Targeted Drug Strategies for Cancer and Inflammation

Targeted Drug Strategies for Cancer and Inflammation

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Ann L. Jackman    Christopher P. Leamon ●

Editors

Targeted Drug Strategies for Cancer and Inflammation

Editors Ann L. Jackman Section of Medicine Institute of Cancer Research 15 Cotswold Road, Sutton Surrey SM2 5NG UK [email protected]

Christopher P. Leamon Endocyte, Inc. 3000 Kent Avenue, Suite A1-100 West Lafayette, IN 47906 USA [email protected]

ISBN 978-1-4419-8416-6 e-ISBN 978-1-4419-8417-3 DOI 10.1007/978-1-4419-8417-3 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011928253 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

In 1999, a book entitled Antifolate Drugs in Cancer Therapy (Humana/Springer) focused on existing and emerging cancer drugs that inhibited folate-dependent enzymes. Several chapters in that volume provided evidence suggesting that the effectiveness and tolerability of antifolate therapy could be further increased by (a) understanding and exploiting some of the molecular determinants of drug sensitivity or (b) by reducing exposure of normal proliferating tissues to these agents. Now, a decade later, we can address the latter subject by reviewing the biological properties of a contemporary class of “targeted agents” that functionally exploit a tumor-associated folate transport protein called the folate receptor (FR). The FR is a glycosylphosphatidyinositol-linked protein that captures its ligands from the extracellular milieu and transports them inside the cell via a nondestructive, recycling endosomal pathway. FRs have restricted expression in normal tissues, and they are not generally exposed to the bloodstream; however, elevated expression occurs in many human malignancies, especially when associated with aggressively growing cancers. These factors help define “FR targeting” as a viable tumor-targeting strategy. Agents that target the FR range in size from small molecule antifolate drugs and folate-drug conjugates to monoclonal antibodies and nanoparticles. In some cases, the agent need only bind to the FR to elicit a biochemical effect (e.g., diagnostic imaging or immunotherapy); in other cases, such as for high affinity antifolates and folate conjugates of small molecule therapeutics, internalization by the FR/endosomal apparatus and subsequent cytosolic delivery is required for biological activity against intracellular targets. The discoveries highlighted in this book parallel the emergence of innovative “molecular targeted” small molecules and monoclonal antibodies, i.e., agents that target proteins within highly activated signal transduction pathways that control proliferation. However, many of the tumor-targeted strategies described within cross the boundaries between what is considered to be “molecular-targeted” vs. conventional systemic therapy. Obviously, for these novel agents to be effective, tumors must express a functional form of the FR. But in contrast to the targets of signaling inhibitors, tumor growth is not necessarily dependent on FR expression; rather, this cell surface receptor imparts key therapeutic specificity. Thus, while the

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pharmacologic targets of FR-guided drugs and folate-drug conjugates are ­frequently those of conventional therapy, the selectivity realized through restricted tissue expression of the FR biomarker reduces the adverse effects against untargeted normal tissues. Regardless, both the cellular and molecular targeting approaches share the goal in shifting the paradigm from that of generalized chemotherapy to that of personalized medicine. Beyond cancer research, FRs are also receiving attention from researchers of inflammatory disorders. Recent discoveries have shown that proinflammatory, activated human monocytes and macrophages express a functional FR isoform. Preclinical and clinical proof has already emerged showing how this marker can be used to identify sites of inflammation (e.g., arthritis) using folate-targeted radiodiagnostic imaging agents, and efforts for therapeutic exploitation are already underway (see Chaps. 9, 10). Clearly, it is only a matter of time before novel FR-targeted anti-inflammatory therapies reach clinical practice. From a historical and complementary viewpoint, advances in our understanding of other folate transport proteins, such as the reduced folate carrier and the protoncoupled folate transporter, are also reviewed in this book (Chap. 1); however, the main theme of this volume is the FR, with much of the content focused on its basic biology and regulation (Chaps. 2, 3) as well as its exploitation for targeted therapy and diagnostic imaging (Chaps. 4–8). The contributors to this volume are all highly regarded in their fields, and we are very grateful to them for devoting so much time and effort into their excellent contributions. Both of us have benefited tremendously from reviewing their chapters, and we wish for their continued success. Surrey, UK West Lafayette, IN

Ann L. Jackman Christopher P. Leamon

Contents

1  Biological Role, Properties, and Therapeutic Applications of the Reduced Folate Carrier (RFC-SLC19A1) and the Proton-Coupled Folate Transporter (PCFT-SLC46A1)................................................................. Larry H. Matherly, Ndeye Diop-Bove, and I. David Goldman

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2  Folate Receptors and Therapeutic Applications..................................... Barton A. Kamen

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3  Hormonal Control of Folate Receptor Genes.......................................... Mesfin Gonit, Marcela D’Alincourt Salazar, Juan Zhang, Hala Elnakat, Suneethi Sivakumaran, and Manohar Ratnam

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4  Folate Receptor-Targeted Radionuclide Imaging Agents...................... Cristina Müller and Roger Schibli

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5  Folate Receptor Targeted Thymidylate Synthase Inhibitors................. Ann L. Jackman, Gerrit Jansen, and Matthew Ng

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6  Discovery of Novel Antifolate Inhibitors of De Novo Purine Nucleotide Biosynthesis with Selectivity for High Affinity Folate Receptors and the Proton-Coupled Folate Transporter Over the Reduced Folate Carrier for Cellular Entry...................................................................................... 119 Larry H. Matherly and Aleem Gangjee 7  Folate Receptor Targeted Cancer Chemotherapy.................................. 135 Joseph A. Reddy and Christopher P. Leamon 8  Anti-FR Antibody Generation and Engineering: Development of New Therapeutic Tools.................................................. 151 Silvana Canevari and Mariangela Figini vii

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  9  Folate Receptor Positive Macrophages: Cellular Targets for Imaging and Therapy of Inflammatory and Autoimmune Diseases......................................... 181 Michael J. Hansen and Philip S. Low 10  Targeting Activated Macrophages Via a Functional Folate Receptor for Potential Treatment of Autoimmune/Inflammatory Disorders.............................................. 195 Yingjuan Lu and Christopher P. Leamon Index.................................................................................................................. 217

Contributors

Ndeye Diop-Bove Departments of Molecular Pharmacology and Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA Silvana Canevari Unit of Molecular Therapies, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Hala Elnakat Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, OH 43614, USA Mariangela Figini Unit of Molecular Therapies, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Aleem Gangjee Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, USA I. David Goldman Departments of Molecular Pharmacology and Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA Mesfin Gonit Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, OH 43614, USA

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Contributors

Michael J. Hansen Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA Ann L. Jackman Section of Medicine, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK Gerrit Jansen Department of Rheumatology, VU Institute for Cancer and Immunology, VU University Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Barton A. Kamen Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA Christopher P. Leamon Endocyte, Inc., 3000 Kent Avenue, Suite A1-100, West Lafayette, IN 47906, USA Philip S. Low Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA Yingjuan Lu Endocyte, Inc., 3000 Kent Avenue, Suite A1-100, West Lafayette, IN 47906, USA Larry H. Matherly Development Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue; Department of Oncology; Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA Cristina Müller Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland Matthew Ng Section of Medicine, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK Manohar Ratnam Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, OH 43614, USA

Contributors

Joseph A. Reddy Endocyte, Inc., 3000 Kent Avenue, Suite A1-100, West Lafayette, IN 47906-1075, USA Marcela D’Alincourt Salazar Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, OH 43614, USA Roger Schibli Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland; Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland Suneethi Sivakumaran Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, OH 43614, USA Juan Zhang Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, OH 43614, USA

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Chapter 1

Biological Role, Properties, and Therapeutic Applications of the Reduced Folate Carrier (RFC-SLC19A1) and the Proton-Coupled Folate Transporter (PCFT-SLC46A1) Larry H. Matherly, Ndeye Diop-Bove, and I. David Goldman Abstract  The mechanisms by which folates are transported across cell membranes have been an area of research interest for nearly five decades. Major transport systems include the facilitative carriers, the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT), and the high affinity folate receptors (FRs) a and b which transport folates by endocytosis. RFC is the major transport system in mammalian cells and tissues for folate cofactors and clinically relevant antifolate drugs including methotrexate, raltitrexed, pemetrexed, and pralatrexate. PCFT was identified in 2006 as the mechanism by which folates are transported across the apical brush border of the proximal small intestine. Whereas both PCFT and RFC are widely expressed in tumors, PCFT differs from RFC in its acidic pH optimum which favors transport at the low pH commonly found in the hypoxic microenvironment of solid tumors. Reflecting tumor-specific patterns of expression and/or function, recent studies have focused on the identification of folate-targeted therapeutics with selective transport by PCFT and FRs over RFC. The goal is to circumvent RFC and the potentially toxic consequences of drug transport by RFC in normal tissues. RFC in tumor cells can also influence the pharmacologic activity of PCFT and FR-selective agents by transporting physiological folates which compete for polyglutamylation and binding to intracellular targets. This review focuses on the facilitative pathways of (anti)folate transport, including RFC (SLC19A1) and PCFT (SLC46A1) in relation to their molecular properties, and their physiological and pharmacological roles.

L.H. Matherly (*) Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201, USA and Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201, USA and Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA e-mail: [email protected] A.L. Jackman and C.P. Leamon (eds.), Targeted Drug Strategies for Cancer and Inflammation, DOI 10.1007/978-1-4419-8417-3_1, © Springer Science+Business Media, LLC 2011

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Keywords  Proton-coupled folate transporter • Reduced folate carrier • Hereditary folate maladsorption • Antifolate • Folate Abbreviations AICAR AICARTase ALL BCRP CNS CSF 5-FormylTHF FR GARFTase GlpT HFM LacY 5-MethylTHF MFS MRP mTOR MTX OAT PCFT RFC RTX SCAM THF TMD UTR

5-Amino-4-imidazolecarboxamide ribonucleotide 5-Amino-4-imidazolecarboxamide ribonucleotide formyltransferase Acute lymphoblastic leukemia Breast-cancer resistant protein Central nervous system Cerebrospinal fluid 5-Formyltetrahydrofolate Folate receptor Glycinamide ribonucleotide formyltransferase Glycerol phosphate/inorganic phosphate antiporter Hereditary folate malabsorption Lactose/proton symporter 5-Methyltetrahydrofolate Major facilitator superfamily Multidrug resistance-associated protein Mammalian target of rapamycin Methotrexate Organic anion transporters Proton-coupled folate transporter Reduced folate carrier Raltitrexed Substituted cysteine accessibility methods Tetrahydrofolate Transmembrane domain Untranslated region

1.1 Introduction The mechanisms by which folates are transported across cell membranes have been an area of research interest for nearly five decades. Folate cofactors as vitamins are available only from exogenous sources. Reflecting this, there has been a longstanding interest in the mechanism by which these compounds are absorbed in the small intestine (Halsted 1979; Selhub and Rosenberg 1981; Said 2004). Studies on transport of antifolates date from mid- to late 1960s when it was recognized that membrane transport of methotrexate (MTX) is carrier-mediated and is an important determinant of MTX chemotherapeutic activity, and that tumor cells commonly develop resistance to MTX due to an acquired defect in cellular uptake (Sirotnak et al. 1968; Goldman et al. 1968; Hakala 1965).

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The first of the folate transporters to be understood at the kinetic and thermodynamic levels was the reduced folate carrier (RFC) (Matherly et al. 2007). Initially characterized in detail in the late 1960s (Goldman et al. 1968), it was nearly 30 years later that this transporter was cloned (Dixon et al. 1994) and its regulation and structure– function understood at the molecular level (Matherly et al. 2007; Matherly and Hou 2008; Zhao et al. 2009a). RFC is a major mode of transport of all the classical antifolate drugs used in the treatment of cancer (Matherly et al. 2007). The second transport mechanism to be described was an endocytic process mediated by high affinity folate receptors (FRs). While the presence of “folatebinding proteins” was recognized in many tissues and secretions dating back to the 1960s, it was some years later when their role in folate delivery to cells was recognized and characterized (Antony 1992, 1996). This eventually led to the cloning of two endocytic proteins termed folate receptor a (FRa) and folate receptor b (FRb) in the late 1980s (Elnakat and Ratnam 2004). The initial focus of FR research from a pharmacological perspective involved the role of these receptors in the delivery of MTX into tumor cells. However, this avenue of research proved to be unproductive because of the comparatively poor substrate activity of MTX for FRs, the ubiquitous presence of RFC in tissues and tumors, and the high rates of MTX transport by RFC relative to rates of FR-mediated endocytosis (Sierra et  al. 1995; Spinella et al. 1995). However, what evolved over time was the concept of utilizing FRs highly expressed in certain tumor types to deliver a variety of structurally unrelated agents linked to folic acid for therapeutic and diagnostic purposes (Leamon 2008; Hilgenbrink and Low 2005; Salazar and Ratnam 2007) and, more recently, as a vehicle for the selective delivery of cytotoxic antifolates with very low affinities for RFC (Gibbs et al. 2005; Deng et al. 2008a, 2009; Theti et al. 2003; Wang et al. 2010) – a major theme of this book. Most recently, a third folate transport system was discovered – the proton-coupled folate transporter (PCFT) (Qiu et al. 2006). PCFT is the mechanism by which folates are transported across the apical brush border of the proximal small intestine and operates optimally in an acid environment, a feature that distinguishes it from RFC. Indeed, the properties of PCFT are fully consistent with those previously reported for intestinal folate absorption and for transport of folates and antifolates at the low pH commonly found within the hypoxic microenvironments of human solid tumors (Helmlinger et al. 1997; Raghunand et al. 1999). Based on the latter, novel antifolates are being developed with specificity for PCFT over RFC (Wang et al. 2010; Kugel Desmoulin et al. 2010; Matherly and Gangjee 2011), so as to selectively target solid tumors while minimizing toxicity to normal tissues that express RFC and are exposed to a neutral pH. PCFT is also critical to the transport of folates and antifolates across the blood–choroid plexus barrier into the cerebrospinal fluid (CSF) and may contribute to folate/antifolate export from acidified endosomes during FR-mediated endocytosis (Zhao et al. 2009a, b; Zhao and Goldman 2007). While the role of membrane transport in the antitumor activities of antifolate drugs has been extensively reviewed (Matherly et al. 2007; Assaraf 2007; Goldman and Matherly 1985; Chattopadhyay et al. 2007; Zhao and Goldman 2003; Goldman et al. 2010), the recent development of novel antifolates, designed for selective transport via FRs or PCFT over RFC (Gibbs et al. 2005; Deng et al. 2008a, 2009; Theti et al. 2003;

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Wang et al. 2010; Kugel Desmoulin et al. 2010; Matherly and Gangjee 2011), makes it particularly timely to revisit the features of the parallel transport routes mediated by the endocytotic FRs vs. these facilitative carriers that exist in most tumor cells. In addition to its role in transporting many antifolates, facilitative transport by RFC is also important for physiological folates, thereby influencing pharmacologic activity of FRor PCFT-targeted antifolates by regulating intracellular folate pools which, in turn, modulate formation of their antifolate polyglutamate derivatives and interactions with target enzymes. Likewise, for antifolate substrates of FRs or PCFT that also preserve RFC substrate activity, RFC provides a route of drug uptake into normal cells with potentially toxic consequences, as noted above. This chapter will focus on facilitative pathways of (anti)folate transport, including RFC (SLC19A1) cal and pharmacological roles. The biology of the FRs will be considered by Kamen (2011) in Chapter 2.

1.2 Role of Membrane Transport in Folate Homeostasis Folates are a family of B9 vitamins that differ in oxidation of the pteridine ring, the nature of the one-carbon substituent at the N5 and N10 positions, and the extent of g glutamate conjugation. The major folate in the diet and in the blood of mammals is 5-methyl tetrahydrofolate (5-methylTHF). Within cells, this folate is, in part, oxidized to dihydrofolate during the synthesis of thymidylate and then fully reduced to tetrahydrofolate (THF) with the subsequent formation of a variety of THF cofactors. The biological importance of reduced folates derives from their roles in one-carbon transfers leading to thymidylate, purine nucleotides, serine, and methionine, and in supporting biological methylation reactions from S-adenosylmethionine encompassing both small molecules (e.g., phosphatidylethanolamine) and macromolecules (e.g., DNA, histones) (Stokstad 1990; Chiang et al. 1996). Glutamate conjugation, catalyzed by folylpoly-g-glutamyl synthetase, confers enhanced cellular retention, as folate polyglutamates are poor substrates for folate transporters, and increased rates of one-carbon transfer over monoglutamyl folates, since these derivatives are preferred substrates for folate-requiring enzymes (Shane 1989; Schirch and Strong 1989). Mammals cannot synthesize folates de novo. Hence, to achieve intracellular folate levels sufficient to meet one-carbon biosynthetic needs requires adequate folate intestinal absorption followed by uptake into systemic cells and transport across epithelial barriers into tissue compartments such as the central nervous system (CNS). Major transport systems for folate uptake include the facilitative carriers, RFC and PCFT, that are widely expressed (Qiu et al. 2006; Zhao et al. 2009a; Whetstine et  al. 2002a) but exhibit disparate pH optima (Matherly et  al. 2007; Zhao et  al. 2009a; Zhao and Goldman 2007; Wang et al. 2004). Other uptake systems include FRa and FRb, which mediate uptake of folates by endocytosis (Elnakat and Ratnam 2004; Salazar and Ratnam 2007), and the organic anion transporters (OATs, OATPs) that are expressed in epithelial tissues (e.g., kidney, intestine) and transport a broad spectrum of organic ions (e.g., probenecid, bromosulfophthalein) in addition to folates (Rizwan and Burckhardt 2007; Shibayama et  al. 2006; Matherly and

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Goldman 2003; Masuda 2003). ABC-cassette transporters such as multidrug resistance-associated proteins (MRPs) 1–5 and 8, and ABCG2 (breast cancer resistant protein [BCRP]) also transport folate substrates (Assaraf 2007; Kruh and Belinsky 2003; Kruh et al. 2007), thus exerting opposing effects on the concentrative uptake of these compounds. The impact of these efflux pumps on net transport becomes clear in the presence of energy inhibitors or competitive substrates which result in marked increases in transmembrane gradients for (anti)folate substrates (Hakala 1965; Goldman 1969). Dietary folates are absorbed in the duodenum and proximal jejunum mediated by PCFT within an acid microenvironment (pH 5.8–6.0) at the cell surface (Zhao et al. 2009a). The critical role that PCFT plays in this process was established by the severe systemic folate deficiency that occurs in patients with hereditary folate malabsorption (HFM) who are null for this transporter (Zhao et al. 2007, 2009a; Qiu et al. 2006; Lasry et al. 2008; Min et al. 2008; Shin et al. 2010, 2011). Although RFC is expressed at the apical brush-border membrane along the entire intestine (Wang et al. 2001), RFC does not likely contribute to folate absorption under physiological conditions, even in intestinal segments in which the pH is more favorable to its function such as the distal small intestine. RFC may, however, contribute to folate absorption when pharmacological doses of folate are administered orally to subjects with HFM (Zhao et al. 2009a). Transport of folates across the basolateral membrane of the jejunum appears to be mediated in part by MRP3 (Kitamura et al. 2008). Hence, in this case, PCFT and MRP3 act in concert to achieve vectorial transport (absorption) across the intestinal epithelium. Folates absorbed in the intestine are delivered to the liver by the hepatic portal vein where both RFC and PCFT are expressed at the sinusoidal membrane (Wang et  al. 2001; Horne 1990; Horne and Reed 1992); the pH at this interface would determine the extent to which each transporter ­contributes to folate uptake into hepatocytes. Folate secretion into the bile at the canalicular membrane is mediated by MRP2 such that in MRP2-null animals, there is a marked defect in the elimination of MTX via the bile (Masuda et al. 1997). Folates are filtered at the glomerulus and then reabsorbed in the proximal renal tubule. FRa, along with PCFT, is expressed at the apical brush-border membrane and RFC at the basolateral membrane of the proximal renal tubule (Zhao et  al. 2009a). In addition, a variety of OATs with much lower specificities for folates may contribute to folate reabsorption in the proximal tubule. These include OATP1 at the apical brush-border membrane and OAT1 and OAT3 at the basolateral membrane (Rizwan and Burckhardt 2007; Masuda 2003; Russel et al. 2002). Folates are the only, or one of the very few, substrates that are concentrated in the CSF (Geller et  al. 2002). To account for this requires active folate transport across the choroid plexus. FRa, RFC, and PCFT are all expressed at the choroid plexus. FRa is expressed primarily at the apical brush-border membrane and to a much lesser extent at the basolateral membrane (Kennedy et  al. 2003; Weitman et al. 1992a, b; Selhub and Franklin 1984; Patrick et al. 1997). RFC is expressed at the apical membrane (Wang et al. 2001) and PCFT is expressed at the basolateral membrane (Zhao et al. 2009b). It is now clear that both PCFT and FRa are required for the delivery of folates into the CSF. In HFM, folate is usually undetectable in the CSF and remains quite low even when the folate blood level is normalized

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(Geller et  al. 2002; Mahadeo et al. 2010a). This abnormality is detected shortly after birth in early infancy. A recent report established that the FRa-null phenotype in humans is also associated with very low CSF folate levels and a clinical ­syndrome of cerebral folate deficiency (Steinfeld et al. 2009). The mechanism by which PCFT contributes to folate transport into the CSF is unclear. PCFT may contribute to export of folates from endosomes during FRa-mediated endocytosis (Zhao et al. 2009b). Sodium/proton exchangers present at the basolateral membrane of choroid plexus ependymal cells may produce an acidic local microenvironment  that provides the driving force for PCFT-mediated transport at this site (Segal 2000). In Sects. 1.3 and 1.4, we consider the structure, function, and regulation of RFC and PCFT as a prelude to understanding their roles as determinants of antifolate drug response and resistance in cancer. In Sect. 1.5, we examine the roles of these physiologically important facilitative transporters in antifolate chemotherapy, in general, and in relation to applications of folate-based therapeutics with tumor targeting via FRs and PCFT.

1.3 Reduced Folate Carrier 1.3.1 RFC Functional and Structural Characteristics Properties of RFC have been characterized in a wide assortment of (mostly tumor) cell culture models (both rodent and human). Transport by RFC is temperature dependent, sodium independent, and is characterized by a neutral pH optimum (Matherly et al. 2007). RFC substrates are structurally diverse with modifications of the ring systems including aromaticity and/or substituents, along with the presence or absence of heteroatoms, the length and composition of the bridge linker between the rings, and replacement of the terminal l-glutamate (Jansen 1999; Westerhof et al. 1995) (Fig. 1.1). The major circulating folate form, 5-methylTHF, is an excellent RFC O NH2 N H2N

N

O

COOH N H

N

H COOH N H2N N N H O Pemetrexed (PMX) COOH

N CH3 COOH N Methotrexate (MTX) H N

O N

O HN H3C

N

GW1843U89

COOH COOH H N 2

N N

N N

H3C

N H

N H PT523

N S HN CH3

HN

O NH2

O

O

HN

N

Raltitrexed (RTX) COOH

COOH

HN

O COOH

Fig. 1.1  Antifolate structures

COOH

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substrate as is the active (6S)5-formyl tetrahydrofolate (5-formylTHF) isomer in pharmacologic formulations of folate (i.e., (6R,S)5-formylTHF or leucovorin). Transport by RFC is not stereospecific for 5-methylTHF (White et al. 1978), in contrast to 5-formylTHF for which the (6S) stereoisomer is preferred over the (6R) form (Sirotnak et al. 1979). Classical antifolates such as MTX, pemetrexed, and raltitrexed (RTX) (Fig.  1.1) are all RFC substrates (Matherly et  al. 2007). These reduced folate and antifolate substrates show saturability at low micromolar concentrations. By contrast, the synthetic form, folic acid, has been generally reported as a poor RFC substrate (Kt > 200  mM), representing a distinguishing feature between RFC and FRs or PCFT that have high affinity for folic acid. The benzoquinazoline antifolate GW1843U89 (Smith et al. 1999) and the hemiphthaloylornithine antifolate PT523 (Rosowsky 1999) (Fig. 1.1) are the best RFC substrates known with binding affinities (Kt and Ki) in the submicromolar range and a complete lack of substrate activity with PCFT (Deng et  al. 2009; Zhao and Goldman 2007). Pralatrexate (10-propargyl-10-deazaaminopterin) (Sirotnak et al. 1987) was recently approved for treatment of relapsed or refractory peripheral T-cell lymphoma (Thompson 2009), based on potent antitumor effects, reflecting efficient transport by RFC, high affinity for folylpolyglutamate synthetase, resulting in rapid and extensive metabolism to its polyglutamate derivatives. The most consistent structural feature of RFC substrates relates to their anionic character. Folates are negatively charged at physiologic pH, resulting from ionized a and g carboxyl groups. Some modifications of the glutamate moiety (e.g., 2-amino-4-phosphonobutanoic acid, l-homocysteic acid, ornithine) are not conducive to RFC binding and transport (Westerhof et al. 1995). Likewise, ICI198583-gd-glutamate is a poor transport substrate for RFC, in contrast to the l-isomer (Westerhof et  al. 1995). Conversely, modifications of the glutamate-g-carboxyl (e.g., valine, 2-aminosuberate) are surprisingly well tolerated and both ZD9331 and PT523 are excellent RFC substrates (Jansen 1999; Westerhof et  al. 1995). For diaminofuro[2,3-d]pyrimidine antifolates with substituted a or g carboxyl groups, analogs with a single a but no g carboxyl group bind avidly to RFC, whereas analogs with a single g but no a carboxyl, or without both a and g carboxyl groups, do not bind appreciably to RFC (Deng et  al. 2008b). Collectively, these results imply that only the a carboxyl group of folate substrates is essential for substrate binding and transport by RFC. Although RFC generates only small transmembrane chemical gradients, when considered in light of the dianionic character of folates and the membrane potential, RFC produces substantial electrochemical–potential differences across cell membranes. Cellular uptake of folates by RFC is not directly linked to hydrolysis of ATP, nor is it sodium or proton dependent (Henderson and Zevely 1983; Goldman 1971). Rather, the driving force for concentrative uptake of folates appears to involve large gradients for organic phosphates across cell membranes which inhibit folate export via RFC, resulting in uphill folate transport into cells (Goldman 1971). Consistent with this model are findings that phosphorylated derivatives of thiamine are good RFC substrates. Their presence

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in cells inhibits MTX export and their efflux is enhanced in cells with elevated RFC levels (Zhao et al. 2001, 2002). RFC is a member of the major facilitator superfamily (MFS) of proteins comprising of more than 2,000 sequenced members including transporters of amino acids, sugars, vitamins, nucleosides, and organic phosphates, along with neurotransmitters (Matherly et al. 2007; Saier et al. 1999). By computer hydropathy analysis based on the predicted amino acid sequence from cloned RFC cDNAs from various species (Matherly et al. 2007; Matherly and Hou 2008), the carrier conforms to a structure typical of MFS proteins including two bundles of six transmembrane domains (TMDs) connected by a large loop domain between TMDs 6 and 7 and internally oriented N- and C-termini (Fig.  1.2). Much of this topology has been experimentally confirmed by hemagglutinin epitope insertion and cysteinescanning mutagenesis and accessibility studies (Ferguson and Flintoff 1999; Flintoff et al. 2003; Cao and Matherly 2004; Liu and Matherly 2002). Glycosylation of the single N-glycosylation consensus site at asparagine 58 in human RFC establishes the TMDs 2–3 connecting loop as extracellular (Liu and Matherly 2002). On SDS gels, human RFC appears as a broadly migrating high molecular weight (~85  kDa) species, which shifts to 65  kDa upon treatment with N-glycosidase F (Wong et al. 1998, 1999). Mutation of Asn58 to Gln, abolishing N-glycosylation at this position, has only a nominal effect on surface targeting or membrane transport of human RFC (Wong et al. 1998). There is 64–66% conservation of amino acid sequence between human and rodent RFCs, with somewhat higher homology in TMDs 1–5, 7, and 8, lower homology for TMDs 6 and 9–12, and several of the connecting loops (Matherly and Hou 2008). Both N- and C-termini exhibit low homology. The RFC C-terminus in primates is 50–86 residues longer than that of other species. RFC structure and function have been studied extensively using state-of-the-art molecular and biochemical techniques for polytopic membrane proteins (Matherly and Hou 2008). Deletions of the N- and C-termini of RFC from hamsters and humans had only minor impact on membrane targeting and transport activity (Sadlish et al. 2002a; Sharina et al. 2002). Deletions of 49 or 60 amino acids of the connecting loop between TMDs 6 and 7 of human RFC abolished activity, whereas replacement of the deleted segments with the nonhomologous loop from the MFS protein SLC19A2 restored transport (Liu et al. 2003). Human RFC was reconstituted in cells from coexpressed TMDs 1–6 and 7–12 RFC half-molecules which co-fold and traffic to the cell surface to restore transport with characteristic properties ranging from kinetics to capacity for trans-stimulation (Witt et al. 2004). Thus, neither the N- or C-termini, nor the TMDs 6–7 loop domain appears to directly participate in substrate binding and translocation of folate substrates. Rather, the primary role of the connecting loop between TMDs 6 and 7 is to provide the requisite spacing between two 6 TMD segments. By exhaustive cysteine-scanning mutagenesis and substituted cysteine accessibility methods (SCAM) of a cysteine-less human RFC, TMDs 4, 5, 7, 8, 10, and 11 were identified as forming the membrane translocation pathway for anionic folates (Hou et  al. 2005, 2006). Of 282 Cys substitutions, only ten were inactivating

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T T N P S D A V R G L PV E V T L R N I K G Y Extracellular F W Q R N S F F L E G V S S E I Q G T I T A D L Q L V P M P A S L H R M Y R H V T R H P 360 S 436 S 355 H 418 V 40 Q I 178 G 186 N 288 V 117 A 73 S L 310 D 123 E I Y A S A Y A L Y V V V T Y F A L Y V V I S M I S F T F I S L W S G Y F Y S H T Y V L T I F L L L L A L C L G Y V L K L L A V I L T A G Y Q V L V L F Y S L G A G L L L A N S M A L G I A T A F I L V I T A A C F I R V V F V P F F Y W F I Q A L V S S W T F V S Y T T S A L Y V V N F W S T G L F L C S S F T S F L S L R F V A V F G L V Y W A V L G V M T A A L L R Q S G F G L L I G V A L L L L G Y D S R A D Q L G L F Q L C R I F Y 1 3 4 7 5 6 8 11 12 2 L 9 10 P L L L F V V V W L L L 337 L 379 R 264 K 204 K 328 R 456 A 159 E 394 24 S R 145 V 95 91 R Y T P R A R V K I R K H P L L R S P E P R S A C Q V P S L I S K W A RW Q R D P K A S S DG L E R L R Y R Y T P V E M S G E PG F Q I A G Q L P G R V R A G G H F A A V K P S L P H F L S P V M 1 G E A A S R L G Q A P P Q P R P V N NM R E H E S R K A L D D A A Q A L S V Q D K G L G G L Q P A Q L E Cytosol S G D R T S A S G V A G L S D E P S L P P R V C E L S A P E A G L F Q E A A R C Q P A A P S R Q S D P Y L A Q P V T S P T C L P C A S T A Q E D A A E P G S T S D G C P Q L A V H P P G V S K L G L Q C L P V QN V N Q

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Fig. 1.2  Topology model for human RFC monomer showing conserved residues. A topology model for human RFC, depicting the 12 TMDs, internally oriented N- and C-termini, the N-glycosylation site at Asn-58 and a cytosolic loop connecting TMDs 6 and 7. Amino acids conserved between RFCs from different species including Homo sapiens (human), Pan troglodytes (chimpanzee), Gallus gallus (chicken), Danio rerio (zebrafish), Bos taurus (cow), Rattus norvegicus (Norway rat), Cricetulus griseus (Chinese hamster), Mus musculus (mouse), and Xenopus laevis (African clawed frog) (Matherly and Hou 2008) are depicted as black circles

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including a stretch in TMD 4 (Arg133, Ile134, Ala135, Tyr136, Ser138), Tyr281 in TMD 7, Ser313 in TMD 8, and Arg373 in TMD 10, suggesting structural or functional importance (Hou et al. 2005, 2006). Arg133, Arg373, and Ser313 were previously identified as possibly mechanistically important from mutant studies (Liu and Matherly 2001; Sadlish et  al. 2002b; Zhao et  al. 1999; Sharina et  al. 2001). While the g carboxyl group of folate substrates was not essential for ­substrate binding to RFC, by N-hydroxysuccinimide [3H]MTX radioaffinity labeling of human RFC, Lys411 was nonetheless found to bind this region (Deng et  al. 2008b). From biochemical data for RFC, and solved structures for the bacterial MFS proteins, lactose/proton symporter (LacY) (Abramson et  al. 2003) and glycerol-3-phosphate/inorganic phosphate antiporters (GlpTs) (Huang et al. 2003), a three-dimensional homology model for the 591 amino acid human RFC was generated including a membrane translocation pathway comprised of TMDs 1, 2, 4, 5, 7, 8, 10, and 11, and functionally important roles for Ser281, Ser313, and Arg373 (Hou et al. 2006).

1.3.2 RFC Gene Structure and Regulation of RFC Expression and Function RFCs from humans and rodents are subject to elaborate regulation involving both transcriptional and posttranscriptional mechanisms (Matherly et  al. 2007). The human RFC gene maps to chromosome 21q22.2 (Moscow et al. 1995). The gene includes five major coding exons with conserved intron–exon boundaries and up to six alternative noncoding regions and promoters (designated A1/A2, A, B, C, D, and E) (Matherly et al. 2007; Whetstine et al. 2002a; Flatley et al. 2004). A, B, C, D, and E represent noncoding exons, whereas the A1/A2 noncoding sequence is fused to the first coding exon. Promoter activity was confirmed for the 5¢ regions proximal to five of the noncoding regions (A1/A2, A, B, C, and D) and for four of these, both tissue-specific (e.g., Ap2, C/EBp, Ikaros) and ubiquitously expressed (e.g., SP, USF) transcription factors and cis elements were identified (Matherly et  al. 2007; Flatley et  al. 2004; Whetstine and Matherly 2001; Whetstine et al. 2002b; Liu et al. 2004; Payton et al. 2005a, b). Thus, net RFC levels achieved in tissues are likely the combined result of levels and posttranscriptional modifications of these factors that determine the transcriptional activities of the multiple RFC promoters. This may be impacted by promoter polymorphisms (see below) and CpG methylation (Worm et al. 2001), as well as by chromatin remodeling. The upstream noncoding exons for the human RFC gene are alternately spliced to generate heterogenous transcripts comprising of (up to 15) unique untranslated regions (UTRs) linked to a common RFC coding sequence (Matherly et al. 2007; Whetstine et  al. 2002a). RFC 5¢ UTR diversity results in differences in 5¢ CAPdependent translation and transcript stabilities (Matherly et al. 2007; Payton et al. 2007).

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For two 5¢ UTRs (A1/A2 and A), upstream AUGs exist in-frame with the RFC coding sequence and result in N-terminally modified RFC proteins with 62 and 22 additional N-terminal amino acids, respectively (Flatley et al. 2004; Payton et al. 2007). However, the biological significance of these N-terminally modified RFC proteins is not well established. While human RFC transcripts and transport are reported to decrease in breast cancer and T-cell acute lymphoblastic leukemia (ALL) cell lines with folate deprivation (Ifergan et al. 2008), it is not clear whether this effect is transcriptional or posttranscriptional. The human RFC gene is polymorphic and includes high frequency polymorphisms involving nucleotide substitutions, deletions, and insertions in the RFC coding region (G80A, results in R27H in TMD 1), the A1/A2 promoter and noncoding region, and promoter A (Matherly et al. 2007; Flatley et al. 2004; Whetstine et al. 2001, 2002b). While the functional impact and broader health significance of these polymorphisms remain uncertain or even controversial, the 61 bp repeat polymorphism in promoter A is associated with increased promoter activity in reporter assays (Whetstine et al. 2002b). As noted above, transcript variants for human RFC were identified including a CATG insertion at position 191 that generates a frame shift and early translational stop at position 1176 in an MTX-resistant ALL cell line and primary ALL specimens (Wong et al. 1999; Whetstine et al. 2001). Additional human RFC transcript splice variants were reported, involving a 625 bp deletion from exon 7 (positions 1569–2193) and a 988 bp deletion (positions 1294–2281) including all of TMD 12 (Wong et al. 1995; Zhang et al. 1998a; Drori et al. 2000). The former encoded a variant RFC (Wong et  al. 1995) that was competent for transport whereas the latter encoded an inactive protein that nonetheless appeared to modulate wild-type RFC activity (Drori et al. 2000). Although posttranslational regulatory mechanisms involving RFC have been implied including RFC phosphorylation (Kumar et  al. 1997), this has not been confirmed. Studies have shown that 5-amino-4-imidazolecarboxamide ribonucleoside, a precursor of 5-amino-4-imidazole carboxamide ribonucleotide (ZMP), potentiates uptake of MTX and 5-formyl THF by CCRF-CEM ALL cells, presumably by RFC (McGuire et al. 2006). The mechanism is unclear.

1.3.3 Human RFC is a Homo-oligomer While considerations of RFC structure and mechanism have generally focused on RFC monomeric structures, human RFC was recently identified as a homo-oligomer (Hou and Matherly 2009). Thus, (a) crosslinking RFC with a homobifunctional crosslinker resulted in higher order complexes with molecular masses approximating those of dimers, trimers, and tetramers. (b) When coexpressed in RFC-null cells, RFC proteins with different epitope tags (Myc and hemagglutinin) were coimmunoprecipitated with epitope-specific antibodies. (c) In coexpression experiments between wild-type and inactive mutant RFC, a dominant-negative phenotype was

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demonstrated involving substantially decreased cell surface RFC of both wild-type and mutant carrier due to profoundly impaired cellular trafficking. Most recently, the operational significance of human RFC oligomerization and the “minimal functional unit” for transport were studied by negative-dominance experiments in which multimeric transporters composed of different ratios of active and inactive RFC monomers were coexpressed and by expressing covalent RFC dimers composed of active and inactive RFC monomers (Hou et  al. 2010). The results strongly support the notion that each RFC monomer comprises a single translocation pathway for anionic folate substrates and functions independently of other monomers. Hence, in spite of an oligomeric structure, human RFC functions as a monomer. Additional studies are clearly warranted to establish the broader mechanistic and/or regulatory features of RFC oligomerization, including the possibility that RFC oligomerization may have therapeutic implications. Oligomerization can regulate RFC trafficking from the endoplasmic reticulum to the cell surface (Hou et al. 2010) and thus may contribute to antifolate resistance in tumors expressing wild-type and mutant RFCs. Oligomerization may also have regulatory significance as a means of acutely responding to levels of extracellular folates via effects on intracellular trafficking. Whereas no unique biological roles for the Arg27His substitution resulting from the G80A polymorphism in human RFC (Matherly et  al. 2007) or N-terminally modified human RFC proteins (Flatley et  al. 2004; Payton et  al. 2007) have been established (see above), the possibility that these modifications may impact RFC function via effects on carrier oligomerization is not unreasonable. Likewise, no obvious biological significance has been ascribed to naturally occurring human RFC transcript variants (Wong et  al. 1995; Zhang et al. 1998a; Drori et al. 2000), although their encoded proteins can be envisaged to act as dominant-negative inhibitors via oligomerization with wild-type RFC, resulting in decreased levels of surface wild-type RFC protein.

1.4 Proton-Coupled Folate Transporter 1.4.1 Identification of the Molecular Entity Responsible for Low-pH Transport in Mammalian Cells RFC-mediated transport is a process with optimal activity at neutral pH (Matherly et al. 2007). Yet, an unexplained folate transport activity optimal at low pH had been recognized for decades. This was a characteristic of folate absorption in the small intestine and was noted for folate and antifolate transport into a variety of human (Selhub and Rosenberg 1981; Zhao et al. 2009a; Vincent et  al. 1985; Mason et  al. 1990; Mason and Rosenberg 1994; Horne et al. 1993; Zhao et al. 2004a), rat (Rajgopal et al. 2001; Said et al. 1997), and hamster (Assaraf et  al. 1998) cell lines. Indeed, a modest low-pH transport

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activity was observed in murine L1210 leukemia cells that had long been an invaluable model for studying the transport properties of RFC (Sierra et  al. 1997; Henderson and Strauss 1990). For the lack of a better explanation, the low-pH transport activity was attributed to functionally distinct alternatively spliced or translated RFC forms (Kumar et  al. 1998; Chiao et  al. 1997). However, no such species was identified that could account for this activity. With time, evidence accumulated indicating that the low-pH folate transport activity must be RFC independent. Most convincing were studies demonstrating that this activity was fully preserved even in the complete absence of genomic RFC (Zhao et  al. 2004a), in cell lines in which there were profound loss-of-function mutations of RFC (Chattopadhyay et  al. 2006; Wang et  al. 2005) or when the RFC gene was silenced (Zhao et al. 2005). Ultimately, this conundrum was resolved with the cloning of PCFT, designated as SLC46A1 in the solute carrier group of integral membrane transport proteins. PCFT was identified using a data mining cloning strategy in which genes with very low homology to RFC across species were identified and then screened by assessing their expression in two HeLa cell lines, both of which lacked genomic RFC, only one of which expressed the low-pH transport activity (Qiu et  al. 2006). The human PCFT gene is located on chromosome 17q11.2 and consists of five exons and encodes 459 amino acids (Fig. 1.3). The human protein shares 91% similarity and 87% identity to both the mouse and rat proteins.

1.4.2 A Comparison of the pH Dependence of PCFT-Mediated and RFC-Mediated Transport Figure 1.4 illustrates the pH profiles of tritiated MTX influx in HeLa cells that lack endogenous transporters and were stably transfected with either RFC or PCFT to achieve levels of expression comparable to those in wild-type HeLa cells. The pH profiles of these transporters are quite distinct. There is little RFC activity below pH 6.5, although a shoulder of residual activity is consistently observed at low pH (Wang et al. 2004). There is little PCFT activity above pH 7.0 when MTX is the transport substrate. The decline in RFC activity as the pH is reduced is due almost entirely to a decrease in influx Vmax; there is a minimal change in influx Km over a pH range of 7.4–5.5 (Wang et al. 2004). On the other hand, the decline in PCFT transport activity as the pH is increased is due to both an increase in influx Km and decreased influx Vmax (Qiu et al. 2006). Notably, changes in PCFT-mediated transport with pH depend on the transport substrate. For instance, while these changes are marked for MTX and folic acid, they are more modest for PCFTmediated pemetrexed transport so that sufficient delivery of this drug is achieved at neutral pH to maintain its activity even in the absence of RFC (Zhao et al. 2008). Similar findings of pH-dependent binding and substrate specificity were recently reported for novel pyrrolo[2,3-d]pyrimidine antifolate substrates for human PCFT (Wang et al. 2010; Kugel Desmoulin et al. 2010).

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INSIDE

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26721661 ]

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Fig. 1.3  Human PCFT genomic organization, predicted secondary structure, and functionally important residues. Human PCFT is located on chromosome 17q11.2 (UCSC genome browser, top panel) and consists of five exons. Exons 1–5 are shown in the middle panel. NM_080669.3 and NP_542400.3 are NCBI accession numbers for the mRNA and the protein sequence for human PCFT, respectively. NC_000017.10 represents the genomic sequence. A two-dimensional predicted topology model of human PCFT is shown at the bottom of the figure. Twelve TMDs are shown with the N- and C-termini located intracellularly. There are two confirmed N-linked glycosylation sites on the first extracellular loop at asparagines 58 and 68 (Unal et  al. 2008). Arrows indicate residues that play an important role in function as described in the text

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14 L.H. Matherly et al.

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[3H] MTX Influx (pmol/mg protein/2 min)

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pH Fig. 1.4  The pH profile of MTX influx mediated by PCFT or RFC. Influx of [3H]MTX influx (1 mM; 2 min) was assessed in transport buffers of different pHs. HBS buffer (20 mM HEPES, 5 mM dextrose, 140 mM NaCl, 5 mM KCl, and 2 mM MgCl2) was used to assess drug uptake at pH 7.0, 7.5, and 8.0. MBS buffer (20 mM MES, 5 mM dextrose, 140 mM NaCl, 5 mM KCl, and 2 mM MgCl2) was used for measuring drug influx at pH 5.0, 5.5, 6.0, and 6.5

1.4.3 PCFT-Mediated Transport Is Electrogenic and Proton-Coupled PCFT-mediated transport is electrogenic, as assessed by folate substrate-­dependent currents generated in Xenopus oocytes microinjected with PCFT cRNA. As the voltage gradient across the oocyte membrane is increased, inside negative, current increases. Likewise, current increases, as the extracellular pH is decreased. However, even when the extracellular pH is 7.4, voltage-dependent current ­persists (Qiu et al. 2006; Umapathy et al. 2007). This process is proton-coupled; that is, as a folate molecule is transported by PCFT, it is accompanied by protons and cellular acidification (Unal et al. 2009a). The current measured represents the flow of positive charges or protons. If it is assumed that the folate molecule is anionic over the pH range that PCFT operates in  vivo, based upon the pKa values of the glutamate moieties, the ratio of protons to folate transported must be greater than 1. Based upon an analysis of the relationship between proton concentration and PCFT-induced currents, a Hill coefficient of 0.6 was obtained; to account for this, the transported folate species was proposed to exist in a Zwitterionic form (Umapathy et  al. 2007). Definitive quantitation of proton coupling will require the direct and simultaneous measurement of current and uptake of a folate species in Xenopus oocytes. PCFT can also function as a ­proton channel. Hence, with the application of a large pH gradient across the oocyte membrane, current flows into the cell can be detected in the absence of folate substrate (Unal et al. 2009a).

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1.4.4 PCFT Functions at Neutral pH in the Absence of a pH Gradient While PCFT transport is proton-coupled, a proton gradient is not absolutely required for function. The evidence for this is compelling. (a) As indicated in Sect.  1.4.3, even in the absence of a pH gradient, folate transport and current is generated in Xenopus oocytes in the presence of a voltage gradient (Qiu et al. 2006; Zhao et al. 2008; Umapathy et al. 2007). This is relevant to mammalian cells which always sustain a transmembrane voltage gradient, inside negative. (b) In RFC-/ FR-null cells, PCFT expression at physiological levels is sufficient to provide folate substrate to meet growth requirements. Indeed, PCFT is the major route of folic acid transport into cells, even at pH 7.4 and in the presence of RFC (Zhao et al. 2008). (c) PCFT mediates pemetrexed growth inhibition in RFC/FR-null cells growing in vitro at neutral pH (see below) (Zhao et al. 2008). The ability of PCFT to deliver a cytotoxic dose of other classical antifolate drugs will depend on the level of PCFT expression and the local pH. Potent PCFT-selective antifolate substrates have been described (Wang et  al. 2010; Kugel Desmoulin et al. 2010; Matherly and Gangjee, 2011).

1.4.5 PCFT Structure: Homology Modeling PCFT has the predicted structure of a member of the solute carrier group of integral membrane transport proteins (Fig. 1.3). Using hydropathy predictive programs, and based upon the localization of the N- and C-termini to the cytosol as established by immunocytochemistry, there must be an even number of TMDs with a large intracellular loop separating an equal number of TMDs on either side (Unal et al. 2008; Qiu et  al. 2007). Studies using SCAM to localize extracellular and intracellular loops confirmed the predicted topological model with 12 TMDs (Zhao et al. 2010). There are two N-linked glycosylation sites in the first extracellular loop between the first and second TMDs. These residues are not required for PCFT targeting to the plasma membrane of HeLa cells nor for transport function (Unal et al. 2008). Homology modeling has been used in an attempt to characterize the threedimensional structure of eukaryotic solute carriers based upon the known crystal structures of bacterial transporters even when there is minimal sequence identity (Lemieux 2007). GlpT was found independently by two groups to be a best fit for PCFT (Unal et al. 2009a; Lasry et al. 2009). These models are, of course, hypothetical in the absence of experimental verification. One model predicted that the conserved His247 in the large intracellular loop was in hydrogen bond distance to a Ser172 in the third intracellular loop (Unal et al. 2009a). Experimental observations supported this prediction. Mutation of either residue to Ala produced the same unusual functional change, a decrease in the influx Km, consistent with an

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increase in the affinity of the carrier for its folate substrates. This was accompanied by a loss of selectivity among a variety of folates (folic acid, 5-methylTHF, and 5-formylTHF). From these results, it was proposed that these residues were located at the cytoplasmic opening of the translocation pathway and served as a “tether” that limited access to the folate-binding pocket. When the bond was severed, access was unimpeded and the selectivity of binding diminished. Little more can be said at this point regarding the validity of the models proposed. Further validation will require considerable additional experimental testing and verification.

1.4.6 The Structural Specificity of PCFT: Comparison with Other Folate Transporters PCFT has a specificity profile that distinguishes it from RFC. PCFT has a high affinity (Km ~ 1 mM) and RFC a very low affinity (Km ~ 200 mM) for folic acid at their optimal pH. Both have comparable good affinities for MTX and reduced folates. The affinity of PCFT for pemetrexed is lower than that of RFC at pH 7.4. RFC has a very high affinity (Km ~ 0.3 mM) and PCFT a very low affinity (Km > 50 mM) for PT523 at their optimal pH (Wang et al. 2004). These differences in affinities, two to three orders of magnitude, make it possible to use these agents to fully block transport mediated by one of these transporters in the presence of the other. The benzoquinazoline antifolate GW184389, with higher affinity for RFC, has also been reported to show very poor substrate activity for PCFT (Deng et al. 2009). Of particular importance are differences in antifolate affinities for PCFT at neutral pH. At pH 7.4, the influx Km for pemetrexed is 12  mM, while the influx Kis for MTX, RTX, and PT523 are 100, 90, and 250  mM, respectively (Zhao et  al. 2004b). Thus, PCFT maintains its selective and relatively high affinity for pemetrexed at neutral pH. Like RFC, PCFT is specific for folate monoglutamates (Qiu et al. 2007). While pemetrexed shows selectivity for PCFT over RFC, until recently, no analogous PCFT-selective substrate was reported. Very recent studies have identified novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with absolute PCFT selectivity over RFC (Wang et  al. 2010; Kugel Desmoulin et al. 2010) (see Matherly and Gangjee 2011). For the most potent of this series, Kis at both acidic and neutral pHs approximated those for pemetrexed. Potent growth inhibitory activity was due to inhibition of the folate-dependent de  novo purine nucleotide biosynthetic enzyme, glycinamide ribonucleotide formyltransferase (GARFTase) (Wang et al. 2010). Discrimination between PCFT and FRa based upon substrate selectivity is more challenging. FRa has a much higher affinity for folic acid than pemetrexed, while PCFT has a higher affinity for pemetrexed than folic acid. At neutral pH, the high affinity of FRa for folic acid is retained, but the affinity of PCFT for

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pemetrexed is decreased. Low concentrations of folic acid can be used to block FRa-mediated transport with minimal effect on pemetrexed transport mediated by PCFT irrespective of pH. While thieno[2,3-d]pyrimidine antifolates have been identified that are selective for FRs over PCFT and RFC (Deng et  al. 2009), pyrrolo[2,3-d]pyrimidine antifolate substrates without RFC activity were substrates for both FRs and PCFT (Deng et al. 2008a; Kugel Desmoulin et al. 2010; Wang et al. 2010) (see Matherly and Gangjee 2011).

1.4.7 Residues and Domains That Are Critical to PCFT Function Information is emerging on PCFT residues that are important to function (Fig. 1.3). Glu185 in human PCFT has been implicated in proton coupling. When Glu185 is mutated to Ala, transport activity is markedly impaired at pH 5.5, but is fully ­sustained at pH 7.4. Likewise, this mutant transporter was capable of undergoing hetero- and autoexchange at low and neutral pH, characteristic of a fully functional carrier (Unal et  al. 2009b). This persistence of function in the absence of a pH gradient indicated that the defect at low pH was due to impaired proton coupling. While it is possible that other residues are involved in PCFT-mediated translocation of protons across the cell membrane, it is clear that Glu185 is an irreplaceable component of this process. There are three fully conserved histidines in PCFT, and two of these play important functional roles (Fig. 1.3). His247 in the large central cytoplasmic loop, at the cytoplasmic opening of the putative translocation pathway, appears to play a role in modulating access of folates to their binding pocket. Data suggest that this is associated with an interaction between this residue and Ser172, another fully conserved residue in the third intracellular loop (Unal et al. 2009b). His281, located close to the extracellular opening of the translocation pathway in the seventh TMD, is an important determinant of proton binding which, in turn, allosterically modifies the affinity for folate in the binding pocket. When this residue is mutated to Ala, the influx Km for folate is markedly increased but this can be substantially reversed by a decrease in pH below the optimal level for wild-type PCFT (Unal et al. 2009b). Other residues, critical for PCFT function, have been identified in patients with hereditary folate malabsorption (see below). Hence, Arg113 in the second intracellular loop and Arg376 in the tenth TMD are critical for function (Zhao et al. 2007; Lasry et al. 2008). While substitution of Arg113 with a like-charged residue (i.e., Lys) restores a very low level of activity ( 100 mM). This contrasts with the naturally occurring folate cofactors which, in addition to displaying high ­affinity for the FRa (Kd ~ 1 nM), also display an affinity for the high capacity RFC (Km of ~1 mM) commensurate with it being the most relevant transporter for these cofactors. Analogs of CB3717, and compounds in related chemical series from other laboratories, had followed, but they contrasted by having higher affinity for the RFC (Jackman et  al. 2007). The TS inhibitors raltitrexed, pemetrexed, plevitrexed (ZD9331; BGC 9331; Fig. 5.1), and BW1843U89, as well as the GARFT inhibitor lometrexol (DDATHF; Fig. 5.1), were all shown to display similar high affinity for FRa; they were also found capable of being transported via either transporter in one or more members of a panel of L1210 mouse and CCRF-CEM human leukemic subcell lines that expressed one or both of the transporters, and in a variety of low or subphysiological folate conditions (Westerhof et  al. 1995b). Other ­compounds showed a preference for just one of the transporters, but those without the classical glutamate or related amino acid sidechain, for example, nolatrexed (Thymitaq; AG337) had very low affinity for both transporters. Studies in human KB epidermoid and IGROV-1 ovarian cancer cell lines, which naturally express

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both ­transporters, evaluated a subset of these compounds in various folate conditions to confirm the low pharmacological importance of FRa for the activity of raltitrexed and lometrexol. However, FRa-mediated activity was demonstrated with CB3717 in the high FRa-expressing KB cell line (Westerhof et al. 1995a). The activity of a range of antifolates in a panel of L1210 sublines is shown in Table 5.3 demonstrating that they can all inhibit the proliferation of RFC-deficient L1210-FBP cells, some very potently, through an FRa-mediated mechanism. L1210 parental cells (FR downregulated) and L1210-1565 (RFC-negative/FR downregulated) display equally low sensitivity to CB3717, consistent with the RFC not being a relevant transporter for CB3717. Note that all the antifolates have low micromolar IC50 in the L1210-1565 cells (nonfunctional RFC) demonstrating that other “low affinity” transporters are present. This is probably the case for most cell lines and this theme is expanded upon later because of the pharmacological relevance of these transporters for FR-targeted antifolates. Theti and Jackman (2004) replaced the supraphysiological levels of folic acid in commercial medium with a physiologically relevant concentration of reduced-folate (20 nM folinic acid) and used this to study the activity of a range of antifolate drugs in the RFC-positive KB, A431 (FR-negative), and A431-FBP (transfected with FRa) human cell lines. Raltitrexed, pemetrexed, plevitrexed (BGC 9331; ZD9331; Fig.  5.1), and lometrexol were three- to fivefold more potent in A431-FBP than A431 cells, or one- to eightfold in KB cells compared with KB cells in which 1 mM folic acid was added

Table 5.3  Inhibition of the proliferation of a panel of mouse L1210 leukemia cell lines with different functional capacity for RFC or FR-mediated transport by antifolates Inhibition of proliferation, IC 50 (mM) L1210 L1210-1565 L1210-FBP L1210-FBP + 1 mM FA Antifolate (primary target) RFC+/FR− RFC−/FR− RFC−/FR++ RFC−/FR− Methotrexate (DHFR) 0.011 1.1 0.002 1.6 Lometrexol (GARFT) 0.11 4.7 0.00036 1.2 CB3717 (TS) 5.0 a 3.8 a 0.0015 a 1.6 Raltitrexed (TS) 0.0088 0.76 0.00032 0.83 Pemetrexed (TS) 0.034 2.0 0.00057 1.1 Plevitrexed (9331) (TS) 0.024 b 1.4 b 0.00077 b 0.95 b b b b ONX 0801 (TS) 7.6  6.4  0.00002  1.1 b L1210 and L1210-1565 cells were grown in standard commercial RPMI media (supraphysiological concentration of folic acid, FA; 2.2) which downregulates the FR. L1210-1565 do not have a functional RFC but survive because FA can be taken up by non-RFC-mediated mechanisms when concentrations are unnaturally high. L1210-FBP cells (RFC-negative) were cultured in the more biologically relevant synthetic reduced-folate cofactor, folinic acid, making them dependent on FR upregulation for folate uptake and survival. The experiments were performed with a low concentration (2 nM) to maintain the FR in a highly upregulated state. FA (1 mM) was added in some experiments to competitively inhibit binding of antifolates to the FR. IC50 were determined after 72 h exposure to antifolates. CB3717 and ONX 0801 are the only antifolates that display a profile consistent with selective FR-mediated transport a Jackman et al. (2004) b Gibbs et al. (2005)

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to competitively and selectively inhibit binding of the antifolates to the FRa (RFC binding is unaffected by this concentration of folic acid) (Table 5.4). It was therefore concluded that tumors with very high FRa expression could be a marginally more sensitive subset. Recently, a study failed to demonstrate a correlation between FRa protein expression and clinical response to pemetrexed in mesothelioma (Nutt et  al. 2010). This may be a consequence of low expression levels in this disease reported by at least some investigators (Table  5.1). Interestingly, two studies in advanced NSCL cancers have demonstrated increased efficacy of pemetrexed in adenocarcinomas compared to squamous cell carcinomas (Scagliotti et  al. 2008; Ciuleanu et  al. 2009). FR expression was not examined in these studies but ­interestingly FRa expression is reported to be higher in adenocarcinomas compared to squamous cell carcinomas in NSCLC (Franklin et al. 1994; Iwakiri et al. 2008). Similar studies may be warranted in ovarian cancer. Nevertheless, because substantial uptake of both of these drugs occurs via the RFC, toxic effects on normal proliferating tissues prevent these drugs exploiting FR overexpression. Some insight into the dynamic process of FRa-mediated antifolate transport and intracellular trafficking in L1210-FBP cells (RFC-negative) revealed that FR binding affinity is generally a dominant determinant to achieve rapid target inhibition (Mauritz et al. 2008). Within a subset of DHFR inhibitors the degree to which polyglutamates formed was also a key feature. On the other hand, the nonpolyglutamatable TS inhibitor plevitrexed was extremely potent at rapidly inhibiting TS in these cells. Theti and Jackman (2004) focussed on trafficking aspects that could potentially influence therapeutic index in vivo. Plevitrexed rapidly inhibited TS in A431, A431-FBP, and KB cells attributable to uptake via the RFC. However, in contrast with the polyglutamatable antifolates, plevitrexed displayed very low Table 5.4  Inhibition of the proliferation of a panel of RFC-expressing human tumor cell lines with different functional capacity for FR-mediated transport by antifolates Inhibition of proliferation, IC 50 (mM) A431-FBP A431 KB KB + 1 mM FA Antifolate (primary target) RFC+/FR++ RFC+/FR− RFC+/FR++ RFC+/FR− Methotrexate (DHFR) a 0.027 0.032 0.023 0.020 Lometrexol (GARFT) a 0.0024 0.0091 0.043 0.087 CB3717 (TS) 0.25 1.3 0.0067 0.58 Raltitrexed (TS) a 0.00073 0.0031 0.0011 0.0012 Pemetrexed (TS) a 0.014 0.040 0.0065 0.055 Plevitrexed (9331) (TS) a 0.016 0.086 0.0036 0.011 0.0011 6.6 0.0033 4.8 ONX 0801 (TS) b A431 and KB epidermoid tumor cell lines express the RFC and are grown in a physiological concentration of a reduced-folate cofactor, folinic acid (20 nM). A431-FBP cells have been transfected with the FR and KB cells naturally overexpress the FR. IC50 were determined after 72 h exposure to antifolates. ONX 0801 and CB3717 are the only antifolates that display a profile consistent with selective FR-mediated transport in both FR-positive cell lines, with ONX 0801 being the most potent a  Theti and Jackman (2004) b  Gibbs et al. (2005)

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antiproliferative potency in A431 cells when exposure was shortened to 24 h or less (IC50 ³ 100 mM) (achieved by replacing the medium with drug-free medium at various times and measuring the IC50 at 72 h) (Theti and Jackman 2004). This is a property of nonpolyglutamatable antifolates such as plevitrexed which are able to efflux freely out of cells because TS needs to be inhibited for approximately a generation time (~24  h) before an irreversible commitment to apoptosis is induced (Jackson et al. 1983; Webley et al. 2000, 2001). Surprisingly, IC50 were two to three orders of magnitude lower for plevitrexed in A431-FBP cells, and this led to the hypothesis that the high affinity FRa allows the drug to accumulate in the FR/endosomal apparatus leading to continued trafficking into the cytosolic compartment and inhibition of TS. These data suggested to us that an FRa-targeted TS inhibitor would display a more advantageous therapeutic index if it was not polyglutamated, so that when concentrations are high following intravenous (i.v.) bolus/short infusion, and consequently if it becomes driven into normal proliferating tissues via non-FR mechanisms, it would not become trapped as a polyglutamate form.

5.5 Discovery of FR-Targeted Thymidylate Synthase Inhibitors Although the folate-based TS inhibitors described can display elements of FR-mediated tumor targeting, their use as targeted agents is complicated by their uptake via the high capacity and ubiquitously expressed RFC. However, some ­properties of CB3717 (low RFC affinity) and plevitrexed (potent TS inhibitor that is not polyglutamated) were seen as attractive starting points for the design of FR-targeted TS inhibitors that would accumulate less in normal proliferating tissues and therefore be less toxic than conventional antifolates. Several members of a series of quinazoline-based TS inhibitors, constructed with l-glu-g-d amino acid dipeptide ligands in place of the glutamate normally found in folate or folate antagonists, were very potent inhibitors of TS (Ki ~ 1 nM) with low affinity for the RFC; however, data partly implicated the RFC in their cytotoxic activity (Bavetsias et  al. 1996; Jackman et  al. 2004). The dipeptide ligands used were stable to ­enzymatic hydrolysis in mice and there was evidence that they also prevented ­polyglutamation (Bavetsias et al. 1996, 2001). FRa-mediated activity was demonstrated in L1210-FBP cells for some compounds in this series, such as CB30523 and CB30901 (Jackman et al. 2004). A further series of cyclopenta[g]quinazolines with dipeptide ligands proved to be particularly interesting in that potency for TS inhibition was increased by an order of magnitude and RFC-mediated activity was low or absent, leading to low antiproliferative potency in tumor cells in which FRs were not expressed (Bavetsias et al. 2000). CB300638 (l-glu-g-d-glu ligand) was a lead compound from this series (TS Ki 0.24 nM) that displayed a consistently high level of FRa-mediated activity in L1210-FBP, A431-FBP, and KB cells (Theti et al. 2003; Jackman et  al. 2004). Its affinity for FRa is similar to that of folic acid (~50%) and indeed similar to the conventional antifolate TS inhibitors described above. Later the 2-methyl group was replaced with 2-hydroxymethyl (CB300945;

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BGC 945; ONX 0801; Fig. 5.1), maintaining high affinity for FRa but reducing the TS inhibitory potency fivefold to 1.2 nM (Gibbs et al. 2005; Henderson et al. 2006). Surprisingly, this was not associated with reduced antiproliferative potency compared with CB300638 in FRa positive tumor cell lines, and we concluded that ONX 0801 was trafficked via the FRa endosomal apparatus more efficiently than was CB300638. RFC-mediated uptake was virtually eliminated with this 2-hydroxymethyl modification as evidenced by very low affinity for the RFC (Km > 250 mM) and preservation of antiproliferative activity in a mouse L1210 subline (L1210-1565) that does not express the RFC and is, therefore, resistant to conventional antifolates (Table  5.3). ONX 0801 displays an order of magnitude decrease in potency compared with CB300638 in non-FRa-expressing cell lines, predicting for a large therapeutic window in vivo (Fig. 5.3).

5.6 ONX 0801 (BGC 945) 5.6.1 In Vitro Properties ONX 0801 binds to FRa with similar very high affinity in A431-FBP, L1210-FBP, and KB cells (Gibbs et  al. 2005; van der Heijden et  al. 2009) (Table  5.2). Consequently, ONX 0801 potently inhibits the proliferation of FRa-expressing tumor cell lines. Mouse L1210-FBP cells (RFC-negative) were found to be highly sensitive with an IC50 value of 0.02 nM but remarkably the IC50 increased to 1.1 mM when 1 mM folic acid was added to saturate FRs on the cell surface, providing the first evidence that the FRa could deliver ONX 0801 into tumor cells (Gibbs et al. 2005) (Table  5.3). In human tumor cell lines that additionally express the RFC, such as A431-FBP, KB, IGROV-1, and JEG-3, the 72 h exposure IC50 range from 0.001 to 0.32 mM (potency related to FR number) but increase to 2–7 mM when 1 mM folic acid is added, similar to the 7 mM IC50 for the FR-negative A431 cells (Table  5.3) (Gibbs et  al. 2005). This suggests a concentration “threshold” of ~2–7  mM at and above which non-FR-mediated uptake becomes increasingly ­relevant, with its associated potential to cause toxicity in vivo if these ­concentrations were maintained. It is unknown what the additional transport mechanism(s) is, but it is probably one of the non-RFC ­mechanisms used by a range of other antifolate drugs at these concentrations. However, because TS needs to be inhibited for approximately 24  h before cells become committed to apoptosis (Jackson et  al. 1983; Jackman et al. 1984; Webley et al. 2000, 2001), short-term exposure to high ONX 0801 concentrations in vivo was considered unlikely to be a problem, particularly as the drug was designed not to be polyglutamated so that it could readily efflux from non FR-expressing cells. This was modeled in  vitro using cultured FR-negative A431 cells. TS activity was not significantly inhibited when A431 cells were exposed to 1 mM ONX 0801 for 4 h (Fig. 5.4a) which contrasts sharply with the potent activity of RFC-mediated TS inhibitors such as plevitrexed

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Fig. 5.4  Inhibition of TS by 1 and 10 mM ONX 0801 in KB and A431 cells – 4 h exposure. Cells were exposed in vitro to 1 and 10 mM ONX 0801 for 4 h, and TS activity measured in intact cells. Consistent with FR-mediated uptake being a slow process, 1 mM inhibited TS by just ~50% in KB cells and this was prevented by the co-addition of 1 mM FA. No inhibition was observed in A431 FR negative cells. In contrast, 10 mM ONX 0801 substantially inhibited TS in both cell lines and this was not prevented by FA, demonstrating that non-FR-mediated uptake occurs at this concentration Table 5.5  Sensitivity of human tumor cell lines to short exposure ONX 0801 Exposure Inhibition of proliferation, IC 50 (mM) – measured at 72 h A431-FBP (FR+++) A431 KB (FR+++) KB + FA JEG-3 (FR+) JEG-3 + FA time (h) >30 a 4 1 >30 20 >100 >30 a b b a 24 0.002  8.0    0.8 30 >30  >30 a 48 0.002 b 5.0 b   0.003 8.0 – – 72 0.001 6.9   0.003 4.8 0.32 1.9 Cells were exposed to ONX 0801 for 4, 24 and 48 h, the medium was replaced with drug-free medium and the incubation continued until a total time of 72 h had elapsed. The IC50 were determined using an MTT assay as the endpoint. In addition, KB and JEG-3 cells were incubated with 1 mM folic acid to inhibit binding of ONX 0801 to FRa. The data are published in Gibbs et al. (2005) except where indicated a  Unpublished data b  Theti (2002)

(0.01% of control at 0.01 mM). However, TS activity was inhibited by two orders of magnitude when A431 cells were exposed to 10  mM ONX 0801 (Fig.  5.4b), consistent with non-FR-mediated uptake being relevant at the higher dose. TS activity recovered when the medium was replaced with drug-free medium (data not shown) and consequently no cytotoxicity was induced (i.e., no inhibition at 30 mM) (Table  5.5). These data predict that, for i.v. doses of ONX 0801 that generate plasma levels of ³10 mM in the first few hours, rapid and substantial TS inhibition will occur in tumor (regardless of FR status) and normal proliferating tissues, driven by non-FR-mediated uptake of the drug. However, provided that plasma levels fall to levels that allow at least some recovery of TS activity within ~24 h, no significant adverse effects should be observed in normal proliferating tissues. The 24 h exposure IC50 of ONX 0801 for inhibition of A431 cells is 8 mM.

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In contrast with A431 cells, FR-positive tumor cells are highly sensitive to FR-mediated effects of ONX 0801 although, because FR-mediated transport is low capacity and slow, the timescale for both TS inhibition and TS recovery is slower than that observed with the non-FR-mediated effects described above. TS activity in KB cells was only inhibited by 50% after 4  h exposure to 1  mM ONX 0801 (Fig.  5.4a), whereas it was inhibited by two orders of magnitude by 0.03  mM plevitrexed (data not shown). However, TS was also inhibited by this degree after 24  h exposure to 0.03  mM ONX 0801 (Fig.  5.5). Although this is sufficient to inhibit the proliferation of KB cells via FR-mediated mechanisms (24 h exposure IC50, 0.8  mM), increasing the length of exposure to increase the time that TS is inhibited markedly increases the sensitivity (48  h exposure IC50, 0.003  mM) (Table 5.5). The low FR-expressing JEG-3 cells are less sensitive to FR-mediated TS inhibition induced by ONX 0801, and although 0.3 mM ONX 0801 inhibits TS by ~80% at 24 h, the duration of inhibition is not sufficient to inhibit proliferation (24 h exposure IC50, >30 mM) (Fig. 5.5 and Table 5.5). The longer that JEG-3 cells are exposed to ONX 0801 the greater their sensitivity to its effects. Significant levels of FR-mediated inhibition of proliferation and induction of apoptosis were seen following 120 h exposure to 0.2 and 1 mM ONX 0801 without introducing any ­non-FR-mediated effects (Gibbs et  al. 2005). This predicts that a schedule that gives prolonged plasma levels of ONX 0801 in approximately the 0.1–1 mM range should be associated with activity in cancer patients with FR-positive tumors, ­without inducing TS inhibition in normal proliferating tissues. Furthermore, lower exposure (concentration and/or duration) would be expected to have activity in high FR-expressing tumors such as many ovarian cancers. These data suggest that plasma drug levels in Phase 1 clinical trial should be targeted to avoid prolonged non-FR-mediated uptake while achieving prolonged FR-mediated uptake selectively in tumors.

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Fig. 5.5  Inhibition of TS by 0.03 and 0.3 mM ONX 0801 in KB and JEG-3 tumor cell lines – 24 h exposure. Cells were exposed in vitro to 0.03 and 0.3 mM ONX 0801 for 24 h and TS activity measured in intact cells (unpublished data). No inhibition was observed in A431 FR-negative cells and the extent of inhibition in the FR-positive cells was dependent on the level of FR expression. The 3H-FA binding capacity: KB = 91 pmol/107 cells; JEG-3 = 3.1 pmol/107 cells

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5.6.2 Pharmacokinetics (PK) and Pharmacodynamics (PD) in Mice Plasma folate levels are ~5-fold higher in mice than in humans, most likely a result of the very high level of folic acid supplementation in standard mouse chow (Schmitz et al. 1994; Mathias et al. 1996; Leamon et al. 2008). This can antagonize the activity of antifolate drugs, but the problem can be addressed by feeding mice with chow without this excessive supplementation so that blood folate mirrors that in humans. More difficult to deal with is an issue specific to TS inhibitors. Rodents have very high plasma thymidine (dThd) that is ~100-fold higher than that observed in human plasma (Benepal et  al. 2003; Li et  al. 2007). This provides a bypass mechanism for thymidylate synthesis via the activity of thymidine kinase (TK) so that even when TS is inhibited, TS inhibitors display no proliferating tissue toxicity in mice or activity in human tumor xenografts after single bolus dosing (Fig. 5.6a). Repeating dosing for 5–14 days daily can increase sensitivity, but cannot be safely used to guide a starting dose or schedule in a Phase 1 clinical trial, at least with respect to TS-related effects. ONX 0801 administered at 200 mg/kg/day daily × 14 induced some growth delay in the human IGROV-1 tumor xenograft without toxicity (Ng et al. 2008). Dogs have lower dThd and consequently display higher sensitivity to antifolates and can be used to provide the safety data for regulatory studies. Mice have been useful for generating pharmacokinetics (PK) and pharmacodynamics (PD) relationship data with respect to TS inhibition in normal proliferating tissues and tumors. ONX 0801 is cleared very rapidly from mouse plasma (t1/2 = 2.1 h; 8.3- to 74-fold, respectively, compared to negative controls). Compounds 1, 2, and 4–6 were all completely inert toward PC4310 cells up to 1 mM, establishing a lack of RFC transport activity for this series. The 3-carbon analog of this series, compound 3, showed a low level activity toward PC43-10 cells. Compounds 2–6 were all active toward FRa-expressing RT16 cells in the order 3 = 4 > 5 > 6 > 2 (Table 6.1). For PCFT-expressing R2/PCFT4 cells, only compounds 3 and 4 inhibited growth, with 3 > 4 by ~9-fold. The potent growth inhibition by 3 and 4 toward PCFT-expressing cells can at least in part be explained by the reduced pH of the tissue culture media (to ~pH 6.7) over 4 days in culture (not shown) which favors cellular uptake by PCFT (Kugel Desmoulin et al. 2010). Additional series of analogs were tested including novel 6-substituted thieno [2,3-d]pyrimidine antifolates with an isosteric thieno for pyrrolo ring replacement and a 2–8 carbon bridge between the thieno[2,3-d]pyrimidine and benzoyl portions (compounds 7–13, respectively) (Deng et  al. 2009), and pyrrolo[2,3-d] pyrimidine thienoyl antifolates with an isosteric thienoyl for benzoyl replacement and 4–6 bridge carbons (compounds 14–16, respectively) (Wang et  al. 2010) (Fig. 6.2). Again, initial testing was based on the isogenic CHO cell line panel with established transport characteristics. Of the thieno[2,3-d]pyrimidine compounds 7–13, four were identified with FRa-targeted activity (8 = 9 > 10 > 11) (Table  6.1).

Table 6.1  In vitro cytotoxicities, FR affinities, and in situ GARFTase inhibitions for classical and novel antifolates Growth inhibition (IC50s, nM) PCFT RFC FRa Analog PC43-10 R2 RT16 RT16 (+FA) R2/PCFT4 R2(VC) Pyrrolo/1C/benzoyl >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 1 2 Pyrrolo/2C/benzoyl >1,000 >1,000 200(34) >1,000 >1,000 >1,000 3 Pyrrolo/3C/benzoyl 649(38) >1,000 4.1(1.6) >1,000 23.0(3.3) >1,000 4 Pyrrolo/4C/benzoyl >1,000 >1,000 6.3(1.6) >1,000 213(28) >1,000 5 Pyrrolo/5C/benzoyl >1,000 >1,000 54(21) >1,000 >1,000 >1,000 6 Pyrrolo/6C/benzoyl >1,000 >1,000 162(18) >1,000 >1,000 >1,000 7 Thieno/2C/benzoyl >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 8 Thieno/3C/benzoyl >1,000 >1,000 13.0(3.4) >1,000 >1,000 >1,000 9 Thieno/4C/benzoyl >1,000 >1,000 9.0(2.9) >1,000 >1,000 >1,000 10 Thieno/5C/benzoyl >1,000 >1,000 56.0(9.8) >1,000 >1,000 >1,000 11 Thieno/6C/benzoyl >1,000 >1,000 108(17) >1,000 >1,000 >1,000 12 Thieno/7C/benzoyl >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 13 Thieno/8C/benzoyl >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 14 Pyrrolo/4C/thienoyl >1,000 >1,000 1.8(0.3) >1,000 43.4(4.1) >1,000 15 Pyrrolo/5C/thienoyl >1,000 >1,000 4.5(1.6) >1,000 101.4(17.9) >1,000 Pyrrolo/6C/thienoyl 16 >1,000 >1,000 137(40) >1,000 >1,000 >1,000 FRa affinity 0.04 ND 0.59 0.81 0.75 0.65 0.48 0.95 0.93 0.85 0.84 0.95 0.77 1.2 0.93 0.59

(continued)

In situ GARFTase (IC50, nM) >20 ND 18 6.8 7.2 8.6 32.3 13.8 13.3 23.6 26.6 ND ND 0.63 7.7 ND

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Growth inhibition (IC50s, nM) RFC FRa PC43-10 R2 RT16 RT16 (+FA)

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FRa affinity

In situ GARFTase (IC50, nM)

MTX 12(1.1) 216(8.7) 114(31) 461(62) 120.5(16.8) >1,000 0.01 ND PMX 138(13) 894(93) 42(9) 388(68) 13.2(2.4) 974.0(18.1) 0.01 30 RTX 6.3(1.3) >1,000 15(5) >1,000 99.5(11.4) >1,000 ND ND LMTX 12(2.3) >1,000 12(8) 188(41) 38.6(5.3) >1,000 0.39 14 Growth inhibition assays for RT16, R2, R2/PCFT4, and R2(VC) were performed as described (Deng et al. 2008, 2009; Wang et al. 2010). For measurements of inhibitory effects on cell proliferation, the cell lines were cultured with the assorted antifolates over a range of concentrations (up to 1 mM) in standard RPMI1640 (PC43-10 cells), or with folate-free RPMI1640 supplemented with 3 nM (RT16) or 25 nM (R2/hPCFT4) leucovorin [(6R,S)5-formyl tetrahydrofolate], all with 10% dialyzed fetal bovine serum. Relative cell numbers were determined after 96 h with a fluorescence-based readout (Cell TiterBlue™) and results for growth inhibitions expressed as IC50 values (in nM). With RT16 CHO, KB, and IGROV1 cells, FR-mediated drug uptake was confirmed with a parallel culture treated with drugs and excess (200 nM) folic acid to block the FRs. For the PC43-10 and R2/hPCFT4 CHO sublines, results were compared to those for parental R2 cells or to vector-control R2 cells transfected with empty pcDNA3.1 vector [designated R2(VC)]. The data shown are mean values from 3 to 10 experiments (±standard errors in parentheses). IC50 data for classical antifolate compounds including MTX, RTX, LMTX, and PMX, along with the novel pyrrolo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine antifolates, are shown. As described in the text, growth inhibition assays were also performed with KB and IGROV1 human tumor cells cultured with antifolates as noted above for the RT16 CHO cells. Results are also summarized for relative binding affinities for FRa in RT16 cells by competitive binding assays with [3H]folic acid where the affinity for unlabeled folic acid is assigned a value of 1. These methods are described in our previous publications (Deng et al. 2008, 2009; Wang et al. 2010). The in situ GARFTase assays were performed in KB cells and involved measuring incorporation of [14C]glycine into formyl GAR (Deng et al. 2008, 2009; Wang et al. 2010). ND not detected

Analog

Table 6.1  (continued)

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None of this series (at 1  mM, the highest concentration tested) showed any growth inhibition toward RFC-expressing PC43-10 or PCFT-expressing R2/ PCFT4 cells. The finding of FR- but not PCFT-specificity for this series is particularly interesting given the report of possible direct functional coupling between these transport systems (Zhao et al. 2009b). Our results with thieno[2,3-d]pyrimidine antifolate substrates for FR establish that, should FR–PCFT coupling occur, this must not be obligatory. The pyrrolo[2,3-d]pyrimidine thienoyl antifolate 14 (4 carbon bridge) is the most potent agent yet identified with a fifty percent inhibition (IC50) at 1.8 nM with FRa-expressing RT-16 CHO cells (Table  6.1). Activity progressively decreased with the 5-carbon (compound 15) and 6-carbon (compound 16) analogs. Interestingly, growth inhibition toward PCFT-expressing R2/PCFT4 cells followed the same ­pattern as for FRa, i.e., 14 > 15 > 16. To extend results in the CHO models to human tumor cells, we tested lead ­compounds from each series, 3, 9, and 14, in human tumor cells that express FRa along with PCFT and RFC, including KB (cervical) and IGROV1 (ovarian) tumor cells (Fig.  6.1). Relative inhibitory potencies paralleled those in RT16 CHO cells (and for compounds 3 and 14, R2/PCFT4 cells) with 14 > 3 > 9 within a ninefold range for KB cells and a sixfold range for IGROV1 cells. For compound 14, IC50s of 0.55 and 0.97 nM, respectively, were recorded with KB and IGROV1 cells. Growth inhibitory effects were completely abolished in the presence of excess folic acid (200 nM). A similarly potent inhibition of colony formation for 14 was measured in KB cells (IC50 ~ 0.3 nM) over a long-term (10 day) drug exposure. For FRs, surface binding is reflective of cellular uptake by this mechanism and is best assessed by competition for high affinity binding with [3H]folic acid (Jansen 1999; Deng et  al. 2008, 2009; Wang et  al. 2010). With the antifolate drugs, relative substrate affinities were calculated with RT16 cells and were expressed as inverse molar ratios of unlabeled ligand required to inhibit binding of [3H]folic acid by 50% and were normalized to folic acid (assigned a value of 1). For most of the novel agents, relative affinities were higher than those for classical antifolates (e.g., LMTX) and only slightly less than the affinity for folic acid (Table 6.1). Importantly, these results show that while cellular uptake by FRa is an important determinant of drug effects, relative antiproliferative activities are not necessarily reflected as differences in binding affinities for FRa. None of the potent analogs 3, 9, and 14 showed evidence of substrate activity for RFC (reflected in competition for binding with [3H]MTX transport at pH 7.2 with 10 mM antifolate in RFC-expressing PC43-10 cells) (not shown). Analogous to PMX, compounds 3 and 14 were potent inhibitors of [3H]MTX transport by PCFT in R2/PCFT4 cells at pH 5.5. Both PCFT transport activity (not shown) and relative transport inhibition (Fig. 6.3) dramatically decreased with increasing pH from pH 5.5 to 7.2. Kis for compounds 3 and 14 at pH 5.5 and 6.8 in comparison with PMX are in Table 6.2. The potent RFC ­substrate PT523 (at 10 mM) did not inhibit transport by PCFT at any pH.

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Percent of Control

100

50

0 pH 5.5

pH 6.0

pH 6.5

pH 6.8

Compound 3

Pemetrexed

Compound 14

PT523

pH 7.2

Fig.  6.3  pH dependence for PCFT transport inhibition by pemetrexed (PMX) and the novel pyrrolo[2,3-d]pyrimidines 3 and 14. R2/PCFT4 cells were assayed for [3H]MTX (0.5  mM) at assorted pHs in MES- (pH 5.5–6.5) and HEPES- (pH 6.8 and 7.2)buffered saline in the presence of 10  mM inhibitors. Results are normalized to relative transport activity in the absence of any inhibitor and expressed as percent (as means ± SEM; n = 3) Table 6.2  Kinetic constants for human PCFT Substrate Parameter pH 5.5 pH 6.8 MTX 0.280 ± 0.022 4.52 ± 0.19 Kt (mM) 31.23 ± 4.31 13.72 ± 2.26 Vmax (pmol/mg/min) Vmax/Kt 111.5 3.0 PMX Kt (mM) 0.124 ± 0.014 1.33 ± 0.26 Vmax (pmol/mg/min) 27.11 ± 4.27 27.59 ± 6.38 Vmax/Kt 218.6 20.7 Compound 3 Ki (mM) 0.223 ± 0.017 4.07 ± 0.34 Compound 14 Ki (mM) 0.13 ± 0.01 1.95 ± 0.02 0.0960 ± 0.012 1.54 ± 0.17 PMX Ki (mM) Kinetic constants for MTX (Kt and Vmax) and PMX (Kt and Vmax) were determined with [3H]MTX and [3H]PMX, respectively, and calculated from Lineweaver Burke plots with R2/PCFT4 cells. Ki values were determined by Dixon plots with [3H]MTX as substrate and a range of inhibitor concentrations in R2/hPCFT4 cells. Results are presented as mean values ± standard errors from three experiments. These data were previously published (Wang et al. 2010; Kugel Desmoulin et al. 2010)

6.4 Identification of GARFTase as the Primary Cellular Target for 6-Substituted Pyrrolo- and Thieno[2,3-d]pyrrolopyrimidine Antifolates Since nucleoside salvage mechanisms circumvent biosynthetic requirements for reduced folates and growth inhibitory effects of classical antifolates such as MTX, we tested excess adenosine and thymidine for their capacities to abolish the growth

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inhibitory effects of the lead compounds from each series (compounds 3, 9, and 14). Adenosine (60 mM) completely obviated the growth inhibitory effects of all these analogs toward KB cells (up to 1 mM antifolate), whereas thymidine (10 mM) was ineffective, thus establishing de novo purine nucleotide biosynthesis as the targeted pathway (not shown). Since de novo purine nucleotide biosynthesis involves two folate-dependent steps, catalyzed by GARFTase and 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) formyltransferase (AICARFTase), additional protection experiments used 5-amino-4-imidazole carboxamide (AICA), a precursor of AICAR, which circumvents the step catalyzed by GARFTase (Fig. 6.4). For compounds 3 and 14, AICA (192–320 mM) protected cells from up to 1 mM antifolate. For 9, AICA was similarly protective at low and medium drug concentrations (£50 nM), whereas at higher drug concentrations, growth was progressively inhibited even in the presence of AICA. Thus, growth inhibitory effects of compounds 3, 9, and 14 must originate at the level of de novo purine nucleotide biosynthesis. For these analogs, GARFTase is likely the primary target, although at higher concentrations of 9, a secondary target (most likely AICARFTase) seems likely. To confirm GARFTase inhibition and to begin to establish structure–activity relationships for GARFTase inhibition, we used an in situ GARFTase assay with KB cells treated with the novel antifolate inhibitors (Table 6.1). A few analogs were also directly tested for their capacities to inhibit one-carbon transfer from 10-formyl-5,8-dideazafolic acid to b-GAR with recombinant mouse GARFTase (forming 5,8-dideazafolic acid and formyl glycinamide ribonucleotide [FGAR]). For the in situ assay, KB cells were labeled with [14C]glycine in the presence of azaserine such that [14C]FGAR accumulations were measured by an ion-exchange method (Deng et al. 2008, 2009; Wang et al. 2010). Results were compared to those for PMX and LMTX. For the active agents, IC50 values for in situ GARFTase inhibition of KB cells approximated those for cell growth inhibition (Table 6.1). Again, the most potent inhibitor was compound 14, which gave an IC50 value ~10-fold lower than its pyrrolo[2,3-d]pyrimidine benzoyl counterpart 4 and 20-fold more potent than the thieno[2,3-d]pyrimidine inhibitor 9. These differences in potency were at least partly preserved with purified GARFTase (IC50s of 60, 150, and 5,510 nM, for 14, 4 and 9, respectively). Differences in relative inhibitory potencies by the in situ cell-based assay vs. those measured with the in vitro cell-free assay likely reflect synthesis of antifolate polyglutamates and increased affinities for GARFTase by polyglutamyl over monoglutamyl antifolates within cells (see below). Thus, for GARFTase inhibition, the nature of the bicyclic heterocycle, the chain length of the bridge region, and the nature of the side chain aromatic ring all play important roles. Of the analogs in this study, 14 with a pyrrolo[2,3-d]pyrimidine and a 4-carbon bridge connecting a thieno ring is clearly optimal.

6.5 In Vivo Antitumor Efficacy with Compound 14 As proof-of-concept that in vivo antitumor efficacy can result from FR- and PCFTtargeting and inhibition of GARFTase, an in vivo efficacy trial was performed with severe combined immunodeficient (SCID) mice implanted with subcutaneous KB

O

N H

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−

Fumarate

OH

OH

H

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ATP ADP+PPi H2O

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NH2 N10-formyl-THF THF

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FGAM

HN R

OHC

H N

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O H

NH2

NH2

N CHO H

NH2

CO2

H2O

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OH

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Asp ATP ADP+PPi

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NH2

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Gly ATP ADP+PPi H3N

FAICAR

N R

N

O

AIR

N R

N

5-Phospho-β -D-ribosylamine

O Glu PPi O P O O

Fig. 6.4  De novo purine nucleotide biosynthesis pathway. The de novo purine nucleotide biosynthetic pathway from phosphoribosyl pyrophosphate (PRPP) to AMP is shown. There are two folate-dependent reactions in which 10-formyl tetrahydrofolate serves as the one carbon donor, GARFTase and AICARFTase. AICA can be metabolized to AICAR, thus circumventing the reaction catalyzed by GARFTase. For the in situ GARFTase assay, incorporation of [14C]glycine into formyl GAR (FGAR) in the presence of azaserine is used as a direct measure of GARFTase activity in cells (Deng et al. 2008, 2009; Wang et al. 2010). Previously undefined abbreviations are: FGAM 5¢-phosphoribosyl-N-formyl-glycinamidine; AIR 5¢-phosphoribosyl-5-aminoimidazole; CAIR 5¢-phosphoribosyl5-aminoimidazole-4-carboxylic acid; SAICAR 5¢-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole; FAICAR 5¢-phosphoribosyl-4-carboxamide5-formamidoimidazole; IMP inosine 5¢-monophosphate

COO

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Gln

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Glu ATP ADP+PPi

H O O H O P P O O O OH

PRPP

OH

H

O

NH2 N R SAICAR

N

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FGAR

HN R

OHC

H N

O O P O O

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Compound 14:180mg/kg/inj IV Days of Injection: 4, 8, 12, 16 or 29, 33, 37, 41 Median Tumor Burden (in mg)

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No Treatment Early Stage

1000 Late Stage 500

0

0

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30 40 50 60 70 Time Post Tumor Implant (Days)

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Fig. 6.5  In vivo efficacy trial with compound 14. Female ICR SCID mice (12 weeks old; 21 g average body weight) were maintained on a folate-deficient diet ad libitum for 27 days prior to subcutaneous tumor implant to decrease serum folate to a concentration approximating that in human serum. Human KB tumors were implanted bilaterally and mice were nonselectively randomized into 5 mice/ group. Compound 14 (dissolved in 5% ethanol (v/v), 1% Tween-80 (v/v), 0.5% NaHCO3) was administered on a Q4dx4 schedule intravenously (0.2 mL/injection) on days 4, 8, 12, and 16 for early stage disease or on days 29, 33, 37, and 41 for advanced stage disease beginning when the KB tumor burdens were 600–689 mg. Mice were observed and weighed daily; tumors were measured twice per week. For the experiment shown, activity was significant for both early (3.5 log kill, 1/5 cures; T-C = 47 days) and late (3.7 log kill, 4/5 complete remissions; T-C = 9 days) stage tumors

tumors (Wang et al. 2010). Mice were maintained ad libitum on a folate-deficient diet to reduce the serum folate concentration to a level approximating that in human serum. For early stage disease, compound 14 was administered intravenously on a Q4dx4 schedule (180 mg/kg/injection) on days 4, 8, 12, and 16 post-implantation. Advanced stage disease was included as a separate arm with median tumor burdens of 600–689 mg with 14 administered on days 29, 33, 37, and 41 (180 mg/kg/injection). Appreciable antitumor activities were measured for both early (3.5 log kill, 1/5 cures, T-C = 49 days) and advanced stage (3.7 log kill, 4/5 complete remissions, T-C = 49 days) tumors (Fig. 6.5). Minor weight losses were completely reversible and there were no other adverse symptoms up to 145 days. These results demonstrate the potent antitumor activity for compound 14 in vivo associated with significant transport by FRs and PCFT and a lack of transport by RFC.

6.6 Conclusions This chapter describes three related series of novel antifolates including 6-substituted pyrrolo- (1–6, 14–16) and thieno[2,3-d]pyrimidine (7–13) antifolates with bridge lengths from 1 to 8 carbons connecting the aromatic ring systems (Deng et  al.

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2008, 2009; Kugel Desmoulin et al. 2010; Wang et al. 2010) (Fig. 6.2). Compounds 1–13 include a side chain benzoyl ring, whereas the pyrrolo[2,3-d]pyrimidines 14–16 include a side chain isosteric thienoyl for benzoyl replacement resembling that in LY309887 (Mendelsohn et al. 1999), AG2034 (Boritzki et al. 1999), and RTX (Hughes et  al. 1999). The pyrrolo[2,3-d]pyrimidine thienoyl antifolate 14 including a 4-carbon bridge between the pyrrole and thiophene rings was the most potent agent toward cells expressing FRa and/or PCFT without detectable RFC activity. When comparing 14 to the benzoyl analogs (including both pyrroloand thieno[2,3-d]pyrimidines) with a 4-carbon bridge (compounds 4 and 9, respectively), 14 was ~3–9-fold more potent as an inhibitor of KB and IGROV1 human tumor cells (Deng et al. 2008, 2009; Wang et al. 2010). In vivo, with subcutaneous KB cells in SCID mice, 14 was highly active toward both early and late stage tumors. Differences in drug activities among the different antifolates were not reflected in relative affinities for FRa but rather in their inhibitory effects on GARFTase. Again, 14 was the most potent inhibitor of GARFTase, showing ~10-fold increased activity over its pyrrolo[2,3-d]pyrimidine benzoyl (4) counterpart in the in situ cellbased GARFTase assay. The dramatically increased potency of 14 in the in situ assay vs. the cell-free GARFTase enzyme assay likely reflects synthesis of polyglutamate derivatives of 14 and increased affinities for GARFTase by these conjugated drug forms in intact cells. The remarkably potent inhibition of nonpolyglutamyl 14 toward the isolated trifunctional GARFTase implies that this agent should be less impacted by polyglutamylation status than the other GARFTase inhibitors which are less potent in their monoglutamyl forms. Accordingly, 14 would be expected to inhibit GARFTase in cells even in its monoglutamyl form and be active toward drug-resistant tumors with decreased folypolyglutamate synthetase activity (Zhao and Goldman 2003). The pyrrolo[2,3-d]pyrimidines 3 and 14 (but not the thieno[2,3-d] pyrimidines) were also substrates for PCFT, as reflected in growth inhibition of the engineered R2/PCFT4 CHO cells, and in competitive inhibition of [3H]MTX uptake at pH 5.5 in this subline, to extents resembling that for the best-known PCFT substrate, PMX (Zhao and Goldman 2007; Deng et  al. 2009; Kugel Desmoulin et al. 2010). However, unlike PMX, 14 does not appear to be a substrate for RFC. For compound 3, there was a low level of activity toward RFC-expressing PC43-10 cells. Increased bridge carbons above 4 completely abolished PCFT but not FRa transport activity. The selectivity of these novel antifolates for FRs over RFC, as described in this chapter, is a paradigm for selective tumor targeting given the association of FRs with tumor cells such as ovarian or cervical cancers. Likewise, PCFT expression is abundant in many tumors such that therapeutic selectivity for cytotoxic PCFT substrates could result from a lack of RFC activity and optimal transport by PCFT at relatively low pH values approximating those in the interstitium of solid tumors. Clearly, drugs that target FRa and PCFT yet are not substrates for the ubiquitously expressed RFC, have the potential to selectively target tumor cells, and decrease toxicity to normal tissues, thus affording viable clinically useful antitumor agents with substantial advantages over chemotherapy drugs currently in use.

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Acknowledgments  This work was supported in part by grants from the National Institutes of Health, National Cancer Institute, CA53535 (LHM), CA125153 (AG), and CA152316 (LHM and AG), a grant from the Mesothelioma Applied Research Foundation (LHM), and a pilot grant from the Barbara Ann Karmanos Cancer Institute (LHM). We acknowledge the contributions of present and past members of the Matherly and Gangjee laboratories who contributed to the studies described in this chapter. Special thanks go to Dr. Lisa Polin of the Karmanos Cancer Institute who performed the in vivo mouse experiments with compound 14.

References Boritzki TJ, Zhang C, Bartlett CA, Jackson RC (1999) AG2034. A GARFT inhibitor with selective cytotoxicity to cells that lack a G1 checkpoint. In: Jackman AL (ed) Anticancer development guide: antifolate drugs in cancer therapy. Humana Press, Totowa, pp 281–292 Deng Y, Wang Y, Cherian C, Hou Z, Buck SA, Matherly LH, Gangjee A (2008) Synthesis and discovery of high affinity folate receptor-specific glycinamide ribonucleotide formyl transferase inhibitors with antitumor activity. J Med Chem. 51:5052–5063 Deng Y, Zhou X, Kugel Desmoulin S, Wu J, Cherian C, Hou Z, Matherly LH, Gangjee A (2009) Synthesis and biological activity of a novel series of 6-substituted thieno[2,3-d]pyrimidine antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors over the reduced folate carrier and proton-coupled folate transporter for cellular entry. J Med Chem 52:2940–2951 Diop-Bove NK, Wu J, Zhao R, Locker J, Goldman ID (2009) Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal transcriptional regulatory region in an antifolate-resistant HeLa cell line. Mol Cancer Ther 8:2424–2431 Elnakat H, Ratnam M (2004) Distribution, functionality and gene regulation of folate receptor isoforms: implications in targeted therapy. Adv Drug Deliv 56:1067–1084 Gangjee A, Zeng Y, McGuire JJ, Mehraein F, Kisliuk RL (2004) Synthesis of classical, threecarbon-bridged 5-substituted furo[2, 3-d]pyrimidine and 6-substituted pyrrolo[2, 3-d]pyrimidine analogues as antifolates. J Med Chem 47:6893–6901 Gangjee A, Zeng Y, McGuire JJ, Kisliuk RL (2005) Synthesis of classical, four-carbon bridged 5-substituted furo[2, 3-d]pyrimidine and 6-substituted pyrrolo[2, 3-d]pyrimidine analogues as antifolates. J Med Chem 48:5329–5336 Gibbs DD, Theti DS, Wood N, Green M, Raynaud F, Valenti M, Forster MD, Mitchell F, Bavetsias V, Henderson E, Jackman AL (2005) BGC 945, A novel tumor-selective thymidylate synthase inhibitor targeted to alpha-folate receptor-overexpressing tumors. Cancer Res 65:11721–11728 Gonen N, Bram EE, Assaraf YG (2008) PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells. Biochem Biophys Res Commun 376:787–792 Helmlinger G, Yuan F, Dellian M, Jain RK (1997) Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med 3:177–182 Hughes LR, Stephens TC, Boyle FT, Jackman AL (1999) Raltitrexed (Tomudex™), a highly polyglutamatable antifolate thymidylate synthase inhibitor. In: Jackman AL (ed) Anticancer development guide: antifolate drugs in cancer therapy. Humana Press, Totowa, pp 147–165 Jackman AL, Jansen G, Ng M (2011). Folate receptor targeted thymidylate synthase inhibitors. Targeted drug strategies for cancer and inflammation. Springer, New York Jansen G (1999) Receptor- and carrier-mediated transport systems for folates and antifolates. Exploitation for folate chemotherapy and immunotherapy. In: Jackman AL (ed) Anticancer development guide: antifolate drugs in cancer therapy. Humana Press, Totowa, pp 293–321 Kugel Desmoulin S, Wang Y, Wu J, Stout M, Hou Z, Fulterer A, Chang M-H, Romero MF, Cherian C, Gangjee A, Matherly LH (2010) Targeting the proton-coupled folate transporter for selective delivery of 6-substituted pyrrolo[2, 3-d]pyrimidine antifolate inhibitors of de novo purine biosynthesis in the chemotherapy of solid. Mol Pharm 78:577–587

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Leamon CP, Reddy JA, Vlahov IR, Westrick E, Dawson A, Dorton R, Vetzel M, Santhapuram HK, Wang Y (2007) Preclinical antitumor activity of a novel folate-targeted dual drug conjugate. Mol Pharm 4:659–667 Lu Y, Wu J, Gonit M, Yang X, Lee A, Xiang G, Li H, Liu S, Marcucci G, Ratnam M, Lee RJ (2007) Role of formulation composition in folate receptor-targeted liposomal doxorubicin delivery to acute myelogenous leukemia cells. Mol Pharm 4:707–712 Matherly LH, Hou Z, Deng Y (2007) Human reduced folate carrier: translation of basic biology to cancer etiology and therapy. Cancer Metastasis Rev 26:111–128 Mendelsohn LG, Worzalla JF, Walling JM (1999) Preclinical and clinical evaluation of the glycinamide ribonucleotide formyltransferase inhibitors lometrexol and LY309887. In: Jackman AL (ed) Anticancer development guide: antifolate drugs in cancer therapy. Humana Press, Totowa, pp 261–280 Muller C, Forrer F, Schibli R, Krenning EP, de Jong M (2008) SPECT study of folate receptorpositive malignant and normal tissues in mice using a novel 99mTc-radiofolate. J Nucl Med 49:310–317 Raghunand N, Altbach MI, van Sluis R, Baggett B, Taylor CW, Bhujwalla ZM, Gillies RJ (1999) Plasmalemmal pH-gradients in drug-sensitive and drug-resistant MCF-7 human breast carcinoma xenografts measured by 31P magnetic resonance spectroscopy. Biochem Pharmacol 57:309–312 Reddy JA, Haneline LS, Srour EF, Antony AC, Clapp DW, Low PS (1999) Expression and functional characterization of the beta-isoform of the folate receptor on CD34(+) cells. Blood 93:3940–3948 Taylor EC (1993) Design and synthesis of inhibitors of folate-dependent enzymes as antitumor agents. In: Ayling JE, Nair MG, Baugh CM (eds) Chemistry and biology of pteridines and folates, vol 338. Plenum Press, New York, pp 387–408 Theti DS, Bavetsias V, Skelton LA, Titley J, Gibbs D, Jansen G, Jackman AL (2003) Selective delivery of CB300638, A cyclopenta[g]quinazoline-based thymidylate synthase inhibitor into human tumor cell lines overexpressing the alpha-isoform of the folate receptor. Cancer Res 63:3612–3618 Trédan O, Galmarini CM, Patel K, Tannock IF (2007) Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst 99:1441–1454 Wang L, Cherian C, Desmoulin SK, Polin L, Deng Y, Wu J, Hou Z, White K, Kushner J, Matherly LH, Gangjee A (2010) Synthesis and biological activity of a novel series of 6-substituted pyrrolo[2, 3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry. J Med Chem 53:1306–1318 Zhao R, Goldman ID (2003) Resistance to antifolates. Oncogene 22:7431–7457 Zhao R, Goldman ID (2007) The molecular identity and characterization of a proton-coupled folate transporter – PCFT; biological ramifications and impact on the activity of pemetrexed. Cancer Metastasis Rev 26:129–139 Zhao R, Gao F, Hanscom M, Goldman ID (2004) A prominent low-pH methotrexate transport activity in human solid tumors: contribution to the preservation of methotrexate pharmacologic activity in HeLa cells lacking the reduced folate carrier. Clin Cancer Res 10:718–727 Zhao R, Matherly LH, Goldman ID (2009a) Membrane transporters and folate homeostasis; intestinal absorption, transport into systemic compartments and tissues. Expert Rev Mol Med 11:e4 Zhao R, Min SH, Wang Y, Campanella E, Low PS, Goldman ID (2009b) A role for the protoncoupled folate transporter (PCFT-SLC46A1) in folate receptor-mediated endocytosis. J Biol Chem 284:4267–4274

Chapter 7

Folate Receptor Targeted Cancer Chemotherapy Joseph A. Reddy and Christopher P. Leamon

Abstract  The membrane-bound folate receptor (FR) is overexpressed on a wide range of human cancers. The vitamin folic acid is a high affinity ligand of the FR which retains its receptor binding and receptor-mediated endocytosis properties when conjugated to other molecules. Consequently, folate targeting technology has successfully been applied for the delivery of various chemotherapeutic agents to FR-positive cancers. Together with optimized spacers and self-immolative linkers, these folate-drug delivery systems have produced major enhancements in cancer cell-specific and selective potency over their nontargeted drug counterparts. Hence, it is hopeful that this targeting strategy will lead to improvements in the safety and efficacy of clinically-relevant anticancer therapeutic agents. The focus of this chapter will be to highlight the current status of folate-drug technology with particular emphasis on the recent advances in this field. Keywords  Folate receptor • Targeted chemotherapy • EC145 • Endocytosis

7.1 Introduction The vitamin folic acid (FA; folate) is a high affinity ligand of the folate receptor (FR), which maintains its strong binding property when conjugated to other molecules. As a result, “folate targeting” has been successfully applied towards the delivery of a wide variety of anticancer agents to FR-positive cancers (Leamon 2008). Compared to their nontargeted counterparts, FA-bearing drugs and delivery systems have repeatedly shown greater cancer cell specificity and selectivity in numerous preclinical studies. Hence, this targeting strategy leads to improvements in the safety and efficacy of anticancer agents, resulting in an increased therapeutic advantage.

J.A. Reddy (*) Endocyte, Inc., 3000 Kent Avenue, Suite A1-100, West Lafayette, IN 47906-1075, USA e-mail: [email protected] A.L. Jackman and C.P. Leamon (eds.), Targeted Drug Strategies for Cancer and Inflammation, DOI 10.1007/978-1-4419-8417-3_7, © Springer Science+Business Media, LLC 2011

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Fig. 7.1  Schematic representation of the internalization of FA-drug conjugates by the FR-mediated endocytosis pathway. FA-drug conjugates bind specifically to the FR protein with high affinity. The plasma membrane invaginates around the conjugate:FR complex to form an intracellular vesicle (early endosome). As the lumen of the maturing endosome acidifies to ~ pH 6, the receptor changes conformation and releases the conjugate. Eventually, the fates of the FA-drug cargo and the FR are determined during a sorting process within late endosomal elements. The reduced folate carrier (RFC), which is an anion transporter, can shuttle unmodified reduced folate molecules inside the cell; FA-drug conjugates, however, are not substrates for the RFC

As illustrated in Fig. 7.1, FA-drug conjugates bind to externally oriented FRs on the plasma membrane of cancer cells, and they are internalized by these cells via a receptor-mediated endocytic mechanism (Leamon and Low 1991, 1993). The endocytosis process begins with the invagination of the surrounding plasma membrane to form a distinct internal vesicle (called an early endosome) within the cytoplasm. An internal proton gradient then acidifies the endosomes (Lee et  al. 1996; Yang et  al. 2007) and promotes the release, or undocking of the FA-drug conjugate from its membrane anchored receptor. The fate of the released conjugate is dependent upon its chemical composition, but cytosolic entry is possible through membrane permeation, localized transporters, or by simple leakage during imperfect membrane fusion events (Turek et al. 1993). While numerous articles on FR expression (Hartmann et al. 2007; Markert et al. 2008; Parker et al. 2005; Shia et al. 2008), FR endocytosis (Sabharanjak and Mayor 2004) and folate-targeted technology (Leamon 2008; Leamon and Jackman 2008; Low and Kularatne 2009) have been published, we will focus the next sections of this chapter on techniques related to the delivery of small molecule chemotherapeutic agents.

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7.2 First Generation Conjugates for Targeted Chemotherapy Several groups have reported on the effects of conjugating FA to known ­chemotherapeutic agents using fairly stable chemical bonds. Such compounds were determined to be moderately active against cancer cells in vitro, but activity against animal tumor models was not proven. The first agent to highlight is a FA conjugate of 5-fluoro-2’-deoxyuridine-5’-O-monophosphate (FdUMP, a thymidylate synthase (TS) inhibitor), which was designed to treat 5-fluorouracil resistant cancers that generally overexpress TS. The FA-FdUMP molecule was used to synthesize a 10-mer FdUMP oligodeoxynucleotide conjugate (Fig.  7.2, Structure 1) (Liu et  al. 2001).

Fig. 7.2  Structures of published FA-drug conjugates. 1, FA-FdUMP 10-mer ODN conjugate; 2, FA-phopharamidate prodrug; 3, FA-PEG3000-carboplatin; 4, FA-PEG-7-Taxol; 5, FA-DM1

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When evaluated in  vitro, the activity of the FA-FdUMP 10-mer was found to be tenfold more cytotoxic than the nonconjugated 10-mer towards H630 (human colorectal cancer) cells, and 25-fold more cytotoxic towards H630-10 cells, the latter having > twofold increased expression of TS and a 26-fold higher expression of FR. The presence of 1 nM free FA had reduced this agent’s activity threefold, but it did not change the cytotoxicity of the untargeted FdUMP 10-mer, thereby ­confirming the conjugate’s specificity towards FR-expressing cells. Unlike the FA-FdUMP agent, conjugation to FA can (in some cases) diminish the potency of the parent drug. For example, when FA was conjugated to a nitroheterocyclic bis(haloethyl) phosphoramidite prodrug (Fig.  7.2, Structure 2), it was 10–400-fold less cytotoxic than the unconjugated prodrug in cell culture (Steinberg and Borch 2001). In another example, a FA-targeted PEG-3000 carboplatin conjugate (Fig. 7.2, Structure 3) significantly elevated the Pt content in FR-positive M109 (murine lung cancer) cells as compared to the nontargeted PEG-Pt (6.7 ng vs. 4.3 ng Pt/106 cells). However, despite the higher cellular uptake of FA-PEG-Pt, it produced less Pt-DNA adducts than the nontargeted control (4.3 ng vs. 9.7 ng Pt/mg DNA) and was overall 1.7-fold less potent than the PEG-Pt construct (Steinberg and Borch 2001). Importantly, since the FA-PEG-Pt conjugate lacked an intrinsic mechanism to release free Pt into the cytosol, sustained entrapment of the conjugate within various endosomal elements likely caused the observed decrease in cytotoxicity. Notably, a FA-Taxol prodrug (Taxol-7-PEG-FA; Fig.  7.2, Structure 4) was designed to release Taxol via hydrolytic cleavage of an intramolecular ester, but it was determined to be 50-fold less cytotoxic towards FR-positive KB (human nasopharyngeal cancer) cells when compared to free Taxol (Lee et  al. 2002). Unfortunately, the hydrophilic property of the associated PEG spacer was not enough to sufficiently dissolve the Taxol conjugate in an aqueous medium without the help of a Cremophor/ethanol excipient. In addition, the hydrolysis rate of the ester bond within the conjugate was found to be very slow (t1/2 ~197 h) at endosomal pH. Such shortcomings of the aforementioned approaches, namely: (1) low water solubility of the FA-drug conjugate, and (2) lack of efficient intracellular releasable linkers, had prompted the design of more effective, second generation conjugates which are discussed in the following section.

7.3 Second Generation Conjugates for Targeted Chemotherapy 7.3.1 Folate-Maytansinoid Conjugates The major limitation of the first generation FA-drug conjugates, namely moderate potency, appeared to have been overcome when highly cytotoxic anticancer drugs were linked to FA via an intramolecular disulfide-based linker. Similar to natural heterodimeric protein-based toxins (e.g., ricin, pseudomonas exotoxin etc.)

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(Madshus and Collier 1989), placement of a bioreducible disulfide bond in-between the FA and drug moieties was predicted to afford efficient release of the active drug cargo within the endosomal milieu (Leamon et al. 1993). Since most chemotherapeutic drugs are particularly lipophilic, the released drug was expected to easily traverse the endosome membrane and diffuse throughout the cytosol to locate its pharmacological target. This hypothesis was indeed confirmed when FA was ­disulfide-linked to a synthetically modified ansamacrolide maytansine (FA-DM1; Fig. 7.2, Structure 5) (Ladino et al. 1997). The FA-DM1 conjugate was shown to retain high specific affinity of FA towards the FR in a competitive binding assay. Immunofluorescence microscopy with an antimaytansinoid mAb suggested that the FA-DM1 bound to KB cells and was then internalized into these FR-positive cells but not into FR-negative A375 (human malignant melanoma) cells. The conjugate was then shown to kill up to 96% of a panel of FR-expressing cancer cells such as KB, SKOV-3 (human ovarian cancer), LoVo (human colon carcinoma), HeLa (human cervical cancer), and SW620 (human colorectal adenocarcinoma) at 1 nM with IC50 values ranging from 10-11 to 10-10 M. In contrast, the same drug conjugate was found to be 100-fold less toxic to the FR-negative cell lines LS174T (human colon carcinoma), SK-BR-3 (human breast cancer) and A375, suggesting that attachment of the cytotoxic moiety to FA severely reduced its ability to enter cells nondiscriminately. This remarkable specificity was further supported by the fact that both excess free FA as well an anti-FR antibody blocked activity. Although this FA-maytansinoid conjugate was found to be extremely potent in vitro, it was never tested in vivo due to poor water solubility. The exciting results with FA-DM1 suggested that potent FA-targeted agents could be assembled if the constructs contained: (1) a highly potent drug, (2) an efficient linker for intracellular drug release, and (3) adequate water solubility. In fact, as shown in Fig.  7.3, a modular synthetic approach that incorporates these features is currently being used to develop clinically tested FA-drug conjugates by Endocyte, Inc. (West Lafayette, IN). Here, the high affinity FA ligand typically functions as Module 1, while a potent drug is placed in the Module 4 position. Module 2 functions as a spacer to optimally separate the drug from FA and to

Fig.  7.3  Modular design of FA-drug conjugates. There are four modules in the design of a FA-drug conjugate. Module 1 represents the ligand element (FA) and Module 2 is a hydrophilic spacer. Module 3 is a biocleavable bond (CB), while Module 4 is the drug payload

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provide ample hydrophilic properties. Module 3 contains a “cleavable bond” (CB) which allows for release of the active drug from folate following endocytosis. To circumvent the poor water solubility of FA-DM1 mentioned above, EC131 (Fig.  7.4, Structure 1) was assembled by attaching FA to DM1 through a polar peptide unit (Asp-Arg-Asp-Cys) in addition to the disulfide bond (Reddy et al. 2007a; b). When tested on cells in culture, EC131 was found to retain high affinity towards the FR. Thus, linkage of the maytansinoid molecule or the water soluble spacer did not significantly alter the vitamin’s intrinsic binding affinity to its receptor. EC131 was next determined to be highly cytotoxic towards various FR-positive KB, ID8Cl-15 (murine ovarian cancer) and M109 cells with IC50s less than 25 nM. This activity was effectively blocked in the presence of excess free FA. In addition, EC131 was not found to be cytotoxic towards the FR-negative 4T1 (mouse breast cancer), CHO (Chinese hamster ovary), 24JK (murine ­sarcoma), or A549 (human lung cancer) cells. Most importantly, when evaluated in the syngeneic M109 tumor model (subcutaneous tumors in Balb/c mice), intravenous (i.v.) administration of EC131 at 1  mmol/kg following a twice weekly (BIW) schedule for 4 weeks yielded 1/5 complete response (CR) and 3/5 partial responses (PR’s). In contrast, no ­significant antitumor activity (0/5 CR and PR) was observed in EC131-treated animals that were co-dosed with an excess of FA, thus demonstrating the targeted specificity of the observed in  vivo activity. EC131 therapy using 1.5  mmol/kg dose level and a 3 times per week (TIW) schedule for 3 weeks also showed striking antitumor activity (4/5 CR’s and 1/5 PR’s) in a subcutaneous KB xenograft tumor model, but not against FR-negative A549 tumors. Furthermore, this therapeutic effect occurred in the apparent absence of weight loss or noticeable organ degeneration in both tumor models. In contrast, therapy with the free maytansinoid drug (in the form of DM1-S-Me) was not effective (0/5 CR and PR) in the KB tumor model when administered at its MTD (0.12 mmol/kg, daily x 5, 1 week schedule). From a historical perspective, these data proved for the first time that observations made with FA-drug conjugates in vitro could translate into effective targeted therapies in vivo and that it was possible to preserve antitumor activity while eliminating or significantly decreasing nontarget organ toxicities.

7.3.2 Folate-DAVLBH Conjugates Success with EC131 led to additional research involving other microtubule inhibitors. EC140 (Fig. 7.4, Structure 2) and EC145 (Fig. 7.4, Structure 3) are novel FA conjugates of the microtubule destabilizing agent, desacetylvinblastine monohydrazide (DAVLBH; a derivative of the natural product vinblastine) (Leamon et al. 2006, 2007b). Like vinblastine, DAVLBH is a Vinca alkaloid that is capable of disrupting the formation of the mitotic spindle, thereby inhibiting cell division and causing cell death. DAVLBH was chosen as the drug moiety for both EC145 and EC140 because it contains a modifiable hydrazide functional group to which one

Fig. 7.4  Structures of pharmacologically active FA-drug conjugates. 1, EC131; 2, EC140; 3, EC145

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could attach a hydrophilic folate-peptide compound (e.g., FA-Asp-Arg-Asp-Asp-Cys) via disulfide linker or acylhydrazone groups, respectively (Vlahov et al. 2006). Similar to EC131, chemical modification of FA with the DAVLBH drug motif had only minimally altered the vitamin’s intrinsic affinity for the FR, since both EC140 and EC145 were experimentally determined to have high binding affinity for FRs (Leamon et al. 2007b). Following a short 2 h exposure, EC140 and EC145 were both shown to kill KB cells in a concentration dependent manner with IC50’s of ~ 11 nM and 9 nM, respectively. Furthermore, such activity was effectively blocked in the presence of excess free FA. These results indicated that at least in vitro, under well-controlled conditions, both the acid-sensitive acylhydrazone and the reducible disulfide linkers afforded facile intracellular release of the active DAVLBH moiety. EC140 and EC145 were also evaluated for antitumor activity against the KB tumor xenograft model. Each agent was administered i.v. at 5 mmol/kg dose level following a TIW, 3-week schedule. During this study, four of five EC140-treated animals experienced PR’s (LCK of 2, 161% T/C), and 1 of 5 mice experienced a CR. Animals in the EC145-treated cohort fared better, with all 5 of the treated mice experiencing CRs. Based on these comparative in vivo studies, EC145 was judged to be the better of the two FA-Vinca alkaloid agents (Leamon et  al. 2007b). The likely explanation for this difference related to the cleavable linkers found within each construct. For example, recently published studies using FRET-based conjugates have indicated that acylhydrazone linkers, such as that found in EC140, may not be as efficiently cleaved inside endosomes as compared to their disulfide bondcontaining counterparts (Yang et al. 2006). The activity of EC145 against the KB tumor model was further assessed by administering the drug 9 days posttumor cell inoculation (PTI) using a lower 2 mmol/ kg dose level and following a brief TIW, 2-week schedule. Beginning with nude mice bearing ~100 mm3 tumors, these treatment conditions led to CR’s in 5/5 mice and cures (i.e., remission without a relapse for >90 days PTI) in 4/5 mice (Fig. 7.5, Panel A). In contrast, when co-dosed with a modest 20-fold molar excess of a benign water soluble folate analog (EC20), EC145 failed to produce any meaningful antitumor activity (0 CR’s and 2 PRs) (Reddy et al. 2007a). Since EC145 therapy was shown to eradicate well-established subcutaneous tumors under conditions that produced little to no toxicity (Fig.  7.5, panels A and B), it was important to assess the therapeutic advantage that this targeted ­molecule may have over the untargeted base drug (DAVLBH). Thus, mice bearing KB tumors were treated with three different dose levels (0.5, 1 and 2 mmol/kg) of DAVLBH following a TIW, 2-week regimen. As shown in Fig. 7.5C, no activity was observed when DAVLBH was dosed at a seemingly nontoxic level (0.5 mmol/ kg), and only PRs were noted at the 1 mmol/kg maximum tolerated (MTD) level where mice lost ~14% of their weight (Fig. 7.5, Panels C and D). In this animal model, DAVLBH was not found to be tolerable at the 2 mmol/kg level when given more than 3 times. Thus, unlike EC145, DAVLBH clearly has a limited, suboptimal therapeutic range (Reddy et al. 2007a). EC145 is now being tested at several clinical sites in and outside the United States. A Phase 1 trial of EC145 for treatment of refractory solid tumors (Li et al. 2009),

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Fig. 7.5  Antitumor effects of EC145 vs. its nontargeted counterpart (DAVLBH) and their toxicities in the KB tumor nu/nu mouse model. KB tumor cells (1 × 106) were inoculated s.c. into nude mice and therapy started on randomized mice with tumors in the 70–110  mm3 range. Panel A consists of control tumor volumes (filled square) and those of mice treated with EC145 at 2 mmol/kg following a TIW, 2-week schedule (open circle). Panel C shows the effect of 0.5 (filled square) and 2 (open circle) mmol/kg DAVLBH administered following a TIW, 2-week schedule. Each curve in Panels A and C represents the growth of a single tumor in an individual mouse. The average (±SD) weights of five mice for each treatment cohort are shown in Panel B (filled square, control; open circle, EC145 2  mmol/kg) and Panel D (filled square, DAVLBH at 0.5  mmol/kg; open circle, DAVLBH at 2 mmol/kg)

and Phase IIA trials in patients with chemo-refractory ovarian cancer and progressive adenocarcinoma of the lung have recently been completed. A Phase IIb trial for the evaluation of Doxil and EC145 combination therapy in platinum-resistant ovarian cancer (PRECEDENT) is currently in progress. Results from the PRECEDENT trail are expected in late 2010.

7.3.3 Folate-Tubulysin B Conjugate Knowing that tumor cells within naturally occurring human malignancies may have varied sensitivities (or resistance) to certain agents, it was reasoned that agents more powerful than and distinct from EC145 may be needed to eradicate chemoresistant tumors. So, building on the preclinical success of the EC145 program, effort

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was devoted towards the discovery of FA-drug conjugates with higher targeted potencies, such as those constructed with a semisynthetic analog of the microtubule inhibitor, tubulysin B (Vlahov et  al. 2008). Much like the Vinca class of agents, tubulysins mimic the tubulin destabilizing ability; however, their in vitro GI50’s are typically 20–1,000-fold greater (Steinmetz et al. 2004). EC0305 was the first reported FA-tubulysin conjugate (Fig.  7.6, Structure 1), and its affinity towards the FR was determined to be nearly identical to that of FA (Leamon et  al. 2008). When tested in  vitro, EC0305 was found to specifically inhibit the growth of FR-positive KB and RAW 267.4 (murine macrophage) cells, with IC50 values of 1.5 and 2.6 nM (Leamon et al. 2008). EC0305’s potency was also confirmed against the KB tumor model in nude mice. Here, 100% of animals treated with ³1 mmol/kg on a TIW, 2 week regimen were tumor-free shortly after therapy was stopped, and no significant weight loss or major organ tissue degeneration was observed (Fig. 7.7, Panels A and B). In contrast, antitumor activity was completely abolished in EC0305-treated animals that were co-dosed with a 40-fold excess of Rhenium-EC20 (a benign folate analog), thereby confirming that this agent’s antitumor effect was mediated by FRs (Leamon et al. 2008). The advantage provided by FA conjugation was further proven by the untargeted free drug (tubulysin B hydrazide) and the natural (tubulysin B) drug, which were both found to be completely inactive, even at highly toxic dose levels (Fig. 7.7, Panels C and D) Thus, both nontargeted drugs produced no significant antitumor effect over these dose ranges; instead, animals experienced dose-dependent toxicity as measured by progressive weight loss and near-moribund behavior (at the highest dose level). Importantly, animals in the 1 mmol/kg tubulysin B hydrazide cohort could only tolerate two consecutive doses while animals in the 0.1, 0.2, and 0.5 mmol/kg tubulysin B cohort could tolerate 4, 3, and 1 doses, respectively. Since no antitumor effect was observed, and only toxicity had occurred, both tubulysin B and its hydrazide counterpart were declared therapeutically null (Leamon et al. 2008; Reddy et al. 2009). Since tubulysins have been previously shown to retain their high cytostatic activity even against multidrug resistant cell lines (Kaur et  al. 2006), EC0305’s activity was compared with that of EC145 against more chemoresistant FR-expressing M109 and 4T1-cl2 tumor models. Here, EC0305 displayed superior antitumor activity to EC145. Mice bearing M109 tumors were treated with EC145 at two different doses and schedules: 2 mmol/kg TIW for 2 weeks, and 4 mmol/kg TIW for 3 weeks. The 2 mmol/kg EC145 cohort yielded 1 CR and 1 cure, while the 4  mmol/kg dose resulted in a modest 2 of 5 cures. In contrast, when mice were treated with EC0305 at only 2 mmol/kg, TIW for 2 weeks (i.e., one-third the total dose of the 4 mmol/kg EC145 cohort), tumors in all the five treated mice quickly regressed with 4/5 cures and only one relapse (PR) by the end of the study. Furthermore, in the highly drug-resistant 4T1-cl2 tumor model, EC145 given at 4 mmol/kg, TIW for 2 weeks did not produce any antitumor effect, whereas EC0305 at half that total dose (2  mmol/kg, TIW for 2 weeks) produced significant tumor regressions or disease stabilizations (Reddy et  al. 2009). Importantly, EC0305’s superior antitumor activity was not at the cost of an associated increase in toxicity, since the maximum average weight loss in the EC145 cohort (~9%) was similar to

Fig. 7.6  Structures of recently published FA-drug conjugates. 1, EC0305; 2, BMS-753493; 3, EC0225

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Fig. 7.7  Antitumor effects of EC0305 vs. nontargeted tubulysin B and their toxicities in the KB tumor/ nu/nu mouse model. KB tumor cells were implanted s.c. into nu/nu mice, and 11 days later, mice were randomized and treatments given following a TIW, 2-week schedule. Panel A consists of mice treated with 1 mmol/kg EC0305 (open circle) and 1 mmol/kg EC0305 plus 40 mmol/kg of Re-EC20 (filled square), while Panel C shows effect of tubulysin B (filled square, 0.1 mmol/kg; open circle, 0.2 mmol/kg). Each curve in Panels A and C represents the growth of a single tumor in an individual mouse. Panels B (open circle, EC0305 1 mmol/kg; filled square, EC0305 1 mmol/ kg + Re-EC20 40  mmol/kg) and D, (0.1  mmol/kg (filled square); 0.2 mmol/kg (open circle) tubulysin B) show the average (±SD) weights of five mice, for each treatment cohort

that of the EC0305 cohort (~10%) (Reddy et al. 2009). Collectively, these results show that EC0305 has significant antiproliferative activity against FR-expressing tumors, including those which were generally found to be more chemoresistant.

7.3.4 Folate-Epothilone Conjugate BMS-753493 (Fig. 7.6, Structure 2) is a folate-epothilone conjugate being developed by Bristol-Myers Squibb in collaboration with Endocyte (Covello et  al. 2008). The epothilones are a new class of microtubule stabilization agents with potent antitumor activity, especially against taxane-resistant cancers (Harrison and Swanton 2008). BMS-753493 was found to induce potent cytotoxicity in a clonogenic assay with a panel of FR-positive KB, IGROV (human ovarian), HeLa, and M109 cells. Similar to previous folate conjugates, this cytotoxic effect was abolished when

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excess FA was present and also against FR-negative cells. BMS-753493 also demonstrated impressive antitumor activity against several different FR-positive in vivo tumor models. When combined with other chemotherapeutic agents, such as bevacizumab, cisplatin, and ixabepilone, BMS-753493 demonstrated synergistic antitumor effect. Importantly, BMS-753493 did not display clinical signs of neuropathy or enteropathy when dosed at its MTD (Covello et al. 2008), which are side-effects frequently associated with antimicrotubule cytotoxic agents. A multicenter Phase 1/2 study of BMS-753493 in subjects with advanced cancer is now in progress.

7.4 Third Generation FA-Drug Conjugates 7.4.1 Multidrug Conjugates FA’s targeting power has been tested by simultaneously delivering more than one type of cytotoxic agent to tumors. In this third generation conjugate, folate was tethered to two different drug molecules, with distinct biological mechanisms of action, to produce EC0225 (Fig. 7.6, Structure 3). This agent is constructed with a single FA molecule, extended by a hydrophilic peptide-based spacer, Asp-Asp-Asp-bDprCys, which is in turn attached to a DNA alkylating drug moiety (N 7-mercaptoethylmitomycin C) and a Vinca alkaloid unit via two separate disulfide-containing linkers (Vlahov et al. 2007). EC0225’s cell killing activity was found to be concentration dependent with an IC50 of ~5  nM. Furthermore, a regimen consisting of 2 mmol/kg EC0225 given i.v. and following a TIW, 2-week schedule was found to produce 5/5 cures against KB tumors. A 2  mmol/kg TIW, 3-week regimen of EC0225 was also found to be highly effective against M109 tumors (4 of 5 CR’s) under conditions where animals once again did not appreciably lose weight. Even mice bearing tumors as large as 750 mm3 in volume were curable following brief i.v. therapy with EC0225 (Leamon et al. 2007a). Notably, treatment with the untargeted drug mixture of DAVLBH + MMC at their respective MTDs (e.g., 1 mmol/ kg each when in combination) was found to be ineffective against the M109 tumor, which added further support that the observed activity with EC0225 was due to FR targeting. Overall, EC0225’s impressive activity enabled its selection for clinical development, and this molecule is being tested in a Phase 1 clinical trial for the treatment of refractory or metastatic tumors.

7.5 Conclusions The FR is significantly upregulated in a large number of solid and hematopoietic human cancers. Since the magnitude of tumor cytotoxicity depends on the cumulative amount of therapeutic agent delivered to the cancer cell, the ability of the FR to recycle and to deliver multiple FA-drug conjugates per cell represents a tremendous advantage.

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As supported by the many examples presented within this chapter, FA-drug conjugates display high selectivity for cancer cells in vivo since: (1) their activities are far superior to unconjugated drugs, and (2) they are severely compromised when co-dosed with an excess of folate competitors. The attachment of FA clearly improves a drug’s therapeutic index by enabling a greater amount of total drug to be administered and by allowing more aggressive dosing regimens to be followed. In context of a targeting ligand, FA constitutes an attractive alternative to antibodies and other proteins/peptides. This ligand is much smaller (MW – 441) than monoclonal antibodies (MW – 160,000) and hence can penetrate tumor tissues much more rapidly and completely (Vlashi et  al. 2009). FA is also nonimmunogenic, whereas even humanized monoclonal antibodies can elicit a neutralizing immune response. FA is relatively easy to conjugate to a wide variety of drug molecules, whereas antibody conjugation can be nonselective and relatively inefficient. FA is stable to mild acids/bases and a variety of solvents, temperatures, and storage conditions, whereas antibodies must be handled carefully to avoid their denaturation. FA is also inexpensive to procure, whereas monoclonal antibodies can be costly to produce. Clearly, much depends on the ongoing human clinical studies before the full potential of FA-drug conjugates can be accurately assessed; however, the results to date point to the fact that folate targeting will soon find an important niche in the treatment of cancer. Moreover, since many FR-positive cancers are associated with poor clinical outcomes (Hartmann et  al. 2007; Toffoli et  al. 1997), FA-based chemotherapeutics may offer new options to patients having few alternatives. Acknowledgements  We are grateful to everybody in the Discovery group at Endocyte, Inc. for creating and testing the novel FA-targeted agents described in this chapter.

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Leamon CP, Low PS (1993) Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells. Biochem J 291(Pt 3):855–860 Leamon CP, Pastan I, Low PS (1993) Cytotoxicity of folate-Pseudomonas exotoxin conjugates toward tumor cells. Contribution of translocation domain. J Biol Chem 268:24847–24854 Leamon CP, Reddy JA, Vetzel M et al (2008) Folate targeting enables durable and specific antitumor responses from a therapeutically null tubulysin B analogue. Cancer Res 68:9839–9844 Leamon CP, Reddy JA, Vlahov IR et al (2006) Synthesis and Biological Evaluation of EC140: A Novel Folate-Targeted Vinca Alkaloid Conjugate. Bioconjug Chem 17:1226–1232 Leamon CP, Reddy JA, Vlahov IR et al (2007a) Preclinical antitumor activity of a novel folatetargeted dual drug conjugate. Mol Pharm 4:659–667 Leamon CP, Reddy JA, Vlahov IR et  al (2007b) Comparative preclinical activity of the folatetargeted Vinca alkaloid conjugates EC140 and EC145. Int J Cancer 121:1585–1592 Lee JW, Lu JY, Low PS et al (2002) Synthesis and evaluation of taxol-folic acid conjugates as targeted antineoplastics. Bioorg Med Chem 10:2397–2414 Lee RJ, Wang S, Low PS (1996) Measurement of endosome pH following folate receptor-mediated endocytosis. Biochim Biophys Acta 1312:237–242 Li J, Sausville EA, Klein PJ et al (2009) Clinical pharmacokinetics and exposure-toxicity relationship of a folate-Vinca alkaloid conjugate EC145 in cancer patients. J Clin Pharmacol 49:1467–1476 Liu J, Kolar C, Lawson TA et al (2001) Targeted drug delivery to chemoresistant cells: folic acid derivatization of FdUMP[10] enhances cytotoxicity toward 5-FU-resistant human colorectal tumor cells. J Org Chem 66:5655–5663 Low PS, Kularatne SA (2009) Folate-targeted therapeutic and imaging agents for cancer. Curr Opin Chem Biol 13:256–262 Madshus IH, Collier RJ (1989) Effects of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A. Infect Immun 57:1873–1878 Markert S, Lassmann S, Gabriel B et al (2008) Alpha-folate receptor expression in epithelial ovarian carcinoma and non-neoplastic ovarian tissue. Anticancer Res 28:3567–3572 Parker N, Turk MJ, Westrick E et al (2005) Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem 338:284–293 Reddy JA, Dorton R, Dawson A et al (2009) In vivo structural activity and optimization studies of folate-tubulysin conjugates. Mol Pharm 6:1518–1525 Reddy JA, Dorton R, Westrick E et  al (2007a) Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate. Cancer Res 67:4434–4442 Reddy JA, Westrick E, Santhapuram HK et al (2007b) Folate receptor-specific antitumor activity of EC131, a folate-maytansinoid conjugate. Cancer Res 67:6376–6382 Sabharanjak S, Mayor S (2004) Folate receptor endocytosis and trafficking. Adv Drug Deliv Rev 56:1099–1109 Shia J, Klimstra DS, Nitzkorski JR et al (2008) Immunohistochemical expression of folate receptor alpha in colorectal carcinoma: patterns and biological significance. Hum Pathol 39:498–505 Steinberg G, Borch RF (2001) Synthesis and evaluation of pteroic acid-conjugated nitroheterocyclic phosphoramidates as folate receptor-targeted alkylating agents. J Med Chem 44:69–73 Steinmetz H, Glaser N, Herdtweck E et al (2004) Isolation, crystal and solution structure determination, and biosynthesis of tubulysins–powerful inhibitors of tubulin polymerization from myxobacteria. Angew Chem Int Ed Engl 43:4888–4892 Toffoli G, Cernigoi C, Russo A et al (1997) Overexpression of folate binding protein in ovarian cancers. Int J Cancer 74:193–198 Turek JJ, Leamon CP, Low PS (1993) Endocytosis of folate-protein conjugates: ultrastructural localization in KB cells. J Cell Sci 106(Pt 1):423–430 Vlahov IR, Santhapuram HK, Kleindl PJ et al (2006) Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part 1: EC145, a folic acid conjugate of desacetylvinblastine monohydrazide. Bioorg Med Chem Lett 16:5093–5096 Vlahov IR, Santhapuram HK, Wang Y et  al (2007) An assembly concept for the consecutive introduction of unsymmetrical disulfide bonds: synthesis of a releasable multidrug conjugate of folic Acid. J Org Chem 72:5968–5972

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Vlahov IR, Wang Y, Kleindl PJ et al (2008) Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part II: Folic acid conjugates of tubulysins and their hydrazides. Bioorg Med Chem Lett 18:4558–4561 Vlashi E, Sturgis JE, Thomas M et al (2009) Real time, noninvasive imaging and quantitation of the accumulation of ligand-targeted drugs into receptor-expressing solid tumors. Mol Pharm 6:1868–1875 Yang J, Chen H, Vlahov IR et al (2006) Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging. Proc Natl Acad Sci USA 103:13872–13877 Yang J, Chen H, Vlahov IR et al (2007) Characterization of the pH of folate receptor-containing endosomes and the rate of hydrolysis of internalized acid-labile folate-drug conjugates. J Pharmacol Exp Ther 321:462–468

Chapter 8

Anti-FR Antibody Generation and Engineering: Development of New Therapeutic Tools Silvana Canevari and Mariangela Figini

Abstract  The discovery of the methodology to raise mouse monoclonal antibodies (mAbs) represents a milestone in the history of medicine and has opened the way to antibody therapy development. In the oncologic field, antibody-based therapy seems an attractive strategy for those tumors, such as epithelial ovarian cancer and glioblastoma, for which the existing treatment options are still not sufficient. Initial clinical trials with mouse mAbs enlighten, as major limitations, their xenogenic origin and their dimension. Thus, in order to optimize mAb clinical therapeutic applications, genetic engineering was developed to: (1) generate chimeric, humanized, and human mAbs starting from mouse mAbs; (2) reshape antibody format; (3) increase antibody efficacy. The history of anti-human folate receptor (FR)a mAb generation and its modification paralleled that of genetic engineering of mAbs. At least three anti-FRa mAbs (MOv18, MOv19, and LK26) and their derivatives have reached advanced levels of development. In this chapter, the most relevant preclinical and clinical results obtained with them are widely discussed. Also, published data related to anti-FRb mAb are reported. Full exploitation of the described anti-FRa antibodybased reagents, however, awaits the confirmation of promising drug safety and clinical efficacy from well-designed, randomized clinical trials. Keywords  Antibody • Antibody fragments • Folate receptor • Ovarian cancer • Therapy

S. Canevari (*) Unit of Molecular Therapies, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy e-mail: [email protected] A.L. Jackman and C.P. Leamon (eds.), Targeted Drug Strategies for Cancer and Inflammation, DOI 10.1007/978-1-4419-8417-3_8, © Springer Science+Business Media, LLC 2011

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8.1 Introduction 8.1.1 Role of Folate Receptor in the Oncologic Field The alpha isoform of the folate receptor (FRa) is upregulated in a variable ­percentage of various carcinomas. Tumors that specifically express FRa on the highest percentage of cells and with the highest percentage of positive cases (>80%) are epithelial ovarian carcinoma (EOC), followed by endometrial cancer and malignant pleural mesothelioma, where the target molecule is expressed at a medium-high level in more than 50% of cases (Salazar and Ratnam 2007). The beta isoform of the folate receptor (FRb) is elevated in acute myelogenous leukemia cells and activated macrophages present in inflamed tissues (Salazar and Ratnam 2007). Furthermore, the FRb is expressed on macrophages infiltrating human glioblastomas (Nagai et al. 2009).

8.1.2 Rational for Using Antibody Therapy Against FR-Expressing Cells 8.1.2.1 FRa as an Appropriate Target for Cancer Immunotherapy with mAb-Based Reagents EOC is the deadliest of the gynecological malignancies. Ovarian cancer is often without overt or specific symptoms until late in its development and, therefore, most women are diagnosed with advanced-stage disease (Jemal et  al. 2009). Surgery followed by chemotherapy is currently the most prevalent treatment, but it affords merely temporary remission with exceedingly unpleasant and occasionally dangerous side effects. A major problem in clinical management of patients with this neoplasm is the largely unpredictable response to first-line treatment and the occurrence of relapse after complete response to initial treatments, associated with broad cross-resistance to even structurally dissimilar drugs. This problem points to the need for anticancer treatments with mechanisms of action different from those of the currently available chemotherapeutic agents. FRa is an appropriate target on epithelial cancer cells, and in particular for EOC immunotherapy with mAb-based reagents, for the following reasons: (1) FRa is largely absent in normal tissues and, when present, such as in kidney proximal tubules, breast, and choroid plexus, its cellular localization is restricted to the apical (luminal) surface of polarized epithelial cells where it is not exposed to the blood stream; (2) on the basis of published and unpublished data, the expression of the FRa is stable or even up-modulated during cancer progression and acquisition of drug resistance (e.g., drugs tested in EOC: platinum-containing compounds at both preclinical and clinical levels; taxanes and doxorubicin at preclinical level) (Ottone et al. 1997; Toffoli et al. 1997); (3) FRa gene transfection confers a proliferative advantage to cells (Bottero et al. 1993); and (4) functional down-­regulation of the

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membrane expression of FR in ovarian cancer cells is accompanied by a partial reversion of the transformed phenotype (Figini et al. 2003). 8.1.2.2 FRb as an Appropriate Target for Immunotherapy with mAb-Based Reagents FRb is an appropriate target for immunotherapy of inflammatory diseases, such as rheumatoid arthritis, or tumors highly infiltrated by macrophages, such as glioblastoma, using mAb-based reagents for the following reasons: (1) expression of the receptor is limited to synovial macrophages, which are implicated as major contributors to the pathogenesis of rheumatoid arthritis, and tumor-infiltrating macrophages; (2) it is theoretically possible to raise anti-FRb antibodies exhibiting a binding affinity higher than that of the natural ligand, i.e., folic acid; (3) therapeutic interventions directed to the tumor environment result in as much, or even more, activity than those directed to the tumor itself.

8.1.3 Humoral Immune Response to FR A pilot study in 2004 (Rothenberg et  al. 2004) reported that a high percentage of women with a history of neural-tube-defect (NTD)-affected pregnancies showed autoantibodies against FRa in comparison to a low percentage in controls. Since then, several studies were conducted to determine the binding characteristics and the ­etiology of these autoantibodies, their prevalence in different populations, and their potential pathogenic roles in subfertility (Berrocal-Zaragoza et al. 2009a), ­NTD-affected pregnancies (Cabrera et al. 2008; Molloy et al. 2009), and oral-cleft-affected pregnancies (Bille et al. 2009). Overall, these reports indicate that: (1) anti-FRa IgM and IgG autoantibodies could be detected at different titers and in a variable percentage of pregnant women, and also of never-been-pregnant women, and of men; (2) the antiFRa antibodies are able to bind to both bovine milk folate-binding protein and FRa isolated from human placenta, supporting the hypothesis that cow’s milk is the source of cross-reactive antigen activating the breaking of tolerance (Berrocal-Zaragoza et al. 2009b); and (3) those autoantibodies that block the folic acid-binding could interfere with folate uptake. On the contrary, there is no agreement about the pathogenic relevance of FRa autoantibodies, since a large matched-case control study found no significant association between the presence or titer of FR autoantibodies and NTD-affected pregnancy (Molloy et al. 2009). Although further studies are needed to better understand the pathogenic significance of FR autoantibodies in birth defects, these studies clearly indicate the potential antigenicity of this molecule. Tolerance to self-antigen could be broken either by cross-reactivity, as in the case of FRa by a high milk intake, or by overexpression, as suggested for tumor-associated antigens (TAA). In agreement with this later mechanism, the overexpression of FRa in EOC and breast cancer patients was associated with the development of a specific T-cell immunity and to the increased levels of circulating anti-FRa antibodies (Knutson et al. 2006). At present, no data about immune response against FRb are reported in the literature.

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8.2 Antibody Therapy Antibody therapy was first indirectly proposed by Paul Ehrlich in 1900 as a magic bullet, and for this, he received the Nobel Prize in 1908. However, it took over 90 years before the first antibody had been approved by the Food and Drug Administration (FDA) for human use in 1997. At present, more than 28 antibodies are FDA-approved, and hundreds of new antibodies and antibody-derived reagents are in clinical trials with at least two thirds of them proposed as therapeutic agents in the oncologic field.

8.2.1 Antibody Structure and Function One of the more versatile components of the acquired immunity is the antibody molecule which has evolved to bind specifically, and with high affinity, to a wide range of antigens. On the basis of differences in the structure of their heavy chain constant regions (a, b, e, g, m), also called isotypes, human antibodies have been divided into five antibody classes (IgM, IgD, IgG, IgA, and IgE) in order to cope with exogenous and endogenous agents by different effector functions. The prototypic model structure of the immunoglobulin (see Fig. 8.1a), i.e., the IgG molecule, is a tetrameric molecule with a symmetric structure composed of two identical heavy (H) chains and two identical light (L) chains joined by disulphide bonds to form a “Y” shaped molecule. Both heavy and light chains contain a variable domain, called VH and VL respectively, containing three hypervariable regions called complementarity determining regions (CDRs). CDRs differ in length and sequence among the different antibodies and are mainly responsible for the specificity (recognition) and affinity (binding strength) of the antibodies to the antigen. These regions called CDR1–CDR3 are spaced by less variable regions called framework regions (FR1–FR4), which support CDR regions. The tridimensional structure indicates that the CDRs are displayed on the antibody surface creating a pocket to receive the antigen. Heavy chains contain three or four constant (C) domains numbered sequentially from amino terminus to carboxyl terminus (CH1–CH3 domains), while light chains contain a single C domain, called CL domain. Proteolysis experiments have demonstrated that the antigen recognition ­functions and the effector domains of the antibodies are spatially segregated. The aminoterminal variable regions of both heavy and light chains participate in antigen recognition, in particular the third hypervariable region (CDR3) has the most extensive contact with bound antigen, and instead the C regions of the heavy chain mediate effector functions. Antibodies are considered as natural therapeutic agents for their ability to: (1) neutralize toxins and viruses either by direct binding or by triggering other components of the immune system; and (2) eliminate nonself or altered-self cells,

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such as tumor cells. The antitumor effect can be exerted either by blocking a vital growth factor, activating an inappropriate cell signaling through the target antigen, or recruiting to the tumor cell surface other components of the immune system, such as the complement system, natural killer cells, or macrophages.

8.2.2 From Mouse to Human Antibodies In 1975, Kohler and Milstein invented hybridoma technology (Kohler and Milstein 1975; Milstein 1980) and for this invention they received the Nobel Prize in 1985. Mouse hybridomas, generated by stable fusion of immortalized myeloma cells with B cells from immunized mice, acquire, from the first fusion partner, the ability to

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grow indefinitely in culture and, from the second, the capacity to produce antibodies with predetermined specificity, called monoclonal antibodies (mAbs). The discovery of mAbs represents a milestone in the history of medicine. However, initial clinical trials with these reagents enlighten: (1) the need of large amounts of the therapeutic reagent because of the relative short half-life of mouse antibodies in humans; (2) the induction of a human anti-mouse immune response (HAMA) that caused enhanced clearance of the injected antibodies from the patient’s serum, allergic reactions, and the formation of immune complexes in circulation blocking their therapeutic potential. Genetic engineering allowed the generation of chimeric, humanized, and human antibodies starting from these mouse mAbs (Fig. 8.1). The modular arrangement of immunoglobulin domains, associated with the PCR technique as discovered by Kary Mullis in 1983 and recognized with a Noble Prize in 1993, greatly facilitated the engineering of antibodies. The gene segments encoding the domains of interest can be isolated from the mRNA of a culture of hybridoma cells, amplified by using PCR, and then cloned into expression vectors which contain genes encoding the human constant domains. In this way, by transplanting the domains of interest of a mouse antibody into the constant domains of human antibodies, it was possible to build chimeric and humanized antibodies which greatly refined and expanded the therapeutic potential of the modality of treatment (Boulianne et al. 1984; Morrison et al. 1984). When used in human clinical trials, chimeric mAbs, containing antigen-binding variable regions from mouse mAb and human constant regions, and humanized mAbs, containing mouse CDRs grafted onto an extensive human ­antibody framework and all human constant regions, generally showed longer ­half-life and less immunogenicity than mouse mAbs. However, in the case of chimeric mAbs, patients eventually tended to develop levels of HAMA comparable with those observed with mouse mAbs. The choice of IgG1 isotype for chimeric and humanized mAbs stems from the pioneering experiments of Waldmann and coworkers, who compared the efficacy of chimeric mAb of all the antibody classes in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Bindon et al. 1988; Dyer et al. 1989). However, there are multiple mechanisms of ADCC, mediated by several types of effector cells, differing in expression of class-specific Fc receptors, cell killing modalities, and anatomical locations. Thus, targeting of a particular type of cancer may depend on where it is located, as well as the potential mechanisms of ADCC. Despite recent technologic advances (Traggiai et al. 2004), there are still considerable difficulties in making stable and high-producing human hybridomas secreting human mAbs of the required specificity. Therefore, alternative approaches based on transgenic mice or in phage display were developed to generate fully human mAbs. Thanks to the increasing ability in manipulating the mouse genome and in cloning large-sized DNA fragments, it became feasible to exploit the natural strategies of the immune system, i.e., the natural recombination and the affinity maturation processes to obtain mouse strains with a large and diverse V gene repertoire that, in a full

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immunocompetent context and upon immunization, are able to produce high-affinity human IgGk and IgGl antibodies (Lonberg 2005). Furthermore, the hybridoma technology, well-established in mice, can be easily applied to B cells obtained from these animals enabling efficient and rapid selection of human mAbs. An alternative way of making fully human antibodies was demonstrated in 1990 by John McCafferty et al. using phage display technology (McCafferty et al. 1990; Hoogenboom 2005). Antibody phage display technology consists in the selection of antibody fragments from combinatorial libraries displayed on the surface of filamentous phage. The immunoglobulin repertoire (antibody library) can be derived from the antibody genes expressed in a variety of B cells (e.g., peripheral blood lymphocytes, bone marrow, or spleen). Large libraries of Fab fragments or scFv (see Fig. 8.2) can be constructed from nonimmunized or immunized individuals and then used for the selection of binders to a desired target. A crucial advantage of this technology is the linkage of displayed antibody phenotype with its encapsulated genotype which allows the evolution of the selected binders into optimized molecules. Considering the aforementioned examples, it should be noted that a specific suffix-based nomenclature has been established to provide a format for mAb classification. Thus, mouse mAb will end with the suffix momab, chimeric mAbs will end with ximab, humanized mAbs will end with zumab, and human mAbs will end with umab (Fig. 8.1).

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8.3 Antibody Engineering 8.3.1 Antibody Fragments Today, protein engineering can be used to reshape antibody molecules and devise strategies to obtain suitable antibody fragments and, thanks to advances in molecular biology, it is now possible to produce recombinant antibodies using bacterial cells (in particular Escherichia coli). The modular domain architecture of immunoglobulins has been exploited to create a wide variety of antibody formats that differ in molecular-weight and in valency (Fig. 8.2). The Fab2 and Fab (fragment antigenbinding) formats, obtainable by proteolytic cleavage, chemical cross-linking, or protein engineering are composed of a pair of a single two-chain structure, VH + CH1 and VL + CL, linked at the terminal of the constant domain by an interchain disulfide bond. At present, the building block that is most frequently used to create novel antibody formats is the single-chain variable (V)-domain (scFv) consisting of a single polypeptide (30  kDa) formed by the variable regions of the heavy and light chains and joined together by a peptide-based linker consisting of up to ~15 amino acid residues. Alternatively, the variable heavy and light domains can be assembled as Fv, without a linker peptide, exploiting their natural hydrophobic interactions. ScFv can also be engineered into a diabody format by using a short (5aa) linker to allow the pairing between chains of the same polypeptide. In the diabody, VH and VL pairing occurs between complementary domains of two different chains creating a stable noncovalent bivalent antibody. The smallest antibody-derived binding structure is the so-called single domain (also named VHH, dAb, nanobody). In the case of camelids, these domains typically display long surface loops, which allow penetration of cavities of target antigens and exhibit good stability in physiologic solvents; isolated VH domains from other mammals are generally less soluble and should be stabilized by masking hydrophobic surface patches (Jespers et al. 2004).

8.3.2 Protein Engineering Other antibody manipulations in practice consist of: (1) affinity maturation using different methodologies, such as chain shuffling (Clackson et al. 1991; Kang et al. 1991; Marks et al. 1992), or random point mutation (Hawkins et al. 1992) obtained both by using an error-prone polymerase or a mutator strain; and (2) engineering of monovalent structures (e.g., Fab, scFv, single domains) in multivalent structures to increase functional affinity (or avidity). If required, scFv or Fab fragments can be grafted onto an Fc (Burtrum et  al. 2003; Powers et al. 2001, Valadon et al. 2006); thus, it is possible to reconstitute an entire antibody molecule and choose the isotope more suitable for the desired purpose. Moreover, during the last couple of decades, the knowledge of antibody

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structure has improved so much that today it is possible to use antibody engineering to improve, or decrease, ADCC, CDC and binding to FcgRs and FcRns. Also, glycoengineering of antibody-producing host cells to express mAbs with enhanced effector functions has been attempted with success. The engineering of the Fc of a mAb to tailor antibody Fc property was extensively reviewed (Yamane-Ohnuki et al. 2004; Carter and Brooks 2006; Kanda et al. 2007; Liu et al. 2008).

8.4 Antibody-Derived Therapeutic Reagents Beside the already described effector functions related to physiological antibody activity, other strategies have been developed to increase antibody efficacy in order to optimize their clinical therapeutic application; indeed, they could also be used as toxic molecule-delivering agents. The antibody-drug conjugate concept is based on using the specificity of an antibody to deliver a cytotoxic agent selectively to a target, such as a TAA. MAbs have been conjugated with a- or b-emitting radionuclides, cytokines, cytotoxic drugs, or enzymes and toxins which have cytostatic or cytotoxic effects (Fig. 8.3). The targeting specificity of mAbs directed to TAAs may also be retained by smaller antibody constructs, like scFv as previously described. Importantly, the scFv fragment displays many of the required characteristics including a better tumor microdistribution in comparison with the intact IgG, which accumulates into the perivascular regions of the tumor (Yokota et al. 1992).

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8.4.1 Immunocytokines Immunocytokines are a class of chemically linked or fusion proteins that combine targeting molecules with cytokines as effectors. These bifunctional molecules increase the tumor-killing activity of the antibodies and activate a secondary antitumor immune response by stimulating a broad variety of immune cells. A very detailed review of the applications of cytokine fusion proteins has been recently published (Schmidt 2009). The advantage of cytokine fusion proteins is that they concentrate the cytokine in the tumor microenvironment and increase the direct antitumor effect of the antibody without causing the severe toxic side effects of systemic high-dose cytokine administration. Taking advantage of the targeting specificity of mAbs directed to TAAs, fusion proteins have been constructed and applied in clinical studies with three different cytokines: IL-12, IL-2, and TNFa (Halin et al. 2003; Niculescu-Duvaz 2004; Lo et al. 2007; Johnson et al. 2008; Wagner et al. 2008).

8.4.2 Immunotoxins The paradigm of the magic bullet, selectively binding and destroying only ­malignant cells, is the idealistic concept that led to the construction of toxin fusion proteins. Initially, toxins were chemically conjugated to the purified IgG molecules. This caused considerable problems with regard to reproducible stoichiometry between the binder and toxin and involved labor-intensive production including steps for the coupling and removal of unbound toxins. Nowadays, it is possible to build the immmunotoxin using recombinant technology. The general principle of immunotoxins is the targeting of cell-surface TAA and their subsequent internalization by endocytosis. After cleavage in the endosome and translocation of the catalytic toxin domain to the cytosol, protein synthesis is inhibited. The arrested protein synthesis then leads to cell death by inducing apoptosis. A recent review provides an excellent overview of the mechanism of action of immunotoxins (Pastan et al. 2006). Currently, four antibody-based immunotoxins are in clinical trials targeting receptors that are primarily expressed on cancer cells.

8.4.3 Bispecific Antibodies In the absence of clear evidence of an efficacious antitumor T-cell immunity, an alternative approach to engage T cell-mediated cytotoxicity at the tumor site are antibodies, which are bispecific for a surface TAA on cancer cells and for CD3 present on T cells. These bispecific antibodies (BsAbs) (Fig.  8.3) are capable of connecting any kind of cytotoxic T cell to a cancer cell, independently of T-cell receptor specificity or peptide antigen presentation. The targeting domain may also

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be linked to a triggering arm that is specific for a cell-surface molecule capable of mediating a phagocytic or lytic response by other effector cells, such as macrophages or natural killer cells. Various strategies, which can be classified into two major approaches, have been used to prepare BsAbs. In one approach, bispecific molecules are generated by two or more different polypeptide chains capable of heterodimerization, resulting in bivalent or multivalent proteins. Production of such hetero-oligomeric BsAbs requires: (1) the simultaneous expression of two or more antibody chains, in equal amounts and in the same bacterial or eukaryotic cell; or (2) laborious refolding from inclusion bodies containing individually expressed polypeptides. The second approach, in contrast, embodies the principle “one gene – one product”. In this case, VH and VL from two antibodies of different specificity are fused together as a single polypeptide chain, and functional Fv modules are either formed from the complementary domains of the same polypeptide chain or are created by homodimerization of the single-chain molecule. The different methods to produce bispecific antibodies have been extensively reviewed (Kipriyanov and Le 2004). Bispecific antibodies are currently in preclinical and clinical development for the treatment of various cancers.

8.4.4 T-Bodies/CIRs “T-bodies” are genetically engineered T cells armed with chimeric receptors whose extracellular recognition unit is comprised of an antibody-derived recognition domain and whose intracellular region is derived from lymphocyte-stimulating moiety (ies). Nowadays, T-body, also known as chimeric immune receptor (CIR), is built using preferentially a scFv antibody fragment (Fig.  8.4a). At present, the most frequent lymphocyte activation moieties utilized include a T-cell triggering (e.g., CD3 zeta or gamma) moiety in tandem with a T-cell costimulatory (e.g., CD28) domain. By arming effectors lymphocytes (such as T cells and natural killer cells) with such chimeric receptors, the engineered cells are redirected with a predefined specificity to any desired target antigen, in a non-HLA restricted manner (Hwu et al. 1995; Alvarez-Vallina 2001; Willemsen et al. 2003).

8.4.5 Intracellular Antibodies/Intrabodies Genetic engineering of antibodies has opened new avenues of therapeutic intervention and the possibility to examine in detail the pathophysiological role of some cellular/tumor markers. Many antibody targets are localized in intracellular compartments such as the cytosol, endosomes, lysosomes, the Golgi complex, the endoplasmic reticulum

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(ER), mitochondria, and the nucleus. In certain cases, the antibody target is ­distributed in several cellular compartments, but resides in an active conformation in only one compartment. Targeting antibodies to these locations is a challenge, but the normal cellular machinery is able to overcome such challenges by making use of sorting signals. Intracellular antibody expression in mammalian cells may target the different specific compartments depending on the presence/absence and the amino acid sequences of some common C-terminal- and N-terminal-targeting signals (Canevari et al. 2002) (Fig. 8.4b). These signals allow for the localization of the intracellular antibody to the nucleus, endoplasmic reticulum, mitochondria, cytoplasm, or even to be secreted, without affecting the specific binding of the scFv to its target molecule. The use of this method to functionally knockout proteins with a relevant role in oncologic processes has given rise to various phenotypic effects in tumor cells. Rational approaches to engineering antibody regions suited for optimal expression in the desired intracellular compartment, together with improved vector design and strategies that engage bystander mechanisms, hold the promise of enhancing the feasibility and efficacy of intracellular antibody gene therapy (Lo et al. 2008).

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8.4.6 Virus Retargeting Viral vector systems are widely being used in the development of new genetic approaches for a variety of human diseases. Oncolytic viruses have shown great potential as cancer therapeutics. For example, live attenuated measles viruses have promising oncolytic activity against a variety of tumor cells and xenografts, and they are being tested in phase 1 clinical trials for patients with recurrent ovarian cancer, glioblastoma, and multiple myeloma (Russell and Peng 2009). Measles virus uses its coat protein, hemagglutinin (H), to attach to a cell surface viral receptor and the fusion (F) protein to mediate virus entry and subsequent virus spread by cell-to-cell fusion (Dorig et al. 1993; Tatsuo et al. 2000). Thus, a unique feature of measles virus tumor cell killing is an extensive cytopathic effect of ­syncytial formation, which, in addition to viral replication, significantly increases bystander killing of neighboring cells by the agent. To minimize virus sequestration by nontarget cells and collateral damage to normal tissues, the tropism and cytopathic activity of an oncolytic virus should ideally be restricted to tumor cells. Virus attachment, entry, and subsequent intercellular fusion between infected and uninfected neighboring cells are mediated via the two measles receptors. Mutations in the H protein have been engineered to ablate virus interactions with its native receptors, and a virus rescue system has been established using a pseudoreceptor (His-6 tag) that allows for the rescue and propagation of a virus-receptor blinded, fully retargeted measles virus displaying scFv against TAA (Fig. 8.4c).

8.5 MAbs against the Alpha Isoform of FR and their Applications 8.5.1 History Soon after the development of the hybridoma technology, numerous research groups applied it in the attempt to generate antibodies directed to TAA. Due to the technological constrains and the limited knowledge of tumor biology at that time, the major antigenic source was the tissue of interest without or only with limited fractionation, and the specificity was searched by adopting rigorous screening approaches. In the case of EOC, two independent research groups, applying quite similar approaches, generated hybridomas using as immunogen the membrane preparations from surgical specimens of ovarian cancer or total protein extracts from a choriocarcinoma cell line (Miotti et al. 1987; Rettig et al. 1985), followed by selection using a large panel of human tumors and cell lines. Binding assays showed that the momabs, called MOv18 (IgG1k), MOv19 (IgG2ak) (Miotti et al. 1987) and LK26 (IgG2ak) (Garin-Chesa et al. 1993), bound with high affinity (KA in the range of 108–109 M−1) to ovarian tumor cells. All three mAbs were directed against the same 38–40  kDa glycoprotein, with epitopes recognized by MOv19 and LK26 being overlapping, but

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independent from that recognized by MOv18 (unpublished data). The molecule recognized by MOv18 and MOv19 was characterized as a GPI-anchored molecule (Alberti et al. 1990) and subsequently identified by screening of a cDNA library as a member of the family of homologous proteins that bind folic acid with high affinity, i.e., the alpha isoform of FR (Coney et al. 1991). The possibility of engagement of two different epitopes on the same molecule enabled the impact and relevance of simultaneous or subsequent binding of MOv18 and MOv19 on FRa internalization (Casalini et  al. 1991), and it allowed for the development of a double-determinant ELISA (Mantovani et al. 1994), suited for the measurement of soluble circulating FRa. None of the above-mentioned mAbs, MOv18, Mov19, and LK26, proved to be adequate for the target molecule detection in formalin-fixed, paraffin-embedded archived material. Few other anti-FRa mAbs were generated and successfully developed for such immunohistochemical applications (Hartmann et  al. 2007; Smith et al. 2007; Brown et al. 2008). The main characteristics and the level of development reached for MOv18, MOv19, and LK26 (and their derivatives) are summarized in Table 8.1, while the preclinical and clinical results obtained with them are widely commented on in the next subsections.

8.5.2 MOv18 and MOv19 -Momab, -Ximab, and Their Derivatives At the preclinical level, mAbs MOv18 and MOv19, which were selected from the same fusion process, have been frequently developed in parallel; therefore, the reported preclinical applications described below are applicable to both mAbs. 8.5.2.1 Preclinical Data -Momab and Derivatives The original mouse mAbs were initially evaluated for their ability to target radioisotopes to tumors (see Sect. 8.4), starting with 131I, the most widely used isotope in clinical practice for radioimmunotherapy due to its well-established protein iodination chemistry and its availability. The obtained radio-immunoconjugates presented a level of tumor uptake sufficient to enable radiographic detection (Gadina et  al. 1991), thus opening the way to the clinical exploitation of these reagents (see Sect. -Momab and Derivatives below). MOv18 was subsequently radiolabeled with other a- or b-emitting radionuclides, such as 211At (Andersson et al. 2000), 90Y (Coliva et al. 2005), and 177Lu (Zacchetti et al. 2009); and, in all cases, a therapeutic effect was recorded, assessed as tumor growth control in either subcutaneous or intraperitoneal animal models. To identify the optimal radioconjugate with b-emitting nuclides, 131I-, 90Y-, and 177Lu-radiolabeled MOv18 were compared in a well-defined animal model. This study allowed for the

8  Anti-FR Antibody Generation and Engineering Table 8.1  Anti-FRa mAbs and their derivatives: state of the art Reached level Name Origin Structure of development MOv18 and its derivatives MOv18 Momab Entire IgG1 OCTR Momab anti-FR/antiCD3 Bi-F(ab)2 Mov-g Momab scFv T-body MV-alphaFR Momab Viral-fusion protein, scFv CHI-MOv18 Ximab Entire IgG1 MOv18-IgE Ximab Entire IgE AFRA1/AFRA2/ Umab Fab AFRA3 MOv19 and its derivatives MOv19 Momab Entire IgG2a Mov19/VD4 Momab anti-FR/antiCD16 Bi-mAb MOV19 Momab scFv intrabody scFvE-er IL-2/MOV19 Momab scFv fusion protein MOV19 scFv-g Momab scFv T-body chain CHI-MOv19 Ximab Entire IgG1 C4 Umab scFv DFM-AFRA5.3 Umab Chemical Fab2 LK26 and its derivatives LK26 Momab Farletuzumab Zumab

Entire IgG2a Entire IgG1

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Key references

Clinical: phase I/II Clinical: phase I/II

Crippa et al. (1995) Canevari et al. (1995b)

Clinical: phase I Preclinical in vivo

Hasegawa et al. (2006) Kershaw et al. (2006)

Clinical: phase I Preclinical in vivo Preclinical in vitro

Molthoff et al. (1997) Karagiannis et al. (2003) Figini et al. (2009)

Preclinical in vitro Preclinical in vitro

Mantovani et al. (1994) Ferrini et al. (1992)

Preclinical in vivo

Figini et al. (2003)

Preclinical in vivo

Melani et al. (1998)

Preclinical in vitro

Mezzanzanica D. (unpublished data) Macor et al. (2006) Figini et al. (1998) Figini et al. (2009)

Preclinical in vitro Preclinical in vitro Preclinical in vivo

Preclinical in vivo Clinical: phase III ongoing a  See also http://www.clinicaltrials.gov/ identifier NCT00318370

Ebel et al. (2007) Spannuth et al. (2010)a

identification of 177Lu as the radionuclide with the best therapeutic window and one more suited for eradicating small tumor masses expressing the antigen of interest (Zacchetti et al. 2009). The good therapeutic index of 177Lu-radiolabelled murine mAb in a nonimmunocompetent system prompted further study to gain insight about this radioimmunotherapy approach by evaluating this radioimmunoconjugate in an immunocompetent transgenic animal model. This double transgenic model expresses human FRa under the control of MMTV on mouse HER2-mammary cancers, and it allowed, on one side, for the monitoring of side effects on the immune system and, on the other side, the evaluation of the induced antitumor immune response following treatment. Repeated treatments were possible without severe side effects, and depending on the time of treatment, a delay in tumor onset, a reduced tumor multiplicity, and impairment in tumor growth were observed. Furthermore, the comparison of the radionuclide-induced cytotoxicity and the engagement of immune effector

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functions, exerted by 177Lu-MOv18 and the entire momab, respectively, enabled the demonstration that only the radiolabeled reagent induced tumor lysis by activating both humoral and cellular responses against the other TAA expressed by mammary cancer cells (HER2); whereas the antitumor effect exerted by the momab induced only an anti-HER2 humoral response (Melani et al., manuscript in preparation). Several different derivatives of MOv18 and/or MOv19 were evaluated in different therapeutic applications. For example, to provide a new tool for the immunotherapy of human ovarian carcinoma, a fusion protein between interleukin-2 (IL-2) and the MOV19 scFv was constructed under the control of the murine IgK promoter (see Fig.  8.3). This small molecule, combining the specificity of MOV19 with the immuno-stimulatory activity of IL-2, was believed to improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. In a syngeneic mouse model, due to its fast clearance from the body, IL-2/MOV19 scFv specifically targeted FRa gene-transduced metastatic tumor cells without accumulating in normal tissues. Furthermore, treatment with the fusion protein, but not with recombinant IL-2, significantly reduced the volume of subcutaneous tumors (Melani et al. 1998). Different formats of bispecific antibodies (see Fig. 8.3), able to engage cytotoxicity of T or natural killer cells to tumor site, were built (Ferrini et al. 1992), and a preclinical model of adoptive immunotherapy based on tumor retargeting of activated T cells by OCTR (an anti-CD3/MOv18 bispecific antibody) was developed. The very encouraging results obtained in the animal model (Mezzanzanica et al. 1991), and the ability to produce a highly purified reagent under GMP conditions, constituted the rational basis for the OCTR to enter into the clinic (see -Momab and Derivatives below). As described above, T cells can be redirected to the antigen of interest by CIR. A MOv18-g CIR (MOv18 scFv-human FcRI g chain) has been used to retrovirally transduce murine tumor-infiltrating lymphocytes, and the treatment with MOv18-gtransduced T cells significantly increased the survival of tumor-bearing mice with respect to mice treated only with saline or nontransduced tumor-infiltrating ­lymphocytes (Hwu et al. 1995). Using the same construct, transduced human cytotoxic T cells from EOC patients were reported to lyse FRa positive cells even after extended in vitro culture times; furthermore, stable MOv18-g CTL clones could be isolated on the basis of effector activity and specific CIR-induced cytokine production profiles (Parker et al. 2000), thus opening the way to a phase 1 clinical trial (see Sect. -Momab and Derivatives below). To gain insight into the role of FR in ovarian cancer progression, the specificity of MOV19 was exploited to construct a scFv intrabody (see Sect.  8.4.5) and the effects of functional down-regulation of FRa membrane expression on ovarian tumor cells were tested. Intrabody-induced FRa downmodulation strongly affected cell proliferation and adhesion, reduced colony-forming ability in soft agar, and was accompanied by morphological change of the cells and the inability to grow in multilayers in three-dimensional organotypic cultures. In fact, anti-FRa intrabodytransfected ovarian cancer cells grew as a single-ordered layer, reminiscent of

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normal ovarian surface epithelium growth in vivo. Taken together, these data suggest that the anti-FRa intrabody reverses the transformed phenotype in ovarian cancer cells, and it may provide an efficient means to inhibit selectively the growth of these cells (Figini et al. 2003). To minimize potential toxicity, e.g., due to measles virus-associated immunosuppression and infection of nontarget tissues (see Sect. 8.4.6), an FRa-exclusive ovarian cancer-targeted oncolytic virus was generated genetically by engineering MOv18 scFv on the tropism-modified viral attachment protein of the measles virus (MV-alphaFR). Tropism and fusogenic activity of MV-alphaFR were redirected exclusively to FRa with no background infectivity on normal human cells. The FRa-targeted measles virus was found to infect and destroy FRa-positive tumors efficiently and exclusively via the displayed scFv. MV-alphaFR, assessed in two models of human ovarian cancer (subcutaneous and intraperitoneal locations), was found to be as therapeutically effective as the original virus, but the latter was only active against FRa-expressing cells, thus introducing a new modality for EOC treatment (Hasegawa et al. 2006).

-Ximab and Derivatives The murine k, g1 (MOv18), or g2a (MOV19) genes were substituted with the genes encoding the human k chain and g1 constant regions (Coney et al. 1994). The IgG1 -ximabs bound to FRa with the same affinity as the original -momabs and were active in ADCC to ovarian tumor cells in in vitro assays. Chi-MOv18, upon production under GMP conditions, entered in phase 1 studies (see Sect.  -Ximab and Derivatives below). Furthermore, the availability of chimeric versions of both MOv18 and MOv19, recognizing two nonoverlapping and spatially closed epitopes on FRa, enabled the finding that effective CDC was possible only when the two epitopes were simultaneously engaged by the mixture of MOv18 and MOv19 -ximabs while in the ­presence of reagents capable of neutralizing membrane ­complement regulatory proteins (Macor et al. 2006). In the case of MOv18, the murine k, g1 genes were substituted with the genes encoding the human k chain and e constant regions, and the resulting IgE -ximab had the expected affinity for FceRI (Gould et al. 1999). This chimerization represents the first example for using the IgE constant region and was based on the hypothesis that IgE should be superior to IgG1 in preventing the growth of solid tumors. In fact, although CHI-MOv18 (IgG1) was more efficient than the corresponding IgE version in the killing of ovarian tumor cells in vitro, the MOv18-IgE gave significantly greater protection against tumor growth in mouse models of ovarian carcinoma (Gould et al. 1999; Karagiannis et al. 2003). When the mechanisms by which MOv18-IgE may exert its antitumor activities were analyzed, it was found that monocytes contributed to both cytotoxicity and phagocytosis, while eosinophils were found to be potent effectors only for cytotoxicity (Karagiannis et al. 2007).

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8.5.2.2 Clinical Data -Momab and Derivatives In the early nineties, the momab, Mov18, as entire molecule, was produced at clinical grade and then clinically evaluated as a radiolabeled reagent for imaging or immunotherapy applications. Scintigraphic images were acquired in more than 100 patients to evaluate the diagnostic potential of 131I-MOv18 or a two-step procedure (e.g., nonradioactive, biotinylated momab followed by 131I-avidin) in comparison to other instrumental evaluations or to laparotomic histopathologic confirmation (Crippa et al. 1991; Paganelli et al. 1992). The obtained data indicated that lesions expressing the target antigen were detected with high specificity (97%), independently of the injection route, and a high sensitivity (mean 87%, range 61–95%), which was more dependent on the isotope than of the injection route. These data proved that the mAb could reach its target in  vivo and provide the basis for its therapeutic exploitation. A phase 1–2 study to evaluate the therapeutic potential of 131 I-MOv18 later was conducted in an adjuvant setting in advanced-stage patients with minimal residual disease after first-line treatment (i.e., debulking surgery and chemotherapy). Clinical follow-up and/or third-look evaluation performed 90 days after a single intraperitoneal administration of 370  GBq (100  mCi) of activity resulted in complete long-term remission in 5 of 16 treated patients and stable disease in six patients (Crippa et al. 1995). On the basis of preclinical data, and since high-energy b emitters, such as 90Y, are more suitable for larger tumors due to the high penetration of their radiation throughout large tumor masses, a pilot trial with 90Y-MOv18 was planned in patients bearing advanced-stage ovarian cancer. Biodistribution studies performed with 111In-MOv18 indicated a significant uptake of the radiolabeled antibody in the tumor, while a normal distribution was observed in the other target organs (Fig. 8.5). Despite the good preclinical and clinical biodistribution results, the study was not feasible since a substantial proportion of patients had mobilized suboptimal amounts of CD34+ cells (to be stored prior to treatment to counteract bone marrow toxicity consequent to 90Y treatment), thus only two out of ten enrolled patients were eligible for leukapheresis (unpublished data). The major limitation of all the studies with momab was the development of a HAMA response. In fact, in agreement with literature data (Khazaeli et al. 1994), and depending upon the assay used to monitor the HAMA response, 60–90% of cancer patients injected with intact murine MOv18 developed a humoral response against the murine Ig, even after a single dose (Crippa et al. 1995). The bispecific antibody OCTR entered in phase 2 studies where activated autologous T cells were coated in vitro with OCTR and were reinjected intraperitoneally (Canevari et al. 1995b) or both intravenously and intraperitoneally (Canevari et al. 1995a). In a total of 26 ovarian carcinoma patients with advanced stages of disease, an overall response rate of 27% was observed and toxicity was found to be transient and only mild to moderate in severity. The major limitations were the ­difficulty in recirculation of activated autologous T cells retargeted by bispecific

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Fig.  8.5  111In-MOv18 scintigraphy. A scintigraphic image acquired 48  h postinjection of 111 In-MOv18 in an EOC patient with a recurrent pelvic lymph-node (anterior view)

anti CD3/anti-FRa F(ab)2 fragments and the need for in vitro preactivation of the T cells (Canevari et al. 1995b). Interestingly, we observed that: (1) OCTR induced a HAMA response in 86% of the patients by the end of treatment where an antiidiotypic response was most consistent; and (2) a significantly longer survival was recorded in patients with high HAMA levels, even when the subgroup of nonresponder patients was considered (Miotti et al. 1999). These observations, although preliminary and requiring further validation, are consistent with some clinical data reported by others (DeNardo et al. 2003).

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A phase 1 study was conducted which was aimed to evaluate the feasibility and toxicity of the treatment with T cells transduced with MOv18-g CIR. FRa-specific T cells could be produced from all 14 EOC patients entered in the study. The treatment was well tolerated; however, there was no evidence of antitumor responses in any patient. Poor trafficking of T cells to tumor, low persistence of transferred T cells in patients, and the development of T cell inhibitor activity (potentially a HAMA response to the mouse scFv component of the chimeric receptor) in serum of 50% of tested patients may account for the absence of patient responses (Kershaw et al. 2006). -Ximab and Derivatives With the aim of reducing the immunogenicity of the antibody-based reagent in the radioimmunodiagnostic or radioimmunotherapeutic applications, 131I-CHI-MOv18 was tested. Imaging ability and dosimetric analysis from both intravenous and intraperitoneal routes of administration confirmed the therapeutic potential of 131I-CHIMOv18 (van Zanten-Przybysz et al. 2000). The pharmacokinetics and efficacy of a therapeutic dose of 131I-CHI-MOv18 (3 GBq) were studied in three patients with ovarian cancer. All patients experienced stabilization of disease for 2–6 months, as assessed by CT scans and serum CA125 measurements (van Zanten-Przybysz et al. 2000). The chimeric MOv18 was also tested in a phase 1 trial to evaluate its potential for in vivo activation of ADCC, and up to 75 mg of the ximAb could be administered without evident toxicity or minor side effects (Molthoff et al. 1997). In contrast to the murine MOv18, the immunogenicity of chimeric MOv18-IgG was low or undetectable (Buist et al. 1995, van Zanten-Przybysz et al. 2000). A chimeric version of OCTR was generated to decrease immunogenicity and to allow more extended schedules. Of the two patients who were treated with the Fab2 fragments of the chimeric OCTR, only one developed a low-transient HAMA response just above background level (Luiten et al. 1997).

8.5.3 LK26 Momab, Its Conversion to Zumab and Its Optimization for Ovarian Cancer Therapy The LK26 momab was tested for its in vivo antitumor activity in a subcutaneous xenograft model (Ebel et al. 2007). Thanks to the IgG2a isotype, the momab was very effective in recruiting the endogenous mouse natural immunity, and depending on the dosing and the schedule of treatment, a decrease in tumor growth, ranging from 40 to 70 %, was obtained. These data, together with the evidence of in  vitro growth inhibitory effect against EOC cell lines, gave the proof-of-principle of the suitability of the binding specificity of LK26 for immunotherapy.

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8.5.3.1 Generation of Farletuzumab The initial humanization process of LK26 resulted in a strong decrease in affinity that required the application of a “whole cell genetic evolution platform” (Nicolaides et al. 2005). The adopted procedure is based on the ability to modulate the mismatch repair process (referred to as morphogenics) in model systems to create genetic diversity within defined cellular systems. Morphogenics has been successfully applied to the LK26-derived, zumabproducing cell line to yield subclones producing antibodies with enhanced binding affinities for therapeutic use, as well as to derive subclones with enhanced titers that are suitable for scaleable manufacturing. The optimized -zumab, called farletuzumab, has an affinity similar to the original momab (nanomolar range), a tissuebinding profile consistent with the target antigen distribution, and demonstrated in  vitro ability to mediate cell cytotoxicity to EOC cell lines through CDC and ADCC (Ebel et al. 2007).

8.5.3.2 Farletuzumab Preclinical Data In a subcutaneous preclinical mouse model, effective systemic targeting of tumors has been achieved with both 131I- and 111In-labeled farletuzumab, and the clearance rate from normal organs such as liver, kidney, and spleen was similar to the blood clearance (Smith-Jones et al. 2008). Farletuzumab (MORAb-003) was the first anti-FRa zumab tested in cynomolgus monkeys. Four toxicology studies were performed: an escalating dose study, a high-dose tolerance study, a 28-day repeat dose study with a 28-day recovery period, and a 24-week repeat dose study. Consistent with administration of a foreign protein, splenomegaly was observed; however, altogether these studies demonstrated no apparent toxicity up to a total dose >120 mg/kg over 28 days (Ebel et al. 2007).

8.5.3.3 Farletuzumab Clinical Data In a pilot study, the biodistribution and tumor targeting of 111In-labeled farletuzumab was assessed in three patients undergoing treatment with unlabeled zumab, and the images demonstrated good tumor uptake and retention; for these patients, blood clearances were biphasic with an average beta biological halflife that was quite long (>120  h) and comparable to that observed in mice (Smith-Jones et al. 2008). Clinical trials, published or presented at international meetings from 2006 to the present, have been conducted and presented in two recent reviews (Kalli 2007; Spannuth et  al. 2010). A first-in-human, open-label, nonrandomized,

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multiple-infusion, dose-escalation (12.5–400  mg/m2) phase 1 trial in patients with platinum-resistant EOC indicated a slow drug clearance and no dose-limiting toxicity, and it suggested a stabilization of the disease in almost half of the treated patients. The open-label, nonrandomized, explorative phase 2 study of farletuzumab as a single agent and in combination with platinum and a taxane had enrolled a total of 58 EOC platinum-sensitive patients at first relapse in the USA and Germany. Forty-three patients, treated either with combination therapy from the beginning of the study or with farletuzumab as single agent until relapse and then with combination therapy, were available for evaluation of objective response. The overall response rate observed (70%) was higherthan-expected relative to the historical expectations and opened the way to the start of an ongoing phase 3 study of farletuzumab in first-relapsed, platinum-sensitive ovarian cancer patients.

8.5.4 Conversion of Momab to Umab and their Optimization for Ovarian Cancer Therapy Completely human Fab fragments against FRa were produced by using phage display and one, named C4, exhibited an estimated Kaff of 200  nM (using Scatchard analysis on entire EOC cells) and a good specificity (Figini et  al. 1998). The development of the C4-Fab fragment for in  vivo clinical use has therefore been envisaged; however, its poor production yield greatly reduced its suitability. New selections using dual-combinatorial library phage from ovary cancer patients were performed applying epitope imprinting selection (Figini et al. 1994; Figini et al. 1998), a method that permits the isolation of antibody, using guided selection, with the same specificity of a preexisting antibody which is of particular interest, but that which is not adequate for in vivo studies (e.g., is of mouse origin). Guided selection of MOv18 or MOv19 resulted in the identification of three (AFRA1–3) and two (AFRA4 and 5) new human Fabs, respectively, that recognize overlapping FRa epitopes. After the selection of the best candidate and the optimization of the lead reagent, a chemical dimer, named AFRA-DFM5.3, was considered suitable for in  vivo preclinical evaluation. The pharmacokinetics parameters of 131 I-AFRA-DFM5.3 supported its potential therapeutic use (Figini et  al. 2009). When the efficacy of human xenografts’ treatments with 131I-AFRA-DFM5.3 was examined in preclinical mouse models, pharmacokinetics, toxicity, and biodistribution analyses highlighted the importance for having a precocious radioimmunotherapy method for allowing easy access to tumor cells with 131I-labeled human antibody fragments. Accordingly, the best antitumor activity was achieved by loco-regional treatment of intraperitoneally growing tumors with more than 50% of treated animals cured (Zacchetti et al., unpublished results).

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8.6 MAbs against the Beta Isoform of FR and their Applications At variance with the large amount of published data at preclinical and clinical levels for anti-human FRa mAb, there are few reports related to anti-mouse FRb mAb generation and to their exploitation in syngeneic preclinical models for therapeutic applications. Mouse and rat mAb were obtained by immunizing with cells ectopically expressing the mouse FRb and then subsequently used for the production of chemical- and recombinant-based immunotoxins (see Sect.  8.4.2), respectively (Nagayoshi et  al. 2005; Nagai et  al. 2009). In both instances, the immunotoxins induced apoptosis in FRb expressing cells; furthermore, the recombinant immunotoxin derived from the momab was also able to bind and to induce apoptosis in synovial mononuclear cells from rheumatoid arthritis patients. In the oncologic field, and in agreement with the role of tumor-associated macrophages in promoting tumor growth, the injection of the recombinant anti-mouse FRb immunotoxin into an experimental glioma model significantly depleted murine tumor-associated macrophages and reduced tumor growth (Nagai et al. 2009).

8.7 Concluding Remarks and Perspectives In the oncologic field, antibody-based therapy seems an attractive strategy for those tumors, such as epithelial ovarian cancer and glioblastoma, for which the existing treatment options are still not sufficient. In addition, the level of development reached by genetic engineering seems to indicate that the antibody molecule could be tailored according to the clinically envisaged application. With the aim to identify a new treatment strategy for ovary cancer, anti-human FRa mAbs were generated and modified, paralleling the history of mAb genetic engineering. The promising results obtained with the mouse mAbs MOv18 and MOv19 and with their derivatives enabled, in some cases, to reach an advanced level of development. In particular, the optimized humanized version of LK26, called farletuzumab, recently entered into a phase 3 trial, and the chemical dimer of a fully human Fab mimicking the MOv19 epitopic recognition, called AFRA-DFM5.3, is almost ready for a first-in-human phase 1 study. The full exploitation of these anti-FRa antibodies awaits confirmation of the promising drug safety and clinical efficacy in further well-designed randomized clinical trials. Furthermore, a series of anti-FRa antibody-based alternative therapeutic approaches, including those based on the new format of bispecific antibody generation, the measles virus retargeting, and the intrabody gene therapy, could be envisaged. Finally, the availability of mAbs directed against two different epitopes of FRa could allow for the development of a double-determinant assay that is potentially useful for the identification of patients accruable in clinical trials based on anti-FRa antibody therapeutic approaches. In the case of anti-FRb mAbs, although at present only a limited

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amount of data is available, the rationale of their application in specific fields, such as the glioblastoma treatment, seems to merit further efforts towards their generation and modification.

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Chapter 9

Folate Receptor Positive Macrophages: Cellular Targets for Imaging and Therapy of Inflammatory and Autoimmune Diseases Michael J. Hansen and Philip S. Low

Abstract  FRb constitutes a unique and selective marker for activated human ­monocytes and macrophages. Based on numerous criteria, most FR+ macrophages appear to be highly inflammatory and important in the development/maintenance of many inflammatory and autoimmune diseases, however, some appear to be activated and anti-inflammatory. Inflammatory pathologies in which FR+ macrophages are commonly enriched include rheumatoid arthritis, Crohn’s disease, atherosclerosis, sarcoidosis, glomerulonephritis, osteoarthritis, organ transplant rejection, ulcerative colitis, Sjogren’s syndrome, diabetes, ischemia/reperfusion injury, impact trauma, microbial infection, prosthesis osteolysis, liver steatosis, and multiple sclerosis. Folate-targeted imaging agents have proven useful in identifying, localizing, and quantifying sites of inflammation in both human patients and animal models of the above diseases. Folate-targeted therapeutic agents offer great promise for the development of highly potent, nontoxic treatment modalities for the same diseases. Keywords  Folate receptor targeting • Activated macrophages • Autoimmune and inflammatory diseases • Rheumatoid arthritis imaging and therapy • Atherosclerosis imaging

9.1 Macrophage: Lineage, Biological Function, and Identification Macrophages originate in the bone marrow from hematopoietic stem cells, which differentiate in response to biological stimuli to become monocytes. Monocytes commonly circulate in the bloodstream for several days before they enter the

P.S. Low (*) Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA e-mail: [email protected] A.L. Jackman and C.P. Leamon (eds.), Targeted Drug Strategies for Cancer and Inflammation, DOI 10.1007/978-1-4419-8417-3_9, © Springer Science+Business Media, LLC 2011

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tissues via a process called diapedesis and then differentiate into macrophages or dendritic cells (see Furth and Cohn 1968; Geissmann and Manz 2010). Because the tissue microenvironment can influence the differentiated state of a macrophage, macrophage properties often differ from tissue to tissue and can change in response to variations in the stimuli to which they are exposed (e.g., cytokines, pathogen-derived molecules, apoptotic cell fragments, etc.) (see Gordon and Taylor 2005). In response to certain stimuli, macrophages become pro-inflammatory, secreting reactive oxygen species (ROS), nitric oxide, metalloproteinases, prostaglandins, and inflammatory cytokines such as TNF-a, IL-1 and IL-6 (see Benoit et al. 2008). They may also express a relatively unique set of cell surface markers including CD80, CD86, Ly6C/G, and CD11c (see Lumeng et  al. 2007; Benoit et al. 2008). In response to other stimuli, macrophages become anti-inflammatory and even immunosuppressive, expressing proteins such as IL-10, IL-4, a mannose receptor, CD23, CD163, and arginase (see Gordon 2003a, b; Benoit et al. 2008; Martinez et  al. 2009). Under still other circumstances, the macrophages can primarily serve a housekeeping function by phagocytosing dying cells and cellular debris. And while references in literature often refer to inflammatory macrophages as M1 macrophages and anti-inflammatory macrophages as M2 macrophages, it is clear that these distinctions are over-simplified and that a continuum of proinflammatory to anti-inflammatory states exists, with some macrophages even simultaneously expressing pro- and anti-inflammatory markers either when transitioning from one activated state to another or when confronted with mixed pro-inflammatory and anti-inflammatory signals (see Fig.  9.1; Buechler et  al. 2000; Stout and Suttles 2004; Porcheray et al. 2005; Lumeng et al. 2007; Zeyda et al. 2007; Benoit et al. 2008; Fuentes-Duculan et al. 2010). To complicate matters further, the morphological and functional plasticity of macrophages has led to their classification with different names in different tissues, being called Kupffer cells in the liver, microglial cells in the brain, osteoclasts in the bone, Langerhans cells in the skin, etc. (see Gordon and Taylor 2005). While macrophages can be generally distinguished from other cell types by their expression of specific cell surface proteins (e.g., CD14, CD11b, F4/80 (mice) EMR1 (humans), Mac-1/Mac-3 and CD68) (see Khazen et al. 2005), there are very few markers that reliably discriminate pro-inflammatory from anti-inflammatory macrophages. One marker that has recently been employed to define the more ­pro-inflammatory phenotype is CD11c, a protein that is upregulated on adipose tissue macrophages and macrophages that actively produce inflammatory cytokines and ROS (see Lumeng et  al. 2007; Patsouris et  al. 2008). A second protein that appears to characterize many pro-inflammatory macrophages is folate receptor b (FRb), an isoform of the folate receptor whose expression is restricted to myeloid cells (see Nakashima-Matsushita et al. 1999; Heijden et al. 2009; Xia et al. 2009). The purpose of this review is to summarize the properties of these FRb positive macrophages and describe efforts aimed at imaging and treating inflammatory diseases with FRb-targeted agents.

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Fig.  9.1  General phenotypes and polarization of macrophages. Phenotypic characteristics overlap continuously between classically activated (M1) and alternatively activated (M2) macrophages, rendering an unambiguous definition of each type of macrophage impossible. The specific phenotype of each macrophage depends on the plethora of environmental stimuli to which it is exposed, which can differ both spatially and temporally within the same tissue. Reproduced with permission from The American Association of Immunologists, Inc. Copyright 2008 (see Benoit et al. 2008)

9.2 Folate Receptor Expression Patterns Folic acid is a vitamin required for the synthesis of nucleotide bases and is consequently essential for the proliferation of all cells. Folates are also required for the production of S-adenosylmethionine, a common substrate used in methylation of DNA, histones, G proteins, and many metabolic building blocks (see Kim 2005; Loenen 2006). Almost all cells take in folates via the reduced folate carrier or proton-coupled folate transporter (see Antony 1992; Matherly and Goldman 2003). A few cells, however, also express a folate receptor that binds folic acid ~100,000 times tighter than the aforementioned transporters, and carries bound folates into cells by receptor-mediated endocytosis (see Antony 1992; Nakashima-Matsushita

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et al. 1999; Turk et al. 2002). Different isoforms of the FR are used by certain cancer cells, activated macrophages, and the proximal tubule cells of the kidney to capture folates from their environment (see Ross et al. 1994; Nakashima-Matsushita et al. 1999; Turk et al. 2002; Parker et al. 2005; Xia et al. 2009). There are four members of the FR family: FRa, FRb, FRg, and FRd (see Elnakat and Ratnam 2004). FRa is expressed on the apical surfaces of a few epithelial cells (primarily proximal tubules of the kidneys and alveolar epithelial cells of the lungs) and is upregulated on a variety of epithelial-derived tumors (see Weitman et  al. 1992; Salazar and Ratnam 2007; Leamon and Jackman 2008). FRb is found on monocytes and macrophages (but not red blood cells, lymphocytes, basophils, platelets, etc.) and is present on myelogenous leukemias (see Ross et al. 1994, 1999; Nakashima-Matsushita et al. 1999). Interestingly, FRb does not bind folic acid on quiescent macrophages until the myeloid cell becomes activated, allowing selective targeting of macrophages with folate conjugates only at sites of inflammation or following their malignant transformation (see Reddy et al. 1999; Paulos et al. 2004; Turk, Waters and Low 2004). FRg is rarely expressed and difficult to detect in vivo (Nagai et al. 2006). FRd is expressed on regulatory T cells, where it exhibits a very low affinity for folic acid (Turk, Paulos, and Low, personal observations). FR in the kidney acts as a salvage receptor where it captures folates from the nascent urine and transcytoses them back across the kidney epithelium for release into the blood (see Weitman et al. 1992; Elnakat and Ratnam 2004; Sandoval et al. 2004).

9.3 Evidence That FRb Constitutes an Activation Marker for Macrophages Several lines of evidence argue that many FR positive macrophages are proinflammatory. First, injection of inflammatory stimuli (live bacteria, thioglycollate, BCG, etc.) into the peritoneal cavities of rodents induces a peritonitis characterized by a large influx of FR+ macrophages (see Xia et al. 2009). These macrophages are enlarged, have irregular morphologies, secrete ROS, express pro-inflammatory markers (CD80, CD86, and Ly6C/G), and exhibit little or no expression of anti-inflammatory markers (e.g., mannose receptor, arginase, and CD23). Second, synovial fluid macrophages from patients with rheumatoid arthritis bind folate-FITC, suggesting that the macrophages enriched in these inflamed joints express FR (see Nakashima-Matsushita et  al. 1999; Xia et  al. 2009). Third, g-scintigraphy images of patients with a variety of inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease, ischemic bowel disease, Sjogren’s syndrome, localized infections, atherosclerosis, organ transplant rejection, etc.) show uptake of 99mTc-EC20 (a folate-peptide metal chelator) at the anticipated sites of inflammation (see Low et  al. 2008; Matteson et  al. 2009; Ayala-López et al. 2010; Rothenbuhler and Low, personal observations). Fourth,

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Fig.  9.2  Macrophage production of reactive oxygen species (ROS) correlates linearly with expression of FR. Macrophages were harvested from the peritoneal cavities of mice that had been injected 1–3 days previously with live bacteria. FR expression and ROS production were then quantitated by flow cytometry, as described elsewhere (Xia et al. 2009). FR expression, as measured by folate-fluorescein binding, was found to correlate linearly with production of ROS, as measured by the fluorescence of 5-(and-6)-carboxy-2¢,7¢-dichlorodihydrofluorescein diacetate. This research was originally published by Xia et al. (2009). Copyright the American Society of Hematology

the level of FRb on macrophages correlates linearly with their production of ROS, and most FRb positive macrophages secrete TNF-a (see Fig. 9.2; Xia et al. 2009). Finally but most importantly, folate-targeted therapies that deplete FR+ macrophages successfully treat the symptoms of various animal models of human inflammatory diseases (Paulos et al. 2006; Varghese et al. 2007; Varghese et al. in press). It is difficult to imagine how depletion of anti-­inflammatory macrophages could cure an inflammatory disease. Despite the above evidence for association of an inflammatory phenotype with FR expression, there is also evidence that FR+ macrophages can be anti-inflammatory. In in vitro studies “Puig-Kroger et al. (2009) showed that monocytes stimulated to differentiate in the presence of GM-CSF (commonly assumed to induce M1 activation) show no expression of FR, whereas monocytes stimulated to differentiate in the presence of M-CSF (commonly assumed to induce M2 activation) promote expression of FR. The same group also examined tumor associated macrophages (TAM’s) isolated from human patients and found some expression of FR on these myeloid cells. Because TAM’s are often assumed to be anti-inflammatory, this observation was cited as evidence that FR+ macrophages are antiinflammatory.” More recently, we examined the characteristics of FR-(beta)+ macrophages from the lungs of mice with experimentally-induced asthma and found them to express anti-inflammatory markers. These data confirm that FR+ macrophages can be activated with either pro-inflammatory or anti-inflammatory properties.

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9.4 Folate Receptors on Activated Macrophages Are Fully Functional FR’s ability to bind and internalize folic acid conjugates is essential for the delivery of folate-conjugated imaging and therapeutic agents. Activated macrophages demonstrate the ability to not only bind but also internalize 3H-folic acid (see Xia et  al. 2009). Binding and internalization of folate conjugates has also been confirmed by confocal microscopy through the use of a near infrared (NIR) dye conjugated to folic acid (see Xia et al. 2009). The functionality of the FR in activated macrophages has been further established by stimulating peritoneal macrophages with thioglycollate and then injecting folate-rhodamine into the intraperitoneal cavity. Harvested macrophages are not only filled with folate-rhodamine, but this folate-rhodamine uptake can be totally blocked by coadministration of an excess of unlabeled folic acid, demonstrating that the uptake is FR-mediated (see Xia et al. 2009). Finally, thin sections of atherosclerotic plaque isolated from 99mTc-EC20 injected ApoE knockout mice show accumulation of the 99mTc-based conjugate specifically in the macrophages of the atheroma (see Ayala-López et al. 2010).

9.5 Macrophage Involvement in Inflammatory Diseases Activated pro-inflammatory macrophages primarily function to protect against opportunistic infections (see Gordon 2003a, b; Geissmann and Manz 2010), but upon misguided or premature activation the same macrophage can play a key role in the development of autoimmune and inflammatory diseases. Thus, activated proinflammatory macrophages have been linked to the development of atherosclerosis, diabetes, ischemia/reperfusion injury, lupus, psoriasis, rheumatoid arthritis, transplantation rejection, ulcerative colitis, impact trauma, multiple sclerosis, scleroderma, Crohn’s disease, Sjogren’s syndrome, glomerulonephritis, sarcoidosis, and others (see Bresnihan 1999; Foster and Kelley 1999; Dragun et al. 2000; Kinne et al. 2000; Ren et  al. 2003; Khazen et  al. 2005; Bobryshev 2006; Wang et  al. 2006; Gueler et al. 2007; Odegaard et al. 2007; Swirski et al. 2007). Throughout progression of most of the above diseases, the activated macrophage releases cytokines (IL-1, IL-6, TNF-a), chemokines (MCP-1), digestive enzymes (collagenases), prostaglandins, and ROS, which aggravate the healthy tissue and expedite development of disease symptoms.

9.6 Folate Receptor-Targeted Imaging Agents for Inflammation Diagnostic imaging is a powerful tool that can provide essential information regarding the development and progression of a disease. The goal of FR-targeted imaging in inflammation can be envisioned to (1) develop protocols for early diagnosis of

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disease, (2) track disease progression, (3) monitor response to therapy, and (4) identify patients who might benefit from a folate-targeted therapy. Three types of folate-targeted imaging agents have proven effective in imaging inflammatory diseases: radionuclide chelating agents, MRI contrast agents, and NIR dyes. The radiodiagnostic imaging agent, 99mTc-EC20, was the first folatetargeted imaging compound employed to visualize inflammatory diseases in vivo (see Turk et al. 2002). Accumulation of 99mTc-EC20 was found to occur in inflamed joints of rats with adjuvant-induced arthritis (a model of human rheumatoid arthritis), while no concentration of the targeted compound was seen to occur in healthy tissues. Importantly, uptake of 99mTc-EC20 was shown to be linearly proportional to the severity of arthritis symptoms (see Fig. 9.3; Varghese et al. in press), and the signal from 99mTc-EC20 was readily blocked by administration of excess free folic acid, suggesting that 99mTc-EC20 accumulation is specific to the FR (see Turk et al. 2002). 99mTc-EC20 has also been used to visualize sites of atherosclerosis in apolipoprotein E knockout (apoE−/−) mice (see Ayala-López et  al. 2010) and Syrian Golden Hamsters (see Antohe et al. 2005), and it has been successfully employed to localize osteoarthritic joints in Dunkin-Hartley guinea pigs and many other animal models of inflammatory osteoarthritis (Rothenbuhler and Low, unpublished observations). More recently, 99mTc-EC20 has also been employed to image sites of infection in both domestic animals treated at the Purdue University Veterinary Clinic and laboratory animal models injected with infectious pathogens. Finally, 99m Tc-EC20 has been used clinically to image the arthritic joints of human patients

4.5 99mTc-EC20 uptake (% injected dose/gram of liver)

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Severe Arthritis (GPI-0100) -

Arthritis (TiterMax)

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Fig.  9.3  Systemic inflammation correlates with 99mTc-EC20 uptake. Different severities of adjuvant-induced arthritis were induced in rats by administration of different adjuvants and therapies, and/or by waiting for different disease development before analysis (see Varghese et al. 2007 for details). Systemic inflammation was quantitated by measuring 99mTc-EC20 uptake in the liver and spleen. The weight of each paw (a measure of the severity of the arthritis) was found to correlate linearly with the magnitude of 99mTc-EC20 uptake in the liver and spleen (a measure of the abundance of FR+ macrophages in these organs)

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Fig. 9.4  99mTc-EC20 image of a human patient with rheumatoid arthritis. 99mTc-EC20 has been used to image patients with rheumatoid arthritis. Accumulation of 99mTc-EC20 (darker regions in image) is associated with the abundance of activated macrophages in the arthritic joints (arrows). Data provided by Eric Matteson, Mayo Clinic. See also Matteson et  al. (2009) for additional images and analysis

with rheumatoid arthritis, where it has demonstrated great promise for detecting the disease before it can be diagnosed by a trained rheumatologist (see Fig. 9.4; Xia et  al. 2009). Images of patients with other inflammatory diseases (e.g., Crohn’s disease, ischemic bowel disease, Sjogren’s syndrome, localized infections, atherosclerosis, organ transplant rejection, etc.) have also shown uptake of 99mTc-EC20 at sites of inflammation (see Low et al. 2008; Matteson et al. 2009; Ayala-López et al. 2010; Rothenbuhler and Low, unpublished observations), suggesting the imaging agent might find application in detection of multiple human inflammatory and autoimmune diseases. Folate-targeted MRI contrast agents have also demonstrated an ability to image inflamed joints in rats with adjuvant-induced arthritis with significantly greater contrast than nontargeted forms of the same gadolinium complexes ( p