Regression of Established Breast Carcinoma Xenografts with ......Enzyme Prodrug Therapy against c-erbB2 p1851 Suzanne A. Eccies, William J. Court, Gary A. Box, Christopher J Dean,

[CANCER RESEARCH54, 5171-5177, October 1, 19941

ABSTRACT

The enzyme carboxypeptidase G2 (CPG2) was conjugated to the ratIgG2a monoclonal antibody (mAb) ICR12, which recognizes the externaldomain of the human c-erbB2 protooncogene producL The conjugateretained antigen-binding and enzyme activity. Radiolabeled conjugatelocalized efficiently and stably to MDA MB 361 breast carcinomaxenografts, which overexpress the c-erbB2 gene product p185. RadIotracer determinations and plasma enzyme activity studies in nulnu micegave conjugate blood clearance rate half-lives of approximately 4 days.In separate antibody-directed enzyme prodrug therapy regimes, one doseof the 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acidprodrug was administered to nu/nu mice bearing established MDA MB361 tumors (mean volume, 170â€”260mm3). In mice which had receivedICR12-CPG2 12â€”14days previously, sustained dose-dependent tumorstasis or regressions were effected, which in some cases persisted throughout observation periods of up to 90 days. In control mice which hadreceived the isotype-matched irrelevant mAb ICR16-CPG2 conjugate,tumors grew progressively, as did those in mice treated with prodrugalone, or treated simultaneously with ICR12-CPG2 and prodrug at themaximum tolerated dose. Control chemotherapy with conventional drugsproved toxic and induced only minimal growth delays.

INTRODUCTION

ADEPT3 has been proposed as a two-phase approach to targetedchemotherapy of human cancer (1). Briefly, in the first phase amonoclonal antibody conjugated to a nonendogenous enzyme is administered, and time is allowed for localization to tumors and clearance from the blood and other tissues. The second component is anontoxic prodrug which is converted to a cytotoxic drug by the actionof the targeted enzyme localized at the tumor sites. Theoretically, suchan approachshould enhancethe therapeuticindex of chemotherapeutic agents by minimizing systemic toxicity and maximizing drugconcentrations in tumor. In addition, unlike some other antibodytargeting strategies (such as delivery of chemoimmunoconjugates,immunotoxins, or activation of host effector mechanisms) this approach is not limited by tumor antigenic heterogeneity or the need tointernalize conjugates or recruit ancillary effectors (2).

Many different enzyme prodrug systems have been designed, eachwith their own advantages and disadvantages (recently reviewed inRef. 3). We have used the bacterial enzyme CPG2 (which has nomammalian homologue) to activate a glutamic acid prodrug derivativeof a benzoic acid mustard (4). Previous ADEPT experiments havedemonstrated the efficacy of mAb-CPG2-effected prodrug activationin choriocarcinoma (2, 5) and colorectal tumor models (6). Theadvantages of using mustard-alkylating agents as opposed to other

Received 5/23/94; accepted 8/16/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by The Cancer Research Campaign (United Kingdom) andMedical Research Council (United Kingdom).

2 To whom requests for reprints should be addressed.

3The abbreviations used are: ADEPT, antibody-directed enzyme prodrug therapy;CPG2, carboxypeptidase G2; CMDA, 4-[(2-chloroethyl@2-mesyloxyethyl)amino]benzoyl-L-glutamic acid prodrug; DMSO, dimethyl sulfoxide; EGFR, epidermal growthfactor receptor; mAb, monoclonal antibody; PBS, phosphate-buffered saline.

types of drugs is that their cytotoxicity is not cell-cycle specific and isdose related, and their use is not generally associated with the development of induced resistance (7). In ADEPT experiments with othersystems, an anti-colorectal carcinoma mAb conjugated to alkaline

phosphatase has been used with etoposide phosphate as prodrug toeffect tumor regressions (8). The same enzyme has been also provedbeneficial when conjugated to a mAb in a lung xenograft model, usingthe prodrugs mitomycin-phosphate (9) or phenol mustard-phosphate

(10). The enzyme @-lactamaseconjugated to mAbs in colon carcinoma models was effective with a ymca alkaloid-cephalosporin prodrug (11). Likewise, in a colorectal xenograft, a mAb fusion proteinof the enzyme j3-glucuronidase in combination with a doxorubicinglucuronide prodrug had some efficacy (12). To date, no group haspublished on the ADEPT system in a breast xenograft model.

The choice of antigenic target and of the corresponding mAb usedto target the enzyme are obviously of paramount importance. Thec-erbB2 protooncogene is amplified and/or overexpressed in 20â€”30%of many types of epithelial tumors, including those of breast, ovary,bladder, stomach, lung, and cervix; in many cases, this has beenlinked with poor prognosis (13â€”18).The cell surface location of thegene product (p185) and its relatively low expression on normal adult

tissues (19) makes antibody-guided therapy of such tumors an attrac

tive proposition.

We have described previously the production and characterizationof a panel of rat monoclonal antibodies directed against the externaldomain of human c-erbB2 p185 (20, 21). Of these, mAb !CR12 wasoutstanding in its ability to localize stably and specifically to humantumor xenografts overexpressing c-erbB2, (22, 23); a phase I trial ofradiolabeled mAb has proved its ability to localize in primary andsecondary tumors in breast cancer patients (24). In addition, unlikemany of the murine antibodies described (such as 4D5 and TA1),ICR12 does not readily internalize or down-modulate the target antigen but remains stably bound to the surface of cells for prolongedperiods (24). These properties suggested that ICR12 would be a goodcandidate for delivering enzymes capable of activating prodrugs inADEPT protocols; the present study was designed to investigate thefeasibility of this approach.

MATERIALS AND METHODS

Cell Lines and Xenografts. The humanbreastcarcinomacell line MDAMB 361 (which overexpresses the c-erbB2 protooncogene) and the squamous

carcinoma cell line LICR-LON-HN5 (which overexpresses EGFR), weremaintained in Dulbecco's modified Eagle's medium containing 10% heatinactivated fetal calf serum and the antibiotics penicillin, streptomycin, andneomycin. Xenograft tumors were established in female outbred athymic mice5 weeks of age by the s.c. inoculation of 5 X 106cells. Subsequent passagesof tumors were initiated from trocar fragments for up to 10 generations, duringwhich time the continued overexpression of the c-erbB2 oncoprotein wasconfirmed by immunohistochemical examination of randomly selected tumorsamples. Each mouse received two trocar fragments implanted s.c. at the

midpoint of each flank under hypnorm-hypnovel anesthesia. The animals were

housed according to British Home Office and !nstitutional guidelines in filterboxes in Maximiser laminar flow cabinets and fed sterilized food and water.All procedures were carried out in class 1 laminar flow hoods using sterileequipment and reagents.

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Regression of Established Breast Carcinoma Xenografts with Antibody-directedEnzyme Prodrug Therapy against c-erbB2 p1851

Suzanne A. Eccies, William J. Court, Gary A. Box, Christopher J Dean, Roger G. Melton, and Caroline J. Springers

CRC Centre for Cancer Therapeutics [C. J. S.] and immunology Section (5. A. E., W. J. C., G. A. B., C. J. DI, institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey.SM2 5NG; and Centre for Applied Microbiology Research, Popton Down, Wilts (R. G. MI, United Kingdom

on July 6, 2021. © 1994 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

REGRESSION OF TUMORS WITH ADEPT AGAINST c-erbB2

Prodrug. The CMDA prodrug was synthesized and characterized as described previously (25, 26).

Monoclonal Antibodies and Immunoconjugates ICR12 is a rat IgG2amAb raised against the external domain of the human c-erbB2 protooncogeneproduct p185 using the breast carcinoma cell line BT474 as immunogen (20).

ICR16, an isotype-matchedrat mAb directed against the external domain of therelated EGFR was used as an irrelevant mAb control (27). mAbs were purifiedfrom hybridoma culture supemanants by salt precipitation and ion-exchangechromatography as described previously (20). All preparations were dialyzedextensively against PBS, filter-sterilized, and stored frozen until use.

Preparation of Monoclonal Antibody-Enzyme Conjugates. CPG2 (Division of Biotechnology, Centre for Applied Microbiology Research, PortonDown, UK; specific activity, 456 units/mg) was conjugated to ICR12 or ICR16using a modification of the method described previously (28). A 5-fold molar

excess of 2-iminothiolane (Sigma Chemical Co., Poole, UK; 2.2 mg/ml inDMSO; 100 s.d) was added to the mAb (10 mg/ml). CPG2 (1 1 mg/ml; 23 ml)

in PBS was reactedwith a 5-fold molar excess of N-succinimidyl-4-(j-maleimidophenyl) butyric acid (Sigma; 6.7 mg/ml in DMSO; 100 pi). Themixtures were left for 30 mm. Excess 2-iminothiolane or N-succinimidyl-4-(-maleimidophenyl) butyric acid was removed on Sephadex 025 PD1Oaolumns and the modified proteins (1.5 mg/mI in PBS) were mixed and left for 12h at 4Â°C.Solid N-ethylmaleimide (1 mg; 30 mm) was added to terminate thereaction; then 2-mercaptoethanol (5 p.1)was added to cap off reactive thiol andmaleimide residues.

The conjugate was purified on a Superdex 0200 column in PBS on a fastprotein liquid chromatography system (Pharmacia). Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the fractions using 4â€”15%gradient Phastgels (data not shown). Peak fractions correspending in molecular weight to both M, 233,000 and 316,000 conjugates (1:1

and 2:1, respectively) were pooled, concentrated, and rerun under the sameconditions. Analysis of fast protein liquid chromatography traces indicated atypical ratio of 60% (1:1) and 40% (2:1) conjugate. This equates to an overall

ratio of approximately 1.3 CPG2 molecules/IgO. The final samples were filtersterilized and stored frozen until use. Two batches of ICR12-CPG2 and onebatch ofICRl6-CPG2 were assayed in the experiments described in this paper.

Radiolabeling of Antibodies and Immunoconjugates. â€˜@I(Nal; specificactivity, 100 mCi/mM) was obtained from ICN Biomedicals, inc. and was usedto radiolabelmonoclonalantibodiesor immunoconjugatesto a specific activityof 1 @Ci/@gusing the lodogen method (29). Potency of the labeled ICR12-CPG2 conjugate was determined by Scatchard analysis on MDA MB 361target cells in comparison with radiolabeled ICR12.

Quality Control of Immunoconjugates. Prior to their use in vivo, antibody-enzyme conjugates were tested to ensure that the coupling procedure hadnot compromised antigen-binding or enzymic activity. Briefly, the conjugateswere assessed for their ability to bind target cells in a competitive radioimmunoassay in comparison with unconjugated antibodies, and immunoreactivity was assessed as described previously (20) using Sepharose 4B beads coated

with an excess of antigen (p185, or EGFR for the control antibody). Enzymeactivity of the conjugates was determined by a spectrophotometric assay asdescribed previously (5).

In Vitro Cytotoxicity Assays. Sensitivity of the targetcells MDA MB 361to the CMDA prodrug and its CPG2-generated active derivative was tested invitro in a modification of the method described previously (4). Briefly, MDAMB 361 carcinoma cells were seeded onto 96-well microtiter plates at S X 10'cells/well and allowed to adhere overnight in a humidified CO2 incubator.Prodrug was dissolved in DMSO immediately prior to use, diluted in Dulbecco's modified Eagles's medium, adjusted to pH 7.4, and added to the wells atvarying concentrations between 1 @LMand 1 mst in triplicate wells. CPG2 (finalconcentration, 6 units/ml) was added to test wells in parallel with each dose of

prodrug to achieve activation. The treatment was repeated twice more at 24-hintervals. Controls consisted of untreated cells, or those receiving equivalentdoses of DMSO or enzyme alone. Cell viability was assessed after 7 days.Viable cells were fixed by a 15-mm incubation with 20 pi glutaraldehyde;nonviable cells were washed off and the remainder were stained by addition of100 @.d0.05% methylene blue. After 30 mm, the cells were washed and airdried, and the stain was solubilized with 0.33 N HO. The absorbance was readat 620 nm on a Titertek Multiscan, and the results were expressed as percentage of control cell values.

In Vivo Biodistribution of ICR12-CPG2 Conjugates. Two differentbatches (A and B) of antibody-enzyme conjugate were labeled with â€˜@â€˜lasdescribed above, and approximately 10â€”12@gof antibody (18â€”20,.&gtotalprotein; 0.83 and 0.66 units of enzyme activity, respectively) were injected i.v.into mice bearing MDA MB 361 breast carcinoma xenografts 3â€”4mm indiameter. Groups of 2â€”6mice were bled at intervals and at various times upto 14 days; randomly selected mice were killed by CO2 asphyxiation and theradioactivity in weighed samples of whole blood, normal tissues, and tumorswas determined in a Packard autogamma spectrometer. Results were expressedas the percentage of injected dose/g of tissue, and as tumor:normal tissueratios.

In VivoADEPT Therapy of EstablishedBreast CarcinomaXenografts.Groups of mice were implanted with MDA MB 361 breast carcinoma xcnografts bilaterally in the flanks as described above. After 14â€”16days ofgrowth, when the tumors had achieved a mean diameter of 3â€”4mm, mice wererandomized to some (in the pilot experiment) or all of the following treatmentgroups: (a) untreated controls; (b) ICR12-CPG2 conjugate alone; (câ€”e)prodrug alone at 600, 900, and 1200 mg/kg; (fâ€”h) ICR12-CPG2 conjugatefollowed later by administration of prodrug at 600, 900, and 1200 mg/kg; (i)irrelevant ICR16-CPG2 conjugate followed later by administration of prodrugat 1200 mg/kg; and (j) ICR12-CPG2 conjugate plus concomitant administration of prodrug at the maximum tolerated dose (systemically activated drug,non-pretargeted).

The conjugates were thawed and enzyme activity was determined prior touse. Micewereinoculatedi.v. via anexposedjugularveinunderhypnormhypnovel anesthesia at a standard dose of 50 units of CPO2/animal; this wasassociated with approximately 300â€”400 @gof ICR12 and 300 @gof ICRI6mAb, respectively. Control (group a) animals received i.v. inoculations of anequivalent volume of PBS under anesthesia. Group j mice received no treatment at this time.

A further group of â€œsentinelâ€•animals was also inoculated with conjugatecontaining 50 units of enzyme activity. These mice were bled at intervals, andenzyme levels were determined on fresh and/or frozen plasma samples.

Prior to CMDA prodrug administration in the second experiment, mice weregiven a short course of antibiotics to prevent any possible untoward activationof prodrug by caecal microflora, which has been observed previously (30).Briefly, the mice received fresh benzylpenicillin (0.5 g/liter) in their drinkingwater for S days, combined with streptomycin (2.5 mg/mI) on days 5 and 1.Prodrug was prepared immediately prior to use by dissolving in DMSO andthen 1.26% bicarbonate solution (1:20), and was administered i.p. to groupscâ€”cand fâ€”h.Mice were weighed, and the volumes injected were adjusted togive accurate individual doses on a mg/kg basis. Control animals received i.p.injections of vehicle at the maximum volume used. A pilot experiment haddetermined that the maximum tolerated dose of prodrug that could be administered concurrently with ICR12-CPG2 conjugate was 400 mg/kg. Accordingly, group j mice were anesthetised and inoculated i.v. with ICR12-CPG2from the same batch used in the other groups, and within 30 mm also receivedprodrug i.p. at 400 mg/kg. All mice were observed daily, and body weights andtumor measurements were recorded at frequent intervals. Tumor volume wascalculated according to the formula:

V = 4/3 ir[(d1 + d2)4J3

where d' and d2 = perpendicular diameters.Response of Established MDA MB 361 Xenografts to Conventional

Chemotherapeulic Agents. In order to compare the efficacy of the ADEPTprotocol with conventional chemotherapy, mice were transplanted with xcnograft MDA MB 361 tumors as before, and after 26â€”28days ofgrowth, whentumors reached 6â€”7mm in diameter (matching those at the time of prodrugadministration), the animals were randomized to receive one of a variety ofdrug treatments. The current standard adjuvant treatment for breast cancer is acombination of cyclophosphamide + methotrexate + 5-fluorouracil, given asseveral courses comprising both bolus and infusional administration. Clearly,such a protocol is impractical in small immunocompromized rodents, and thedifferential drug sensitivity of the two species (particularly to antifolates)renders the design of an equivalent therapeutic regime difficult. We attemptedtwo schedules of combined therapy comprising cyclophosphamide + methotrexate + 5-fluorouracil at 200/40/63 mg/kg and 130/20/50 mg/kg administered i.p. as a single bolus, but both proved highly toxic and led to deaths of

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with the parallel radiotracer study. Plasma samples which were snapfrozen and thawed gave results that were not significantly different

T from those obtained with fresh material, and blood samples fromnormal mice gave negative results in all cases.

Tumor and Normal Tissue Distribution ofRadiolabeled ICR12-CPG2 Conjugates. The biodistribution of ICR12-CPG2 conjugate Bassayed at 7, 11, and 14 days following administration is shown inFig. 4. Tumor localization at 7 days was >10% of the injected dose/gand by 14 days significant levels remained tumor associated (2.42%i.d./g), despite the fact that tumor volume had increased 2â€”3-foldduring this period. Tumor to normal tissue ratios are illustrated in Fig.5, and ranged between 1.7 (blood) and 14.1 (gut) at day 14 whenprodrug was administered in the parallel ADEPT therapy experiment.The results obtained with ICR12-CPG2 conjugate A were almostidentical (data not shown).

10 20 30 40 50 60

REGRESSION OF TUMORS WITH ADEFF AGAINST c-erbB2

all mice treated. As an alternative, mice were treated with the maximumtolerated doses of drugs which have shown activity against breast cancer cells,namely doxorubicin, cisplatinum, and methotrexate. Doxorubicin was administered i.p. as 5 daily doses of 3.5 or 4.0 mg/kg, cisplatinum as 2 weekly dosesof 4.5 or 5.8 mg/kg, and methotrexate as 2 weekly doses of 40 mg/kg; eachexperimental group comprised 6â€”7mice. The animals were weighed, andtumor measurements taken as before.

RESULTS

In Vitro Activity of Conjugates. The immunoreactivity of radiolabeled ICR12-CPG2 conjugates was estimated to be between 70 and76%, which compares favorably with the value obtained for ICR12alone (79%). Scatchard analysis using MDA MB 361 cell monolayerscomparing binding of ICR12 with ICR12-CPG2 (batch B) is shown inFig. 1. Unlabeled ICR12-CPG2 was found to compete with radiolabeled ICR12 for binding to target antigen as effectively as ICR12alone at equivalent molar ratios of antibody (data not shown).

MDA MB 361 target cells were tested for their susceptibility to thecytotoxic effects of prodrug and the activated derivative produced bythe action of CPG2 in vitro; the results are illustrated in Fig. 2.Minimal cytotoxicity was evident with doses of prodrug up to 800 p.M.whereas significant cell killing was obtained at all doses down to 100p.M with concomitant addition of CPG2 and prodrug.

Blood Clearance of Radiolabeled ICR12-CPG2 Conjugates andPlasma Enzyme Determinations from Sentinel Mice. The bloodclearance of two different radioiodinated ICR12-CPG2 conjugates isillustrated in Fig. 3, and compared with the rate of loss of enzymeactivity of unlabeled conjugates in plasma samples from sentinelmice. In spite of the fact that the unlabeled conjugate was administered at therapy levels approximately 30 times the antibody dose usedin the radiotracer studies (300â€”400 p.g versus 10â€”12p.g), the rate ofclearance in both assays was in most cases in good agreement,yielding half-life estimations of approximately 4 days.

In the pilot experiment (conjugate A), each plasma enzyme determination was from a single mouse, which may explain the onespurious point (0) at day 12. Once it became evident that 50 p.1 ofplasma would provide sufficient material for assay, we were able totake small repeated blood samples from groups of mice, and forconjugate B (mean of 5 mice/point), the results show excellent accord

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Fig. 2. in vitro cytotoxicity of CPG2-activated prodrug against MDA MB 361 breastcarcinoma cells assayed at day 7. 0, prodrug alone; â€¢,CPG2 plus prodrug; A, vehiclealone control. Points, mean; bars, SD.

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â€˜@I-ICR12-CPG2(â€¢)using MDA MB 361 breast carcinoma cells. Points, mean;bars, SD.

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in Vivo ADEPT Treatment of Established MDA MB 361 BreastCarcinoma Xenografts. A pilot experiment was performed withconjugate A and a full therapy experiment with conjugate B. In thefirst experiment, on day 0, tumors were implanted, on day 16, ICR12-CPG2 conjugate A was administered i.v. to 3 groups of mice, and on

day 28 prodrug was administered i.p. at 600, 900, and 1200 mg/kg tothese animals and 3 parallel groups of mice which had not receivedconjugate. Two further groups of mice received vehicle inoculationsonly or ICR12-CPG2 conjugate only and served as controls. Therewere 3â€”5mice each bearing 2 tumors in each group. The mean tumorvolume at the time of prodrug treatment was 262 Â±41 mm@.

All of the mice treated with ICR12-CPG2 conjugate followed by1200 mg/kg prodrug died within days of treatment, indicating thatsystemic activation of prodrug by conjugate remaining in the blood 12days after administration was sufficient to induce toxicity (jlasmaenzyme levels, 0.59 units/mi based on measurements in sentinelmice). All remaining groups of mice which received prodrug (with or

without conjugate) sustained transient body weight losses of between5 and 12%. For this reason in the second experiment, a prophylacticantibiotic course of treatment was introduced.

In all mice treated with conjugate followed by the two lower dosesof prodrug, tumors regressed and reached a nadir of 28â€”30%of theirinitial volumes at 22 days post-drug injection, as shown in Fig. 6. At

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ICR12-CPG2 (conjugate A) ADEPT. , vehicle controls (a); 0, ICR12-CPG2 conjugatealone (b); 0, prodrug alone at 600 mg/kg (c); i@,prodrug alone at 900 mgfkg (d); V,prodrug alone at 1200 mghg(e); â€¢,ICR12-CPG2 ADEPT with pmdrug at 600 mgtkg(f);and A, ICR12-CPG2 ADEfl with prodrug at 900 mg/kg (g). Letters in parentheses referto treatment groups described in text.

35 days, most tumors began to regrow; the ADEPT treatments wereshown to have induced growth delays of 50â€”55days, and the experiment was terminated 64 days following prodrug administration (day92 of tumor growth). All control groups were killed when tumorsreached 10â€”12mm mean diameter (500â€”700mm@volume), whichoccurred at days 41â€”43of tumor growth (13â€”15days post-prodrug).There was no significant effect on tumor growth of ICR12-CPG2conjugate or prodrug alone at three dose levels.

In the full ADEPT therapy trial, the experimental groups were asdescribed above but with the addition of an antibody specificitycontrol (ICR16-CPG2) and a systemically-activated prodrug control(â€œMaterialsand Methods,â€•groups i and j, respectively). Each groupcomprised 5â€”6mice with 2 tumors each; conjugates (ICR12-CPG2batch B or ICR16-CPG2) were administered on day 14 of tumorgrowth, and prodrug (and conjugate in the case of group j mice) onday 28 when mean tumor volumes were 170 Â±26 mm3. Assays on agroup of 5 sentinel mice which had received ICR12-CPG2 indicatedenzyme levels of 0.32â€”0.35 units/mI plasma at this time. All micereceived antibiotics p.o. prior to prodrug administration.

Fig. 7 shows that all 3 ADEPT treatment groups responded byinhibition of tumor growth in a dose-dependent manner. Tumors inmice treated with conjugate plus 600 mg/kg prodrug remained static

for about 12 days and then regrew more slowly than controls, givinga growth delay of the order of 30â€”35 days. Tumors in mice treated

with ADEPT protocols incorporating 900 and 1200 mg/kg prodrugregressed to 24 and 4% of initial volumes, respectively. In the lattergroup no progressive growth of residual nodules was seen during aposttreatment observation period of 90 days; in the former group slowresumption of growth occurred after 65 days but tumors did notachieve their pretreatment volumes until 85 days post-prodrug. Toxicity as evidenced by body weight loss was minimal (maximum 6%).

ICR12-CPG2 or prodrug alone at 3 dose levels (as in the pilotexperiment) again had no significant effects on tumor growth (data

omitted for clarity). Treatment of mice with an irrelevant antibodyenzyme conjugate (ICR16-CPG2) prior to administration of the highest drug dose produced a growth delay of only 7 days. Cleavage ofprodrug to maximum tolerated levels of active drug in the systemiccirculation by concomitant administration of ICR12-CPG2 also

yielded tumor growth delays of only 7â€”9days, and 2 of 5 mice died2 days after treatment.

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Fig. 4. Biodistribution of radiolabeled ICR12-CPG2 in nu/nu mice bearing MDA MB361 breast carcinoma xenografts. Percentage injected dose of radiolabeled conjugate perg of tissue on day 7 (0), day 11 (0), and day 14 (@). Columns, mean; bars, SD.

3.

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Fig. 5. Tumor: normal tissue ratios ofradiolabeled ICR12-CPG2 in nu/nu mice bearingMDA MB 361 breast carcinoma xenografts on day 7 (i), day 11 (,CJ),and day 14 (a).

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DrugDosage mg/kgWt lossToxicity(%)â€œDeathsTumorGrowth

Delay(d)@'Doxorubicin4.0

(X5)222/66Doxorubicin3.5(X5)151/74Cia-platinum5.8(X2)231/73Cis-platinum4.5(X2)140/71Methotrexate40

(X2)80/70CMF200/40/63(xl)9/9NMCCMF130/20/50(Xl)12/12NM'

pies illustrate the feasibility and flexibility of the ADEPT system in avariety of tumor models. However, all these examples required at leasttwo courses of conjugate and prodrug in a treatment schedule to effecttumor regressions or stasis. This is the first experimental study on theuse of an ADEPT protocol which uses only one administration ofconjugate followed by one injection of prodrug to obtain long lastingtumor regressions in large established tumors. We are also the first toshow the efficacy of ADEPT in a breast tumor model that is resistantto conventional chemotherapy.

Amplification and overexpression of the c-erbB2 oncogene hasbeen unequivocally associated with poor prognosis in node-positive,and in some studies, node-negative breast cancer patients (3 1, 32).This is manifest in shorter relapse-free intervals, a higher probabilityof distant metastases [notably in brain and viscera (33)], and lower

overall survival (reviewed in Refs. 34 and 35). In addition, elevatedexpression of the c-erbB2 oncoprotein has been associated with alower probability of response to endocrine therapy or chemotherapy(36, 37). This factor alone does not account for poor patient survival,however, since a large retrospective study in Finland in patients whoreceived no adjuvant therapy also concluded that HER-2 (c-erbB2)overexpression was an independent indicator of poor prognosis (38).These data suggest that c-erbB2 overexpression defines a subpopulation of breast cancer patients at high risk of recurrent disease but inwhom conventional treatments are likely to be of limited value.

A variety of monoclonal antibodies have been raised against thec-erbB2 protein p185. Some of these have been shown to haveintrinsic growth-inhibitory activity against human tumor cells overexpressing the target antigen or to sensitize cells to the effects of drugs(e.g., cisplatinum) or cytokines (e.g., tumor necrosis factor a) (39â€”41). Other investigations have demonstrated the possibility of usingsuch mAbs or recombinant constructs to target toxins, radionuclides,or cytotoxic effector cells (42â€”44).To our knowledge this study is thefirst to demonstrate in a preclinical in vivo model the feasibility ofusing anti-c-erbB2 mAbs to target an enzyme for activation of prodrug in ADEPT protocols.

The rat monoclonal antibody selected for study has several important properties which differentiate it from the antibodies describedabove and render it particularly suitable for ADEPT. ICR12 recognizes the aglycosyl protein core of the external domain of c-erbB2p185, to which it binds with high affinity (0.2 nM). The complex is notshed or internalized to any appreciable extent, leading to long-termstable expression at the cell surface both in vitro and in vivo. We havenot been able to demonstrate any significant reactivity with shedreceptor or cross-reactive normal epitopes in sera or tissues, as hasbeen demonstrated for several murine antibodies (44â€”46), whichwould compromise tumor-associated prodrug activation in ADEPT.

In the present study, we utilized the human breast carcinoma cellline MDA MB 361. This line was derived from a brain metastasis andis inherently relatively chemoresistant. It contains 2â€”4copies of the

c-erbB2 gene and expresses about 9 X i0@ p185 molecules/cell,approximately 17-fold higher than normal levels. A recent paper (14)quantified c-erbB2 expression in breast cancers and demonstratedelevations of 11â€”19-fold,with the risk of recurrence directly proportional to the degree of overexpression. Thus, we conclude that MDAMB 361 is appropriate for studies concerning the feasibility of targeted therapy for breast cancer, since the levels of target antigenexpressed are within the range encountered clinically.

In separate experiments, we were able to show that pretargetedICR12-CPG2 conjugates could activate sufficient prodrug at the tumor site to lead to significant growth-inhibitory effects on large, wellestablished tumors without undue systemic toxicity. The stability ofthe tumor conjugate localization was such that administration of thesingle dose of prodrug could be delayed for 14 days to minimize

REGRESSIONOF TUMORSWITHADEPTAGAINSTc-erbB2

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Iâ€”

Days Post Prodrug AdministrationFig. 7. Growth of MDA MB 361 breast carcinoma xenografts in mice treated with

ICR12-CPG2 (conjugate B) ADEPT. Key to treatment groups: t vehicle controls (a);â€¢,ICR12-CPG2 ADEPT with prodrug at 600 mg/kg (f); A, ICR12-CPG2 ADEFF withprodrug at 900 mg/kg (g); V, ICR12-CPG2 ADEFF with prodrug at 1200 mg/kg (h); *,irrelevant ICR16-CPG2 ADEFF with prodrug at 1200 mg/kg (I); 0 , ICR12-CPG2 plusconcomitantly administered prodrug at 400 mg/kg (j). Letters in parentheses refer totreatment groups described in text.

in Vivo Chemotherapy of Established MDA MB 361 BreastCarcinoma Xenografts. Table 1 shows the outcome of attempts toinhibit the growth of established MDA MB 361 breast carcinomaxenografts using a variety of drugs and dosage regimes. All treatmentswere associated with severe toxicity, and in no cases did the tumorgrowth delay induced exceed 6 days.

DISCUSSION

Other authors have utilized different enzymes to activate distinctprodrug systems in ADEPT (3). Notably, antitumor effects wereobserved when alkaline phosphatase-antibody conjugates were usedin combination with phosphorylated etoposide and mitomycin C pro

drugs (8). This group has also reported encouraging data with antibody-enzyme conjugates of cytosine deaminase, penicillin V amidase,penicillin G amidase, and @3-lactamasein combination with a varietyof different prodrug classes in ADEPT (reviewed in Ref. 3). Promis

ing results have been reported by using @3-lactamasewith a vinblastinederivative prodrug in colorectal tumor xenografts (1 1). These exam

Table 1 Effect of conventional therapeutic agents on nu/nu mice bearing establishedMDA MB 361 breast carcinoma xenografts

Mice bearing established tumor xenografts were dosed with single or combinedtherapeutic agents as shown. Therapeutic effects were assessed by mean tumor growthdelay and toxicity by weight loss and death.

0 30 10 30

a Maximum body weight loss as % of initial weight.b Difference in time (d) taken for mean test and control tumor volumes to increase 3

fold.C Not measurable.

5175




activation in blood without the need for â€œclearingâ€•agents, such asthose described by Sharma et a!. (47). Our protocol is in accord withthe pharmacokinetic model of Yuan et aL (48), which predicts that thelonger the time delay between the antibody-enzyme conjugate and theprodrug administrations, the higher the therapeutic index. An alternative protocol currently being explored utilizes antibody fragmentslinked to CPG2 to reduce the time delay between conjugate andprodrug administration.

Previous experiments with radioiodinated ICR12 had indicated thatdehalogenation of the protein was not a significant problem (22, 23);the present study using radiolabeled ICR12-CPG2 conjugates alsosuggested that loss of the label was minimal. Because of this, wefound that radiotracer estimates of blood clearance of conjugateagreed closely with direct measurements of plasma enzyme activity.Toxicity was evident in the first study where the highest dose ofprodrug was administered 12 days following conjugate but not in themain study when administration was delayed until day 14(CPG2 = 0.35 units/ml). These figures provide a guide to the levelsof enzyme required for safe administration of prodrug and suggest thatin this system, concurrent radiotracer studies could be used to predictthe optimum interval between the first and second phases of treatment.

In summary, an ADEPT protocol utilizing CPG2 targeted toc-erbB2-overexpressing tumors with rat mAb ICR12 showed greatlyimproved antitumor activity compared with conventional chemotherapeutic regimes, and with less associated toxicity. The results highlight the importance of careful selection of antibody for the precisebiological properties required. Many previously described murinemonoclonal antibodies recognizing p185 are rapidly internalized

and catabolized, and their binding leads to down-regulation of thetarget antigen (43, 49), properties at variance with those ideallyrequired for ADEPT but suitable for some other applications asdescribed above. It is critical that generalizations regarding the potential applications of new mAbs directed against a particular targetare not drawn from studies on existing individual antibodies, since itis becoming clear that the epitope recognized, the isotype, and other

as yet undefined characteristics of each mAb will all contribute to theresults obtained (50).

In conclusion, c-erbB2 represents an appropriate target for ADEPTtreatment, using the ICR12 mAb in combination with the CPG2enzyme and the CMDA prodrug. The results of the ADEPT experiments described herein suggest that these components are ideallysuited for a clinical trial in breast carcinoma patients that overexpress c-erbB2. A clinical trial using the CMDA prodrug in cobrectal carcinoma patients with the anticarcinoembryonic antigenmAb A5B7, conjugated to CPG2, is in progress with promisingresults (51, 52).

ACKNOWLEDGMENTS

We thank Professor K. R. Harrap for support and helpful discussions,Professor K. D. Bagshawe for his careful review of the paper, Professor T. A.Connors for advice, and Dr. G. Rogers for useful information on conjugation.

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1994;54:5171-5177. Cancer Res Suzanne A. Eccles, William J. Court, Gary A. Box, et al. p185

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