[CANCER RESEARCH 50, 3540-3544, June 15, 1990]

Ant ¡tumorActivity of the Monoclonal Antibody- Vinca Alkaloid Immunoconjugate

LY203725 (KSl/4-4-Desacetylvinblastine-3-carboxhydrazide) in a Nude Mouse

Model of Human Ovarian CancerLynn D. Apelgren,1 Dianna L. Zimmerman, Stephen L. Briggs, and Thomas F. Bumol

Department of Connective Tissue and Monoclonal Antibody Research [L. D. A., D. L. Z., T. F. BJ and Department of Chemical, Cancer, and Virology Research [S. L.BJ, Lilly Research Laboratories, Indianapolis, Indiana 46285

ABSTRACT

The activity of the conjugate of monoclonal antibody KS1/4 with 4-desacetylvinblastine-3-carboxhydrazide (KS1/4-DAVLB-HY) was explored in the OVCAR-3 human ovarian xenograft tumor model. Multipleschedules of KS1/4-DAVLB-HY administration were employed, including a comparison of i.p. and i.v. routes of treatment. When inoculates of6x10' OVCAR-3 cells were injected i.p. into female athymic nude mice,untreated control animals had a mean survival of 18-34 days, with thedevelopment of massive ascites and large intraabdominal tumors. Significant increases in survival were observed in KS1/4-DAVLB-HY conjugate-treated animals with all schedules utilized. Parallel therapy withequivalent doses of free DAVLB-HY or a non-antigen-binding immuno-conjugate did not significantly increase the survival of the animals. Thesedata demonstrate that the immunoconjugate KS1/4-DAVLB-HY significantly increases the survival of OVCAR-3 tumor-bearing mice andindicates that this immunoconjugate may be useful in the treatment ofhuman ovarian cancer.

INTRODUCTION

Ovarian cancer is one of the most common fatal gynecologicalmalignancies. The OVCAR-3 human ovarian carcinoma cellline grown i.p. in female athymic nude mice provides a modelsystem suitable for studying ovarian cancer (1-5). The progression of disease in this model closely parallels the human disease,with animals developing intraabdominal tumors and asciteswhich lead to death from bowel obstruction and respiratorycompromise (3). Since the disease remains confined to theperitoneal cavity throughout most of its course, it has beensuggested by a number of investigators that ¡.p.therapy withimmunoconjugates may be a useful treatment of this disease (1,2, 5-8).

Monoclonal antibodies have been successfully used in thediagnosis of gynecological carcinoma (9) and in radiotherapy(8, 10). Site-directed therapy of carcinoma with monoclonalantibody-drug conjugates or monoclonal antibody-toxin conjugates has been an area of intense investigation in recent years[for reviews see Pimm (7) and Pastan et al. (11)]. Fitzgerald etal. (I, 2) have documented antitumor activity in the OVCAR-3ovarian cancer model with Pseudomonas exotoxin or ricin Acoupled to a monoclonal antibody to the human TFR.2 In theseexperiments, the median survival time of the immunotoxin-treated animals was extended from 34 days for untreated control animals to 111 days with anti-TFR-ricin A (1) and from52 days for untreated control animals to 99 days with anti-JFR-Pseudomonas exotoxin (2). Monoclonal antibodies conjugated with either the Vinca alkaloid desacetylvinblastine (12)or DAVLB-HY (13-17) have been shown to regress human

Received 12/27/89; revised 2/28/90.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.JThe abbreviations used are: TFR, transferrin receptor; DAVLB-HY, 4-

desacetylvinblastine-3-carboxhydrazide; LERFI, linear equivalent mean fluorescence intensity.

adenocarcinoma or squamous carcinoma xenografts in athymicnude mice in several preclinical models, in an antigen-mediateddose-dependent manner. Since the KS1/4 antigen is highlyexpressed on human ovarian carcinoma (18), we investigatedthe effect of the monoclonal antibody- Vinca alkaloid immunoconjugate KS1/4-DAVLB-HY in the OVCAR-3 human ovarian carcinoma nude mouse model. The experiments reportedhere show that the immunoconjugate KS1/4-DAVLB-HY significantly prolongs the life of tumor-bearing animals, in a dose-dependent fashion, and suggest that it may be a potentialtherapeutic agent for ovarian cancer.

MATERIALS AND METHODS

Cell Line. The human ovarian cancer cell line OVCAR-3 [generouslyprovided by Dr. I. Pastan of the NCI (4, 11)] was routinely maintainedin vivo by serial transfer of ascites fluid in female athymic nude mice(outbred CD1; Charles River). Generally, 0.5 ml of ascites fluid containing approximately 6x10" cells (equal to 0.17 ml of packed cells)

was injected i.p. into the recipient mice. The mice were housed inisolator cages (Lab Products, Maywood, NJ) and all handling of theanimals was done under laminar flow hoods.

Monoclonal Antibodies. The hybridoma cell line KS1/4 (19) wasobtained through Scripps Clinic and Research Foundation. Monoclonalantibody KS1/4 defines an adenocarcinoma-associated antigen whichappears to be associated with epithelial malignancies and certain normalepithelial surfaces (18). The hybridoma cell line 9.2.27 (20) was alsoobtained from Scripps Clinic and Research Foundation. Monoclonalantibody 9.2.27 recognizes a chondroitan sulfate proteoglycan which iswidely regarded as a well defined tumor-associated antigen, with excellent expression in a high percentage of malignant melanomas, and itdoes not react with epithelial malignancies.

Immunoconjugate Preparation. DAVLB-HY was provided by G. J.Cullinan (Lilly Research Laboratories). KS1/4-DAVLB-HY (Lilly Research Laboratories serial number LY203725) and 9.2.27-DAVLB-HYwere prepared as described previously (16). KS1/4-DAVLB-HY is acovalent conjugate of the monoclonal antibody KS1/4 with 4-desace-tylvinblastine-3-carboxhydrazide, a derivative of the Vinca alkaloidvinblastine. In some experiments, KS1/4-007B (a single secretor cloneof the KS1/4 hybridoma) was used in the preparation of the immunoconjugate. Briefly, the monoclonal antibody (10 mg/ml) was dialyzedagainst 0. l M sodium acetate buffer, pH 5.6, oxidized by treatment ofthis solution with 160 HIMsodium metaperiodate at 0°C,and subse

quently purified by Sephadex G-25 chromatography in 0.1 M sodiumacetate buffer, pH 5.6. This material was then coupled with DAVLB-HY (5 mM, 4°C,24 h) and purified by Sephadex G-25 chromatography

in phosphate-buffered saline, pH 7.4.Characterization of KS1/4-DAVLB-HY. The functional reactivity of

KS1/4-DAVLB-HY has been documented utilizing flow cytometricanalyses (21). Various aspects of the K.S1/4-DAVLB-HY conjugationchemistry and representative in vitro and in vivo activities have beenreported previously (16). In general, immunoconjugates with a molarconjugation ratio of 3.5 to 5.5 mol of Vinca alkaloid/mol of antibodywere used.

Immunofluorescence Assays. OVCAR-3 cells were harvested fromcarrier mice, washed in Dulbecco's modified Eagle's medium (GIBCO

Laboratories, Grand Island, NY) containing 10% fetal bovine serum3540

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ANTITUMOR ACTIVITY OF LY203725

(GIBCO), and resuspended in the same medium at a concentration of1 to 2 x IO6cells/ml. Samples containing 2 x IO5cells in 0.1 ml wereincubated with the immunoconjugates KS1/4-DAVLB-HY or 9.2.27-DAVLB-HY, at a concentration of 10 Mg/ml, for 30 min at 4°C.The

samples were washed 3 times with medium and then incubated withrabbit anti-mouse IgG-fluorescein isothiocyanate (Kirkegaard & PerryLaboratories, Inc., Gaithersburg, MD) for 30 min at 4°C.Finally, the

cells were washed 3 times, resuspended in 0.1 ml of medium, andexamined using a Olympus fluorescent microscope equipped with ax40 objective.

Survival Experiments. On day 0 of each experiment, cells wereharvested from carrier mice, washed in normal saline, and immediatelyinjected into female athymic nude mice. The typical inoculate containedapproximately 6 x IO7OVCAR-3 cells, which is equivalent to 0.17 ml

of packed cells. The use of ascites fluid as the test inoculate leads to aninherent variability in this assay due to the issue of cell viability, sinceOVCAR-3 cells grow in large clusters, making a viable cell countdifficult. Since each experiment is set up with the same ascites inoculate,test groups within the experiment are directly comparable to each other.When comparing different experiments, however, the possible variability of the assay must be kept in mind. Untreated control animals havea mean survival of 18 to 34 days after inoculation. Dosing schedulesusing four or six treatments with the immunoconjugate KS1/4-DAVLB-HY, free DAVLB-HY, or the non-antigen-binding immunoconjugate 9.2.27-DAVLB-HY were employed and are indicated in"Results." Treatment was by either i.p. or i.v. injection in 0.2 ml, as

indicated in the text. The date each experiment was terminated isindicated in "Results." Statistical analysis was done for all in vivo

experiments, using a FORTRAN program for computing various statistical tests relating proportions to dose described by Thomas et al.(22). In brief, this statistical analysis utilizes the Kaplan-Meier survivalcurve analysis (23), which compares the survival of the various testgroups.

RESULTS

Immunoreactivity of KS1/4-DAVLB-HY. The antigen-binding capacity of KS1/4-DAVLB-HY and free KS1/4 monoclonalantibody, as a measurement of the LERFI by flow cytometrywith OVCAR-3 target cells, is shown in Fig. 1. LERFI iscomputed by converting the fluorescent intensity of cells collected by logarithmic amplifiers to the linear fluorescent equivalent units (24). These data indicate that essentially 100% ofthe functional reactivity of the unconjugated antibody is maintained in the immunoconjugate KS1/4-DAVLB-HY. The im-munoreactivity of KS1/4-DAVLB-HY was also assayed by livecell immunofluorescent microscopy, which generally gave aheterogeneous staining of moderate intensity against the OV-

ccLU

CONCENTRATION (ug/ml)Fig. 1. Flow cytometric analysis of KS1/4-DAVLB-HY binding to OVCAR-

3 target cells. The LERFI of unconjugated KS1/4 IgG is compared to theimmunoconjugate KS1/4-DAVLB-HY. See "Results" for an explanation of

LERFI.

CAR-3 target cell (data not shown). The immunoconjugate9.2.27-DAVLB-HY, as well as the unconjugated monoclonalantibody 9.2.27, did not react with the OVCAR-3 target cell in

this assay.In Vivo Comparison of KS1/4-DAVLB-HY and Free DAVLB-

HY. The in vivo efficacy of KS1/4-DAVLB-HY, as comparedto free DAVLB-HY, was evaluated in female athymic nudemice which had been inoculated i.p. with OVCAR-3 humanovarian cancer cells. The mice were given i.p. injections of KS1/4-DAVLB-HY or free DAVLB-HY at dose levels of 1 and 0.5mg/kg (based on mg of Vinca alkaloid per kg of animal bodyweight, i.e., Vinca alkaloid content of the immunoconjugate)on days 3, 6, 9, and 12 after inoculation of the OVCAR-3 cells(Fig. 2). Both the immunoconjugate KS1/4-DAVLB-HY andthe free drug DAVLB-HY were equally effective with thisprotocol. Based on this observation, therapy in all other experiments was delayed at least 10 days after inoculation of OVCAR-3 cells, to allow establishment of small tumors and ascitesfluid in the peritoneal cavity. A second protocol, with therapyon days 12, 15, 19, and 23, was also used with KS1/4-DAVLB-HY and DAVLB-HY and is shown in Fig. 3. KS1/4-DAVLB-HY was more effective in increasing the survival of the animalsthan the free drug DAVLB-HY at both dose levels tested,indicating a conjugate-mediated effect with this immunoconjugate. These data suggest that the OVCAR-3 xenograft must

A.

100 U

80-

> 60-

rrw 40 H

20-

é-

CONTROL

O— KS1/4-HY

--••-- DAVLB-HY

1 MG/KG (VINCA CONTENT)RX DAYS 3.6.9.12

20 40 60 80 100

DAY AFTER IMPLANT

120

B.

100

co

80-

60

40-

20-

CONTROL

KS1/4-HY

DAVLB-HY

0.5 MG/KG (VINCA CONTENT)RX DAYS 3.6.9.12

20 40 60 80 100 120

DAY AFTER IMPLANTFig. 2. Comparison of KS1/4-DAVLB-HY and free DAVLB-HY against

OVCAR-3 ovarian carcinoma. The animals (n = 10) were treated on days 3, 6, 9,and 12 after inoculation with OVCAR-3 cells, 1.0 mg/kg (Vinca content) (A) and0.5 mg/kg (B). This experiment is summarized in Table 1, experiment 1.

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ANTITUMOR ACTIVITY OF LY203725

A.

100

DAY AFTER IMPLANT

100 200

DAY AFTER IMPLANT

300

Fig. 3. Comparison of KS1/4-DAVLB-HY and DAVLB-HY against OV-CAR-3 ovarian carcinoma. The animals (n= 10) were treated on days 12, 15. 19,and 23 after inoculation with OVCAR-3 cells, with 1.0 mg/kg (Vinca content)(A) and 0.5 mg/kg (B). This experiment is summarized in Table 1. experiment 2.

be allowed to establish in the peritoneal cavity prior to administration of the immunoconjugate or the free drug; otherwise,the xenograft is equally susceptible to either agent. A statisticalsummary of the results is shown in Table 1. The results indicatethat a 5-fold increase in survival time with KS1/4-DAVLB-HYis obtained over that with free DAVLB-HY, compared tocontrol animals.

In Vivo Comparison of KS1/4-DAVLB-HY and 9.2.27-DAVLB-HY. To further evaluate the conjugate-mediated effectof KS1/4-DAVLB-HY in the OVCAR-3 ovarian carcinomanude mouse model, a non-antigen-binding, subisotype-equiva-lent immunoconjugate, 9.2.27-DAVLB-HY, was tested. Whenthe immunoconjugates 9.2.27-DAVLB-HY and KS1/4-DAVLB-HY were administered i.p. with a five-dose treatmentschedule, a significant increase in survival was obtained withKS1/4-DAVLB-HY at 0.25 and 0.5 mg/kg (Fig. 4). A significant increase in survival was not observed at 0.5 mg/kg with9.2.27-DAVLB-HY with this therapy schedule. A statisticalsummary of this experiment is shown in Table 1. These datasuggest that at least a 2-fold increase in survival time is obtainedwith KS1/4-DAVLB-HY over that with 9.2.27-DAVLB-HY,compared to untreated control animals.

in Vivo Comparison of i.p. and i.v. Therapy with KS1/4-DAVLB-HY. In order to compare the effect of the route ofadministration in the OVCAR-3 ovarian carcinoma nudemouse model, KS1/4-DAVLB-HY at 0.25, 0.5, and 1.0 mg/kg

(Vinca alkaloid content) was administered 4 times, either i.p.or i.v., on days 10, 13, 16, and 19 after inoculation of OVCAR-3 cells (Fig. 5). Both routes of administration produced significant survival of the test groups over the untreated controlanimals at all dose levels tested. There was no significantdifference between the i.p. and i.v. routes of therapy at the doselevels and treatment schedule used. A statistical summary ofthese results is shown in Table 1. These results suggest that a3- to 4-fold increase in survival time is obtained with eitherroute of therapy, compared to untreated control animals.

DISCUSSION

These experiments have described the effect of the immunoconjugate KS1/4-DAVLB-HY on the survival of athymic nudemice with OVCAR-3 human ovarian carcinoma xenografts.Initial experiments documented that animals with minimaltumor burden could be effectively treated with either freeDAVLB-HY or the KS1/4-DAVLB-HY chemoimmunoconju-gate (Fig. 2). These data suggest that animals with minimumtumor burden and a low level of ascites fluid are extremelysensitive to the oncolytic activity of free Vinca alkaloid with i.p.therapy administration, and no advantage was observed with asite-directed chemotherapy strategy. However, if sufficient timewas allowed for the establishment of small tumor lesions andascites development in the peritoneal cavity after inoculationwith the OVCAR-3 cells (7 to 10 days), the immunoconjugateKS 1/4-DAVLB-HY was significantly more effective than freeDAVLB-HY in increasing the survival of the animals, whencompared to the untreated control animals (Fig. 3). These datasuggest that the antitumor efficacy of KS1/4-DAVLB-HY isdue to a conjugate-mediated interaction with the OVCAR-3xenografts. Because the KS1/4-DAVLB-HY conjugate hasbeen characterized to release Vinca alkaloid, due to an inherentreversibility of the chemical linkage, it is possible that the effectsobserved were simply due to a carrier effect, in that the conjugate effected a nonspecific drug depot in the peritoneal cavity.However, antigen-mediated chemoimmunoconjugate effectswere subsequently demonstrated in experiments comparing thenon-antigen-binding immunoconjugate 9.2.27-DAVLB-HY toKS1/4-DAVLB-HY, where a significant increase in survivalcould be demonstrated with KS1/4-DAVLB-HY. With a five-dose treatment schedule, a significant difference between thetwo immunoconjugates was seen at the 0.5 mg/kg dose level(Fig. 4). These data support the hypothesis that the significantincrease in survival in the animals treated with the immunoconjugate KS1/4-DAVLB-HY is a target antigen-mediated conjugate response.

The efficacy of the immunoconjugate KS1/4-DAVLB-HY inthe OVCAR-3 xenograft model is comparable or superior tothat attainable with the immunotoxin conjugates using Pseu-domonas exotoxin or ricin A chain coupled to a monoclonalantibody to the human transferrin receptor (1, 2). These datamay reflect possible pharmacokinetic advantages of chemoim-munoconjugates over toxin subunit conjugates. Alternatively,the topography of the target antigen and the residence time ofthe chemoimmunoconjugate at the tumor site may play a critical role in the observed efficacy of KS1/4-DAVLB-HY. Despite these significant survival results, the durability of theefficacy observed with KS1/4-DAVLB-HY is currently understudy, to examine whether selection has occurred to antigenicvariants of the KS1/4 antigen or whether privileged sites existin the mouse which resist therapeutic intervention. Multiple-course therapy is also planned to examine these questions.

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Table 1 Inhibition of OVCAR-3 xenografts by KS1/4-DA VLB-HYEach animal was given an i.p. injection of 6 x IO7OVCAR-3 cells on day 0. The animals (n = 10) were treated on the days indicated in the treatment schedule.

The dose received was in mg/kg, based on the Vinca content of the ¡mmunoconjugate. Inhibition of xenograft growth was determined by increased survival time ofthe treated animals compared to the untreated control animals.

Experiment1234TreatmentControlKS1/4-HY'KS1/4-HYDAVLB-HYDAVLB-HYControlKS1/4-HYKS1/4-HYDAVLB-HYDAVLB-HYControlKS1/4-HYKS1/4-HY9.2.27-HVControlKS1/4-HYKS1/4-HYKS1/4-HYKS1/4-HYKS1/4-HYKS1/4-HYRouteDose(mg/kg)i.p.1.00.51.00.5i.p.1.00.51.00.5i.p.0.50.250.5i.v.i.v.

1.0i.v.0.5i.v.0.25i.p.1.0i.p.0.5i.p.

0.25Treatment

Mean Day ofschedule (day)fl survival (days)termination3,6,9,12

36 ±13110110+0''100±

22d102±IS"102±24d12,15,19,23

35 ±10233210±42"162

+70*40

±2260±25"12,15,18,21,24

27+12175139+44''119+49''76±5513,17,21,26

37 ±13176119±34'114±17'93

±38*137+34'119

±27'91±14'

" Days after implant of OVCAR-3 cells.* On the day of termination, all surviving animals were sacrificed and survival was statistically evaluated.' KS1/4-HY, KS1/4-DAVLB-HY (LY203725).* P < 0.001 as compared to control, in a x2 paired analysis.' 9.2.27-HY, 9.2.27-DAVLB-HY.

rr

V)

60-

40-

20-

0.5MG/KG (VINCA CONTENT)RX DAYS 12.15.18.21.24

50 100 150 200

DAY AFTER IMPLANTFig. 4. Comparison of KS1 /4-DA VLB-HY and 9.2.27-DAVLB-HY against

OVCAR-3 ovarian carcinoma with a five-dose treatment protocol. The animals(n = 9) were treated with 0.5 mg/kg (Vinca content) on days 12, 15, 18, 21, and24 after inoculation with OVCAR-3 cells. This experiment is summarized inTable 1, experiment 3.

Several investigators have examined the pharmacokinetics ofmonoclonal antibodies after i.p. or i.v. injection (25-27) intoanimals with gynecological tumors. Colcher et al. (25) haveshown that only a minor component of i.p. administered monoclonal antibody is found in the plasma. Furthermore, a complementation of i.p. and i.v. administration of monoclonal antibodies may occur, since they demonstrated that the monoclonalantibody B72.3 targeted peritoneal carcinoma métastasesafteri.p. administration and targeted hematogenously spread andregional lymph node métastasesafter i.v. administration. Preliminary data from this laboratory suggest that equivalent bloodlevels are achieved within 72 h after i.v. or i.p. administrationof [35S]methionine-labeled KS1/4.3 The pharmacokinetics and

3L. D. Apelgren et al., manuscript in preparation.

--- , 1 MG/KG (VINCA CONTENT)

I RX DAYS 13.17.21 26

so 100 150 200

DAY AFTER IMPLANTFig. 5. Comparison of i.p. and i.v therapy with KSI/4-DAVLB-HY against

OVCAR-3 ovarian carcinoma. The animals (n = 10) were treated either i.p. ori.v. on days 13, 17, 21, and 26 after inoculation with OVCAR-3 cells, with 1.0mg/kg (Vinca content). This experiment is summarized in Table 1, experiment4.

toxicology profiles of KS1/4-DAVLB-HY are currently beingexamined with i.p. and i.v. administration, to correlate theoptimal therapy schedule observed with the in vivo biodistribution of the chemoimmunoconjugate. As indicated in "Materials and Methods," ascites development is dependent on the

time after tumor cell inoculation, and it is possible that a largeamount of ascites fluid and accompanying tumor burden mayinhibit egress of the immunoconjugate to the circulation. Theinitial data presented in this manuscript, which compared i.p.therapy with i.v. therapy of KS1/4-DAVLB-HY, showed significant efficacy at all dose levels examined (Fig. 5). These datasuggest that both routes of administration can be efficaciousbut other parameters such as the toxicology profile of bothroutes and ultimate therapeutic index require further experimentation.

The route of administration of KS1/4-DAVLB-HY in the

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immunotherapy of ovarian carcinoma may be important withrespect to the normal tissue and marrow toxicities of the Vincaalkaloid. Previous studies have demonstrated that a key dose-limiting toxicity of the Vinca alkaloid desacetylvinblastine isleukopenia (28). Studies with the closely related Vinca alkaloidDAVLB-HY indicate a similar toxicology profile.4 If leuko

penia is indeed the dose-limiting clinical toxicity in therapywith KS1/4-DAVLB-HY, this toxicology parameter may bemodified by i.p. administration. Additional preclinical toxicology studies are currently in progress.

The studies reported here document that the immunoconju-gate KS1/4-DAVLB-HY is highly effective in increasing survival of athymic nude mice with the human ovarian carcinomacell line OVCAR-3 xenograft tumor. This activity was superiorto free drug administration and irrelevant chemoimmunocon-jugate controls. This report has also shown that i.p. and i.v.administration of the immunoconjugate were equally effectivein the OVCAR-3 model and suggests that either i.p. or i.v.therapy with the immunoconjugate KS1/4-DAVLB-HY may

be an effective treatment of human ovarian cancer.

ACKNOWLEDGMENTS

The authors would like to acknowledge Philip Marder for his excellent flow cytometric analyses. We also thank Dr. Ira Pastan for generously supplying the OVCAR-3 cell line.

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1990;50:3540-3544. Cancer Res   Lynn D. Apelgren, Dianna L. Zimmerman, Stephen L. Briggs, et al.   Mouse Model of Human Ovarian Cancer(KS1/4-4-Desacetylvinblastine-3-carboxhydrazide) in a NudeImmunoconjugate LY203725

AlkaloidVincaAntitumor Activity of the Monoclonal Antibody-

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