[CANCER RESEARCH 60, 3384–3388, July 1, 2000]

Advances in Brief

Tumor-inhibitory Antibodies to HER-2/ErbB-2 May Act by Recruiting c-Cbl andEnhancing Ubiquitination of HER-21

Leah N. Klapper, Hadassa Waterman, Michael Sela, and Yosef Yarden2

Departments of Immunology [L. N. K., M. S.] and Biological Regulation [H. W., Y. Y.], The Weizmann Institute of Science, Rehovot 76100, Israel

Abstract

Overexpression of HER-2/ErbB-2, a homologue of the epidermalgrowth factor receptor, is associated with poor prognosis, and an ErbB-2-specific antibody is therapeutic when administered to patients withmetastatic breast cancer. To understand the mechanism underlying im-munotherapy, we concentrated on antibody- and epidermal growth fac-tor-induced degradation of ErbB-2. We show that enhanced degradationis preceded by poly-ubiquitination of ErbB-2. This process necessitatesrecruitment of the c-Cbl ubiquitin ligase to tyrosine 1112 of ErbB-2.Consequently, mutagenesis of this site retards antibody-induced degrada-tion. Thus, the therapeutic potential of certain antibodies may be due totheir ability to direct ErbB-2 to a c-Cbl-regulated proteolytic pathway.

Introduction

HER-2/ErbB-2 is a receptor tyrosine kinase that is a member of theEGF3-receptor family. By serving as a shared coreceptor for multiplestromal growth factors, ErbB-2 cooperates with ErbB-1 (EGF recep-tor) and the neuregulin receptors (ErbB-3 and ErbB-4; Ref. 1). Unlikelow expression in normal adult cells, ErbB-2 is overexpressed in;20–35% of human breast, gastric, lung, and prostate cancer. ErbB-2overexpression in breast carcinomas is correlated with poor prognosis,especially of node-positive cases (reviewed in Ref. 2). Extensive workin animal models has indicated that blocking ErbB-2 with mAbs canreduce the rate of tumor growth. These studies led to successfulclinical tests of a humanized anti-ErbB-2 mAb (3). Furthermore,patients with metastatic breast cancer showed improved responseswhen treated with a combination of chemotherapy and the mAb (4).Two potential mechanisms may underlie immunotherapy: (a) mAbscan inhibit the interaction of ErbB-2 with other family members (5);and (b) they can accelerate ErbB-2 degradation (6, 7).

Accelerated degradation of ErbB-1 following EGF binding (“down-regulation”) involves receptor ubiquitination and sorting of internal-ized molecules in the early endosome (8). The specific ubiquitin ligaseinvolved in this process has been recently identified as c-Cbl (9, 10),a major substrate of many tyrosine kinases (11). Because mAb-induced down-regulation of ErbB-2 is related to the process promotedby EGF binding to ErbB-1, we addressed the possibility that immu-notherapy targets ErbB-2 to a c-Cbl-regulated ubiquitination/degra-dation pathway. Here, we show that a tumor-inhibitory mAb canenhance a Cbl-mediated process of ubiquitination and degradation ofErbB-2.

Materials and Methods

Materials. mAbs directed against ErbB-2 have been described previously(5). A polyclonal rabbit antiserum was raised against a synthetic peptidecomprising the 15 COOH-terminal amino acids of the human ErbB-2. EGF(human, recombinant) and an anti-HA mAb were purchased from BoehringerMannheim. Antibodies were radiolabeled with Na125I (Amersham) by usingthe chloramine T method. The isolation of a recombinant v-Cbl-GST proteinand its subsequent binding to gluthatione-agarose has been described previ-ously (10). The lysis buffer contained 25 mM Tris (pH 7.5), 150 mM NaCl,0.5% Na-deoxycholate, 1% NP40, 0.1% SDS, 1 mM phenylmethylsulfonylfluoride, 0.5 mM Na3VO4, 5 mg/ml leupeptin, and 5mg/ml aprotinin. Washingsolution contained 25 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100,and 10% glycerol.

Construction and Transfection of Expression Vectors.To generate mu-tated ErbB-2 proteins, we used an oligonucleotide-directed mutagenesismethod with a site-directed mutagenesis kit (QuickChange; Stratagene, LaJolla, CA). Tyrosine 1112 was mutated to a phenylalanine using the oligonu-cleotide 59-CTCTACAGCGGTTCAGTGAGGACCC. The transmembranalvaline 659 was replaced by a glutamic acid with the oligonucleotide59-GTCTCTGCGGTGGAG GGCATTCTGCTG. The double mutant EF wasgenerated by introducing the V659E mutation into a receptor construct con-taining the Y1112F mutation. Expression vectors were introduced into CHOcells by using the Lipofectamine transfection method (Life Technologies, Inc.,Bethesda, MD) and into HEK-293T cells by using the calcium phosphateprecipitation method.

Determination of mAb Effect on Tumor Growth in Vivo. N87 cells(5 3 106) were injected s.c. into CD1/nude mice, followed by three i.p.injections of the mAbs on days 3, 7, and 10. Tumor parameters were measuredonce a week with calipers, and tumor volume was calculated according to theformula: tumor volume5 length3 width 3 height.

Receptor Down-Regulation.Cells grown in 24-well plates were incubatedat 37°C for various time intervals with or without mAb L26 (20mg/ml) inbinding buffer (0.01% albumin in DMEM). Cells were then rinsed three timesin ice-cold binding buffer. Surface-bound antibody molecules were removedby use of low pH wash (8). The level of receptor residing on the cell surfacewas then determined by incubating the cells at 4°C with a radiolabeled mAbL26 for 1.5 h.

Detection of Ubiquitinated Proteins. The ubiquitinated form of ErbB-2was detected in immunoprecipitates prepared from cells that were cotrans-fected with a plasmid encoding a HA-tagged ubiquitin [a gift from DirkBohmann (European Molecular Biology Laboratory, Heidelberg, Germany)]and the appropriate ErbB-2 construct. The receptor was immunoprecipitatedfrom whole cell lysates with a rabbit serum. Ubiquitin levels were determinedby immunoblotting with anti-HA antibodies.

Receptor Turnover. Confluent cell monolayers grown in 90-mm plateswere biosynthetically labeled with35S-methionine for 16 h. The cells werethen chased at 37°C for different time intervals, in the presence or absence ofmAb L26. ErbB-2 was immunoprecipitated and separated by gel electrophore-sis. Labeled proteins were detected by directly exposing the dried gel to anX-ray film.

In Vitro Binding of v-Cbl to ErbB-2. HEK-293T cells grown in 90-mmplates were transfected with pCDNA3 plasmids encoding either ErbB-1 (1mg)or ErbB-2 (2mg). Twenty-four h later, cultures were split into two plates, andforty-eight h after transfection cells were treated with EGF (10 min at 37°C).Cell lysates were then prepared, cleared from debris, and incubated withgluthatione-agarose beads that were precoupled to a v-Cbl-GST fusion protein.

Received 1/13/00; accepted 5/22/00.The costs of publication of this article were defrayed in part by the payment of page

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

1 Supported in part by NIH Grant CA-72981 and a grant from Genentech Inc.2 To whom requests for reprints should be addressed, at Department of Biological

Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel. Phone: 972-8-9343974; Fax: 972-8-9344116; E-mail: yosef.yarden@weizmann.ac.il.

3 The abbreviations used are: EGF, epidermal growth factor; CHO, Chinese hamsterovary; GST, gluthatione S-transferase; HA, hemagglutinin A; mAb, monoclonal antibody;WT, wild-type.

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After 2 h of incubation at 4°C, the beads were washed twice and boundproteins were dissociated in boiling sample buffer prior to gel electrophoresis.

Results and Discussion

The L26 mAb Down-Regulates ErbB-2 from the Surface ofHuman Cancer Cells and Inhibits Their Tumorigenic Growth inMice. We have previously shown that antibodies directed at severaldistinct epitopes of ErbB-2 inhibit tumor growthin vivo (5). Althoughthis suggests a variety of mechanisms underlying the inhibitory effect,the ability of mAbs to increase receptor turnover seems common tomany tumor-inhibitory mAbs (6, 7). To elucidate the underlyingmechanism, we selected antibody L26 as an example for mAbscapable of strong tumor inhibition. This previously described mAb (5)decreased the growth, in nude mice, of human gastric carcinoma cells(N87) overexpressing ErbB-2, in a dose-dependent manner (Fig. 1A).Mice receiving a total dose of 2 mg of purified L26 demonstrated analmost undetectable tumor growth along a period of 35 days. Becauseit has previously been shown that tumor-inhibitory mAbs can induce

the internalization of antibody-receptor complexes whereas a nonin-hibitory mAb cannot (7), we tested the ability of L26 to down-regulateErbB-2 from the surface of N87 cells. As shown in Fig. 1B, theamount of surface ErbB-2 declined significantly when cells wereexposed to mAb L26, reaching 50% of the initial level within 2 h. Thissuggests that the enhanced receptor internalization caused by L26reduces the number of surface receptors available for the transmissionof growth signals.

Antibody-induced Degradation of ErbB-2 Is Associated with anIncrease in Receptor Ubiquitination. Treatment with a mAb thatdown-regulates ErbB-2 should be manifested in the total cellularamount of this oncoprotein. Using the same cellular system appliedfor in vivo studies, we could show that cells exposed to the tumorinhibitor gradually lost the expression of ErbB-2 as a function of time(Fig. 2A). Extending the treatment to 4 h caused the complete disap-pearance of the protein from the cellular pool. However, a similarregimen of N28, a mAb that cannot inhibit tumor growth (7), did notsignificantly affect the levels of ErbB-2.

Because several receptor tyrosine kinases, including ErbB-1 (8) andthe platelet-derived growth factor receptor (12), undergo ligand-induced degradation through a mechanism that involves their poly-ubiquitination, we tested the possibility that mAb L26 can elevateubiquitination of ErbB-2. To examine this possibility, we transientlyexpressed ErbB-2 in CHO cells together with a peptide-tagged ubiq-uitin. This revealed basal ubiquitination of ErbB-2 and a moderateincrease on short treatment with L26, in receptor precipitates, as wellas in a corresponding protein band in whole cell lysates (Fig. 2B).Because heterodimerization of ErbB-2 with ErbB-1 has been shown to

Fig. 1. Tumor inhibition and down-regulation of cell surface expressed ErbB-2 by theL26 mAb.A, athymic mice received a s.c. injection of 53 106 N87 human gastric cancercells that overexpress ErbB-2. Three, 7, and 10 days later, mAb L26 was injected i.p. ata total dose of 0.1 mg (f), 0.25 mg (Œ), or 2 mg (E). Tumor volumes were measured atthe end of the indicated time periods. Saline-injected mice were used for control (M). Barsrepresent SDs for groups of six mice.B, N87 cells were grown to confluence in 24-wellplates, washed with binding buffer, and incubated at 37°C for the indicated time intervalsin the presence (F) or absence (E) of mAb L26 (20mg/ml). Monolayers were then rinsedtwice and stripped of surface-bound antibody by using a 7-min long incubation in a lowpH buffer. The level of ErbB-2 molecules that remained at the cell surface was determinedby binding of a radiolabeled mAb L26. Each point represents the average6 SD ofduplicates.

Fig. 2. Antibody- and ligand-induced degradation of ErbB-2 involves increased recep-tor ubiquitination.A, N87 cells were incubated at 37°C for the indicated time intervals inthe presence of mAbs L26 or N28 (each at 20mg/ml). ErbB-2 was immunoprecipitated(IP) by using a mixture of mAbs specific to its extracellular domain and immunoblotted(IB) with a rabbit polyclonal serum. Untreated cells served as controls (laneslabeled —).B, CHO cells were cotransfected with an ErbB-2 expression vector, along with plasmidsencoding a HA-tagged ubiquitin. Forty-eight h later, cell monolayers were treated for 30min at 37°C with or without mAb L26 (20mg/ml). ErbB-2 ubiquitination was detected byIB receptor precipitates (top) or whole cell lysates (bottom) with an anti-AH antibody.C,CHO cells were transfected, as described above, in the presence of a plasmid encodingErbB-1. Following stimulation by EGF (100 ng/ml, 10 min), cells were lysed and ErbB-2was specifically precipitated under conditions that break ErbB-1FErbB-2 heterodimers.The precipitated receptor was examined for ubiquitination, as described above. Whole celllysates were subjected to anti-ErbB-2 IB to compare receptor expression (bottom).

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shunt the endocytic pathways of the two proteins (13, 14), we testedthe ability of EGF to cause similar ubiquitination of ErbB-2. Indeed,ErbB-2 ubiquitination was remarkably increased on short stimulationwith EGF (Fig. 2C). By extensive washing of the immunoprecipitates,we assured that the increase in ubiquitination was attributed toErbB-2, rather than to a coprecipitated ErbB-1. Of relevance, directcomparison revealed that EGF-induced ubiquitination of ErbB-1 issignificantly more extensive than mAb-induced ubiquitination ofErbB-2 (data not shown). Likewise, the effect of EGF on ubiquitina-tion of ErbB-2 was reproducibly higher than that of antibodies toErbB-2. However, due to the short time course of treatment with EGF,we observed no significant effect on ErbB-2 degradation (Fig. 2C,bottom). Notably, ubiquitination of ErbB-2 has previously only beenshown to underlie the effect of neuregulin-1 (15). The addition of EGFand mAbs as mediators of this process suggests it to be a majormechanism that regulates ErbB-2 turnover.

c-Cbl and Its Putative Docking Site at Tyrosine 1112 of ErbB-2Mediate Increased Receptor Ubiquitination. Ligand-induced deg-radation and ubiquitination of ErbB-1 depend on the c-Cbl protein (8,10). However, due to the absence of a high-affinity ligand for ErbB-2,the causative relationships between c-Cbl and increased ErbB-2 ubiq-uitination are difficult to address. To circumvent this, we have intro-duced a point mutation in the transmembrane domain of the humanErbB-2 protein (mutant denoted V659E). A similar carcinogen-induced mutation in the rat ortholog (Neu) activates its transformingpotential, probably because it mimics ligand-induced dimerization ofthe oncoprotein (16). Consistent with the previously observed rapidturnover of the rat mutant, we noted basal ubiquitination of V659Ewhen ectopically expressed in CHO cells. However, ubiquitinationwas significantly increased on overexpression of c-Cbl (Fig. 3A), inline with the observation that homodimerization of ErbB-2 withsynthetic ligands weakly enhances coupling to c-Cbl (17). The rela-tion of Cbl-induced ubiquitination to degradation of ErbB-2 wasconfirmed by a decrease in V659E expression in c-Cbl-overexpress-ing cells, but the level of the WT form of ErbB-2 was not affected(Fig. 3B), conforming with its relatively slow degradation.

Anticipating c-Cbl involvement in the enhancement of ErbB-2ubiquitination and degradation, we next searched a putative Cbldocking site on ErbB-2. The consensus sequence Tyrosine-X-X-X-Proline has previously been characterized as the ZAP-70 binding sitefor c-Cbl (18), and we have shown that a similar motif surroundingtyrosine 1045 of ErbB-1 mediates the effect of Cbl on the EGFreceptor (10). Aligning the amino acid sequence of ErbB-2 with theabove proteins identified tyrosine 1112 as a potential site (Fig. 3C).By using mutagenesis, we replaced this tyrosine with a phenylalanine(mutant denoted Y1112F). To test the effect of the mutation on c-Cblbinding, we coexpressed the Y1112F mutant, or the WT form, to-

Fig. 3. c-Cbl recruitment is involved in ubiquitination of ErbB-2.A, an expressionvector encoding the transforming mutant of human ErbB-2 (V659E) was transfected intoCHO cells. Alongside, plasmids encoding a sequence-tagged ubiquitin, c-Cbl (1), or acorresponding empty vector (2) were also used. Twenty-four h later, receptor moleculeswere immunoprecipitated (IP) and their ubiquitination was detected by using an anti-HAantibody (top). Whole cell lysates were examined for the effect on receptor level (bottom).B, CHO cells were cotransfected with plasmids encoding the WT form of ErbB-2 or the

transforming mutant (V659E), along with a c-Cbl vector (1) or the respective emptyvector (2). Total cell proteins were examined for receptor expression 48 h later byimmunoblotting (IB) with an anti-ErbB-2 antibody.C, amino acid sequence alignment ofthe predicted Cbl-binding sites of ErbB-1 (10), ErbB-2, and ZAP-70 (18) is shown. Theconsensus tyrosine and proline residues are shown inbold. The indicated amino acidnumbering refers to the corresponding human proteins.D, HEK-293T cells were trans-fected with plasmids encoding ErbB-1 and ErbB-2 (either WT or Y1112F), and 24 h latercells were exposed for 10 min to EGF (100 ng/ml). Control cultures (Cont.) weremock-treated prior to cell lysis. Cell extracts were directly analyzed by IB with ananti-ErbB-2 antibody (bottom). Alternatively, extracts were first incubated with an im-mobilized v-Cbl-GST protein and association with ErbB-2 was analyzed by IB (top). Notethe up-smearing of ErbB-2, which is attributed to poly-ubiquitination of the WT form.E,ubiquitination effects of EGF (left) and a bivalent Fab fragment of the L26 mAb (right)were examined in CHO cells. For stimulation by EGF, the WT form of ErbB-2 and aY1112F mutant were introduced along with a plasmid encoding ErbB-1. Antibodystimulation was tested in cells expressing the V659E mutant of ErbB-2, and a doublemutant (V659E and Y112F) denoted EF. Ubiquitination was detected in receptor immu-noprecipitates (top), and the ErbB-2 level was determined in whole cell lysates (bottom).

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gether with ErbB-1. Following cellular stimulation with EGF, theY1112F and WT proteins were isolated and testedin vitro for bindingto a recombinant v-Cbl protein. The latter was preferred over c-Cblbecause its shorter structure excludes interactions that are not medi-ated by the Src homology 2 domain of Cbl (11). As predicted, theY1112F mutant, but not the WT form, lost the ability to bind the Srchomology 2 domain of Cbl (Fig. 3D,top). Concomitantly, this mutantexhibited no electrophoretic up-smearing, suggesting a reduction inpoly-ubiquitination (Fig. 3D). However, reblotting of the filter withcommercially available antiubiquitin antibodies yielded too highbackground (data not shown).

To examine the putative c-Cbl docking site in cells and itsrelevance to receptor poly-ubiquitination, we transiently expressedY1112F, or the WT form, together with ErbB-1. Although basalubiquitination has been observed under these conditions, EGFstimulation was able to enhance ubiquitination of the WT form, butnot of the single residue mutant (Fig. 3E). Thus, tyrosine 1112 isessential for ubiquitination of ErbB-2 in heterodimers. To test thisrequirement in homodimers, we introduced the Y1112F mutationinto the constitutively dimerized transforming mutant V659E (pro-ducing a double mutant, denoted EF). An increase in ubiquitinationof the EF receptor could not be induced by a bivalent fragment ofL26, but ubiquitination of the original V659E receptor, bearing atyrosine at position 1112, was readily induced (Fig. 3E), probablybecause L26 stabilized dimers. A high level of basal ubiquitinationdetected for the double mutant, could be the result of differingexpression levels (see total cellular protein in thebottompanel).Alternatively, additional mechanisms, independent of tyrosine1112, may be involved in ErbB-2 ubiquitination. One example isubiquitination by the benzoquinone geldanamycin that involves aninteraction of ErbB-2 with a member of the HSP90 family of stressproteins (19). Taken together, the results presented in Fig. 3attribute a role to c-Cbl in ligand-, antibody-, and also in mutation-induced ubiquitination of ErbB-2.

Enhanced Turnover of ErbB-2 Is Dependent On the Putativec-Cbl Docking Site. To study the involvement of the putative c-Cbldocking site in the induction of ErbB-2 degradation, we used HEK-293T cells. These cells enable very high expression of transfectedplasmids, which was particularly useful for testing effects on the rateof ErbB-2 turnover. Similar to observations made with N87 tumorcells (Fig. 1Band 2A), mAb L26 induced a decrease in the level of theWT ErbB-2 expressed in HEK-293T cells (Fig. 4A,right). However,mAb treatment of cells expressing the mutant Y1112F could notreproduce this effect (Fig. 4A,left), indicating that the putative c-Cbldocking site is essential for antibody-induced degradation of ErbB-2.Similarly, stimulation by EGF decreased the level of the WT (Fig.4B), but not of the Y1112F, form of ErbB-2 (Fig. 4B). MonitoringErbB-2 turnover by using metabolic labeling confirmed the shorthalf-life of the V659E transforming mutant relative to the WT form(Fig. 4C). We then used this mutant, the ubiquitination in CHO cellsof which was stimulated by mAb L26, to examine the involvement oftyrosine 1112 in antibody-induced ErbB-2 turnover. Treatment withmAb L26 rapidly decreased the amount of the V659E mutant (Fig.4D, right), but double mutant EF molecules harboring a phenylalanineat the putative c-Cbl-docking site were not degraded after mAbtreatment. This confirms the importance of tyrosine 1112 for anti-body-induced degradation of ErbB-2 and suggests that c-Cbl directsmAb-stimulated ErbB-2 molecules to proteasomal/lysosomal degra-dation, as it does for other receptors on stimulation with their naturalligands.

Cbl proteins emerge as pivotal players in the process, leading toinactivation of tyrosine kinase signaling pathways (9, 10). Relativeto other growth factor receptors, the coupling of c-Cbl to ErbB-2seems very weak, which may explain why this interaction escapeddetection by previous studies (17, 20). Nevertheless, the realizationthat breast cancer immunotherapy directs HER-2/ErbB-2 to thismajor pathway of negative regulation is consistent with severalobservations. For example, targeted inactivation of thec-cbl genein mice resulted in mammary hyperplasia, perhaps because c-Cblnegatively regulates the action of many mammary gland-derivedgrowth factors (21). Consistent with negative regulation by c-Cbl,infection of an ErbB-2/Neu-driven neuroblastoma with ac-cbl-

Fig. 4. The Cbl docking site at tyrosine 1112 is essential for antibody-, ligand-, andmutation-induced enhanced turnover of ErbB-2.A, receptor degradation was followed inHEK-293T cells transfected with plasmids encoding ErbB-2 (WT) or its mutant (Y1112F).Cells were incubated with or without mAb L26 (20mg/ml) at 37°C for the indicated timeintervals. Whole cell lysates were immunoblotted (IB) with an anti-ErbB-2 antiserum.B,ErbB-2 degradation was examined as above, except cells were cotransfected with anErbB-1 plasmid. Forty-eight h later, cells were treated with EGF (15 min at 37°C) and thelevel of ErbB-2 was determined in whole cell lysates.C, ErbB-2 turnover was determinedin cells transfected with the indicated constructs. Following biosynthetic labeling withradioactive methionine, cells were chased with fresh medium for the indicated timeintervals, and ErbB-2 was immunoprecipitated (IP). Subsequent to gel electrophoresis,labeled proteins were detected by directly exposing the dried gel to an X-ray film.D,V659E and its corresponding Y1112F double mutant (EF) were compared for their abilityto undergo antibody-induced turnover. Receptor turnover was followed as described inC,following incubation at 37°C with or without the L26 mAb (20mg/ml) for the indicatedtime intervals.

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encoding retrovirus caused tumor retardation in mice.4 The exactmechanism allowing c-Cbl to enhance ubiquitination of targetproteins is unknown, but similar to other ubiquitin ligase com-plexes, it may involve association with still unknown ancillaryproteins. Predictably, detailed understanding of ErbB-2 ubiquiti-nation and degradation by a c-Cbl-mediated process will yieldimprovements in immunotherapy. For example, drugs knownto enhance ubiquitination and degradation of ErbB-2, suchas geldanamycin and herbimycin A (19), may enhance c-Cbl-mediated immunotherapy. Likewise, mAbs that better recruitErbB-2 to the c-Cbl-regulated endocytic pathway are expectedto act as superior therapeutic agents. Future studies will haveto resolve the intricate machinery leading to ErbB-2 degradation.

Acknowledgments

We thank Drs. Wallace Langdon (University of Western Australia, Ned-lands, Australia) for the c-Cbl cDNA and Dirk Bohmann (European MolecularBiology Laboratory, Heidelberg, Germany) for the HA-ubiquitin plasmid.

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