Pharmacology & Therapeutics 83 (1999) 67–123
`
`Associate editor: T.W. Doyle
`Receptor-mediated and enzyme-dependent targeting of
`cytotoxic anticancer drugs
`Gene M. Dubowchik *, Michael A. Walker
`Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, P.O. Box 5100, Wallingford, CT 06492-7660, USA
`
`Abstract
`
`This review is a survey of various approaches to targeting cytotoxic anticancer drugs to tumors primarily through biomolecules expressed
`by cancer cells or associated vasculature and stroma. These include monoclonal antibody immunoconjugates; enzyme prodrug therapies,
`such as antibody-directed enzyme prodrug therapy, gene-directed enzyme prodrug therapy, and bacterial-directed enzyme prodrug therapy;
`and metabolism-based therapies that seek to exploit increased tumor expression of, e.g., proteases, low-density lipoprotein receptors, hor-
`mones, and adhesion molecules. Following a discussion of factors that positively and negatively affect drug delivery to solid tumors, we con-
`centrate on a mechanistic understanding of selective drug release or generation at the tumor site. © 1999 Elsevier Science Inc. All rights
`reserved.
`
`
`
`Keywords: Antitumor; Antibody; Prodrug; Drug delivery; Immunoconjugate; Drug release
`
`Ab, antibody; ADAPT, antibody-directed abzyme prodrug therapy; ADC, antibody-directed catalysis; ADEPT, antibody-directed enzyme
`Abbreviations:
`prodrug therapy; Ag, antigen; AP, alkaline phosphatase; AraC, cytarabine; BDEPT, bacterial-directed enzyme prodrug therapy; BSA, bovine serum albumin;
`CB1954, 5-(1-aziridinyl)-2,4-dinitrobenzamide; CCM, 7-(4-carboxybutanamido)cephalosporin-phenylenediamine; CD, cytosine deaminase; CEA, carcino-
`embryonic antigen; CHO, aldehyde group; Cit,
`
`-citrulline; CMDA, 4-[N
`
`
`-(2-chloroethyl)-N-[2-(mesyloxy)ethyl]amino]benzoyl-
`-glutamic acid; CP, carbox-
`L
`L
`ypeptidase; DAVLB, desacetylvinblastine; DAVLBHYD, 4-desacetylvinblastine-3-carbohydrazide; dCK, deoxycytidine kinase; Dex, dextran; DM1, may-
`9
`tansine derivative; DMT,
`-dimethoxytrityl; DNM, daunomycin; DNR, daunorubicin; DOX, doxorubicin; DSG, 15-deoxyspergualin; DTT, dithiothreitol;
`p,p
`9
`EGF, epidermal growth factor; EP, etoposide phosphate; Fab, monovalent antibody fragment; F(ab
`)
`, bivalent antibody fragment; 5-FdU, 5-fluorodeoxyuri-
`2
`9
`dine; 5-FC, 5-fluorocytosine; FdUR, 5-fluoro-2
`-deoxyuridine; 5-FU, 5-fluorouracil; GB, guanidinobenzoatase; GCV, ganciclovir; GDEPT, gene-directed
`enzyme prodrug therapy; GI, gastrointestinal; GUS, glucuronidase; HAMA, human anti-mouse antibody; HDL, high-density lipoprotein; HPMA,
`-(2-
`N
`g
`hydroxypropyl)methacrylamide; HSA, human serum albumin; HSV, herpes simplex virus; IDA, idarubicin; IgG, immunoglobulin-
`; IL, interleukin; ING-1,
`x
`y
`anti-Epcam; LDL, low-density lipoprotein; Le
` or Le
`, Lewis x or y; mAb, monoclonal antibody; MDR, multidrug resistance; MeP, 6-methylpurine; MeP-dR,
`6-methylpurinedeoxyriboside; MeT,
`-monomethyltrityl; MMC, mitomycin C; MMCDan, anionically charged polymeric prodrug of mitomycin C; MMT,
`p
`
`-monomethoxytrityl; morph-DOX, morpholinodoxorubicin; MR, mole ratio; MTD, maximum tolerated dose; MTX, methotrexate; N
`-AcMEL,
`-acetylmel-
`p
`N
`phalan; NCS, neocarzinostatin; NTR, nitroreductase; PABC,
`-aminobenzylcarbonyl; PDM, phenylenediamine mustard; PEG, polyethylene glycol; PGP,
`p
`5
`
`P-glycoprotein; PHEG, poly[N
`-(2-hydroxyethyl)-
`-glutamine]; PNP, purine nucleoside phosphorylase; Pt, platinum; RGD, Arg-Gly-Asp; SPDP,
`-succin-
`N
`L
`imidyl 3-(2-pyridyldithio)-propionate; Tk, thymidine kinase; TX, 6-thioxanthine; VEGF, vascular endothelial growth factor; XGPRT, xanthine-guanine phos-
`phoribosyltransferase.
`
`CONTENTS
`1.
`Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.
`Properties of tumors that affect drug-carrier therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.1. Tumor-associated antigens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.2.
`Internalization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.3. Tumor blood vessels and drug penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.4. Aspects of tumor metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3. Antigen-targeted therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.1. Monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.2.
`Immune response to antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.3. Chemical conjugation of cytotoxic drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.4. Antibody-drug linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
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`* Corresponding author. Tel.: 203-677-7539; fax: 203-677-7702.
`
`E-mail address: dubowchg@bms.com (G.M. Dubowchik)
`
`0163-7258/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
`
`PII:
`S0163-7258(99)00018-2
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`G.M. Dubowchik, M.A. Walker / Pharmacology & Therapeutics 83 (1999) 67–123
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`3.5.
`
`3.4.1. Nonselective linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.4.2. Acid-cleavable linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.4.3. Lysosomally degradable peptide linkers . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.4.4. Disulfide linkages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Intermediate carriers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.5.1. Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.5.2.
`Synthetic polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.5.3. Human serum albumin carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.5.4. Antibody-targeted liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6. Antibody-directed enzyme prodrug therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.1. Carboxypeptidase G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.2. Alkaline phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.3. Carboxypeptidase A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.4. Nitroreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`b
`3.6.5.
`-Lactamase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.6.
`Penicillin amidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.7. Cytosine deaminase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.8. Glucuronidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.9. Galactosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.6.10. Abzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.7. Bi-specific antibody approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4. Metabolism-based targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.1. Cancer-associated proteases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.2. Adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.3.
`Folate receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.4. Low-density lipoprotein receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.5. Hormone receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5. Genetically induced prodrug activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1. Gene-directed enzyme prodrug therapy (virus-directed
`enzyme prodrug therapy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1.1. Thymidine kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1.2. Nitroreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1.3. Cytosine deaminase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1.4. Cytochrome P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1.5.
`Phosphorylase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1.6. Deoxycytidine kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.1.7. Xanthine-guanine phosphoribosyltransferase . . . . . . . . . . . . . . . . . . . . . . .
`5.1.8. Carboxypeptidase G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5.2. Bacterial-directed enzyme prodrug therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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`1. Introduction
`
`Despite several decades of intensive research in the labo-
`ratory and the clinic, the long-term outlook for cancer pa-
`tients with aggressive disease remains discouraging (Brun
`et al., 1997; Dunton, 1997; Piccart, 1996; Rahman et al.,
`1997). Unlike bacteria and viruses, cancer cells do not con-
`tain molecular targets that are completely foreign to the
`host. As a result, cytotoxic anticancer therapy has relied pri-
`marily on the enhanced proliferative rate of cancer cells, us-
`ing drugs that act on DNA, tubulin, and enzymes such as the
`topoisomerases that are important in DNA replication.
`However, for patients with appreciable tumor burdens, clin-
`ically approved cytotoxics usually only cause remissions of
`
`limited duration and variable degree, followed by regrowth
`and spread of often more malignant and multidrug-resistant
`disease (Eltahir et al., 1998). Part of the reason for this is
`that hypoxic cells in the center of tumors can be essentially
`dormant and much less susceptible to traditional cancer
`drugs (Clarkson, 1974), not only because they are tempo-
`rarily in a growth-arrested state, but also because of limited
`drug penetration (Erlanson et al., 1992) and induced cellular
`resistance mechanisms (Wartenberg et al., 1998). When
`these cells are revived by vascularization, following de-
`struction of the tumor periphery, there is evidence that they
`often have a higher metastatic potential (Young & Hill,
`1990; Young et al., 1988). In addition, aggressive microme-
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`69
`
`tastases and minimal residual disease (Hirsch-Ginsberg,
`1998), often beginning only as vanishingly small popula-
`tions of cells that evade resection of the primary tumor or
`first-line chemotherapy, are often the cause of clinical re-
`lapse (Schott et al., 1998). These stray cells, which are diffi-
`cult to detect, can also be present as contamination in auto-
`logous grafts after high-dose chemotherapy (Ross, 1998).
`Newer approaches to cancer chemotherapy that exploit an-
`giogenesis, tumor suppressors, and other signal transduction
`pathways show promise, but have yet to make an impact in
`the clinic (Alessandro et al., 1996; OReilly, 1997; Schwartz,
`1996; Sebti & Hamilton, 1997).
`It can be argued that many of the shortcomings of cur-
`rently approved cytotoxics are a result of dose-limiting
`toxic side effects, not only toward normally proliferative
`cell populations (Lowenthal & Eaton, 1996), but also, in the
`case of specific classes of chemotherapeutics, organ-spe-
`cific toxicities such as the cardiotoxicity shown by most
`members of the widely used anthracycline family of anti-
`cancer agents (Hortobagyi, 1997; Shan et al., 1996). This
`effectively limits the amount of agent that can be given to
`below the threshold that exposes all the tumor tissue to a
`killing dose, resulting in induction of resistance mecha-
`nisms and metastasis. In the past several decades, various
`approaches toward targeting cytotoxic agents to cancer cells
`have been developed that use conjugated forms of these
`agents with carriers that selectively accumulate in tumors.
`The best of these approaches combine a protective mecha-
`nism for normal tissues that deactivates the agent until the
`tumor is reached, at which time, a tumor-specific mecha-
`nism releases the cytotoxic effect. Therefore, the goal of tar-
`geting is 2-fold: to actively deliver an effective dose of a cy-
`totoxic agent to tumor tissue and to protect the rest of the
`body from its toxic effects.
`This review will survey various approaches to targeting
`cytotoxic drugs to neoplastic tissue, using vehicles that
`show affinity for specific biomolecules expressed on the
`surface of cancer cells or in tumor-associated tissue, such as
`vasculature and stroma. It will emphasize the rational de-
`sign of drug release mechanisms that take advantage of con-
`ditions at the tumor site or within cancer cells. It will not
`cover the following areas, for which the reader is directed to
`recent reviews or leading articles: delivery of protein toxins
`(Ghetie & Vitetta, 1994; Pastan, 1997), radioimmunother-
`apy (Schott et al., 1994), boron-neutron capture therapy
`(Chen et al., 1997; Mehta & Lu, 1996), targeted photody-
`namic therapy (Akhlynina et al., 1997; Peterson et al.,
`1996), electrochemotherapy (Jaroszeski et al., 1997), drug
`delivery using magnetic particles (Devineni et al., 1995;
`Lubbe et al., 1996), T-lymphocyte targeting using bacterial
`superantigens (Giantonio et al., 1997; Hansson et al., 1997)
`and bi-specific antibodies (Abs) (Mokotoff et al., 1996;
`Renner & Pfreundschuh, 1995), and passive targeting using
`liposomes (Ceh et al., 1997; Sharma & Sharma, 1997) and
`polymers (Cummings, 1998; Soyez et al., 1996; Zalipsky,
`1995).
`
`2. Properties of tumors that affect drug-carrier therapy
`
`2.1. Tumor-associated antigens
`
`The delivery of immunoconjugates to tumor-associated
`antigens (Ags) has been the most commonly employed
`method of anticancer targeting in preclinical studies. Cancer
`cells overexpress many proteins in comparison with normal
`tissue, as a result of their transformed state. Modern hybri-
`doma technology has allowed the large-scale production of
`monoclonal antibodies (mAbs) raised to numerous tumor-
`associated Ags (Hellstrom & Hellstrom, 1991, 1997; Urban
`& Schreiber, 1992; Wick & Groner, 1997; Wright, 1984).
`Most Ags used for targeting are expressed to a lesser, vary-
`ing degree in some normal tissues. If these molecules are re-
`ceptors for growth factors or are differentiation related, for
`example, they may also be expressed in normal proliferative
`tissues, such as portions of the lining of the gastrointestinal
`(GI) tract. This expression can show striking interspecies
`differences. As such, these Ags are tumor-selective rather
`than tumor-specific, and therapy will target those normal
`tissues to some extent. In several clinical trials, this cross-
`reactivity with normal tissue has determined the maximum-
`tolerated dose (MTD) (Elias et al., 1990; Sugerman et al.,
`1995).
`One of the first selective tumor markers discovered was
`the carcinoembryonic antigen (CEA), which is most likely a
`cell adhesion molecule (Johnson, 1992). Found in tumors
`associated with the GI tract, as well as some lung and breast
`cancers, CEA has been frequently targeted (Ballesta et al.,
`1995; Siler et al., 1993). Other notable classes of tumor-
`associated Ags that have been used in immunotherapy in-
`a
`clude
`-fetoprotein (Masuda et al., 1994), gangliosides
`(Zhang et al., 1997a) such as the L6 Ag (Fell et al., 1992;
`Hellstrom et al., 1986), blood group carbohydrates (Ragu-
`y
`pathi, 1996; Zhang et al., 1997b) such as Lewis y (Le
`) (rec-
`ognized by the mAbs BR64 and BR96 and possibly related
`to apoptosis [Nagai et al., 1995] or cell migration [Garri-
`gues et al., 1994; Hellstrom et al., 1990]), B-cell differentia-
`tion Ag (Rowland et al., 1993), the transferrin receptor
`(Starling et al., 1988), the adenocarcinoma-related KS1/4
`(Bumol et al., 1988b; Varki et al., 1984), mucins (Hinman et
`al., 1993), selectins (Ravindranath et al., 1997), glycosphin-
`golipids (Hakomori & Zhang, 1997), integrins (Ruoslahti,
`1997), and other adhesion molecules (Chang & Pastan,
`1996; Huang, Y. W. et al., 1997; Lally et al., 1997).
`Ags that are more tumor selective recently have been
`found in oncogenic protein products (Appleman & Frey,
`1996; Curiel, 1997; Halpern, 1997), such as the HER-2/neu
`(or c-erbB2) glycoproteins (Cirisano & Karlan, 1996; Disis
`& Cheever, 1997), or products resulting from chromosomal
`translocations (Rabbitts, 1994). The mutated form of the tu-
`mor suppressor p53 has been shown to be a tumor-specific
`Ag in T-cell targeting (Theobald et al., 1995), and a number
`of heat shock proteins have been found to be overexpressed
`in certain cancers, and may be sites that attract natural killer
`cell activity (Multhoff et al., 1997).
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`The receptor for folic acid (Coney et al., 1994) and the
`iron transport protein transferrin are overexpressed in many
`cancers (Lally et al., 1997; Richardson & Ponka, 1997), and
`the presence of transferrin in the blood-brain barrier has
`also allowed brain penetration of transferrin conjugates
`(Youle, 1996). The multidrug resistance (MDR)-associated
`membrane pump protein P-glycoprotein (PGP) has also
`been shown to be antigenic in a bi-specific Ab approach to
`target T-lymphocytes (Van Dijk et al., 1989) and immuno-
`toxins (Bruggemann et al., 1991) to drug-resistant tumor
`cells. Recently, Ags associated with various cancers, such
`as melanoma (Merimsky et al., 1994), ovarian carcinoma
`(Bast et al., 1994), and gliomas (Kurpad et al., 1995), as
`well as growth factor-associated Ags (Fan & Mendelsohn,
`1998), have been reviewed. In addition, the use of overex-
`pressed cellular receptors for drug targeting has been re-
`viewed (Feener & King, 1998).
`Two problems related to targeting tumor-associated Ags
`are heterogeneity of Ag expression and Ag shedding. The
`origin of the first problem is complex, but may result in part
`from the genetic instability of cells in the necrotic region of
`tumor tissue (Fleuren et al., 1995; Reynolds et al., 1996). A
`given Ag can also be expressed with different glycosylation
`patterns within tumor tissue, leading to diminished or non-
`existent Ab reactivity in certain areas (Hernando et al.,
`1994). Interferons (Guadagni et al., 1994; Murray, 1992;
`Schlom et al., 1990) have been used to enhance expression
`of certain tumor Ags in vitro and in vivo, and Ab-directed
`interleukin (IL)-2 has been proposed as an approach to
`overcome Ag heterogeneity by recruiting a host immune re-
`sponse against the tumor (Becker et al., 1996). Ag heteroge-
`neity may not be a problem in cases where the cytotoxic
`drug is stable enough, and is delivered in sufficient quantity
`to kill Ag-negative cells by the “bystander effect.” This is
`operative when excess drug in dead, Ag-positive cells is
`released into the tumor interstitium to be absorbed nonse-
`lectively (Laguzza et al., 1989; Liu et al., 1996; Niculescu-
`Duvaz et al., 1998). Other workers have addressed the po-
`tential problem of a “binding-site barrier,” where a combination
`of high Ag expression and a tightly binding mAb may lead
`to reduced tumor penetration of an immunoconjugate
`(Shockley et al., 1992; Sung et al., 1992; 1993; van Osdol et
`al., 1991).
`Ag shedding is related to the fact that tumor cells shed
`various biomolecules without the degree of control exerted
`on normal cells (Kiessling & Gordron, 1998). Some of
`these, such as adhesion molecules and proteases, are part of
`the metastatic cascade in which cancer cells dissociate from
`the primary tumor and degrade surrounding basement mem-
`brane (Taylor & Black, 1985). Instances of clinical detec-
`tion of circulating tumor-associated Ag have been reported
`(Maimonis et al., 1990; van Hof et al., 1996), and in the case
`of melanoma, the degree of shedding has been linked to cir-
`culating cytokine levels (Anichini et al., 1993). Shed Ags
`can be expected to compete with tumor cell-bound Ags (van
`Hof et al., 1996), especially since they may be more accessi-
`
`ble to the immunoconjugate. This will decrease the effec-
`tiveness of targeting therapy by the formation of inactive
`conjugate-Ag complexes that are rapidly cleared (Pimm et
`al., 1989).
`
`2.2. Internalization
`
`The importance of internalization of Ag-bound immuno-
`conjugates by receptor-mediated endocytosis (Kato et al.,
`1996; Mellman, 1996), resulting in conjugate processing in
`endosomes and lysosomes, depends on the type of therapy.
`For antibody-directed enzyme prodrug therapy (ADEPT), in
`which an Ab-bound enzyme is localized to the cell surface
`where it unmasks a prodrug, internalization is not desired.
`For delivery of cytotoxic radioisotopes or photosensitizers,
`internalization probably does not matter. However, in gen-
`eral, it has been found that delivery of cytotoxic drugs or
`protein toxins is much more effective when the metabolic
`potential of endosomes and lysosomes can be utilized for
`drug release. The limitations of endocytosis as an entry
`point for drugs into cells depend on such factors as cell sur-
`face Ag density, rate of internalization, and re-expression
`(Kato & Sugiyama, 1997).
`
`2.3. Tumor blood vessels and drug penetration
`
`Tumor blood vessels possess a number of properties that
`differentiate them from those in normal tissue. Vasculature
`in well-differentiated tumors can be close to normal. How-
`ever, in rapidly growing and large solid tumors, new blood
`vessels are often deficient in many ways, including inter-
`rupted or absent basement membranes and endothelial lin-
`ing (Cobb, 1989); tortuous, often spiral-shaped, paths
`(Baish et al., 1996; Jain, 1994); lack of regularity and sys-
`tematic connectivity leaving unvascularized areas, espe-
`cially in the tumor interior, all together resulting in unstable
`blood flow (Eskey et al., 1994; Vaupel et al., 1989). Inter-
`cellular adhesion between tumor vascular endothelial cells
`is often poor, resulting in a high proportion of leaky blood
`vessels. In addition, large solid tumors generally do not de-
`velop a functional lymphatic network and, therefore, do not
`have adequate fluid drainage. One result of this is a sieving
`effect in which large molecules can become trapped in tu-
`mor tissue. These properties have been exploited in the
`“passive targeting” (Maeda, 1992) of polymer-bound drugs
`(Maeda et al., 1992; Seymour et al., 1995, 1996; Steyger et
`al., 1996), liposomes (Forssen, 1997; Gabizon et al., 1997;
`Uchiyama et al., 1995; Unezaki et al., 1996), nanoparticles
`(Hodoshima et al., 1997; Kwon & Okano, 1996), and non-
`specific protein conjugates (Hunerbein et al., 1991; Suda et
`al., 1993). In one study, liposomes up to 400 nm in diameter
`were shown to passively diffuse through gaps in tumor
`blood vessels in LS174T adenocarcinoma xenografts in
`nude mice (Yuan et al., 1995).
`These vascular abnormalities, exacerbated by constant
`angiogenesis, can result in a buildup of microvascular pres-
`sure (Boucher et al., 1996). Combined with a highly prolif-
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`71
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`erative cell population in a restricted space, the result can be
`a sizable positive osmotic intratumoral pressure and a net
`outflow of liquid from solid tumors into the surrounding tis-
`sue (Jain, 1996). This physical stress can also cause the vir-
`tual collapse of blood vessels and any functional lymphatics
`within tumors, further impairing blood flow (Helmlinger et
`al., 1997a). The barrier to the diffusion of molecules from
`the blood vessel, through the interstitium, to tumor cells has
`been shown to increase with tumor size and to be highly de-
`pendent on molecular weight, allowing small molecule
`drugs to penetrate much deeper into the tumor than proteins,
`polymers, or other particles (Yuan et al., 1995). This pres-
`sure gradient in solid tumors, along with the presence of sig-
`nificant areas lacking adequate vascularization, is an impor-
`tant consideration in the choice of, for example, a full-size
`Ab or a smaller Ab fragment as a delivery vehicle or an Ab
`with high or moderate binding affinity to an Ag (Baxter &
`Jain, 1991). Agents such as nicotinamide (Lee et al., 1992),
`pentoxifylline (Lee et al., 1994), hydralazine (Zlotecki et
`a
`al., 1995), and tumor necrosis factor
` (Kristensen et al.,
`1996) have been shown to increase tumor perfusion by re-
`ducing interstitial fluid pressure. In addition, Ab-targeted
`IL-2 was shown to increase tumor vascular permeability in
`mouse models by an unknown mechanism (LeBerthon et
`al., 1991), while IL-2 given alone increased vascular perme-
`ability in all organs.
`Potentially antigenic or otherwise targetable proteins ex-
`pressed in tumor-associated vasculature (Baillie et al.,
`1995) and stroma (Dvorak et al., 1991; Rettig et al., 1992)
`include cellular adhesion molecules
`(Brooks, 1996;
`Griffioen, 1997) and receptors for growth factors (Martini-
`Baron & Marme, 1995). These provide alternative or addi-
`tional targets for therapy that seeks to destroy tumors by
`starving them of nutrients and oxygen (Folkman, 1996; Ol-
`son et al., 1997; Thorpe & Derbyshire, 1997). Progress has
`been made in the elucidation of the genetic and environ-
`mental factors that control tumor angiogenesis (Fan et al.,
`1995), which is especially important in animal model sys-
`tems using xenografted tumors (Damore & Shima, 1996).
`For example, in one study looking at in vivo model systems
`for vascular targeting, human tumor xenografts in mice
`were shown to promote vasculature that expressed mouse,
`not human, antigenic CD31 adhesion molecules (Lehr et al.,
`1997). A mouse model system for testing approaches to vas-
`cular targeting has been developed in which tumors that se-
`g
`crete interferon-
` induce tumor blood vessel expression of
`antigenic major histocompatibility complex Class II,
`whereas normal vasculature expressed Class I (Burrows et
`al., 1992).
`Tumor blood vessels use vascular endothelial growth
`factor (VEGF) as a survival factor because of constant and
`extensive remodeling. Normal vasculature, on the other
`hand, does not require VEGF following embryonic develop-
`ment (Plate et al., 1994). Withdrawal of VEGF leads to apop-
`totic death of tumor-associated vascular endothelial cells,
`while overproduction of VEGF leads to hyper-vascularized
`
`tumors that are less necrotic (Benjamin & Keshet, 1997;
`Yuan et al., 1996). VEGF may also be at least partly respon-
`sible for enhanced tumor vascular permeability (Roberts &
`Palade, 1997; Wang et al., 1996). VEGF has been used as a
`targeting vehicle for truncated diphtheria toxin (Olson et al.,
`1997). A chemically linked conjugate caused delayed
`growth of solid tumors in athymic mice. Histological analy-
`sis showed tumor necrosis originating from vascular injury
`and no effect on well-vascularized, normal tissues. In an-
`other novel approach to vascular targeting, an experimen-
`tally induced Ag in tumor vasculature of large xenografted
`neuroblastomas in mice was targeted with truncated human
`tissue factor through a bi-specific Ab (Huang, X. et al.,
`1997). Blood clots readily formed in tumor blood vessels,
`leading to 38% partial regressions, while thrombolytic ac-
`tivity in normal vasculature was limited.
`
`2.4. Aspects of tumor metabolism
`
`Deficiencies in tumor-associated angiogenesis can leave
`large sections (up to 80%; Leith et al., 1991) of sizable tu-
`mors without adequate vascularization. The resulting lack
`of oxygen and other nutrients forces cells to produce energy
`by glycolysis, leading to a buildup of acidic by-products
`(Brown, 1997; Helmlinger et al., 1997b). To maintain near-
`normal cytosolic pH, cells actively export protons (Boyer &
`Tannock, 1992) so that the extracellular space becomes
`acidified by an average of 0.5 pH units (Stubbs et al., 1994;
`Yamagata & Tannock, 1996). In addition, it is thought that
`some tumors cause a decrease in extracullular pH to allow
`secreted lysosomal proteases to retain activity for basement
`membrane digestion as one of the initial steps in metastasis
`(Montcourrier et al., 1997).
`Preclinical approaches have been reported that aim to ex-
`ploit this pH gradient using bilayer membrane-active agents
`(Boyer et al., 1993; Karuri et al., 1993) or drugs that act on
`1
`1
`/H
` exchanger (Hasuda et al., 1994; Maidorn et al.,
`the Na
`1993; Yamagata & Tannock, 1996) to kill tumor cells by
`defeating active proton transport and acidifying the cytosol.
`These agents are attractive in that they show selectivity in
`cell killing at low pH (Tannock et al., 1995) against cancer
`cell populations that have been shown to be genetically
`more unstable than parental tumor cell lines (Reynolds et
`al., 1996), and are potentially more metastatic once revascu-
`larized (Cuvier et al., 1997; Young & Hill, 1990). Mild
`acidity within tumor tissue has been proposed to contribute
`to the selective localization of porphyrinic photosensitizers
`in photodynamic therapy (Pottier & Kennedy, 1990).
`Tumor cells have been shown to be heterogeneous in
`their ability to survive under hypoxic conditions by down-
`regulating energy consumption (Skoyum et al., 1997) and
`up-regulating enzymes such as mitochondrial hexokinase
`that allow them to optimize energy metabolism (Oudard et
`al., 1997). Cells that express the bcl-2 protein have been
`shown to be able to overcome the apoptotic response that
`normally accompanies hypoxic ATP depletion (Garland &
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`G.M. Dubowchik, M.A. Walker / Pharmacology & Therapeutics 83 (1999) 67–123
`
`Halestrap, 1997). Consequently, the center of a large tumor
`often contains necrotic tissue, as well as populations of cells
`that survive in a state of semi-dormancy. Hypoxia attenu-
`ates the activity of many cytotoxic drugs (Batchelder et al.,
`1996; Teicher, 1994b), as well as radiation therapy (Will-
`son, 1991), and oxygen-carrying blood substitutes have
`shown utility in reversing this resistance (Teicher, 1994a).
`On the other hand, drugs such as mitomycin C (MMC) (Sar-
`torelli et al., 1994; Spanswick et al., 1996), as well as other
`designed bioreductively activated drugs (Denny et al., 1996;
`Jaffar et al., 1998; Wilson & Pruijn, 1995; Workman &
`Stratford, 1993) take advantage of this physiological differ-
`ence between tumor and normal tissue.
`
`3. Antigen-targeted therapy
`
`3.1. Monoclonal antibodies
`
`g
`Most targeted mAbs fall into the immunoglobulin-
`(IgG) class, although IgMs have also been used (Ballou et
`al., 1992), especially for liposome (Ohta et al., 1993) and
`polymer (Flanagan et al., 1993; Hoes et al., 1996) targeting.
`IgGs are symmetric glycoproteins (MW ca. 150,000) com-
`posed of identical pairs of heavy and light chains (Fig. 1).
`At the ends of the two arms are hypervariable regions con-
`taining identical Ag-binding domains. A variable-sized
`branched carbohydrate domain is attached to the comple-
`ment-activating Fc region, and the so-called “hinge region”
`contains especially accessible interchain disulfides (Padlan