selective activation of anticancer prodrugs by monoclonal antibody–enzyme conjugates

Advanced Drug Delivery Reviews 53 (2001) 247–264www.elsevier.com/ locate /drugdeliv

Selective activation of anticancer prodrugs by monoclonalantibody–enzyme conjugates

a , b*Peter D. Senter , Caroline J. SpringeraSeattle Genetics, 21823 30th Dr. SE, Bothell, WA 98021, USA

bCRC Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK

Abstract

A great deal of interest has surrounded the activities of monoclonal antibodies (mAbs), and mAb–drug, toxin andradionuclide conjugates for the treatment of human cancers. In the last few years, a number of new mAb-based reagents havebeen clinically approved (Rituxan, Herceptin, and Panorex), and several others are now in advanced clinical trials. Successfultherapeutic treatment of solid tumors with drug conjugates of such macromolecules must overcome the barriers topenetration within tumor masses, antigen heterogeneity, conjugated drug potency, and efficient drug release from the mAbsinside tumor cells. An alternative strategy for drug delivery involves a two-step approach to cancer therapy in which mAbsare used to localize enzymes to tumor cell surface antigens. Once the conjugate binds to the cancer cells and clears from thesystemic circulation, antitumor prodrugs are administered that are catalytically converted to active drugs by the targetedenzyme. The drugs thus released are capable of penetrating within the tumor mass and eliminating both cells that have andhave not bound the mAb–enzyme conjugate. Significant therapeutic effects have been obtained using a broad range ofenzymes along with prodrugs that are derived from both approved anticancer drugs and highly potent experimental agents.This review focuses on the activities of several mAb–enzyme/prodrug combinations, with an emphasis on those that haveprovided mechanistic insight, clinical activity, novel protein constructs, and the potential for reduced immunogenicity. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Prodrugs; Enzymes; Cancer; Targeting; Monoclonal antibodies

Contents

1. Introduction ............................................................................................................................................................................ 2482. Overview of mAb–enzyme conjugates for prodrug activation..................................................................................................... 248

2.1. Enzyme prodrug combinations .......................................................................................................................................... 2492.1.1. Class 1: enzymes of non-mammalian origin that have no mammalian homologues...................................................... 2492.1.2. Class 2: enzymes of non-mammalian origin with a mammalian homologue ................................................................ 2492.1.3. Class 3: Enzymes of mammalian origin ................................................................................................................... 251

2.2. In vitro activities .............................................................................................................................................................. 2513. Specific illustrations of ADEPT systems ................................................................................................................................... 251

3.1. b-Lactamase (bL): a system with several anticancer prodrugs and recombinant fusion proteins.............................................. 2513.2. Cytosine deaminase (CDase): control of pharmacokinetics can lead to high intratumoral drug concentrations ......................... 255

*Corresponding author. Fax: 1 1-425-527-4109.E-mail address: [email protected] (P.D. Senter).

0169-409X/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 01 )00206-X

248 P.D. Senter, C.J. Springer / Advanced Drug Delivery Reviews 53 (2001) 247 –264

3.3. Carboxypeptidase G2 (CPG2): clinical efficacy.................................................................................................................. 2553.4. Human enzymes: potential for reduced immunogenicity...................................................................................................... 257

4. Mathematical models............................................................................................................................................................... 2595. Conclusions ............................................................................................................................................................................ 259References .................................................................................................................................................................................. 260

1. Introduction has proven to be quite challenging compared tohematologic malignancies, due in part to the barriers

A major limitation in the chemotherapeutic treat- of macromolecule penetration within the tumorment of cancer results from the lack of tumor masses and to the heterogeneity in target antigenspecificity displayed by anticancer drugs. Because of expression [12]. This has prompted considerablethis, a great deal of research has focused on the research into alternative drug delivery strategies thatdevelopment of new chemotherapeutic agents that dissociate the delivery of the mAb from that of theare able to more effectively exploit the differences drug. One such method that has been widely ex-between neoplastic and normal tissues. One approach plored is often known as antibody directed enzyme–has been to prepare inactive drug precursors known prodrug therapy or ADEPT (reviewed in Refs. [13–as prodrugs that are activated by enzymes or physio- 16]). This is a two-step process for drug delivery inlogical conditions associated with cancer cells and which mAbs localize enzymes to tumor cell surfaces.tumor masses (reviewed in Refs. [1–3]). While The enzymes are selected for their abilities topromising agents are in various stages of develop- convert subsequently administered anticancer pro-ment, progress in this field has been hampered by drugs into active antitumor agents. In the past fewdifficulties in defining prodrug activation pathways years, many enzyme/prodrug combinations havethat are restricted to the tumor cell population. been used in this drug delivery strategy. Here, weAlternatively, several drug-targeting strategies are present an overview of the field, along with somebased on the preferential expression of various detailed descriptions of systems that provide insightantigens on tumor cell surfaces. Monoclonal anti- into the capabilities and challenges of this therapeu-bodies (mAbs) against these antigens have been used tic modality.to deliver chemotherapeutic drugs [1,4], potent plantand bacterial toxins [5], and radionuclides [6] totumors. A major advancement in mAb-based target- 2. Overview of mAb–enzyme conjugates foring strategies was made in developing Mylotarg, an prodrug activationanti-CD33–calicheamicin conjugate, which is nowclinically approved for the treatment of acute ADEPT is conceptually illustrated in Fig. 1. Themyelogenous leukemia [7]. Other promising agents first step involves systemic administration of a mAb–that have been through Phase III clinical trials for the enzyme conjugate. The immunoconjugate can betreatment of B-cell lymphomas and are now awaiting prepared by chemically linking the mAb or mAbFDA approval include Zevalin [8] and Bexxar [9], fragment to an enzyme of interest. Alternatively, the

90comprised of anti-CD20 mAbs conjugated to Y and conjugate can be a fusion protein produced recombi-131I, respectively. As yet, there are no clinically nantly with the mAb variable region genes and theapproved immunoconjugates for the treatment of gene encoding the enzyme. Depending on the phar-solid tumors, although the unconjugated mAb Her- macokinetics of the particular conjugate being used,ceptin has demonstrated significant levels of activity it may take anywhere from several hours to severalfor the treatment of metastatic breast carcinoma [10] days for localization to take place within tumors, andand Panorex is approved for use in patients with for clearance to occur from non-target tissues. Dur-colorectal cancer which has spread to the nearby ing this time, it is desirable that the tumor-associatedlymph glands [11]. conjugate remains bound to the outer membrane of

Therapy of solid tumors with mAb-based therapies the target cell population, rather than be taken up

P.D. Senter, C.J. Springer / Advanced Drug Delivery Reviews 53 (2001) 247 –264 249

Fig. 1. Schematic representation of ADEPT. mAb–enzyme conjugates that are bound to antigens on tumor cell surfaces activate anticancerprodrugs. The released drugs penetrate into the tumor mass and enter cells that may and may not have bound the conjugate.

intracellularly. The second step of this therapy is 2.1.1. Class 1: enzymes of non-mammalian originsystemic prodrug administration. Ideally, the prodrug that have no mammalian homologuesshould be non-toxic, resistant to the action of endog- Examples of such enzymes include carboxypep-enous enzymes, and be converted into active drug tidase G2, b-lactamase, penicillin G amidase, andonly by the targeted enzyme. cytosine deaminase. The rationale of using such

Therapy with mAb–enzyme conjugates for pro- enzymes is that prodrugs can be designed that aredrug activation may offer numerous advantages over stable, non-toxic, and that are not substrates forother mAb-based approaches. Since the enzyme endogenous human enzymes. In addition, the Class 1behaves as a catalyst, a single conjugate molecule at enzymes may not be inhibited by substrates ora tumor site should be able to generate a high inhibitors of human origin. Since many of the non-concentration of active drug. Importantly, the drug is mammalian enzymes are bacterial or are easilynot covalently bound to the immunoconjugate. As a expressed in bacteria, they are available in largesmall molecule, it will be free to diffuse throughout quantities. The main disadvantage of such enzymesthe tumor, even if the enzyme conjugate is primarily is that they elicit immune responses in humans.confined to well vascularized regions or is not boundto all of the cells within the tumor mass. 2.1.2. Class 2: enzymes of non-mammalian origin

with a mammalian homologueThe enzymes should be chosen such that low

2.1. Enzyme prodrug combinations levels of their endogenous counterparts are present inthe blood. Examples of Class 2 enzymes include

A summary of the published enzymes for prodrug b-glucuronidase and nitroreductase. One of the ad-activation along with the drugs that they release is vantages of bacterial b-glucuronidase over its humanshown in Table 1. The enzymes for ADEPT can be counterpart is that it has a higher turnover rate and acharacterized into three major classes, designated as pH optimum of 6.8, instead of 5.4 [17,18]. Anotherfollows: enzyme belonging to this category, bacterial nitrore-


Table 1Summary of enzymes and released drugs for ADEPT

Enzyme Released active drug Reference

Alkaline phosphatase Doxorubicin [16]Etoposide [25]Mitomycin [26]Phenol mustard [27]

Aminopeptidase Melphalan [93]

Aryl sulfatase Etoposide [94]Phenol mustard [94]

Carboxypeptidase A Methotrexate [28–30]Antifolates [31,32]

Carboxypeptidase G2 Nitrogen mustards [13,41,66,67,95]

Catalytic antibodies Chloramphenicol [85]5-Fluorodeoxyuridine [86]Phenol mustard [87]Doxorubicin [88]Camptothecin [88]

Cytosine deaminase 5-Fluorouracil [42–45]

a-Galactosidase Doxorubicin [96]

b-Galactosidase 5-Fluorouridine [97]Anthracycline derivatives [98,99]

Glucose oxidase Peroxide, reactive oxygen, and iodine [40]

b-Glucosidase Cyanide [100]

b-Glucuronidase Doxorubicin [17,33,34]Daunorubicin [18]Phenol mustard [35,36]5-Fluorouracil [37]9-Aminocamptothecin [38]Verapamil [39]Quinine [39]Dipyridamole [39]

b-Lactamase Nitrogen mustards [46–49]Doxorubicin [50–52]Mitomycin [53]Vinca derivative [54]Paclitaxel [55]Platinum reagents [56]

Nitroreductase Benzodiazapine derivatives [19]Amino-CBI derivatives [20]Benzamide mustard [21,22]Actinomycin D [23]Mitomycin C [23]Doxorubicin [23]Nitrogen mustard derivatives [23]Enediynes [24]

Penicillin amidase Doxorubicin [101]Melphalan [101]Palytoxin [102]

Xanthine oxidase Peroxide and reactive oxygen species [103]


ductase [19–24], is significantly different from the mAb–alkaline phosphatase and mAb–carboxypep-human counterpart. Consequently, it is possible to tidase G2 conjugates for the activation of etoposidedevelop prodrugs that are selectively activated by the phosphate [25] and benzoyl glutamic acid mustardsbacterial enzyme rather than the endogenous protein. [41], respectively. In both systems, the prodrugsAs with the Class 1 enzymes, these proteins suffer were much less cytotoxic than the drugs they re-from limitations due to immunogenicity. leased upon hydrolysis, and when added to cells that

were pretreated with mAb–enzyme conjugates, im-2.1.3. Class 3: Enzymes of mammalian origin munologically specific activation took place. There

These include alkaline phosphatase [16,25–27], are now numerous other enzyme/prodrug combina-carboxypeptidase A [28–32] and b-glucuronidase tions that have such properties. Cytosine deaminase[17,18,33–39]. It is likely that such enzymes will be has been shown to convert the non-cytotoxic agentmuch less immunogenic than bacterial or fungal 5-fluorocytosine to the approved anticancer drug 5-enzymes, and as a result they can be used for several fluorouracil [42–45]. Carboxypeptidase A enzymesrounds of therapy. However, a major issue surround- activate methotrexate prodrugs [28–30] and thy-ing the use of such proteins for prodrug activation is midylate synthase inhibitors [31,32]. Several inves-that endogenous enzymes may lead to non-specific tigations using bacterial nitroreductase have demon-drug activation. strated that a broad array of non-toxic prodrugs can

As shown in Table 1, the drugs released by the be activated [19–24]. This system differs from thetargeted enzymes can be clinically approved (e.g. others, since reducing cosubstrates like NADH oretoposide, doxorubicin, paclitaxel, methotrexate), NADPH are required for activity. A number ofstructural relatives of approved anticancer drugs (e.g. laboratories have reported the use of b-lactamase fornitrogen mustards, platinum derivatives, vinca de- the activation of relatively non-cytotoxic cephalo-rivatives), or drugs that are too toxic for systemic sporin-containing prodrugs [46–56]. Table 1 listsadministration (e.g. palytoxin, potent nitrogen mus- several other enzymes that have demonstrated utilitytards, cyanide, enediynes). Prodrugs of clinically for anticancer prodrug activation.approved anticancer agents have the potential ofleading to predictable activities and toxicities. Due tohigh tumor to non-tumor mAb–enzyme ratios, 3. Specific illustrations of ADEPT systemsADEPT provides the opportunity to improve thetherapeutic windows of highly active drugs that have 3.1. b-Lactamase (bL): a system with severallittle clinical potential due to non-specific toxicities. anticancer prodrugs and recombinant fusion

proteins2.2. In vitro activities

Significant attention has been directed towards theEfficacy of many mAb–enzyme/prodrug combi- use of bL for prodrug activation, due in part to the

nations has been demonstrated using in vitro models. depth of knowledge surrounding cephalosporin andThe earliest example of a specific prodrug activation penicillin chemistry and the bL enzymes that cleavesystem predated the development of mAbs, and the b-lactam rings within these drug families. Theinvolved a polyclonal antisera conjugate of glucose stage for the design of cephalosporin-containingoxidase [40]. With glucose as a substrate, this anticancer prodrugs was set through the developmentenzyme generates peroxide. It was found that spe- of ‘dual-action cephalosporin’ antibiotics designed tocific cellular cytotoxicity could be obtained by expel potent antibacterial agents when acted upon bycoupling peroxide generation to lactoperoxidase and bL producing bacterial strains [57,58]. It was demon-iodide. This led to the formation of cytotoxic quan- strated that antibiotics attached to the 39-position oftities of iodine. Since this work, the strategy was cephalosporins were eliminated through a 1,4-frag-extended to include mAbs as delivery agents and a mentation reaction. One of the first published exam-panel of different enzymes for the release of an array ples of a cephalosporin-based anticancer prodrugof anticancer drugs. The first examples included concerned cephalosporin nitrogen mustard deriva-


tives that were activated by the broad scale bL The first report of in vivo activity in a mAb–bLenzyme from Enterbacter cloacae [46]. This work system used the bL enzyme from E. cloacae and awas extended in several laboratories to include cephalosporin–vinca alkaloid prodrug [54]. The en-prodrugs of other nitrogen mustards [47–49], doxo- zyme was linked to mAb Fab9 fragments recognizingrubicin [50–52], mitomycin C [53], a vinca alkaloid the CEA, TAG-72 and KS1/4 antigens on tumor[54], paclitaxel [55], and a carboplatinum analogue tissues. The therapeutic effects of each of these[56]. These prodrugs were activated by a diverse mAb–enzyme conjugates in combination with thearray of bL enzymes from E. cloacae, E. coli, and B. vinca prodrug were studied in models of humancereus. The structures of some of the prodrugs for colorectal carcinoma in nude mouse. In all cases, thebL are shown in Fig. 2. therapeutic effects of the antitumor mAb–bL conju-

Fig. 2. Structures of some prodrugs that are activated by targeted b-lactamases (bL). Upon activation, a fragmentation reaction ensues,resulting in the release of the indicated drugs.


gate in combination with the vinca prodrug were conjugate that were demonstrated not to lead tosuperior to drug therapy, prodrug alone, and to non- significant depletion of blood-borne prodrug, in-binding IgG enzyme conjugates with prodrug. The tratumoral doxorubicin concentrations in animalseffects were also superior to those obtained when the receiving the binding mAb–bL conjugate followedvinca drug was attached directly to the mAb. Long- by C-Dox were fivefold higher than systemic doxo-term regressions of established tumors were obtained rubicin treatment. This most likely accounts for thein several dosing regimens, even in animals having pronounced antitumor activities of the mAb–bL/tumors as large as 700 mg at the initiation of prodrug combinations.therapy. One of the interesting aspects of this work The conjugates used in these studies were pre-was that the maximum tolerated doses of the prodrug pared by linking maleimide-substituted bL to cys-and drug were approximately equal. Improved phar- teines on the mAb–Fab9 fragments. mAb fragmentsmacokinetics of the prodrug compared to the drug were used since they provided reasonably high tumormay account for at least part of the activity enhance- to blood ratios approximately 3 days post-administra-ment seen with the mAb–bL/prodrug combinations. tion, the time of prodrug treatment. While the

In related studies, prodrugs of doxorubicin and conjugates were primarily monomeric, SDS–PAGEphenylenediamine mustard were evaluated in combi- indicated that they were heterogeneous. A morenation with an anti-melanoma mAb–bL conjugate uniform chemical conjugation strategy for preparing[59]. Conjugates were formed by linking the bL mAb–Fab9–bL conjugates was described, in whichenzyme to the Fab9 fragment of the 96.5 mAb, which the terminal threonine of bL was oxidized withrecognizes the melanotransferrin antigen present on periodate, forming an aldehyde that was coupled tomost melanomas and several carcinomas. In vitro bL through a bifunctional crosslinking reagent [61].cytotoxicity experiments showed that the doxorubi- The conjugate yield using this approach was threecin prodrug C-Dox (Fig. 2) was approximately times higher than the random chemistry describedninefold less toxic than doxorubicin, while the earlier. In addition, it was found that blood clearancenitrogen mustard prodrug CCM was 26 times less of the site-specific conjugate was more rapid thantoxic than phenylenediamine mustard (PDM, 12). with the random chemistry.Therapy studies in nude mice bearing subcutaneous A more defined method for mAb–bL production3677 human tumor xenografts showed that the 96.5- uses recombinant technology. The gene encoding thebL/C-Dox combination was much more effective variable regions of the anticarcinoma mAb L6 wasthan doxorubicin or a non-binding control conjugate / fused to the gene encoding B. cereus bL, and theC-Dox combination. Systemically administered corresponding protein was expressed in E. coli [62].doxorubicin at the maximum tolerated dose had After purification by affinity chromatography, thenegligible activity. The effects of 96.5-bL with the fusion protein was pure by SDS–PAGE, retainedmustard prodrug CCM were more pronounced than enzymatic activity, and was able to effect prodrugthose obtained with C-Dox [59]. Regressions were activation in vitro. In a related study, a disulfide-observed in 100% of the treated mice at doses that stabilized-Fv–bL fusion protein was produced in E.caused no apparent toxicity. At day 120-post tumor coli [51]. The mAb fragments used in the construc-implant, four of five mice in this treatment arm tion were from a humanized mAb of murine originremained tumor free. Significant antitumor effects and were fused to the E. coli RTEM-1 class A bL

3were even seen in mice that had large (800 mm ) enzyme. The fusion protein recognized the p185tumors before the first prodrug treatment. (HER2) antigen, which is present on many breast

Pharmacokinetic studies using mAb–bL/C-Dox and ovarian carcinomas. One of the several disulfide-combinations provided insight into the therapeutic linked variants investigated in the study bound aseffects obtained. The level of doxorubicin and C- well as the wild-type Fv. In vitro cytotoxicity assaysDox in two lung carcinoma models was undertaken with the combination of the fusion protein and ain animals that received mAb–bL conjugates [60]. In cephalosporin–doxorubicin prodrug demonstrated athis study, HPLC was used to measure both prodrug specific and potent cytotoxic effect towards cellsand drug levels in tissue extracts. At doses of expressing the HER2 antigen. Experiments in nude


mice indicated that the conjugate cleared very quick- obtained 4, 12, and 24 h post L49–sFv–bL injection,ly from the blood (t a 0.23 h, t b 1.27 h). The respectively. Without the assistance of a separate1 / 2 1 / 2

fusion protein was also capable of activating protax clearance step, these are the highest tumor to non-(Fig. 2) resulting in the release of paclitaxel [55]. tumor ratios yet described for a mAb–enzyme

One of the most extensively studied fusion pro- construct. Localization and clearance was vastlyteins for prodrug activation is the anti-melanotran- superior to L49–Fab9–bL, which gave only a 5.6sferrin L49–sFv–bL conjugate [63]. Like the 96.5 tumor to blood ratio 72 h after administration. L49–mAb described earlier, the L49 mAb binds to most sFv–bL/CCM combinations led to pronounced anti-human melanomas and to several carcinomas. Re- tumor activities in 3677 bearing nude mice [63]. Duecombinant L49–sFv–bL, containing the mAb bind- to the rapid kinetics of intratumoral uptake anding regions of L49 fused to E. cloacae bL was systemic clearance, it was possible to effectivelyconstructed, expressed in soluble form in E. coli, and administer CCM within 12 h of the fusion protein.purified to homogeneity in two steps. The resulting The therapeutic effects of L49–sFv–bL and L49–protein had a molecular weight of 63 kDa, which Fab9–bL, both in combination with CCM, werewas approximately 27 kDa less than that of the compared in two models of human renal carcinomaL49–Fab9–bL chemical conjugate. Surface plasmon [64]. While both conjugate /prodrug combinationsresonance, fluorescent activated cell sorting, and were very active, L49–sFv–bL was superior in bothMichaelis–Menten kinetic analyses showed that tumor models. This is illustrated in Fig. 3, whichL49–sFv–bL retained the antigen binding capability shows that both conjugates led to complete tumorof monovalent L49 as well as the enzymatic activity cures when combined with maximum tolerated dosesof bL. As expected, the fusion protein activated of CCM (240 mg/kg per injection and 180 mg/kgCCM (Fig. 2) in an immunologically specific man- per injection CCM in L49–sFv–bL and L49–Fab9–ner. bL, respectively). The differences between the two

Pharmacokinetic studies of L49–sFv–bL were conjugates becomes apparent at much lower prodrugundertaken in nude mice with subcutaneous 3677 doses, where the high activity is maintained withhuman tumor xenografts, and the results were com- L49–sFv–bL, but lost for L49–Fab9–bL. Thus, thepared to chemically prepared L49–Fab9–bL. Inter- fusion protein is uniform in composition, displaysestingly, the fusion protein displayed very high ideal pharmacokinetic characteristics for prodrugtumor to blood ratios within just a few hours of activation, and leads to pronounced antitumor ac-administration. Ratios of 13, 66, and 105 were tivities when combined with antitumor prodrugs.

Fig. 3. In vivo therapeutic efficacy of mAb–bL conjugates in combination with CCM (Fig. 2) on subcutaneous SN12P human renal cellcarcinoma xenografts in nude mice [64]. (A) L49–sFv–bL and (B) L49–Fab9–bL were administered i.v., followed by i.v. CCM (Fig. 2) 24and 72 h later, respectively. The effects were compared with the maximum tolerated dose of the released drug PDM (12). The arrowsindicate time of drug or prodrug treatment.


3.2. Cytosine deaminase (CDase): control of The ratios of 5FC to 5FU were very high in thepharmacokinetics can lead to high intratumoral blood, kidneys, and livers of mice receiving thedrug concentrations L6–CDase /anti-idiotype /5FC combination, suggest-

ing low conversion of 5FC to 5FU in these tissues.As indicated in Section 3.1, efficacy with ADEPT In stark contrast, tumors displayed high 5FU to 5FC

is dependent to a significant degree on achieving ratios, comprising the only tissue where this washigh tumor to non-tumor conjugate ratios at the time seen. Importantly, the intratumoral 5FU concentra-of prodrug administration. This is further illustrated tion was 17- to 25-fold higher than the other tissueswith the enzyme CDase, a protein that converts the tested. Within the 2-h time period in these experi-non-cytotoxic antifungal drug 5-fluorocytosine (5FC) ments, intratumoral drug was significantly higherto the approved anticancer agent 5-fluorouracil than any other tissue measured, suggesting that 5FU(5FU). The human body is devoid of CDase activity, was retained at the site of generation. The area underwhich is why high doses of 5FC are well tolerated the curve of 5FU generated enzymatically waswhen treating fungal infections. greater than 10-fold higher than that achievable by

CDase from yeast was chemically conjugated to systemic administration of 5FU at the maximumwhole L6 mAb, forming a conjugate with a molecu- tolerated dose. Drug generation was most likelylar weight of approximately 195 kDa [42]. The intratumoral, since the total amount of 5FU inL6-CDase was fully active with respect to binding tumors that were L6-antigen positive was aboutand enzymatic activities, and was able to localize in fourfold higher than in L6-antigen negative tumors.human tumor xenografts in nude mice. However, Further evidence for intratumoral 5FU generation

19clearance from the blood was exceedingly slow, and was obtained in real time using F magnetic reso-low tumor to blood ratios were obtained for extended nance imaging, which readily distinguishes theperiods of time. While mAb fragments undoubtedly fluorine atoms present in 5FC and 5FU [45]. Nudewould have cleared more quickly, significant dif- mice with subcutaneous tumor xenografts wereficulties were encountered preparing Fab9–CDase treated with the L6–CDase /antiidiotypic mAband F(ab9) –CDase conjugates with retained binding combination, and intratumoral 5FU generation was2

and enzymatic activities. Consequently, an alterna- detected very shortly after the administration of 5FC.tive strategy was explored in which clearance of The 5FC to 5FU ratio in tumors was comparable tocirculating conjugate was accelerated through the that seen in the biodistribution studies describedformation of immune complexes. Consistent with earlier [44]. There was no evidence for 5FC activa-what had been shown earlier with a mAb–CPG2 tion in control mice.conjugate (reviewed in Ref. [65]), clearance of blood The three-step strategy, while complex and thusborne L6–CDase was facilitated with a mAb against probably not viable for clinical development, pro-CDase [43] or with an anti-idiotypic mAb against L6 vides further evidence that mAb–enzyme/prodrugcomponent of the conjugate [44]. Use of a clearance combinations can lead to higher intratumoral drugstep introduced a third step, and the targeting levels than that achieved through systemic drugstrategy proceeded as follows: L6–CDase was ad- administration. The results emphasize the importanceministered, followed 24 h later by the anti-idiotypic of high tumor to blood conjugate ratios prior tomAb (or the mAb against CDase) and 48 h later by prodrug administration. As illustrated in Section 3.1,treatment with 5FC. With the clearing step, the blood the best way to achieve this is to prepare conjugates,L6–CDase levels dropped 40- to 70-fold, while the such as recombinant fusion proteins, that havetumor levels remained high [44]. As a result, the 5FC appropriate clearance characteristics.dose could be increased from 80 mg/kg per injectionwithout clearance to more than 800 mg/kg per 3.3. Carboxypeptidase G2 (CPG2): clinicalinjection with clearance. efficacy

Biodistribution studies of drug generation wereundertaken using this three-step protocol [44]. After One of the earliest reported enzymes for anti-5FC administration, tissues were excised and 5FC cancer prodrug activation involved the bacterialand 5FU were extracted and quantified by HPLC. enzyme CPG2 [41]. This enzyme is an exoprotease


Fig. 4. Prodrugs (compounds 1–3 and 7–9) and the drugs they release (compounds 4–6 and 10–12) upon hydrolysis of the glutamic acidresidues (glu) by the enzyme carboxypeptidase G2 (CPG2).

that specifically cleaves terminal glutamic acid in cures of established tumors after one course of9amides. Glutamic acid-substituted nitrogen mustards treatment [70]. The Fab fragment of the anti-CEA2

(Fig. 4) were prepared, and analyzed for stability in antigen Ab–A5B7 linked to CPG2 in combinationthe presence and absence of enzyme [41,66–69]. with 2 had significant effects on the outgrowth ofFrom these studies the monomesyl prodrug 2 (4[(2- OvCa-433 ovarian human tumor xenografts [72].chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glu- This work was extended to include a conjugate oftamate) had the highest ratio of CPG2 induced to CPG2 with the rat IgG2a mAb ICR12, recognizingnon-enzymatic hydrolysis rates [68,69]. The product the external domain of the human c-erbB2 proto-formed from enzymatic hydrolysis of 2 was the oncogene product [71]. The combination of ICR12–glutamic acid nitrogen mustard 5. Prodrug 2 (IC 5 CPG2 with 2 induced sustained dose-dependent50

21100 mM, K 5 3 mM, k 5 700 s ) exhibited the regressions of MDA MB361 tumors, persisting up toM cat

best in vivo therapeutic index within the series 90 days after only one course of treatment. Control[68,69]. Several features were incorporated into the chemotherapy in the same tumor model with conven-design of this family of prodrugs. It was anticipated tional drugs at maximum tolerated doses proved tothat the glutamic acid moiety within the prodrug be therapeutically ineffective. More recently, it haswould restrict its ability to transverse cell mem- been shown that the effects of mAb–CPG2/prodrugbranes, but that the much more lipophilic released combinations was significantly enhanced when useddrug would have a minimal barrier to penetration. In in conjunction with an antivascular agent [73].addition, at physiological pH, the carboxylic group Insight into the mechanism of therapeutic activityof the active drug is ionized resulting in an electron was gained through pharmacokinetic analyses, in-donating effect that activates the mustard group [67]. dicating that only in the tumor was there complete

The combination of mAb–CPG2 conjugates with conversion of the prodrug to drug [41,74]. In furtherprodrug 2 displayed significant in vivo therapeutic studies with this prodrug, active drug in the bloodeffects [68,70,71]. Initial studies were undertaken in was measurable in the absence of a pre-injection ofnude mice with subcutaneous human choriocar- the Ab–CPG2 conjugate [75]. This was shown to be

9cinoma xenografts. The combination of the Fab caused by bacteria in the gut flora and was readily2

fragment of the anti-human chorionic gonadotrophin prevented by antibiotic pre-treatment.Ab–W14 conjugated to CPG2 and prodrug 2 resulted In order to fine-tune the chemical reactivity of the


prodrugs, extensive mechanistic and quantitative parable to the values found optimum in nude micestructure activity relationships were examined. The bearing xenografts. Conjugate treatment was fol-factors that most strongly influenced the reactivity of lowed 24–48 h later by administration of a clearing

2the aromatic nitrogen mustards were the electronic agent, anti-CPG2 galactosylated Ab (220 mg/m ).character of the substituent para to the nitrogen The purpose of the clearing agent was to acceleratemustard, substitution on the aromatic ring, and the clearance of the A5B7–F(ab9) –CPG2 from the2

leaving groups of the alkylating moieties [76]. It was blood. In the final phase, 72 h after antibody–en-found that oxycarbonyl and carbamic linkages were zyme conjugate administration, prodrug was injected

2hydrolyzed by CPG2. This led to the development of over 1–5 days up to a total dose of 1.2–10 g/m .phenol mustard prodrugs 7 and 8, and the aniline Oral cyclosporin was associated with fatal toxicity inmustard prodrug, 9, which released the potent phenol two patients, but given i.v. delayed the host antibodyand aniline mustards 10–12 (Fig. 4). The prodrugs response allowing three cycles of therapy. A total ofwere 100- to 200-fold less toxic than the corre- eight of the 17 patients receiving ADEPT protocolssponding active drugs in LoVo colorectal tumor cells with adequate dosage were assessable and five of[77]. Administration of the A5B7–CPG2 conjugate these showed partial remissions or mixed responsesfollowed 3 days later by prodrug 7 led to tumor [80]. All patients developed IgG and IgM antibodiesregressions and growth delays in mice with LoVo to mouse immunoglobulins and carboxypeptidasetumors [78]. The bis-iodomustard 8 was a good G2. Clearly, the immune response against the foreign

21substrate for CPG2 (K 5 1 mM, k 5 30 s ), and protein illustrates a significant limitation in thisM cat

released the bis-iodo mustard, which had an IC particular treatment protocol.50

value of 0.34 mM. The chemical half-life of the Recently, pharmacokinetic studies were reportedactive drug was of the order of a few seconds, thus for colorectal cancer patients treated with the A5B7–minimizing the chances of active drug extravasation CPG2/prodrug combination [81]. CPG2 enzymeout of the tumor upon activation. This prodrug, in levels were determined by direct measurement ofcombination with mAb–CPG2 conjugates, produced enzyme activity and also using quantitative gammalong lasting regressions in the colorectal carcinoma camera imaging. Very high tumor to plasma ratiosLoVo tumor xenograft model [78]. were obtained prior to prodrug administration, but

Following an extensive series of studies in nude this required the use of a conjugate clearing agent asmice with different human xenografts, the first pilot mentioned earlier. Evidence for prodrug activationscale clinical trial of ADEPT was undertaken using was obtained.the prodrug 2 in combination with a conjugate The conjugate used in these studies was heteroge-

9prepared from CPG2 and the Fab fragment of the neous, since it was prepared using chemical cross-2

antiCEA mAb A5B7 [79,80]. During the initial linking reagents. A recombinant fusion protein com-stages of this study, evaluation of the prodrug alone posed of MFE-23 and CEA sFv fragments fused towas achieved in order to determine its safety and the amino terminus of CPG2 has been constructed totoxicity. The trial was carried out on six patients address this issue, and also to improve the phar-with advanced colorectal cancers in a dose escalating macokinetics of tumor uptake and conjugate clear-protocol. The patients received 1–12 doses (200 ance [82]. Recombinant MFE-23–CPG2 cleared

2mg/m , i.v.) of the prodrug 2, over 3 days to a rapidly from the circulation and led to tumor to2maximum total dose of 2400 mg/m . Nausea and plasma ratios of 10–19 within 72 h of conjugate

vomiting occurred as the only discernible toxic administration. This constitutes an improvement overeffects at the higher doses. After this initial phase, 17 the chemical conjugate.patients with advanced colorectal cancers of thelower intestinal tract received the full ADEPT treat- 3.4. Human enzymes: potential for reduced

2ment. Doses of 20 000 enzyme units /m (215 mg/ immunogenicity2m antibody–enzyme protein) of A5B7–F(ab9) –2

CPG2 were administered during the first phase. This Several promising approaches towards usingAb–enzyme dose gave plasma enzyme levels com- human enzymes for ADEPT have been reported. The


first example involved a mAb–b-glucuronidase in vivo. For example, the catalytic mAb described(mAb–GUS) fusion protein for the activation of a for the activation of a D-valine-5-fluorodeoxyuridinedoxorubicin glucuronide prodrug [33,83]. The fusion ester was shown to activate the prodrug, but with 5%protein consisting of a Fab9 fragment of the anti- molar equivalents of mAb compared to prodrug [86].CEA mAb BW431 linked to human placental GUS The observed cytotoxic effects of the catalytic mAb/was expressed in baby hamster ovary cells. Size prodrug combination could be explained by a singleexclusion chromatography indicated an average mo- turnover of the prodrug by each mAb variablelecular weight for the fusion protein of greater than region. Thus, there is no evidence that the mAb even250 kDa, and the binding properties of the com- acted in a catalytic manner. In a more recent set ofponent proteins were preserved. In vivo experiments experiments, a catalytic mAb activated prodrugs ofwere carried out in nude mice with subcutaneous doxorubicin and camptothecin through a sequentialhuman colorectal carcinoma xenografts. The conju- retro-aldol / retro-Michael reaction [88]. Although thegate localized in the tumors, but due to its high prodrugs were activated by the catalytic mAb, themolecular weight, blood and normal tissue clearance amount of mAb needed to achieve substantial drugtook approximately 1 week. Therapy experiments generation was almost stoichiometric. While thesewere conducted by injecting the fusion protein, studies do not exclude the possibility that catalyticfollowed 7 days later by the doxorubicin glucuronide mAbs can someday be used for targeted prodrugprodrug at 1 /6 the maximum tolerated dose. Signifi- activation, they certainly illustrate the need to im-cant therapeutic effects were obtained that were prove the kinetics of drug generation.greater than those achieved by systemic doxorubicin One of the most elegant approaches towards thetreatment. Pharmacokinetic studies indicated that the use of human enzymes for anticancer prodrug activa-BW431–GUS/prodrug combination led to higher tion involved site-directed mutagenesis to formintratumoral and lower normal tissue doxorubicin enzymes that hydrolyzed biologically stable prodrugslevels than treatment with doxorubicin, providing a [30,32]. A series of methotrexate amides wererationale for improved therapeutic efficacy. One of evaluated for stability towards human pancreaticthe interesting offshoots of this work was the discov- extracts, which is a major source for carboxypep-ery that the prodrug had some degree of selective tidase A1 (hCPA1). It was found that methotrexate-antitumor activity on its own, due to the release of phenylalanine was rapidly hydrolyzed, while some oflysosomal GUS from necrotic sites within solid the hindered derivatives, namely methotrexate-a-3-tumors [84]. As a result, the doxorubicin glucuronide cyclobutylphenylalanine and methotrexate-a-3-prodrug is being considered for development in the cyclopentyltyrosine, were stable. In addition, neitheruntargeted form as a tumor-selective prodrug. of these hindered methotrexate amides were hydro-

An exciting prospect for mAb–enzyme/prodrug lyzed by purified hCPA enzymes. Computer modelstherapy concerns the use of catalytic mAbs, known indicated that threonine-268 of the wild-type enzymeas abzymes, which are capable of effecting prodrug projected towards the hindered amide substituents.activation. Typically, these proteins are created by On the basis of this, a site-specific mutant wasimmunization of mice with a transition state ana- prepared in which threonine-268 was replaced withlogue of the reaction that is desired, followed by glycine, forming hCPA1–T268G. The mutated pro-isolation of the mAbs by hybridization techniques. tein was . 99% human and readily accepted both ofThe first report described a catalytic mAb capable of the bulky methotrexate derivatives as substrates.releasing chloramphenicol from an ester prodrug Conjugates of hCPA1–T268G were prepared using[85]. Since then, catalytic mAbs have been shown to standard crosslinking reagents, and were shown tobe capable of activating prodrugs of 5-fluorodeoxy- activate the hindered methotrexate prodrugs in anuridine [86], a nitrogen mustard [87], doxorubicin immunologically specific manner [32]. Since the[88], and camptothecin [88]. Unfortunately, the mutated enzyme is almost entirely of human origin,efficiencies of the catalytic mAbs are orders of it is unlikely to be as immunogenic as the bacterialmagnitude lower than what is necessary for immuno- and fungal enzymes described earlier. In vivo experi-logically specific prodrug activation both in vitro and ments performed in nude mice indicated that the


conjugate localized in human tumor xenografts, Another model was devised to analyze some of theproviding reasonably high tumor to blood ratios critical parameters for conjugate uptake and retention24–72 h post injection. With this information, within solid tumor masses [91]. It was predicted thattherapy experiments were undertaken in which the vascularity, target antigen density, and mAb on andmaximum tolerated dose of methotrexate-a-3- off rates would all strongly influence the amount ofcyclopentyltyrosine prodrug was administered in conjugate that could localize and be retained withinthree separate injections. There was no evidence of the tumor mass. The model was very accurate for theantitumor activity, owing possibly to the instability L49–sFv–bL fusion protein for both blood clearanceof the mutated enzyme within the tumor mass. Thus, and for tumor uptake. Therefore, the model could bealthough the specific approach failed to demonstrate used to design experiments in which the prodrug isin vivo prodrug activation, the in vitro effects administered at the optimal point in time. An addi-validate the concept of using mutated human en- tional study was undertaken using this model tozymes for prodrug activation. This general area predict how the dosing of the fusion protein wouldwarrants further investigation. affect tumor to blood ratios. Three different dosing

regimens were considered: a bolus injection, multipleinjections spaced several hours apart, and continuous

4. Mathematical models infusion. Several hours post conjugate administra-tion, the model predicted that the various dosing

The previous sections illustrate in general terms schedules would lead to similar tumor to bloodhow the localization, clearance, and enzyme kinetics conjugate ratios. This was confirmed experimentallyplay roles in prodrug activation and therapeutic for the bolus versus multiple injection routes.activity. Mathematical modeling of prodrug activa- Theoretical analysis and numerical simulationstion can lead to the identification of critical parame- were also used to elucidate the critical parameters forters for therapeutic efficacy, providing the basis for intratumoral and systemic drug generation [92]. Thedesigning optimized reagents and experimental de- model predicted that prodrug clearance from thesign. The first such analyses were based on a two- blood and the initial prodrug dose constituted thecompartment model comprising the tumor and plas- most important pharmacokinetic parameters. As withma [89]. One of the interesting conclusions of this the previously described mathematical modelwork was that under certain conditions, less efficient [89,90], tumor to blood drug concentration ratio wasenzymes would be expected to produce greater tumor strongly affected by the turnover rate of the targetedto blood drug ratios [90]. Highly efficient enzymes enzyme, such that enzymes with slightly reduced(low K , high V ) will very efficiently convert V values led to higher ratios. One of the interest-M max max

prodrug to drug in the blood, especially since the ing aspects of this study involved the distribution oftumor to blood prodrug ratio is not expected to be drug levels within the solid tumor mass. The rapidlyfavorable. On the other hand, low activity enzymes growing periphery of the tumor and the necrotic corewill result in only a fraction of the blood pool of were predicted to have the lowest drug concen-prodrug being converted to drug and will yield tumor trations. Taken together, these mathematical modelsto blood ratios that are more dependent on the provide a framework for the design of protocols formAb–enzyme distribution. The mathematical model improved therapeutic efficacy.was applied to some previously reported systems inwhich conjugate uptake and clearance, as well asprodrug and drug pharmacokinetics were known 5. Conclusions[90]. A good correlation was found between themodel and the experimental data for three mAb– Several mAb–enzyme/prodrug combinations haveenzyme/prodrug systems, in that the model accu- shown considerable levels of activity in preclinicalrately predicted the tumor and blood levels of mAb– models of human cancer. The active drugs generatedCPG2, mAb–GUS, mAb–CDase, along with their are quite diverse, ranging from clinically approvedrespective prodrugs and released drugs. drugs to agents that would be too toxic for clinical


M. Dahlbom, A. Raubitschek, K. Karvelis, T. Schultheiss,use in the untargeted form. Pharmacokinetic studiesT.E. Witzig, R. Belanger, S. Spies, D.H. Silverman, J.R.showing that active drug is generated intratumorallyBerlfein, E. Ding, A.J. Grillo-Lopez, Phase I / II 90Y-Zevalin

in concentrations that are not achievable through (yttrium-90 ibritumomab tiuxentan, IDEC-Y2B8) radioim-systemic drug administration provide a rationale for munotherapy dosimetry results in relapsed or refractory non-the high antitumor activities obtained. One of the Hodgkin’s lymphoma, Eur. J. Nucl. Med. 27 (2000) 766–

777.9ADEPT systems, A5B7–Fab –CPG2 for the activa-2[9] J.D. Hainsworth, Monoclonal antibody therapy in lymphoidtion of a nitrogen mustard prodrug, has shown signs

malignancies, Oncologist 5 (2000) 376–384.of activity in a Phase 1 clinical trial.[10] J. Stebbing, E. Copson, S. O’Reilly, Herceptin (trastuzamab)

Many challenges must be addressed before in advanced breast cancer, Cancer Treat. Rev. 26 (1998)ADEPT becomes a standard tool for cancer therapy. 287–290.

[11] S. Welt, G. Ritter, Antibodies in the therapy of colon cancer,It will be necessary to economically generate largeSemin. Oncol. 26 (1999) 683–690.quantities of recombinant fusion proteins or highly

[12] R.K. Jain, Delivery of molecular and cellular medicine tohomogeneous chemical conjugates in which thesolid tumors, J. Controlled Release 53 (1998) 49–67.

activities of the component proteins are preserved.[13] C.J. Springer, I. Niculescu-Duvaz, Antibody-directed en-

Ideally, the resulting conjugates should be non-im- zyme prodrug therapy (ADEPT): a review, Adv. Drug Deliv.munogenic, achieve high tumor to non-tumor ratios, Rev. 26 (1997) 151–172.

[14] K.N. Syrigos, A.A. Epenetos, Antibody directed enzymeand be retained in the tumors sufficiently long as toprodrug therapy (ADEPT): A review of the experimental andallow prodrug administration over extended periodsclinical considerations, Anticancer Res. 19 (1999) 605–614.of time. Finally, it is preferable to utilize enzymes

[15] M.P. Hay, W.A. Denny, Antibody-directed enzyme–prodrugthat can activate a wide panel of prodrugs. This will therapy (ADEPT), Drugs Future 21 (1996) 917–931.facilitate the treatment of tumors with agents they are [16] P.D. Senter, Activation of prodrugs by antibody–enzymehighly sensitive to, and will also enable the use of conjugates: a new approach to cancer therapy, FASEB J. 4

(1990) 188–193.prodrug combinations that lead to additive and[17] R.G.G. Leenders, E.W.P. Damen, E.J.A. Bijsterveld, H.W.synergistic activities. These challenges provide the

Scheeren, P.H.J. Houba, I. Van der Meulen-Muileman, H.E.framework for further research in this promisingBoven, H.J. Haisma, Novel anthracycline-spacer-b-glucuro-

therapeutic modality. nide, b-glucoside, and b-galactoside prodrugs for applicationin selective chemotherapy, Bioorg. Med. Chem. 7 (1999)1597–1610.

[18] P.J.J. Houba, R.G.G. Leenders, E. Boven, J.W. Scheeren,References H.M. Pinedo, H.J. Haisma, Characterization of novel anthra-

cycline prodrugs activated by human b-glucuronidase for usein antibody-directed enzyme prodrug therapy, Biochem.[1] G.M. Dubowchik, M.A. Walker, Receptor-mediated andPharmacol. 52 (1996) 455–463.enzyme-dependent targeting of cytotoxic anticancer drugs,

Pharmacol. Ther. 83 (1999) 67–123. [19] M.J. Sagnou, P.W. Howard, S.J. Gregson, E. Eno-Amooquaye, P.J. Burke, D.E. Thurston, Design and synthesis[2] W.A. Denny, The design of selectively-activated prodrugsof novel pyrrolobenzodiazepine (PBD) prodrugs for ADEPTfor cancer chemotherapy, Curr. Pharm. Des. 2 (1996) 281–and GDEPT, Bioorg. Med. Chem. Lett. 10 (2000) 2083–294.2086.[3] A.K. Sinhababu, D.R. Thakker, Prodrugs of anticancer

[20] M.P. Hay, B.M. Sykes, W.A. Denny, W.R. Wilson, A 2-agents, Adv. Drug Deliv. Rev. 19 (1996) 241–273.nitroimidazole carbamate prodrug of 5-amino-1-[4] R.V. Chari, Targeted delivery of chemotherapeutics: tumor-(chloromethyl)-3-[5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2 -activated prodrug therapy, Adv. Drug Deliv. Rev. 31 (1998)dihydro-3H-benz[E]indole (amino-seco-CBI-TMI) for use89–104.with ADEPT and GDEPT, Bioorg. Med. Chem. Lett. 9[5] U. Brinkmann, Recombinant antibody fragments and im-(1999) 2237–2243.munotoxin fusions for cancer therapy, In Vivo 14 (2000)

[21] G.J. Atwell, M. Boyd, B.D. Palmer, R.F. Anderson, S.M.21–27.Pullen, W.R. Wilson, W.A. Denny, Synthesis and evaluation[6] T.M. Illidge, P.W. Johnson, The emerging role of radioim-of 4-substituted analogues of 5-[N,N-bis (2-chloro-munotherapy in haematological malignancies, Br. J.ethyl)amino]-2-nitrobenzamide as bioreductively activatedHaematol. 108 (2000) 679–688.prodrugs using an Escherichia coli nitroreductase, Anticancer[7] E.L. Sievers, Targeted therapy of acute myeloid leukemiaDrug Des. 11 (1996) 553–567.with monoclonal antibodies and immunoconjugates, Cancer

Chemother. Pharmacol. 26 (2000) 18–22. [22] G.M. Anlezark, R.G. Melton, R.F. Sherwood, W.R. Wilson,[8] G.A. Wiseman, C.A. White, M. Stabin, W.L. Dunn, W. Erwin, W.A. Denny, B.D. Palmer, R.J. Knox, F. Friedlos, A.


Williams, Bioactivation of dinitrobenzamide mustards by an J.P. Gesson, J.C. Jacquesy, M. Mondon, B. Renoux, S.E. coli B nitroreductase, Biochem. Pharmacol. 50 (1995) Andrianomenjanahary, S. Michel, M. Koch, F. Tillequin, M.609–618. Gerken, J. Czech, R. Straub, K. Bosslet, Prodrugs of

[23] A.B. Mauger, P.J. Burke, H.H. Somani, F. Friedlos, R.J. anthracyclines for use in antibody-directed enzyme prodrugKnox, Self-immolative prodrugs: candidates for antibody- therapy, J. Med. Chem. 41 (1998) 3572–3581.directed enzyme prodrug therapy in conjunction with a [35] R. Lougerstay-Madec, J.C. Florent, C. Monneret, F. Nemati,nitroreductase enzyme, J. Med. Chem. 37 (1994) 3452– M.F. Poupon, Synthesis of self-immolative glucuronide-3458. based prodrugs of a phenol mustard, Anticancer Drug Des.

[24] M.P. Hay, W.R. Wilson, W.A. Denny, Nitrobenzyl carbamate 13 (1998) 995–1007.prodrugs of enediynes for nitroreductase gene-directed en- [36] T.L. Cheng, S.L. Wei, B.M. Chen, J.W. Chern, M.F. Wu, P.W.zyme prodrug therapy (GDEPT), Bioorg. Med. Chem. Lett. Liu, S.R. Roffler, Bystander killing of tumour cells by9 (1999) 3417–3422. antibody-targeted enzymatic activation of a glucuronide

[25] P.D. Senter, M.G. Saulnier, G.J. Schreiber, D.L. Hirschberg, prodrug, Br. J. Cancer 79 (1999) 1378–1385.¨ ¨J.P. Brown, I. Hellstrom, K.E. Hellstrom, Anti-tumor effects [37] J.L. Guerquin-Kern, A. Volk, E. Chenu, R. Lougerstay-

of antibody–alkaline phosphatase conjugates in combination Madec, C. Monneret, J.C. Florent, D. Carrez, A. Croisy,with etoposide phosphate, Proc. Natl. Acad. Sci. USA 85 Direct in vivo observation of 5-fluorouracil release from a(1988) 4842–4846. prodrug in human tumors heterotransplanted in nude mice: a

[26] P.D. Senter, G.J. Schreiber, D.L. Hirschberg, S.A. Ashe, K.E. magnetic resonance study, NMR Biomed. 13 (2000) 306–¨ ¨Hellstrom, I. Hellstrom, Enhancement of the in vitro and in 310.

vivo antitumor activities of phosphorylated mitomycin C and [38] Y.L. Leu, S.R. Roffler, J.W. Chern, Design and synthesis ofetoposide derivatives by monoclonal antibody–alkaline phos- water-soluble glucuronide derivatives of camptothecin forphatase conjugates, Cancer Res. 49 (1989) 5789–5792. cancer prodrug monotherapy and antibody-directed enzyme

[27] P.M. Wallace, P.D. Senter, In vitro and in vivo activities of prodrug therapy (ADEPT), J. Med. Chem. 42 (1999) 3623–monoclonal antibody–alkaline phosphatase conjugates in 3628.combination with phenol mustard phosphate, Bioconjug. [39] S. Desbene, H.D. Van, S. Tillequin, M. Koch, F. Schmidt,Chem. 2 (1991) 349–352. J.C. Florent, C. Monneret, R. Straub, J. Czech, M. Gerken,

[28] K.S. Vitols, B. Haag-Zeino, T. Baer, Y.D. Montejano, R.M. K. Bosslet, Application of the strategy to the MDR resistanceHuennekens, Methotrexate-a-phenylalanine: optimization of in cancer chemotherapy, Anticancer Drug Des. 14 (1999)methotrexate prodrug for activation by carboxypeptidase A– 93–106.monoclonal antibody conjugate, Cancer Res. 55 (1995) 478– [40] G.W. Philpott, W.T. Shearer, R.W. Bower, C.W. Parker,481. Selective cytotoxicity of hapten-substituted cells with an

[29] M.J. Perron, M. Page, Activation of methotrexate-a-phenyl- antibody–enzyme conjugate, J. Immunol. 111 (1972) 921–alanine by monoclonal antibody–carboxypeptidase A conju- 929.gate for the specific treatment of ovarian cancer in vitro, Br. [41] K.D. Bagshawe, C.J. Springer, F. Searle, P. Antoniw, S.K.J. Cancer 73 (1996) 281–287. Sharma, R.G. Melton, R.F. Sherwood, A cytotoxic agent can

[30] G.K. Smith, S. Banks, T.A. Blumenkopf, M. Cory, J. be generated selectively at cancer sites, Br. J. Cancer 58Humphreys, R.M. Laethem, J. Miller, C.P. Moxham, R. (1988) 700–703.Mullin, P.H. Ray, L.M. Walton, L.A. Wolfe, Toward [42] P.D. Senter, P.C.D. Su, T. Katsuragi, T. Sakai, W.L. Cosand,

¨ ¨antibody-directed enzyme prodrug therapy with the T268G I. Hellstrom, K.E. Hellstrom, Generation of 5-fluorouracilmutant of human carboxypeptidase A1 and novel in vivo from 5-fluorocytosine by monoclonal antibody-cytosinestable prodrugs of methotrexate, J. Biol. Chem. 272 (1997) deaminase conjugate, Bioconjug. Chem. 2 (1991) 447–451.

¨15804–15816. [43] D.E. Kerr, J. Garrigues, P.M. Wallace, K.E. Hellstrom, I.¨[31] C.J. Springer, V. Bavetsias, A.L. Jackman, F.T. Boyle, D. Hellstrom, P.D. Senter, Application of monoclonal antibodies

Marshall, R.B. Pedley, G.M. Bisset, Prodrugs of thymidylate against cytosine deaminase for the in vivo clearance of asynthase inhibitors: potential for antibody directed enzyme cytosine deaminase immunoconjugate, Bioconjug. Chem. 4prodrug therapy (ADEPT), Anticancer Drug Des. 11 (1996) (1993) 353–357.625–636. [44] P.M. Wallace, J.F. MacMaster, V.F. Smith, D.E. Kerr, P.D.

[32] L.A. Wolfe, R.J. Mullin, R. Laethum, T.A. Blumenkopf, M. Senter, W.L. Cosand, Intratumoral generation of 5-Cory, J.F. Miller, B.R. Keith, J. Humphreys, G.K. Smith, flourouracil mediated by an antibody–cytosine deaminaseAntibody-directed enzyme prodrug therapy with the T268G conjugate in combination with 5-fluorocytosine, Cancer Res.mutant of human carboxypeptidase A1: in vitro and in vivo 54 (1994) 2719–2723.studies with prodrugs of methotrexate and the thymidylate [45] E.O. Aboagye, D. Artemov, P.D. Senter, Z.M. Bhujwalla,synthase inhibitors GW1031 and GW1843, Bioconjug. Intratumoral conversion of 5-fluorocytosine to 5-fluorouracilChem. 10 (1999) 38–48. by monoclonal antibody–cytosine deaminase conjugates:

[33] J. Bosslet, J. Czech, D. Hoffmann, Tumor-selective prodrug noninvasive detection of prodrug activation by magneticactivation by fusion protein-mediated catalysis, Cancer Res. resonance spectroscopic imaging, Cancer Res. 58 (1998)54 (1994) 2151–2159. 4075–4078.

[34] J.C. Florent, X. Dong, G. Gaudel, S. Mitaku, C. Monneret, [46] R.P. Alexander, N.R.A. Beeley, M. O’Driscoll, F.P. O’Neill,


T.A. Millican, A.J. Pratt, F.W. Willenbrock, Cephalosporin cures of melanoma xenografts following treatment withnitrogen mustard carbamate prodrugs for ‘ADEPT’, Tetra- monoclonal antibody b-lactamase conjugates in combinationhedron Lett. 32 (1991) 3269–3272. with anticancer prodrugs, Cancer Res. 55 (1995) 3558–3563.

[47] R.P. Alexander, R.W. Bates, A.J. Pratt, J.A.E. Kraunsoe, A [60] H.P. Svensson, V.M. Vrudhula, J.E. Emswiler, J.F. MacMas-N-nitrosochloroethyl-cephalosporine carbamate prodrug for ter, W.J. Cosand, P.D. Senter, P.M. Wallace, In vitro and inantibody-directed enzyme prodrug therapy (ADEPT), Tetra- vivo activities of a doxorubicin prodrug in combination withhedron 16 (1996) 5983–5988. monoclonal antibody b-lactamase conjugates, Cancer Res.

55 (1995) 2357–2365.[48] D.E. Kerr, Z. Li, N.O. Siemers, P.D. Senter, V.M. Vrudhula,Development and activities of a new melphalan prodrug [61] S.D. Mikolajczyk, D.L. Meyer, J.J. Starling, K.L. Law, K.designed for tumor-selective activation, Bioconjug. Chem. 9 Rose, B. Dufour, R.E. Offord, High yield, site-specific(1998) 255–259. coupling of N-terminally modified b-lactamase to a

proteolytically derived single-sulfhydryl murine Fab,[49] V.M. Vrudhula, H.P. Svensson, K.A. Kennedy, P.D. Senter,Bioconjug. Chem. 5 (1994) 636–646.P.M. Wallace, Antitumor activities of a cephalosporin pro-

drug in combination with monoclonal antibody–b-lactamase [62] S.C. Goshorn, H.P. Svensson, D.E. Kerr, J.E. Somerville,conjugates, Bioconjug. Chem. 4 (1993) 334–340. P.D. Senter, H.P. Fell, Genetic construction, expression and

characterization of a single chain anti-carcinoma antibody[50] V.M. Vrudhula, H.P. Svensson, P.D. Senter, Cephalosporinfused to b-lactamase, Cancer Res. 53 (1993) 2123–2127.derivatives of doxorubicin as prodrugs for activation by

monoclonal antibody–b-lactamase conjugates, J. Med. [63] N.O. Siemers, D.E. Kerr, S. Yarnold, M.R. Stebbins, V.M.¨ ¨Chem. 38 (1995) 1380–1385. Vrudhula, I. Hellstrom, K.E. Hellstrom, P.D. Senter, Con-

struction, expression, and activities of L49–sFv–b-lactam-[51] M.L. Rodrigues, L.G. Presta, C.E. Kotts, C. Wirth, J.ase, a single-chain antibody fusion protein for anticancerMordenti, G. Osaka, W.L. Wong, A. Nuijens, B. Blackburn,prodrug activation, Bioconjug. Chem. 8 (1997) 510–519.P. Carter, Development of a humanized disulfide-stabilized

anti-p185HER2 Fv-beta-lactamase fusion protein for activa- [64] D.E. Kerr, V.M. Vrudhula, H.P. Svensson, N.O. Siemers, P.D.tion of a cephalosporin doxorubicin prodrug, Cancer Res. 55 Senter, Comparison of recombinant and synthetically formed(1995) 63–70. monoclonal antibody–b-lactamase conjugate for anticancer

prodrug activation, Bioconjug. Chem. 10 (1999) 1084–1089.[52] D.L. Meyer, L.L. Law, J.K. Payne, S.D. Mikolajczyk, J.Zarrinmayeh, L.N. Jungheim, J.K. Kling, T.A. Shepherd, J.J. [65] S.K. Sharma, Accelerated clearance systems, Adv. DrugStarling, Site-specific prodrug activation by antibody-b-lac- Deliv. Rev. 22 (1996) 315–324.tamase conjugates: preclinical investigation of the efficacy [66] J. Mann, M. Haase-Held, C.J. Springer, K.D. Bagshawe,and toxicity of doxorubicin delivered by antibody directed Synthesis of an N-mustard prodrug, Tetrahedron 46 (1990)catalysis, Bioconjug. Chem. 6 (1995) 440–446. 5377–5382.

[53] V.M. Vrudhula, H.P. Svensson, P.D. Senter, Immunologically [67] C.J. Springer, P. Antoniw, K.D. Bagshawe, F. Searle, G.M.F.specific cephalosporin derivative of mitomycin C by mono- Bisset, M. Jarman, Novel prodrugs which are activated toclonal antibody b-lactamase conjugates, J. Med. Chem. 40 cytotoxic alkylating agents by carboxypeptidase G2, J. Med.(1997) 2788–2792. Chem. 33 (1990) 677–681.

[54] D.L. Meyer, L.N. Jungheim, K.L. Law, S.D. Mikolajzcyk, [68] C.J. Springer, CMDA, an antineoplastic prodrug, DrugsT.A. Shepherd, D.G. Mackensen, S.L. Briggs, J.J. Starling, Future 18 (1993) 212–215.Site-specific prodrug activation by antibody–b-lactamase [69] C.J. Springer, P. Antoniw, K.D. Bagshawe, D.E.V. Wilman,conjugates: regressions and long-term growth inhibition of Comparison of half-lives and cytotoxicity of N-mesylox-human colon carcinoma xenograft models, Cancer Res. 53 yethyl-and N-chloroethyl-4-amino benzoyl compounds,(1993) 3956–3963. products of prodrugs in antibody-directed enzyme prodrug

[55] M.L. Rodrigues, P. Carter, C. Wirth, S. Mullins, A. Lee, B.K. therapy (ADEPT), Anticancer Drug Des. 6 (1991) 4671–Blackburn, Synthesis and b-lactamase-mediated activation of 4679.cephalosporin–taxol prodrug, Chem. Biol. 2 (1995) 223– [70] C.J. Springer, K.D. Bagshawe, S.K. Sharma, F. Searle, J.A.227. Boden, P. Antoniw, P.J. Burke, G.T. Rogers, R.F. Sherwood,

[56] S. Hanessian, J. Wang, Design and synthesis of a cephalo- R.G. Melton, Ablation of human choriocarcinoma xenograftssporin–carboplatinum prodrug activatable by a b-lactamase, in nude mice by antibody-directed enzyme prodrug therapyCan. J. Chem. 71 (1993) 896–906. (ADEPT) with three novel compounds, Eur. J. Cancer 27

[57] C.H. O’Callaghan, R.B. Sykes, S.E. Staniforth, A new (1991) 1361–1366.cephalosporin with a dual mode of action, Antimicrob. [71] S. Eccles, W.J. Court, G.A. Box, C.J. Dean, R.G. Melton,Agents Chemother. 10 (1976) 245–248. C.J. Springer, Regression of established breast carcinoma

[58] S. Mobashery, S.A. Lerner, M. Johnston, Conscripting b- xenografts with antibody-directed enzyme prodrug therapylactamase for use in drug delivery. Synthesis and biological against C-erbB2p185, Cancer Res. 54 (1994) 5171–5177.activity of a cephalosporin C 10-ester of an antibiotic [72] S.K. Sharma, J.A. Boden, C.J. Springer, P.J. Burke, K.D.dipeptide, J. Am. Chem. Soc. 108 (1986) 1685–1686. Bagshawe, Antibody-directed prodrug therapy (ADEPT): A

[59] D.E. Kerr, G.L. Schreiber, V.M. Vrudhula, H.P. Svensson, I. three-phase study in ovarian tumour xenografts, Cell Bio-¨ ¨Hellstrom, K.E. Hellstrom, P.D. Senter, Regressions and phys. 24/25 (1994) 219–228.


[73] R.B. Pedley, S.K. Sharma, G.M. Boxer, R. Boden, S.M. [87] P. Wentworth, A. Datta, D. Blakey, T. Boyle, L.J. Partridge,Stribbling, L. Davies, C.J. Springer, R.H.J. Begent, Enhance- G.M. Blackburn, Toward antibody-directed ‘abzyme’ pro-ment of antibody-directed enzyme prodrug therapy in drug therapy, ADAPT: carbamate prodrug activation by acolorectal xenografts by an antivascular agent, Cancer Res. catalytic antibody and its in vitro application to human tumor59 (1999) 3998–4003. cell killing, Proc. Natl. Acad. Sci. USA 93 (1996) 799–803.

[74] P. Antoniw, C.J. Springer, K.D. Bagshawe, F. Searle, R.G. [88] D. Shabat, C. Rader, B. List, R.A. Lerner, C.F. Barbas III,Melton, G.T. Rogers, P.J. Burke, R.F. Sherwood, Disposition Multiple event activation of a generic prodrug trigger byof the prodrug 4-[bis(2-chloroethyl)amino]benzoyl-L- antibody catalysis, Proc. Natl. Acad. Sci. USA 96 (1999)glutamic acid and its active parent drug in mice, Br. J. 6925–6930.Cancer 62 (1990) 909–914. [89] L.T. Baxter, F. Yaun, R.K. Jain, Pharmacokinetic analysis of

[75] C.J. Springer, P. Antoniw, K.D. Bagshawe, Endogenous the perivascular distribution of bifunctional antibodies andactivation of a prodrug in antibody directed enzyme prodrug haptens: comparison with experimental data, Cancer Res. 52therapy, in: A.A. Epenetos (Ed.), Monoclonal Antibodies: (1992) 5838–5844.Applications in Clinical Oncology, Vol. 1, Chapman & Hall,

[90] L.T. Baxter, R.K. Jain, Pharmacokinetic analysis of theLondon, 1991, pp. 185–191.

microscopic distribution of enzyme-conjugated antibodies[76] C.J. Springer, I. Niculescu-Duvaz, Antibody-directed en-

and prodrugs: comparison with experimental data, Br. J.zyme prodrug therapy (ADEPT) with mustard prodrugs,

Cancer 73 (1996) 447–456.Anticancer Drug Des. 10 (1995) 361–372.

[91] T.L. Jackson, S.R. Lubkin, N.O. Siemers, D.E. Kerr, P.D.[77] D.C. Blakey, D.J. Davies, R.I. Dowell, S.J. East, P.J. Burke,Senter, J.D. Murray, Mathematical and experimental analysisS.K. Sharma, C.J. Springer, A.B. Mauger, R.G. Melton,of localization of anti-tumour antibody–enzyme conjugates,Anti-tumour effects on an antibody-carboxypeptidase G2Br. J. Cancer 80 (1999) 1747–1753.conjugate in combination with phenol mustard prodrugs, Br.

[92] T.L. Jackson, P.D. Senter, J.D. Murray, Development andJ. Cancer 72 (1995) 1083–1088.validation of a mathematical model to describe anti-cancer[78] C.J. Springer, R. Dowell, P.J. Burke, E. Hadley, D.H. Davis,prodrug activation by antibody–enzyme conjugates, J. Theor.D.C. Blakey, R.G. Melton, I. Niculescu-Duvaz, OptimizationMed. 2 (2000) 93–111.of alkylating agent prodrugs derived from phenol and aniline

[93] D.W. Larden, H.T.A. Cheung, Synthesis of a-aminoacylmustards: a new clinical candidate prodrug (ZD2767) forderivatives of melphalan for use in antibody directed enzymeantibody-directed enzyme prodrug therapy (ADEPT), J.prodrug therapy, Tetrahedron 55 (1999) 3265–3276.Med. Chem. 38 (1995) 5051–5065.

[94] P.D. Senter, V. Vrudhula, P.M. Wallace, J.E. Somerville, I.K.[79] K.D. Bagshawe, S.K. Sharma, C. J Springer, P. Antoniw,Wang, D.A. Lowe, Sulfated etoposide and nitrogen mustardAntibody directed enzyme prodrug therapy: a pilot-scaleprodrugs and their activation by Streptomyces arylsulfatase,clinical trial, Tumor Targeting 1 (1995) 17–29.Drug Deliv. 2 (1995) 110–116.[80] K.D. Bagshawe, R.H.J. Begent, First clinical experience with

[95] D.C. Blakey, P.J. Burke, D.H. Davies, R.I. Dowell, S.J. East,ADEPT, Adv. Drug Deliv. Rev. 22 (1996) 365–367.K.P. Eckersley, J.E. Fitton, J. McDaid, R.G. Melton, I.A.[81] M.P. Napier, S.K. Sharma, C.J. Springer, K.D. Bagshawe,Niculescu-Duvaz, P.E. Pinder, S.K. Sharma, A.F. Wright,A.J. Green, J. Martin, S.M. Stribbling, N. Cushen, D.C.J. Springer, ZD2767, an improved system for antibody-O’Malley, R.H. Begent, Antibody-directed enzyme prodrugdirected enzyme prodrug therapy that results in tumortherapy: efficacy and mechanism of action in colorectalregressions in colorectal tumor xenografts, Cancer Res. 56carcinoma, Clin. Cancer Res. 6 (2000) 765–772.(1996) 3287–3292.[82] J. Bhatia, S.K. Sharma, K.A. Chester, R.B. Pedley, R.W.

[96] M. Azoulay, J.C. Florent, C. Monneret, J.P. Gesson, J.C.Boden, D.A. Read, G.M. Boxer, N.P. Michael, R.H. Begent,Jacquest, F. Tillequin, M. Koch, K. Bosslet, J. Czech, D.Catalytic activity of an in vivo tumor targeted anti-CEA-Hoffman, Prodrugs of anthracycline antibiotics suited forscFv-carboxypeptidase G2 fusion protein, Int. J. Cancer 85tumor-specific activation, Anticancer Drug Des. 10 (1995)(2000) 571–577.441–450.[83] K. Bosslet, J. Czech, P. Lorenz, H. Sedlacek, M. Schuer-

[97] R. Abraham, N. Aman, R. Von Borstel, M. Darsley, B.mann, G. Seemann, Molecular and functional characteriza-Kamireddy, J. Kenten, G. Morris, R. Titimas, Conjugates oftion of a fusion protein suited for tumour specific prodrugCOL-1 monoclonal antibody and beta-D-galactosidase canactivation, Br. J. Cancer 65 (1992) 234–238.specifically kill tumor cells by generation of 5-fluorouridine[84] K. Bosslet, R. Straub, M. Blumrich, J. Czech, M. Gerken, B.from the prodrug beta-D-galactosyl-5-fluorouridine, Cell Bio-Sperker, H.K. Kroemer, J.P. Gesson, M. Koch, C. Monneret,phys. 24–25 (1994) 127–133.Elucidation of the mechanism enabling tumor selective

[98] E. Bakina, D. Farquhar, Intensely cytotoxic anthracyclineprodrug monotherapy, Cancer Res. 58 (1998) 1195–1201.prodrugs: galactosides, Anticancer Drug Des. 14 (1999)[85] H. Miyashia, Y. Karaki, M. Kikuchi, I. Fujii, Prodrug507–515.activation via catalytic antibodies, Proc. Natl. Acad. Sci.

USA 90 (1993) 5337–5340. [99] J.P. Gesson, J.C. Jacquest, M. Mondon, P. Petit, B. Renoux,[86] D.A. Campbell, B. Gong, L.M. Kochersperger, S. Yonkovich, S. Andrianomenjanahary, H. Van Dufat-Trinh, M. Koch, S.

M.A. Gallop, P.G. Schultz, Antibody-catalyzed prodrug Michel, F. Tillequin, J.C. Florent, C. Monneret, K. Bosslet,activation, J. Am. Chem. Soc. 116 (1994) 2165–2166. J. Czech, D. Hoffmann, Prodrugs of anthracyclines for


chemotherapy via enzyme–monoclonal antibody conjugates, [102] G.S. Bignami, P.D. Senter, P.G. Grothaus, J.J. Fischer, R.Anticancer Drug Des. 9 (1994) 409–423. Humphreys, P.M. Wallace, N-(49-Hydrox-

[100] T. Yokota, D.E. Milenic, M. Whitlow, J. Schlom, Rapid phenylacetyl)palytoxin: a palytoxin prodrug that can betumor penetration of a single-chain Fv and comparison with activated by a monoclonal antibody–penicillin G amidaseother immunoglobulin forms, Cancer Res. 52 (1992) 3402– conjugate, Cancer Res. 52 (1992) 5759–5764.3408. [103] T. Sawa, J. Wu, T. Akaike, H. Maeda, Tumor-targeting

[101] V.M. Vrudhula, P.D. Senter, K.J. Fischer, P.M. Wallace, chemotherapy by a xanthine oxidase–polymer conjugateProdrugs of doxorubicin and melphalan and their activation that generates oxygen-free radicals in tumor tissue, Cancerby a monoclonal antibody–penicillin-G amidase conjugate, Res. 60 (2000) 666–671.J. Med. Chem. 36 (1993) 919–923.

selective activation of anticancer prodrugs by monoclonal antibody–enzyme conjugates

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