiii 3. Second Clinical Study... 202 3.1. Study Design... 202 3.2. Results... 203 4. Conclusions................................................ 206 References............................................. 207 Chapter 9 Gene-Directed Enzyme Prodrug Therapy (GDEPT) of Cancer Richard J. Knox 1. Introduction................................................ 209 2. Why GDEPT?... 212 3. Enzymes and Prodrugs Used for GDEPT... 213 3.1. Varicella-Zoster Virus Thymidine Kinase... 213 3.2. Herpes Simplex Virus Thymidine Kinase... 216 3.3. Cytosine Deaminase... 221 3.4. Cytochrome P450... 223 3.5. Carboxypeptidase G2... 226 3.6. Nitroreductase... 229 3.7. Other Enzymes... 234 4. Conclusions................................................ 236 References................................................. 238 Appendix Enzymes and Prodrugs Proposed for Cancer Therapy... 245 References................................................ 249 Index... 255