Emerging Cancer Therapy E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Serie: Wiley Series on Biotechnology
• Sprache: Englisch

Seitenzahl: 832

Veröffentlichungsjahr: 2010

Beschreibung

Explores current and emerging applications of microbes as cancer-fighting agents WILEY SERIES IN BIOTECHNOLOGY AND BIOENGINEERING Anurag S. Rathore, Series Editor Today, treatment options for cancer patients typically include surgery, radiation therapy, immunotherapy, and chemotherapy. While these therapies have saved lives and reduced pain and suffering, cancer still takes millions of lives every year around the world. In recent years, researchers have been working on a new strategy: developing microbes and microbial products that specifically attack cancer cells. This book breaks new ground in emerging cancer treatment modalities by presenting recent advances in the use of microorganisms and viruses as well as their products in cancer therapy. Seventeen chapters review the application of live microorganisms, high and low molecular weight products derived from microorganisms, and microbial products fused to cancertargeting molecules. In addition, the book highlights the benefits of a multi-target approach to destroy cancer cells. Readers will not only discover the results and significance of basic and clinical research, but also encouraging results from clinical trials. Emerging Cancer Therapy is divided into three sections: * Section 1: Live/Attenuated Bacteria and Viruses as Anticancer Agents * Section 2: Bacterial Products as Anticancer Agents * Section 3: Patents on Bacteria/Bacterial Products as Anticancer Agents With chapters written by leading pioneers in microbial, biotech, and cancer research, Emerging Cancer Therapy is recommended for biotechnologists, microbiologists, clinical oncologists, medicinal chemists, and biochemists. Readers will not only learn the tremendous potential of microbial and biotechnological approaches to cancer therapy, but also discover new directions of research for effective drug discovery and development.

Leseprobe

Table of Contents

Cover

Table of Contents

Halftitle page

Series page

Title page

Copyright page

PREFACE

CONTRIBUTORS

Part I: LIVE/ATTENUATED BACTERIA AND VIRUSES AS ANTICANCER AGENTS

1 SALMONELLA TYPHIMURIUM MUTANTS SELECTED TO GROW ONLY IN TUMORS TO ERADICATE THEM IN NUDE MOUSE MODELS

INTRODUCTION

CONCLUSION

2 THE USE OF LIVING LISTERIA MONOCYTOGENES AS AN ACTIVE IMMUNOTHERAPY FOR THE TREATMENT OF CANCER

INTRODUCTION

LISTERIAL DISEASE

ADAPTIVE IMMUNITY

NONCLASSICAL IMMUNE FUNCTIONS

RECOMBINANT L. MONOCYTOGENES AS A VACCINE VECTOR

CROSS-PRESENTATION AND EPITOPE SPREADING

LISTERIA LLO-AG FUSIONS AND THE TUMOR MICROENVIRONMENT

CONCLUSIONS

3 BACILLUS CALMETTE–GUERIN (BCG) FOR UROTHELIAL CARCINOMA OF THE BLADDER

THE BURDEN OF BLADDER CANCER AND BACILLUS CALMETTE–GUERIN (BCG) IMMUNOTHERAPY

DIAGNOSIS OF BLADDER CANCER

STAGING OF BLADDER CANCER

TREATMENT OF UROTHELIAL CARCINOMA

HISTORY OF M. BOVIS BCG

GENETIC VARIABILITY OF BCG

CLINICAL USE OF BCG

BCG SIDE EFFECTS

BCG MECHANISM OF ACTION

NEUTROPHILS, TRAIL, AND BCG

GENETIC DETERMINANTS OF THE BCG RESPONSE

GENETIC MODIFICATION OF BCG

CELLULAR COMPONENT THERAPY

COMBINATION WITH CYTOKINES

CONCLUSION

4 LIVE CLOSTRIDIA: A POWERFUL TOOL IN TUMOR BIOTHERAPY

THE GENUS CLOSTRIDIUM IN A NUTSHELL

THE TUMOR MICROENVIRONMENT: RESISTANT TO CONVENTIONAL TUMOR THERAPEUTICS BUT A HAVEN FOR CLOSTRIDIA

THE FIRST ERA OF TUMOR BIOTHERAPY USING CLOSTRIDIUM SPORES

RECOMBINANT CLOSTRIDIA AS TOOL IN TUMOR BIOTHERAPY

C. NOVYI-NT: THE REVIVAL OF NATIVE ONCOLYTIC CLOSTRIDIA FOR BIOTHERAPY PURPOSES

MODULATION OF C. PERFRINGENS TO AN ONCOTHERAPEUTIC MICROBE

CDAT: CLOSTRIDIUM-DIRECTED ANTIBODY THERAPY

ADVANTAGES OF CLOSTRIDIAL SPORES AS ANTITUMOR TOOLS

FURTHER IMPROVEMENTS AND FUTURE PERSPECTIVES

5 BIFIDOBACTERIUM AS A DELIVERY SYSTEM OF FUNCTIONAL GENES FOR CANCER GENE THERAPY

EXPRESSION PLASMIDS IN BIFIDOBACTERIUM AS A DELIVERY SYSTEM OF FUNCTIONAL GENES

BIFIDOBACTERIUM AS AN ORAL DELIVERY SYSTEM OF FUNCTIONAL GENES FOR CANCER GENE THERAPY

BIFIDOBACTERIUM IS A GOOD VECTOR FOR ORAL CANCER GENE THERAPY

BIFIDOBACTERIUM AS A DELIVERY SYSTEM OF FUNCTIONAL GENES FOR CANCER GENE THERAPY AND ITS APPLICATION

BIFIDOBACTERIUM COMBINATION WITH OTHER FACTORS

PROSPECT

6 REPLICATION-SELECTIVE VIRUSES FOR THE TREATMENT OF CANCER

INTRODUCTION

ENGINEERING TUMOR SELECTIVITY INTO VIRUSES

FURTHER ENHANCING ONCOLYTIC VIRUS THERAPIES

SUMMARY

7 ENGINEERING HERPES SIMPLEX VIRUS FOR CANCER ONCOLYTIC VIROTHERAPY

INTRODUCTION

ONCOLYTIC VIRUSES

TARGETING HSV TO CANCER CELLS

COMBINATION THERAPY INVOLVING THE ONCOLYTIC HSV VIRUS

CLINICAL TRIALS WITH ONCOLYTIC HSV

CURRENT LIMITATIONS OF ONCOLYTIC VIROTHERAPY

CONCLUSIONS AND FUTURE PERSPECTIVES

ACKNOWLEDGMENTS

Part II: BACTERIAL PRODUCTS AS ANTICANCER AGENTS

8 PROMISCUOUS ANTICANCER DRUGS FROM PATHOGENIC BACTERIA: RATIONAL VERSUS INTELLIGENT DRUG DESIGN

INTRODUCTION

PROMISCUOUS DRUGS TARGETING MULTIPLE STEPS IN CANCER GROWTH PROGRESSION

ANTICANCER ACTIVITY OF Pa-CASPASE RECRUITMENT DOMAIN AND Pa-ARGININE DEIMINASE

9 ARGININE DEIMINASE AND CANCER THERAPY

INTRODUCTION

CLINICAL STUDIES

MECHANISMS OF ACTION

ASS EXPRESSION

FUTURE DIRECTIONS

10 CYTOSINE DEAMINASE/ 5-FLUOROCYTOSINE MOLECULAR CANCER CHEMOTHERAPY

GENE-DIRECTED ENZYME/PRODRUG CANCER THERAPY

CD-MEDIATED GENE-DIRECTED ENZYME/PRODRUG CANCER THERAPY

GENE DELIVERY SYSTEM

BYSTANDER ACTIVITY OF CD/5-FC THERAPY

THE MOLECULAR MECHANISMS OF RESISTANCE TO 5-FU

COMBINATION OF CD AND UPRT INCREASES EFFECT OF GENE-DIRECTED ENZYME/PRODRUG CANCER THERAPY

MUTATION OF CD SIGNIFICANTLY ENHANCES MOLECULAR CHEMOTHERAPY

yCD

COMBINATION OF CD/5-FC AND HSV-TK/GCV THERAPY

COMBINATION OF CD/5-FC-MEDIATED MOLECULAR CHEMOTHERAPY AND RADIATION THERAPY

CD/5-FC MOLECULAR CHEMOTHERAPY IN CLINICAL TRIALS

CONCLUSION

11 BACTERIAL PROTEINS AGAINST METASTASIS

INTRODUCTION

CHEMOKINES AND THEIR RECEPTORS

GPCR STRUCTURE AND LIGAND BINDING

GPCR ACTIVATION BY CHEMOKINES

GPCR SIGNALING

GPCRS AND CANCER

CHEMOKINE RECEPTOR 4 (CXCR4)

CHEMOKINE RECEPTOR 3 (CXCR3)

CHEMOKINE RECEPTOR 5 (CCR5)

CHEMOKINE RECEPTOR 7 (CCR7)

CHEMOKINE RECEPTOR 1/2 (CXCR1/2)

BACTERIAL GPCR INHIBITORS

BACTERIAL INHIBITOR SSL10 AGAINST CANCER-RELATED GPCR CXCR4

PSGL-1

BACTERIAL PSGL-1 INHIBITOR: SSL5

12 PSEUDOMONAS EXOTOXIN A- BASED IMMUNOTOXINS FOR TARGETED CANCER THERAPY

INTRODUCTION

PSEUDOMONAS AERUGINOSA

PSEUDOMONAS EXOTOXIN A

PSEUDOMONAS EXOTOXIN A-BASED IMMUNOTOXINS

CONCLUSIONS

13 DENILEUKIN DIFTITOX IN NOVEL CANCER THERAPY

DENILEUKIN DIFTITOX: STRUCTURE AND BIOLOGY

MECHANISM OF ACTION

ACTIVITY IN T CELL LYMPHOID MALIGNANCIES AND T CELL-MEDIATED PHENOMENA

OTHER DISEASES

CONCLUSION

14 THE APPLICATION OF CATIONIC ANTIMICROBIAL PEPTIDES IN CANCER TREATMENT: LABORATORY INVESTIGATIONS AND CLINICAL POTENTIAL

INTRODUCTION

MOLECULAR BASIS FOR CANCER CELL TARGETING BY CAPs

MECHANISMS OF CANCER CELL KILLING BY CAPs

MODULATION OF IMMUNE FUNCTION BY CAPs

PRECLINICAL AND CLINICAL INVESTIGATIONS

ENHANCING ANTICANCER ACTIVITY AND CAP STABILITY THROUGH PEPTIDE MODIFICATION

CONCLUSION

15 PRODIGININES AND THEIR POTENTIAL UTILITY AS PROAPOPTOTIC ANTICANCER AGENTS

INTRODUCTION

HISTORY OF PRODIGININES

BIOSYNTHESIS OF PRODIGININES

MUTASYNTHESIS TO GENERATE NOVEL PRODIGININES

THE BIOACTIVE PRODIGININES DISPLAY ANTICANCER ACTIVITY

STRUCTURE–ACTIVITY RELATIONSHIP STUDIES OF BACTERIAL PRODIGININES

PRODIGININES MEDIATE COPPER-DEPENDENT DNA CLEAVAGE

PRODIGININES AS H+/CL− SYMPORTERS AND A ROLE IN INTRACELLULAR ACIDIFICATION

SIGNAL TRANSDUCTION AND MAPKS

CELL CYCLE INHIBITION

CASE STUDY

SUMMARY

16 FARNESYLTRANSFERASE INHIBITORS OF MICROBIAL ORIGINS IN CANCER THERAPY

RAS AND FARNESYLTRANSFERASE INHIBITORS (FTIS)

FTIS OF MICROBIAL ORIGINS

ANTITUMOR ACTIVITY OF FTIS OF MICROBIAL ORIGINS

TARGETS OF FTIS

FUTURE PROSPECTS

ACKNOWLEDGMENTS

17 THE USE OF RNA AND CpG DNA AS NUCLEIC ACID-BASED THERAPEUTICS

TOLL-LIKE RECEPTOR 9 AND CpG DNA SEQUENCE RECOGNITION

TLR7/8 AND RNA SEQUENCE RECOGNITION

HOMEOSTASIS OF TLR RESPONSES: HOW TO DISTINGUISH FOREIGN FROM SELF

TLRs 7, 8, AND 9 SINGLE NUCLEOTIDE POLYMORPHISMS AND SUSCEPTIBILITY TO ATOPIES, INFECTIOUS DISEASES, OR SYSTEMIC LUPUS ERYTHEMATOSUS

CpG DNA MOTIFS AND BEYOND

CELL SURFACE AND CYTOPLASMIC RNA RECEPTORS: TLR3 AND RETINOIC ACID-INDUCIBLE PROTEIN-I-LIKE RECEPTORS

FROM PRECLINICAL STUDIES TO CLINICAL APPLICATION

ACKNOWLEDGMENT

Part III: PATENTS ON BACTERIA/BACTERIAL PRODUCTS AS ANTICANCER AGENTS

18 THE ROLE AND IMPORTANCE OF INTELLECTUAL PROPERTY GENERATION AND PROTECTION IN DRUG DEVELOPMENT

INTRODUCTION

U.S. AND EUROPEAN PATENT LAWS HAVE SIMILARITIES AND DIFFERENCES

EXTENDING THE REACH OF GRANTED PATENTS

PATENTING HUMAN GENES AND PARTS THEREOF

OBVIOUSNESS IN THE ISSUANCE OF PATENTS

INHERENT ANTICIPATION

ARE ALL PATENTS NECESSARILY ENFORCED?

COPYING OR MAKING INCONSEQUENTIAL CHANGES IN A PATENT—THE DOCTRINE OF EQUIVALENTS

WRITTEN DESCRIPTION AND ENABLEMENT

CONCLUDING REMARKS

Index

EMERGING CANCER THERAPY

Wiley Series in

BIOTECHNOLOGY AND BIOENGINEERING

Significant advancements in the fields of biology, chemistry, and related disciplines have led to a barrage of major accomplishments in the field of biotechnology. The Wiley Series in Biotechnology and Bioengineering will focus on showcasing these advances in the form of timely, cutting-edge textbooks and reference books that provide a thorough treatment of each respective topic.

Topics of interest to this series include, but are not limited to, protein expression and processing; nanotechnology; molecular engineering and computational biology; environmental sciences; food biotechnology, genomics, proteomics, and metabolomics; large-scale manufacturing and commercialization of human therapeutics; biomaterials and biosensors; and regenerative medicine. We expect these publications to be of significant interest to practitioners both in academia and industry. Authors and editors were carefully selected for their recognized expertise and their contributions to the various and far-reaching fields of biotechnology.

The upcoming volumes will attest to the importance and quality of books in this series. I would like to acknowledge the fellow co-editors and authors of these books for their agreement to participate in this endeavor. Lastly. I would like to thank Ms. Anita Lekhwani, Senior Acquisitions Editor at John Wiley & Sons, Inc. for approaching me to develop such a series. Together, we are confident that these books will be useful additions to the literature that will not only serve the biotechnology community with sound scientific knowledge, but also with inspiration as they further chart the course in this exciting field.

Anurag S. Rathore

Amgen Inc.

Thousand Oaks, CA, USA

Titles in series

Quality by Design for Biopharmaceuticals: Principles and Case Studies / Edited by Anurag S. Rathore and Rohin Mhatre

Emerging Cancer Therapy: Microbial Approaches and Biotechnological Tools / Edited by Arsenio Fialho and Ananda Chakrabarty

Copyright © 2010 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, [phone], fax [phone], or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, [phone], fax [phone], or online at http://www.wiley.com/go/permission.

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Library of Congress Cataloging-in-Publication Data:

Emerging cancer therapy : microbial approaches and biotechnological tools / edited by Arsenio Fialho and Ananda Chakrabarty.

p. ; cm.

 Includes bibliographical references and index.

 ISBN 978-0-470-44467-2 (cloth); ISBN 978-1-118-03518-4 (ebk)

 1. Cancer–Treatment. 2. Viruses–Therapeutic use. 3. Bacteria–Therapeutic use. 4. Microbial biotechnology. I. Fialho, Arsenio. II. Chakrabarty, Ananda M., 1938–

 [DNLM: 1. Neoplasms–therapy. 2. Antineoplastic Agents. 3. Bacteria–immunology. 4. Biotechnology. QZ 266 E53 2010]

 RC271.V567E64 2010

 616.99′406–dc22

2009051007

PREFACE

The major approaches to cancer therapy today are surgery, radiation therapy, immunotherapy, and chemotherapy including the use of antimetabolites, rationally designed or randomly screened small molecule drugs, monoclonal antibodies, and combination thereof. While these therapies have contributed significantly to the savings of millions of lives and reduced pain and suffering, cancers still take a major toll in our lives as a deadly, mostly incurable, disease.

It has been known for more than a hundred years that microorganisms/viruses, particularly pathogenic bacteria that cause infections in human bodies, allow cancer regression, sometimes with astounding results. Not only live bacteria, but also bacteria-free sterile growth media of such bacteria, allow tumor shrinkage and regression, implying the role of soluble, secreted bacterial products having anticancer activity. Much efforts have, therefore, been expended to develop either live microorganisms/viruses, with or without additional cloned genes that encode toxins targeted specifically to cancer cells, or bacterial products with cancer-killing ability. This book addresses the recent advances in the use of microorganisms/viruses and their products, with or without genetic intervention, in cancer therapy, including human clinical trials. Seventeen different chapters address various facets of the use of live microorganisms, high/low molecular weight products derived from microorganisms, and microbial products fused to cancer-targeting molecules such as monoclonal antibodies or fragments of antibodies.

All therapies, including anticancer drugs, must reach the bedside and the global market to be useful. This requires that the therapies, whether diagnostic agents or drugs, be protected from copying. Such protection is usually afforded through creation of intellectual property rights, often through patenting. Patenting a drug could, however, be tricky and expensive as the criteria for patenting are still debatable, and patent infringement cases abound. Indeed, the U.S. Supreme Court, on June 1, 2009, accepted to consider a case, known as re Bilski, which purports to patent a business method for hedging the risks of commodity trading, for example, the price of natural gas or electricity, for both the suppliers and the consumers. The U.S. Court of Appeals for the Federal Circuit (CAFC), in an en banc decision, rejected the Bilski claims as nonpatentable subject matter involving no machines or material transformations that are believed to be required under U.S. patent laws for process patents. The Supreme Court’s decision to take up the case is thus believed to be important to determine patent eligibility of business methods, including medical diagnostic processes. Thus, the last chapter is devoted to address the fundamental concepts in patenting of drugs, including patent infringement or eligibility cases. Hopefully, this will give the readers a sense of what it takes to bring a drug, including anticancer drugs, to the market. The editors hope that the readers will find all the chapters useful, timely, relevant, and informative.

A.M. Fialho

A.M. Chakrabarty

CONTRIBUTORS

Jozef Anné, Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium

Donald J. Buchsbaum, Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL

Jason S. Buhrman, Massachusetts General Hospital, Harvard Medical School, MA and University of Illinois at Chicago School of Medicine, IL

Ananda M. Chakrabarty, Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL

Suresh Chawrai, Department of Chemistry, University of Cambridge, Cambridge, UK

Tooba A. Cheema, Massachusetts General Hospital, Harvard Medical School, MA

Lin-Chi Chen, Department of Medical Oncology, Nevada Cancer Institute, Las Vegas, NV

Nam H. Dang, Division of Hematology/Oncology, University of Florida Cancer Center Clinical Trials Office, University of Florida, Gainesville, FL

Ursula Elsässer-Beile, Department of Urology, Experimental Urology, University Hospital Freiburg, Freiburg, Germany

Lynn Feun, Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL

Arsénio M. Fialho, Institute for Biotechnology and Bioengineering (IBB), Center for Biological and Chemical Engineering, Instituto Superior Tecnico, Lisbon, Portugal

Geng-Feng Fu, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China

Giulia Fulci, Massachusetts General Hospital, Harvard Medical School, MA

Thomas S. Griffith, Department of Urology, University of Iowa, Iowa City, IA

Ashley L. Hilchie, Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada

Robert M. Hoffman, AntiCancer, Inc. and Department of Surgery, University of California, San Diego, CA

David W. Hoskin, Departments of Microbiology and Immunology, and Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada

Bi Hu, Center for Public Health Research, Medical School, Nanjing University, Nanjing, China

Sergey A. Kaliberov, Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL

Timothy P. Kresowik, Department of Urology, University of Iowa, Iowa City, IA

M. Tien Kuo, Molecular Pathology, M.D. Anderson Cancer Center, Houston, TX

Finian J. Leeper, Department of Chemistry, University of Cambridge, Cambridge, UK

Paulo Cesar Maciag, Advaxis Inc., North Brunswick, NJ

Jingxuan Pan, Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, People’s Republic of China

Yvonne Paterson, University of Pennsylvania Department of Microbiology, Philadelphia, PA

Sandra Rivera, Advaxis Inc., North Brunswick, NJ

John Rothman, Advaxis Inc., North Brunswick, NJ

George P.C. Salmond, Department of Biochemistry, University of Cambridge, Cambridge, UK

Padma Sampath, Departments of Surgery and Immunology, University of Pittsburgh, Pittsburgh, PA

Niramol Savaraj, Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL

Vafa Shahabi, Advaxis Inc., North Brunswick, NJ

Steve H. Thorne, Departments of Surgery and Immunology, University of Pittsburgh, Pittsburgh, PA

Lieve Van Mellaert, Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium

Jörg Vollmer, Coley Pharmaceutical GmbH—A Pfizer Company, Düsseldorf, Germany

Anna Maria Elisabeth Walenkamp, Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

Anu Wallecha, Advaxis Inc., North Brunswick, NJ

Medhi Wangpaichitr, Hematology/Oncology, Sylvester Comprehensive Can­cer Center, University of Miami School of Medicine, Miami, FL

Ming Q. Wei, Division of Molecular and Gene Therapies, Griffith Institute for Health and Medical Research, School of Medical Science, Griffith University, Queensland, Australia

Neil R. Williamson, Department of Biochemistry, University of Cambridge, Cambridge, UK

Philipp Wolf, Department of Urology, Experimental Urology, University Hospital Freiburg, Freiburg, Germany

Chung Jing Wu, Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL

Gen-Xing Xu, Center for Public Health Research, Medical School, Nanjing University, Nanjing and Jiangsu Research Center for Gene Pharmaceutical Engineering and Technology, Suzhou, China

Sai-Ching Jim Yeung, Departments of General Internal Medicine, Ambulatory Treatment and Emergency Care, and Endocrine Neoplasia and Hormonal Disorders, The University of Texas, M.D. Anderson Cancer Center, Houston, TX

Yan Yin, Center for Public Health Research, Medical School, Nanjing University, Nanjing, China

Ming You, Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL

2

THE USE OF LIVING LISTERIA MONOCYTOGENES AS AN ACTIVE IMMUNOTHERAPY FOR THE TREATMENT OF CANCER

John Rothman,1 Anu Wallecha,1 Paulo Cesar Maciag,1 Sandra Rivera,1 Vafa Shahabi,1 and Yvonne Paterson2

1Advaxis Inc., North Brunswick, NJ

2University of Pennsylvania Department of Microbiology, Philadelphia, PA

INTRODUCTION

Listeria monocytogenes is a gram-positive facultative intracellular bacterium responsible for causing listeriosis in humans and animals (1–3). L. monocytogenes is able to infect both phagocytic and nonphagocytic cells (4–6). Due to this intracellular growth behavior, L. monocytogenes triggers potent innate and adaptive immune responses in an infected host that are required for the clearance of the organism (7). This ability, to induce efficient immune responses using multiple simultaneous and integrated mechanisms of action, has encouraged efforts to develop this bacterium as a recombinant antigen delivery vector to induce protective cellular immunity against cancer or infection. Listeria infection also involves other systems which are not essentially a part of the immune system but which support immune function to affect a therapeutic outcome, such as myelopoesis and vascular endothelial cell function (8–16). This chapter discusses the multiple simultaneous mechanisms of action induced by bioengineered iatrogenic Listeria infection and how they may be optimized to induce therapeutic responses as active immunotherapy.

LISTERIAL DISEASE

Pathogenesis

To survive within the host, L. monocytogenes activates a set of virulence genes, which have been identified using biochemical and molecular genetic approaches. These genes include: actA, hly, inlA, inlB, inlC, plcA, and plcB, which are regulated by a pluripotential transcriptional activator, PrfA (17). Listeria lacking prfA are avirulent as they lack the ability to survive within the infected host (18, 19). Several other proteins such as Ami, Auto, ActA, and Vip are also essential for the full virulence of L. monocytogenes (20–22).

L. monocytogenes surface proteins termed as invasins interact with the receptors present on host cell plasma membranes to subvert signaling cascades, leading to bacterial internalization in nonphagocytic cells. Among these are 24 internalins present in the L. monocytogenes genome that are believed to contribute to host cell invasion; of these, internalins A (InlA) and B (InlB) are the most well characterized (23–31).

Listeria must escape the host cell phagolysosome to become virulent. Upon infection, less than 10% of the L. monocytogenes typically escapes into the host cell cytosol. This is mediated by listeriolysin O (LLO), a pore-forming hemolysin (32), and phospholipases (PlcA and PlcB). LLO is a member of a group of cholesterol-dependent cytolysins (CDC) and was the first major virulence factor of L. monocytogenes identified (33–35). In the cytoplasm, L. monocytogenes replicates and uses ActA, another major virulence factor, to polymerize host cell actin, support its motility, and spread from cell to cell (6, 36–38).

Epidemiology

Listeria is a ubiquitous environmental pathogen found in the soil, on leafy vegetables, in meat, and in dairy products; typical exposure to L. monocytogenes does not result in disease (39). Listeria is not laterally transmissible and is only pathogenic when ingested. Globally, listeriosis is a rare disease and its prevalence has declined wherever food control measures have been implemented. In the United States, the attack rate is estimated to be around one per million, leading to 2500 cases and around 700 deaths per year with infection more common in children, the elderly, and pregnant women, who tend to have less competent immune function (1, 2, 40–48).

Unlike most human foodborne infections, which are associated with a high incidence rate counterbalanced by low morbidity and mortality, the situation is opposite for clinically presented human listeriosis, which is a rare but potentially fatal infection associated with a 30% mortality, even when an antimicrobial treatment is administered (2, 48). This is probably because individuals with healthy immune systems eliminate infection before clinical signs are apparent.

Innate Immunity and Listeria Infection

Innate immunity plays an essential role in the clearance of L. monocytogenes and control of the infection at early stages. Severe combined immunodeficiency (SCID) mice have been observed to clear infection with attenuated L. monocytogenes vaccine strains (for instance, Lm-LLO-E7) through innate immune mechanisms (Y. Paterson, unpublished observations). Upon intraperitoneal (IP) or i.v. inoculation, L. monocytogenes are cleared from the blood primarily by splenic and hepatic macrophages (49).

Cytokine, Chemokine, Costimulatory Molecules, and Similar Responses

Hepatic Kupffer cells clear most of the circulating bacteria and are the major source of interleukin (IL)-6 as a consequence of LLO (50, 51). Neutrophils are rapidly recruited to the site of infection by the cytokine IL-6 and other chemoattractants where they secrete IL-8, colony-stimulating factor (CSF)-1, and monocyte chemotaxtic protein 1 (MCP-1), which then attract macrophages to the infection foci (52). Granulocytes are replaced by large mononuclear cells, and within 2 weeks, the lesions are completely resolved (51). Mice in which granulocytes are depleted are unable to survive to L. monocytogenes administration (53–56). Listeria replicates within hepatocytes that are then lysed by the granulocytes, which migrate to the site of infection, releasing the intracellular bacteria to be phagocytosed and killed by neutrophils (53). Mast cells are not infected but are activated by L. monocytogenes and rapidly secrete tumor necrosis factor (TNF)-α and induce neutrophils recruitment, and their depletion results in higher titers of L. monocytogenes in liver and spleen (57).

L. monocytogenes or sublytic doses of LLO in human epithelial Caco-2 cells induce the expression of IL-6 that reduces bacterial intracellular growth (58) and causes overexpression of inducible nitric oxide synthase (NOS) (59). NO appears to be an essential component of the innate immune response to L. monocytogenes, having an important role in listericidal activity of neutrophils and macrophages (60), with a deficiency of inducible NOS (iNOS) causing susceptibility to infection (61).

Impairing the recruitment of myelomonocytic cells by blockade of the type 3 complement receptor (62) or diminishing chemokine receptor 2 (CCR2) (63) results in an enhanced susceptibility to L. monocytogenes infection (62, 63) and significantly decreased levels of IL-6 (50). CD18-deficient mice are more resistant to listeriosis due to neutrophilia, with faster clearance of L. monocytogenes in the liver and spleen, milder inflammatory and necrotizing lesions, and higher levels of IL-1β and G-CSF, leukocytosis, and impaired transendothelial neutrophil migration (64, 65). Similarly, mice deficient in lymphocyte function-associated antigen 1 (LFA-1 or CD11a/CD18) have an increased resistance to L. monocytogenes infection and neutrophilia, and upon infection, they show a higher infiltration of neutrophils in the liver in a LFA-1-independent way (64).

CCR2-deficient mice have impaired macrophage recruitment, which would be induced by the CCR2 ligand MCP-1, and thus, they are very susceptible to listeriosis and rapid death by L. monocytogenes infection. They also lack a subset of dendritic cells (Tip-DCs) that are the predominant source of TNF and iNOS in the spleens of infected mice (13, 63), adding to their inability to clear primary bacterial infection, although CD8+ and CD4+ T cell responses to L. monocytogenes antigens are preserved (13). In the T cell zone in the spleen, Tip-DC can result from monocyte differentiation in the presence of L. monocytogenes (14).

L. monocytogenes-infected macrophages produce TNF-β, IL-18, and IL-12, all of which are important in inducing the production of interferon (IFN)-γ, and subsequent killing and degradation of L. monocytogenes in the phagosome (66). IL-12 deficiency results in an increased susceptibility to listeriosis (67, 68), which can be reversed through administration of IFN-γ (67). Resistance to L. monocytogenes is conferred, in part, through the release of TNF-α and IFN-γ (69, 70), and deficiency in either of these cytokines or their receptors increases susceptibility to infection (71). Natural killer (NK) cells are the major source of IFN-γ in early infection (72). Upon reinfection, memory CD8+ T cells have the ability to produce IFN-γ in response to IL-12 and IL-18 in the absence of the cognate antigen (73). CD8+ T cells colocalize with the macrophages and L. monocytogenes in the T cell area of the spleen where they produce IFN-γ independent of antigen (74). CD8+ T cells are also associated with Lm lesions in the liver (74). IFN-γ production by CD8+ T cells depends partially on the expression of LLO (75).

IFN-γ plays an important role in antitumor responses obtained by L. monocytogenes-based vaccines. Although produced initially by NK cells, IFN-γ levels are subsequently maintained by CD4+ T helper cells for a longer period (76). Dominiecki et al. (77) used a tumor that is insensitive to IFN-γ to show that L. monocytogenes vaccines require IFN-γ for effective tumor regression and that IFN-γ is specifically required for tumor infiltration of lymphocytes but not for trafficking to the tumor. Paterson et al. (unpublished) have demonstrated that the difference between the IFN-γ-insensitive and sensitive TC-1 tumors is the induced upregulation of multiple chemokines and their receptors, which may explain why lymphocytes infiltrate these wild-type tumors more efficiently. IFN-γ also inhibits angiogenesis at the tumor site in the early effector phase following vaccination (76).

IL-18 is also critical to resistance to L. monocytogenes, even in the absence of IFN-γ, and is required for TNF-α and NO production by infected macrophages (78). A deficiency of caspase-1 impairs the ability of macrophages to clear L. monocytogenes and causes a significant reduction in IFN-γ production and listericidal activity that can be reversed by IL-18. Recombinant IFN-γ injection restores innate resistance to listeriosis in caspase-1−/− mice (79). Caspase-1 activation precedes the cell death of macrophages infected with L. monocytogenes, and LLO-deficient mutants that cannot escape the phagolysosome have an impaired ability to activate caspase-1 (80).

LLO secreted by L. monocytogenes causes specific gene upregulation in macrophages, resulting in significant IFN-γ transcription and secretion (81). Cytosolic LLO activates a potent type I IFN response to invasive L. monocytogenes independent of Toll-like receptors (TLR) without detectable activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) (82). One of the IFN I-specific apoptotic genes, TNF-related apoptosis-inducing ligand (TRAIL), is upregulated during L. monocytogenes infection in the spleen (83). Mice lacking TRAIL are also more resistant to primary listeriosis coincident with lymphoid and myeloid cell death in the spleen.

Pathogen-Associated Molecular Patterns

Nucleotide-binding oligomerization domain (NOD) proteins recognize peptidoglycans present in the bacterial cell wall and are believed to recognize bacteria in the cytosol. Degraded L. monocytogenes in the phagolysosomes of macrophages, but not intact bacteria, induce a TLR-independent IFN-β transcriptional response similar to the response observed with cytosolic L. monocytogenes, which is dependent on NOD2 (84). NOD1, however, is crucial for IL-8 production and NF-κB activation initiated by L. monocytogenes in human endothelial cultures (85).

TLRs are also important components of innate immunity, recognizing conserved molecular structures on pathogens and signaling through adaptor molecules, such as MyD88, to induce NF-κB activation and transcription of several proinflammatory genes. They have a role in the recognition of L. monocytogenes at the cell surface. TLR2 recognizes bacterial peptidoglycan lipoteichoic acid and lipoproteins present in the cell wall of gram-positive bacteria, including L. monocytogenes. Thus, TLR2-deficient mice are slightly more susceptible to listeriosis (86). TLR5 recognizes bacterial flagellin and may be involved in L. monocytogenes recognition; however, flagellin expression is downregulated at 37°C in most isolates, and the role of TLR5 in listeriosis in vivo is uncertain, since TLR5 is not required for innate immune activation against this bacterial infection (87).

Although a single TLR has not been shown to be essential in innate immune responses to L. monocytogenes, the adaptor molecule MyD88, which is used by signal transduction pathways of all TLRs, besides IL-1 and IL-18, is critical to the defenses against L. monocytogenes since infection is lethal in MyD88-deficient mice. MyD88−/− mice have a severely impaired ability to produce IL-12, IFN-γ, TNF-α, and NO following infection, and while MyD88 is not required for MCP-1 production or monocyte recruitment following infection, it is essential for L. monocytogenes-induced IL-12 and TNF-α production and monocyte activation (13). Mice deficient in the NOD receptor interacting protein kinase 2 (RIP2) are impaired in their ability to defend against infection and have decreased IFN-γ production by NK and T cells, which is partially attributed to a defective IL-12 signaling (88). NF-κB activates several genes involved in innate immune responses, and mice lacking the p50 subunit of NF-κB are also highly susceptible to L. monocytogenes infections (89).

CpG motifs act as pathogen-associated molecular patterns (PAMPs) and immune stimulators (90–94). In vivo and in vitro studies have shown that treatment with CpG oligodeoxynucleotides (ODN) can improve the resistance of normal neonatal mice and pregnant mice to lethal L. monocytogenes infection (95–96).

ADAPTIVE IMMUNITY

MHC Class I Responses

L. monocytogenes secretes a limited number of proteins into the cytosol of the host cell, which are rapidly degraded by the proteosome, resulting in MHC Class Ia-restricted peptide antigens (97, 98). Certain secreted proteins, such as p60 and LLO, are rapidly degraded because their amino-termini contain destabilizing residues as defined by the N-end rule (99, 100). LLO may also be degraded in a proteosome-dependent fashion as it contains a PEST-like sequence (101). The rapid proteosome-mediated degradation of a potentially toxic protein such as LLO enhances host cell survival and generates peptide fragments that enter the MHC Class I antigen processing pathway.

After intravenous inoculation of L. monocytogenes, MHC Class Ia-restricted T cell responses to reach peak frequencies in approximately 8 days (102). In experiments in which mice were treated with antibiotics to curtail the duration of the infection, it was found that the magnitude of T cell responses is independent of the quantity or the duration of in vivo antigen presentation (103–108), since even with marked differences in the number of viable bacteria and inflammatory response, the expansion and contraction of CD8+ T cells is similar in mice treated with antibiotics 24 h after infection and in mice that are untreated (108). This is consistent with in vitro studies of L. monocytogenes-specific CD8+ T cell proliferation, in which brief antigen exposure is followed by prolonged proliferation that does not require further exposure to antigen (9). It has been speculated that antigen-independent T cell proliferation is driven by cytokines such as IL-2; however, Wong et al. (109) showed that endogenous IL-2 production by CD8+ T cells is required for Ag-independent expansion following TCR stimulation in vitro, but not in vivo.

Cell-Mediated Immune Responses to Heat-Killed and Irradiated Lm

Unlike priming with live infection, heat-killed L. monocytogenes does not induce a protective immune response. It was believed for years that this was due to the inability of killed bacteria to enter the cytosol of phagocytic antigen presenting cell (APC) impairing the access of antigen to MHC Class I pathway. We now know that immunization of mice with heat-killed bacteria does result in proliferation of antigen-specific CD8+ T cells but does not result in the differentiation of primed T cells into effector cells (110). Recently, studies have shown that vaccination with irradiated L. monocytogenes efficiently activated DCs and induced protective T cell responses (111).

CD4+ T Cells Responses

L. monocytogenes infection also results in the generation of robust MHC Class II-restricted CD4+ T cell responses and shifts the phenotype of CD4+ T cells to Th-1 (112–114). Expansion of these cells was found to be synchronous with the expansion of the CD8+ T cell responses (115). CD4+ T cells produce copious amounts of Th-1 cytokines that contribute to bacteria clearance. Immunization with L. monocytogenes has been shown to result in the generation of “high quality” effector CD4+ T cells capable of secreting multiple cytokines such as IFN-γ and TNF-α, or three cytokines such as TNF-α, IFN-γ, and IL-2 (116), coincident with the generation of a memory CD4+ T cell response. CD4+ T cell-mediated protective immunity requires T cell pro­duction of IFN-γ, whereas CD8+ T cells mediate protection independently of IFN-γ (71, 117). Production of IFN-γ from CD4+ T cells likely activates macrophages to become more bactericidal. This is supported by in vitro studies showing that treatment of macrophages with IFN-γ prevents bacterial escape from the phagosome (118).

CD4+ T cell help is required for the generation and maintenance of functional CD8+ T cell memory against L. monocytogenes (119). The kinetics of bacterial clearance and magnitude of the primary antigen-specific CD8+ T cell responses are similar in MHC Class II-deficient mice and wild-type mice, and the kinetics of contraction and numbers of memory CD8+ T cell responses are also similar between the two strains. However, when examined up to 60 days postinfection, a reduction in the number of memory CD8+ T cells, generated in the absence of CD4+ T cell help, was evident at later time points. When rechallenged months after the primary infection, mice that lacked CD4+ T cells were not able to eliminate bacteria due to their inability to mount a vigorous secondary response required for killing infected cells. Memory CD8+ T cells generated in the absence of CD4+ T cells were ineffective (119–122). In viral infection studies, it has been shown that memory cytotoxic T lymphocytes (CTLs) cannot be converted back to effectors without CD4+ cells or exogenous cytokines (123, 124). It is not known whether this help is provided by direct CD4+–CD8+ T cell interaction or by the cytokine milieu generated by the activated CD4+ T cells.

Induction of Tγδ Cells

Skeen et al. (125) has reported that infection of mice intraperitoneally with L. monocytogenes caused a local induction of CD4+ Tγδ cells associated with IL-17 secretion in the peritoneal cavity; however, no changes were observed in the splenic or lymph node T cell populations after these injections. When peritoneal T cells from L. monocytogenes-immunized mice were restimulated in vitro, the induced Tγδ cells exhibited a greater expansion potential than the Tαβ cells. Modifications that abrogate the virulence, such as heat-killed or hly mutations, eliminate the inductive effect for Tγδ cells. Depletion of either Tαβ or Tγδ cells in vivo impairs the resistance to primary infection; however, the memory response is unaffected by the depletion of Tγδ cells, consistent with the presumed effects of IL-17 to shift CD4+ Treg phenotypic cells to Th-17 phenotypes and supporting the hypothesis that this T cell subset forms an important line of defense in innate immunity (125).

NONCLASSICAL IMMUNE FUNCTIONS

Effects of Listeria on Myeloid Cells

Listeria infection stimulates the expansion of myeloid cells and biases myeloid cell lineages to proliferate and mature into effective terminally differentiated immune cells. Accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals, occurs in response to Listeria infection. Cytokines such as GM-CSF, IL-6, IL-8, IL-10, IL-12, and TNF-α were found to be induced by L. monocytogenes infection, which indicates that infection of human stem cells (HSC) affects the differentiation of CD34+ hematopoietic progenitors (11). Bone marrow composition changes dramatically during infection, leading to an increase of myeloid cells, which peak after 1 week of infection (9). In addition, a water-soluble monocytosis-producing activity (MPA) extracted from L. monocytogenes has been found to be able to stimulate proliferation of promonocytes in vivo. An acceleration of both the generation time of monocyte precursors and the half-time of blood monocytes has been observed in L. monocytogenes-treated mice when compared with control mice (126). Elevated levels of various CSFs in the serum were quantitated subsequent to L. monocytogenes infection, with the great bulk of serum colony-stimulating activity represented in M-CSF and G-CSF, and with measurable GM-CSF. The increase in serum CSFs occurred before the peak in bone marrow GM progenitors and before the reduction in bacterial numbers, which follows the onset of specific cell-mediated immunity (8). The rise in serum CSF concentration correlated with monocyte production during L. monocytogenes infection (16).

L. monocytogenes induces the release of IL-2, IL-6, IL-12, and TNF-α from DCs and the subsequent upregulation within these cells of CD40, B7-H1 program death-ligand 1 (PD-L1), CD86 (B7-2), and B7-DC (PD-L2) that results in the maturation and activation of high-affinity T cells (12). In addition, the production of IL-12, IL-6, and TNF-α is most efficiently triggered by cytosolic L. monocytogenes. Costimulatory molecules induced by cytosolic entry regulate T cell proliferation and the number of functional T cells generated. DC-produced cytokines (IL-12 and IL-10) are the major factors determining the proportion of IFN-γ-producing T cells. LLO is required for optimal T cell priming and cytokine production that result in functionally therapeutic CTL responses (127).